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Sample records for active chitinases chitotriosidase

  1. Chitotriosidase activity in the blood serum and organs of mice of various strains under the influence of chitin.

    PubMed

    Monoszon, A A; Cherkanova, M S; Duzhak, A B; Korolenko, T A

    2012-11-01

    Mouse chitotriosidase cleaving chitin belongs to the family of mammalian chitinases, whose biological functions are poorly understood. Chitotriosidase activity in mouse serum was shown to be much higher than in humans. The following interstrain differences were revealed in mouse chitotriosidase activity: GR>C57Bl/6>BALB/c>A/Sn>CBA. Chitotriosidase activity in CBA mice was lowest and practically did not differ from that in C3H/He and ICR mice. No sex-related differences were found in enzyme activity. Hybrids of opposite strains CBA and C57Bl/6 were characterized by dominant inheritance of this sign (elevated activity of chitotriosidase in the serum). Intragastric administration of chitin in a single dose of 100 mg/kg was followed by a decrease in chitotriosidase activity in the lungs, but not in the blood serum and homogenate of gastric cells from CBA mice. These data indicate that intragastric administration of chitin does not induce chitotriosidase in mice.

  2. Immunomodulatory Effects of Chitotriosidase Enzyme

    PubMed Central

    Elmonem, Mohamed A.; van den Heuvel, Lambertus P.; Levtchenko, Elena N.

    2016-01-01

    Chitotriosidase enzyme (EC: 3.2.1.14) is the major active chitinase in the human body. It is produced mainly by activated macrophages, in which its expression is regulated by multiple intrinsic and extrinsic signals. Chitotriosidase was confirmed as essential element in the innate immunity against chitin containing organisms such as fungi and protozoa; however, its immunomodulatory effects extend far beyond innate immunity. In the current review, we will try to explore the expanding spectrum of immunological roles played by chitotriosidase enzyme in human health and disease and will discuss its up-to-date clinical value. PMID:26881065

  3. Plasma chitotriosidase activity in patients with beta-thalassemia.

    PubMed

    Altarescu, G; Rudensky, B; Abrahamov, A; Goldfarb, A; Rund, D; Zimran, A; Elstein, D

    2002-09-01

    Variable increases in chitotriosidase levels have been reported in Italian patients with beta-thalassemia major and intermedia. We measured plasma chitotriosidase levels in Israeli patients with beta-thalassemia to ascertain its use as a universal marker of disease and/or response to therapy. Chitotriosidase levels in 39 adults (16-53 years; 30 with beta-thalassemia major, 9 with intermedia), and in 14 children (0.7-15 years; 12 with beta-thalassemia major, 2 with intermedia) were compared with other measures of disease, such as ferritin, hemoglobin, liver function tests, and genotype. Plasma chitotriosidase levels were normal (0.37 +/- 0.04 mU/mL) in all children. Twelve adults (31%) had elevated levels (>1.33 mU/mL): 11 patients (37%) with thalassemia major and 1 patient (11%) with thalassemia intermedia. A significant correlation was only found between plasma chitotriosidase levels and ferritin levels, and with mean number of transfusions per year. The patient with the highest chitotriosidase (1,440 nmol/mL/hr) had the highest ferritin (5,175 microg/L), required the most transfusions per year (40), and had abnormal liver tests. Normal chitotriosidase levels in the pediatric cohort and increased levels in only some adults may reflect status of iron overload in macrophages; thus there may be a role for monitoring chitotriosidase in patients with beta-thalassemia. Our results confirm results of the Italian cohort; however, in the latter, a more universal correlation was noted and chitotriosidase levels were much higher.

  4. Chitotriosidase Activity and Gene Polymorphism in Iranian Patients with Gaucher Disease and Sibling Carriers

    PubMed Central

    MOZAFARI, Hadi; TAGHIKHANI, Mohammad; KHATAMI, Shohreh; ALAEI, Mohammad Reza; VAISI-RAYGANI, Asad; RAHIMI, Zohreh

    2016-01-01

    Objective Chitotriosidase (CT) activity is a useful biomarker for diagnosis and monitoring of Gaucher disease (GD). Its application is limited by some variants in the CT gene. Two main polymorphisms are 24 bp duplication and G102S led to reduce CT activity. The aim of this study was to determine these variants influencing on plasma CT activity. Materials & Methods Blood samples were collected from 33 patients with GD, 15 sibling carriers and 105 healthy individuals serving as controls. CT activity was measured using 4-methylumbelliferyl-β-D-N,N′,N″triacetylchitotrioside substrate in plasma samples. The CT genotypes of 24 bp duplication and G102S variants were determined using PCR and PCR-RFLP. Results Untreated GD patients had a significantly higher CT activity compared to treated patients (P = 0.021). In addition, chitotriosidase activity in carriers was higher rather than controls. Allele frequencies of 24 bp duplication in GD patients, sibling carriers and controls were 0.21, 0.266 and 0.29 and for G102S were 0.318, 0.366 and 0.219, respectively. Different G102S genotypes had not significant effect on CT activity. Chitotriosidase activity has a positive correlation with age in normal group, carriers, and negative correlation with hemoglobin in GD patients. Using cut-off level of 80.75 nmol/ml/h, sensitivity and specificity of CT activity were 93.9% and 100%, respectively. Conclusion Chitotriosidase activity is a suitable biomarker for diagnosis and monitoring of GD. Determination of 24 bp duplication is helpful for more accurate monitoring the GD patient’s therapy. However, it seems that, specifying of the G102S polymorphism is not required for Iranian GD patients. PMID:27843468

  5. Increased chitotriosidase activity in plasma of patients with type 2 diabetes

    PubMed Central

    Knapik-Kordecka, Maria; Rorbach-Dolata, Anna; Piwowar, Agnieszka

    2016-01-01

    Introduction Chitotriosidase (CHIT1) is a chitinolytic enzyme involved mainly in the immune and inflammatory response. It shows increased activity in many pathologies, including in newly diagnosed type 2 diabetes (T2D). This study aimed to investigate this enzyme's activity in plasma of patients with ongoing T2D and indicate factors related to the increased activity of this enzyme. Material and methods Ninety-one patients and 46 control subjects without abnormalities in carbohydrate metabolism and inflammatory states were enrolled in the study. Plasma CHIT1 activity was measured by a spectrofluorometric method. Routine laboratory parameters such as blood glucose, total cholesterol and HDL fraction, triglyceride, glycated hemoglobin, white blood cell count and C-reactive protein were measured by standard methods. Results We found that the chitotriosidase activity was significantly higher (p < 0.001) in type 2 diabetic patients and positively associated with parameters of glycemic control (levels of glucose and glycated hemoglobin) and blood pressure. Plasma glucose level and systolic blood pressure were independent determinants of increased CHIT1 activity in T2D patients, even after adjustment for disease duration, body mass index, parameters of inflammation and lipid metabolism. We also found that increased CHIT1 activity was associated with occurrence of diabetic angiopathies. Conclusions This investigation indicates a possible role of chitotriosidase in the course of T2D, especially in relation to development of diabetic angiopathies. PMID:27695487

  6. Toxoplasma gondii Chitinase Induces Macrophage Activation

    PubMed Central

    Almeida, Fausto; Sardinha-Silva, Aline; da Silva, Thiago Aparecido; Pessoni, André Moreira; Pinzan, Camila Figueiredo; Alegre-Maller, Ana Claudia Paiva; Cecílio, Nerry Tatiana; Moretti, Nilmar Silvio; Damásio, André Ricardo Lima; Pedersoli, Wellington Ramos; Mineo, José Roberto; Silva, Roberto Nascimento; Roque-Barreira, Maria Cristina

    2015-01-01

    Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that is able to chronically infect almost all vertebrate species, especially birds and mammalians. Chitinases are essential to various biological processes, and some pathogens rely on chitinases for successful parasitization. Here, we purified and characterized a chitinase from T. gondii. The enzyme, provisionally named Tg_chitinase, has a molecular mass of 13.7 kDa and exhibits a Km of 0.34 mM and a Vmax of 2.64. The optimal environmental conditions for enzymatic function were at pH 4.0 and 50°C. Tg_chitinase was immunolocalized in the cytoplasm of highly virulent T. gondii RH strain tachyzoites, mainly at the apical extremity. Tg_chitinase induced macrophage activation as manifested by the production of high levels of pro-inflammatory cytokines, a pathogenic hallmark of T. gondii infection. In conclusion, to our knowledge, we describe for the first time a chitinase of T. gondii tachyzoites and provide evidence that this enzyme might influence the pathogenesis of T. gondii infection. PMID:26659253

  7. GENETIC ASSOCIATION BETWEEN HUMAN CHITINASES AND LUNG FUNCTION IN COPD

    PubMed Central

    Aminuddin, F.; Akhabir, L.; Stefanowicz, D.; Paré, P.D.; Connett, J.E.; Anthonisen, N.R.; Fahy, J.V.; Seibold, M.A.; Burchard, E.G.; Eng, C.; Gulsvik, A.; Bakke, P.; Cho, M. H.; Litonjua, A.; Lomas, D.A.; Anderson, W. H.; Beaty, T.H.; Crapo, J.D.; Silverman, E.K.; Sandford, A.J.

    2013-01-01

    Two primary chitinases have been identified in humans – acid mammalian chitinase (AMCase) and chitotriosidase (CHIT1). Mammalian chitinases have been observed to affect the host’s immune response. The aim of this study was to test for association between genetic variation in the chitinases and phenotypes related to Chronic Obstructive Pulmonary Disease (COPD). Polymorphisms in the chitinase genes were selected based on previous associations with respiratory diseases. Polymorphisms that were associated with lung function level or rate of decline in the Lung Health Study (LHS) cohort were analyzed for association with COPD affection status in four other COPD case-control populations. Chitinase activity and protein levels were also related to genotypes. In the Caucasian LHS population, the baseline forced expiratory volume in one second (FEV1) was significantly different between the AA and GG genotypic groups of the AMCase rs3818822 polymorphism. Subjects with the GG genotype had higher AMCase protein and chitinase activity compared with AA homozygotes. For CHIT1 rs2494303, a significant association was observed between rate of decline in FEV1 and the different genotypes. In the African American LHS population, CHIT1 rs2494303 and AMCase G339T genotypes were associated with rate of decline in FEV1. Although a significant effect of chitinase gene alleles was found on lung function level and decline in the LHS, we were unable to replicate the associations with COPD affection status in the other COPD study groups. PMID:22200767

  8. Differentiation of Chitinase-Active and Non-Chitinase-Active Subpopulations of a Marine Bacterium during Chitin Degradation

    PubMed Central

    Baty, Ace M.; Eastburn, Callie C.; Diwu, Zhenjun; Techkarnjanaruk, Somkiet; Goodman, Amanda E.; Geesey, Gill G.

    2000-01-01

    The ability of marine bacteria to adhere to detrital particulate organic matter and rapidly switch on metabolic genes in an effort to reproduce is an important response for bacterial survival in the pelagic marine environment. The goal of this investigation was to evaluate the relationship between chitinolytic gene expression and extracellular chitinase activity in individual cells of the marine bacterium Pseudoalteromonas sp. strain S91 attached to solid chitin. A green fluorescent protein reporter gene under the control of the chiA promoter was used to evaluate chiA gene expression, and a precipitating enzyme-linked fluorescent probe, ELF-97–N-acetyl-β-d-glucosaminide, was used to evaluate extracellular chitinase activity among cells in the bacterial population. Evaluation of chiA expression and ELF-97 crystal location at the single-cell level revealed two physiologically distinct subpopulations of S91 on the chitin surface: one that was chitinase active and remained associated with the surface and another that was non-chitinase active and released daughter cells into the bulk aqueous phase. It is hypothesized that the surface-associated, non-chitinase-active population is utilizing chitin degradation products that were released by the adjacent chitinase-active population for cell replication and dissemination into the bulk aqueous phase. PMID:10919822

  9. Identification and characterization of a novel chitinase with antifungal activity from 'Baozhu' pear (Pyrus ussuriensis Maxim.).

    PubMed

    Han, Peng; Yang, Chengcheng; Liang, Xiaobo; Li, Lirong

    2016-04-01

    A novel chitinase from the 'Baozhu' pear was found, purified, and characterized in this report. This chitinase was a monomer with a molecular mass of 28.9 kDa. Results of the internal peptide sequence analyses classify this chitinase as a class III chitinase. In the enzymatic hydrolytic assay, this chitinase could hydrolyze chitin derivatives into di-N-acetylchitobiose (GlcNAc2) as a major product in the initial phase, as well as hydrolyze GlcNAc2 into N-acetylglucosamine (GlcNAc), which represents both chitobiosidase and β-N-acetylglucosaminase activity. Biological analyses showed that this chitinase exhibits strong antifungal activity toward agricultural pathogenic fungi. In total, chitinase from 'Baozhu' pear is a novel bifunctional chitinase that could be a potential fungicide in the biological control of plant diseases.

  10. Chitotriosidase deficiency: a mutation update in an african population.

    PubMed

    Arndt, Silke; Hobbs, Angela; Sinclaire, Iain; Lane, Anthony B

    2013-01-01

    Human plasma chitotriosidase activity is a commonly used diagnostic and therapeutic biomarker for non-neuronopathic Gaucher disease. Chitotriosidase deficiency is common in non-African populations and is primarily caused by a 24 bp duplication in the encoding gene (CHIT1). Allele frequencies for the 24 bp duplication range from 20-50 % outside Africa. The present study found chitotriosidase deficiency to be rare in the South African Black population (1.6 %) and the otherwise common 24 bp duplication is absent in this African population. Instead, chitotriosidase deficiency is caused by a 4 bp deletion across the exon/intron 10 boundary (E/I-10_delGAgt) of the CHIT1 gene. The exact position of this mutation was found to differ from the previously reported location. Allele frequencies for six coding variants of CHIT1 (p.G102S, p.G354R, 24 bp duplication, E/I-10_delGAgt, p.A442V/G) were determined and the 4 bp deletion was found to be in complete linkage disequilibrium (LD) with two of the coding variants (p.G354R and p.A442V). The in silico assessments of the two missense mutations in LD predict a protein-damaging nature and functional studies are needed to clarify if one or both abolish the enzyme's activity. Overall, the low frequency of chitotriosidase deficiency in South African Blacks makes chitotriosidase activity an excellent biomarker of choice in this population.

  11. Human Chitotriosidase Is an Endo-Processive Enzyme

    PubMed Central

    Sørlie, Morten; Väljamäe, Priit

    2017-01-01

    Human chitotriosidase (HCHT) is involved in immune response to chitin-containing pathogens in humans. The enzyme is able to degrade chitooligosaccharides as well as crystalline chitin. The catalytic domain of HCHT is connected to the carbohydrate binding module (CBM) through a flexible hinge region. In humans, two active isoforms of HCHT are found–the full length enzyme and its truncated version lacking CBM and the hinge region. The active site architecture of HCHT is reminiscent to that of the reducing-end exo-acting processive chitinase ChiA from bacterium Serratia marcescens (SmChiA). However, the presence of flexible hinge region and occurrence of two active isoforms are reminiscent to that of non-processive endo-chitinase from S. marcescens, SmChiC. Although the studies on soluble chitin derivatives suggest the endo-character of HCHT, the mode of action of the enzyme on crystalline chitin is not known. Here, we made a thorough characterization of HCHT in terms of the mode of action, processivity, binding, and rate constants for the catalysis and dissociation using α-chitin as substrate. HCHT efficiently released the end-label from reducing-end labelled chitin and had also high probability (95%) of endo-mode initiation of processive run. These results qualify HCHT as an endo-processive enzyme. Processivity and the rate constant of dissociation of HCHT were found to be in-between those, characteristic to processive exo-enzymes, like SmChiA and randomly acting non-processive endo-enzymes, like SmChiC. Apart from increasing the affinity for chitin, CBM had no major effect on kinetic properties of HCHT. PMID:28129403

  12. Reduced Chitinase Activities in Ant Plants of the Genus Macaranga

    NASA Astrophysics Data System (ADS)

    Heil, Martin; Fiala, Brigitte; Linsenmair, K. Eduard; Boller, Thomas

    Many plant species have evolved mutualistic associations with ants, protecting their host against detrimental influences such as herbivorous insects. Letourneau (1998) reported in the case of Piper that ants defend their plants principally against stem-boring insects and also reduce fungal infections on inflorescences. Macaranga plants that were experimentally deprived of their symbiotic Crematogaster ants suffered heavily from shoot borers and pathogenic fungi (Heil 1998). Here we report that ants seem to reduce fungal infections actively in the obligate myrmecophyte Macarangatriloba (Euphorbiaceae), while ant-free plants can be easily infected. We also found extremely low chitinase activity in Macaranga plants. The plants' own biochemical defense seems to be reduced, and low chitinase activity perhaps may represent a predisposition for the evolution of myrmecophytism. These plants are therefore highly dependent on their ants, which obviously function not only as an antiherbivore defense but also as an effective agent against fungal pathogens.

  13. Low chitinase activity in Acacia myrmecophytes: a potential trade-off between biotic and chemical defences?

    NASA Astrophysics Data System (ADS)

    Heil, M.; Staehelin, Christian; McKey, D.

    We determined chitinase activity in leaves of four myrmecophytic and four non-myrmecophytic leguminous species at the plants' natural growing sites in Mexico. Myrmecophytic plants (or 'ant plants') have obligate mutualisms with ants protecting them against herbivores and pathogenic fungi. Plant chitinases can be considered a reliable measure of plant resistance to pathogenic fungi. The myrmecophytic Acacia species, which were colonised by mutualistic ants, exhibited at least six-fold lower levels of chitinase activity compared with the non-myrmecophytic Acacia farnesiana and three other non-myrmecophytes. Though belonging to different phylogenetic groups, the myrmecophytic Acacia species formed one distinct group in the data set, which was clearly separated from the non-myrmecophytic species. These findings allowed for comparison between two recent hypotheses that attempt to explain low chitinase activity in ant plants. Most probably, chitinases are reduced in myrmecophytic plant species because these are effectively defended indirectly due to their symbiosis with mutualistic ants.

  14. Effects of induced parturition in goats on immunoglobulin G and chitotriosidase activity in colostrum and plasma and on plasma concentrations of prolactin.

    PubMed

    Castro, N; Capote, J; Batista, M; Bruckmaier, R M; Argüello, A

    2011-05-01

    The effect of induction of parturition with a PGF(2)α analog on plasma concentration of prolactin (PRL) and its effects on colostrum concentration of IgG and chitotriosidase (ChT) activity were studied in 16 pregnant Majorera goats. Treated goats, those in which parturition was induced, had greater concentrations of PRL than control goats 24 h before parturition (P < 0.05) and 48 h after parturition (P < 0.05). Control goats had greater concentrations of PRL than treated goats 96 h after parturition (P < 0.05). Plasma concentration of IgG did not differ between groups during the experimental period, but colostrum concentrations of IgG were greater in control goats than in treated goats at parturition (P < 0.05). Plasma ChT activity decreased during the period 72 h before parturition to 24 h after parturition in control and treated goats. Time evolution after partum affected the colostrum ChT activity, being greater at parturition than after parturition in both groups (P < 0.05). In summary, concentration of IgG in colostrum is slightly diminished if parturition is induced. Induction of parturition causes an early increase in PRL, which is most likely responsible for preterm suppression of IgG transport into mammary secretions.

  15. [The induced increase of chitinase activity in tomato (Lycopersicon esculentum L) cells].

    PubMed

    Emel'ianov, V I; Dmitriev, A P

    2007-01-01

    The levels of chitinase activity induced with elicitors in tomato cells have been detected. It was shown that enzymatic activity depended on degree of polymerization and concentration of biotic elicitors.

  16. Purification and characterisation of a novel chitinase from persimmon (Diospyros kaki) with antifungal activity.

    PubMed

    Zhang, Jianzhi; Kopparapu, Narasimha Kumar; Yan, Qiaojuan; Yang, Shaoqing; Jiang, Zhengqiang

    2013-06-01

    A novel chitinase from the persimmon fruit was isolated, purified and characterised in this report. The Diospyros kaki chitinase (DKC) was found to be a monomer with a molecular mass of 29 kDa. It exhibited optimal activity at pH 4.5 with broad pH stability from pH 4.0-9.0. It has an optimal temperature of 60°C and thermostable up to 60°C when incubated for 30 min. The internal peptide sequences of DKC showed similarity with other reported plant chitinases. It has the ability to hydrolyse colloidal chitin into chito-oligomers such as chitotriose, chitobiose and into its monomer N-acetylglucosamine. It can be used to degrade chitin waste into useful products such as chito-oligosacchaarides. DKC exhibited antifungal activity towards pathogenic fungus Trichoderma viride. Chitinases with antifungal property can be used as biocontrol agents replacing chemical fungicides.

  17. Chitinase producing bacteria with direct algicidal activity on marine diatoms

    PubMed Central

    Li, Yi; Lei, Xueqian; Zhu, Hong; Zhang, Huajun; Guan, Chengwei; Chen, Zhangran; Zheng, Wei; Fu, Lijun; Zheng, Tianling

    2016-01-01

    Chitinase producing bacteria can involve extensively in nutrient cycling and energy flow in the aquatic environment through degradation and utilization of chitin. It is well known that diatoms cells are encased by box-like frustules composed of chitin. Thus the chitin containing of diatoms shall be a natural target of chitinase producing bacteria, however, the interaction between these two organismic groups has not been studied thus far. Therefore, in this study, the algicidal mechanism of one chitinase producing bacterium (strain LY03) on Thalassiosira pseudonana was investigated. The algicidal range and algicidal mode of strain LY03 were first studied, and then bacterial viability, chemotactic ability and direct interaction characteristic between bacteria and diatom were also confirmed. Finally, the characteristic of the intracellular algicidal substance was identified and the algicidal mechanism was determined whereby algicidal bacterial cells showed chemotaxis to algal cells, fastened themselves on algal cells with their flagella, and then produced chitinase to degrade algal cell walls, and eventually caused algal lysis and death. It is the first time to investigate the interaction between chitinase producing bacteria and diatoms, and this novel special interaction mode was confirmed in this study, which will be helpful in protection and utilization of diatoms resources. PMID:26902175

  18. Chitinase producing bacteria with direct algicidal activity on marine diatoms.

    PubMed

    Li, Yi; Lei, Xueqian; Zhu, Hong; Zhang, Huajun; Guan, Chengwei; Chen, Zhangran; Zheng, Wei; Fu, Lijun; Zheng, Tianling

    2016-02-23

    Chitinase producing bacteria can involve extensively in nutrient cycling and energy flow in the aquatic environment through degradation and utilization of chitin. It is well known that diatoms cells are encased by box-like frustules composed of chitin. Thus the chitin containing of diatoms shall be a natural target of chitinase producing bacteria, however, the interaction between these two organismic groups has not been studied thus far. Therefore, in this study, the algicidal mechanism of one chitinase producing bacterium (strain LY03) on Thalassiosira pseudonana was investigated. The algicidal range and algicidal mode of strain LY03 were first studied, and then bacterial viability, chemotactic ability and direct interaction characteristic between bacteria and diatom were also confirmed. Finally, the characteristic of the intracellular algicidal substance was identified and the algicidal mechanism was determined whereby algicidal bacterial cells showed chemotaxis to algal cells, fastened themselves on algal cells with their flagella, and then produced chitinase to degrade algal cell walls, and eventually caused algal lysis and death. It is the first time to investigate the interaction between chitinase producing bacteria and diatoms, and this novel special interaction mode was confirmed in this study, which will be helpful in protection and utilization of diatoms resources.

  19. Enzyme activity determination on macromolecular substrates by isothermal titration calorimetry: application to mesophilic and psychrophilic chitinases.

    PubMed

    Lonhienne, T; Baise, E; Feller, G; Bouriotis, V; Gerday, C

    2001-02-09

    Isothermal titration calorimetry has been applied to the determination of the kinetic parameters of chitinases (EC 3.2.1.14) by monitoring the heat released during the hydrolysis of chitin glycosidic bonds. Experiments were carried out using two different macromolecular substrates: a soluble polymer of N-acetylglucosamine and the insoluble chitin from crab shells. Different experimental temperatures were used in order to compare the thermodependence of the activity of two chitinases from the psychrophile Arthrobacter sp. TAD20 and of chitinase A from the mesophile Serratia marcescens. The method allowed to determine unequivocally the catalytic rate constant k(cat), the activation energy (E(a)) and the thermodynamic activation parameters (DeltaG(#), DeltaH(#), DeltaS(#)) of the chitinolytic reaction on the soluble substrate. The catalytic activity has also been determined on insoluble chitin, which displays an effect of substrate saturation by chitinases. On both substrates, the thermodependence of the activity of the psychrophilic chitinases was lower than that observed with the mesophilic counterpart.

  20. Purification, characterization, and antifungal activity of chitinase from Streptomyces venezuelae P10.

    PubMed

    Mukherjee, G; Sen, S K

    2006-10-01

    Streptomyces venezuelae P(10) could produce extracellular chitinase in a medium containing 0.6% colloidal chitin that was fermented for 96 hours at 30 degrees C. The enzyme was purified to apparent homogeneity with 80% saturation of ammonium sulfate as shown by chitin affinity chromatography and DEAE-cellulose anion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the enzyme showed a molecular weight of 66 kDa. The chitinase was characterized, and antifungal activity was observed against phytopathogens. Also, the first 15 N-terminal amino-acid residues of the chitinase were determined. The chitin hydrolysed products were N-acetylglucosamine and N, N'-diacetylchitobiose.

  1. Chitinase activity on amorphous chitin thin films: a quartz crystal microbalance with dissipation monitoring and atomic force microscopy study.

    PubMed

    Wang, Chao; Kittle, Joshua D; Qian, Chen; Roman, Maren; Esker, Alan R

    2013-08-12

    Chitinases are widely distributed in nature and have wide-ranging pharmaceutical and biotechnological applications. This work highlights a real-time and label-free method to assay Chitinase activity via a quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM). The chitin substrate was prepared by spincoating a trimethylsilyl chitin solution onto a silica substrate, followed by regeneration to amorphous chitin (RChi). The QCM-D and AFM results clearly showed that the hydrolysis rate of RChi films increased as Chitinase (from Streptomyces griseus) concentrations increased, and the optimal temperature and pH for Chitinase activity were around 37 °C and 6-8, respectively. The Chitinase showed greater activity on chitin substrates, having a high degree of acetylation, than on chitosan substrates, having a low degree of acetylation.

  2. Purification, characterization, and antifungal activity of chitinases from pineapple (Ananas comosus) leaf.

    PubMed

    Taira, Toki; Toma, Noriko; Ishihara, Masanobu

    2005-01-01

    Three chitinases, designated pineapple leaf chitinase (PL Chi)-A, -B, and -C were purified from the leaves of pineapple (Ananas comosus) using chitin affinity column chromatography followed by several column chromatographies. PL Chi-A is a class III chitinase having a molecular mass of 25 kDa and an isoelectric point of 4.4. PL Chi-B and -C are class I chitinases having molecular masses of 33 kDa and 39 kDa and isoelectric points of 7.9 and 4.6 respectively. PL Chi-C is a glycoprotein and the others are simple proteins. The optimum pHs of PL Chi-A, -B, and -C toward glycolchitin are pH 3, 4, and 9 respectively. The chitin-binding ability of PL Chi-C is higher than that of PL Chi-B, and PL Chi-A has lower chitin-binding ability than the others. At low ionic strength, PL Chi-B exhibits strong antifungal activity toward Trichoderma viride but the others do not. At high ionic strength, PL Chi-B and -C exhibit strong and weak antifungal activity respectively. PL Chi-A does not have antifungal activity.

  3. Chitinase from Paracoccidioides brasiliensis: molecular cloning, structural, phylogenetic, expression and activity analysis.

    PubMed

    Bonfim, Sheyla M R C; Cruz, Aline H S; Jesuino, Rosália S A; Ulhoa, Cirano J; Molinari-Madlum, Eugênia E W I; Soares, Célia M A; Pereira, Maristela

    2006-03-01

    A full-length cDNA encoding a chitinase (Pbcts1) was cloned by screening a cDNA library from the yeast cells of Paracoccidioides brasiliensis. The cDNA consists of 1888 bp and encodes an ORF of 1218 bp corresponding to a protein of 45 kDa with 406 amino acid residues. The deduced PbCTS1 is composed of two signature family 18 catalytic domains and seems to belong to fungal/bacterial class. Phylogenetic analysis of PbCTS1 and other chitinases suggests the existence of paralogs of several chitinases to be grouped based on specialized functions, which may reflect the multiple and diverse roles played by fungi chitinases. Glycosyl hydrolase activity assays demonstrated that P. brasiliensis is able to produce and secrete these enzymes mainly during transition from yeast to mycelium. The fungus should be able to use chitin as a carbon source. The presence of an endocytic signal in the deduced protein suggests that it could be secreted by a vesicular nonclassical export pathway. The Pbcts1 expression in mycelium, yeast, during differentiation from mycelium to yeast and in yeast cells obtained from infected mice suggests the relevance of this molecule in P. brasiliensis electing PbCTS1 as an attractive drug target.

  4. Chitinase mRNA levels by quantitative PCR using the single standard DNA: acidic mammalian chitinase is a major transcript in the mouse stomach.

    PubMed

    Ohno, Misa; Tsuda, Kyoko; Sakaguchi, Masayoshi; Sugahara, Yasusato; Oyama, Fumitaka

    2012-01-01

    Chitinases hydrolyze the β-1-4 glycosidic bonds of chitin, a major structural component of fungi, crustaceans and insects. Although mammals do not produce chitin or its synthase, they express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). These mammalian chitinases have attracted considerable attention due to their increased expression in individuals with a number of pathological conditions, including Gaucher disease, Alzheimer's disease and asthma. However, the contribution of these enzymes to the pathophysiology of these diseases remains to be determined. The quantification of the Chit1 and AMCase mRNA levels and the comparison of those levels with the levels of well-known reference genes can generate useful and biomedically relevant information. In the beginning, we established a quantitative real-time PCR system that uses standard DNA produced by ligating the cDNA fragments of the target genes. This system enabled us to quantify and compare the expression levels of the chitinases and the reference genes on the same scale. We found that AMCase mRNA is synthesized at extraordinarily high levels in the mouse stomach. The level of this mRNA in the mouse stomach was 7- to 10-fold higher than the levels of the housekeeping genes and was comparable to that the level of the mRNA for pepsinogen C (progastricsin), a major component of the gastric mucosa. Thus, AMCase mRNA is a major transcript in mouse stomach, suggesting that AMCase functions as a digestive enzyme that breaks down polymeric chitin and as part of the host defense against chitin-containing pathogens in the gastric contents. Our methodology is applicable to the quantification of mRNAs for multiple genes across multiple specimens using the same scale.

  5. A fast, sensitive and easy colorimetric assay for chitinase and cellulase activity detection

    PubMed Central

    2014-01-01

    Background Most of the current colorimetric methods for detection of chitinase or cellulase activities on the insoluble natural polymers chitin and cellulose depend on a chemical redox reaction. The reaction involves the reducing ends of the hydrolytic products. The Schales’ procedure and the 3,5-dinitrosalicylic acid (DNS) method are two examples that are commonly used. However, these methods lack sensitivity and present practical difficulties of usage in high-throughput screening assays as they require boiling or heating steps for color development. Results We report a novel method for colorimetric detection of chitinase and cellulase activity. The assay is based on the use of two oxidases: wild-type chito-oligosaccharide oxidase, ChitO, and a mutant thereof, ChitO-Q268R. ChitO was used for chitinase, while ChitO-Q268R was used for cellulase activity detection. These oxidases release hydrogen peroxide upon the oxidation of chitinase- or cellulase-produced hydrolytic products. The hydrogen peroxide produced can be monitored using a second enzyme, horseradish peroxidase (HRP), and a chromogenic peroxidase substrate. The developed ChitO-based assay can detect chitinase activity as low as 10 μU within 15 minutes of assay time. Similarly, cellulase activity can be detected in the range of 6 to 375 mU. A linear response was observed when applying the ChitO-based assay for detecting individual chito-oligosaccharides and cello-oligosaccharides. The detection limits for these compounds ranged from 5 to 25 μM. In contrast to the other commonly used methods, the Schales’ procedure and the DNS method, no boiling or heating is needed in the ChitO-based assays. The method was also evaluated for detecting hydrolytic activity on biomass-derived substrates, that is, wheat straw as a source of cellulose and shrimp shells as a source of chitin. Conclusion The ChitO-based assay has clear advantages for the detection of chitinase and cellulase activity over the conventional

  6. Chitotriosidase is a Biomarker for the Resistance to World Trade Center Lung Injury in New York City Firefighters

    PubMed Central

    Cho, Soo Jung; Nolan, Anna; Echevarria, Ghislaine C.; Kwon, Sophia; Naveed, Bushra; Schenck, Edward; Tsukiji, Jun; Prezant, David J.; Rom, William N.; Weiden, Michael D.

    2013-01-01

    Purpose World Trade Center (WTC) exposure caused airflow obstruction years after exposure. Chitinases and IgE are innate and humoral mediators of obstructive airway disease. We investigated if serum expression of chitinases and IgE early after WTC exposure predicts subsequent obstruction. Methods With a nested case-control design, 251 FDNY personnel had chitotriosidase, YKL-40 and IgE measured in serum drawn within months of 9/11/2001. The main outcome was subsequent Forced Expiratory Volume after one second/Forced Vital Capacity (FEV1/FVC) less than the lower limit of normal (LLN). Cases (N=125) had abnormal FEV1/FVC whereas controls had normal FEV1/FVC (N=126). In a secondary analysis, resistant cases (N=66) had FEV1 (≥107%) one standard deviation above the mean. Logistic regression adjusted for age, BMI, exposure intensity and post-exposure FEV1/FVC modeled the association between early biomarkers and later lung function. Results Cases and Controls initially lost lung function. Controls recovered to pre-9/11 FEV1 and FVC while cases continue to decline. Cases expressed lower serum chitotriosidase and higher IgE levels. Increase in IgE increased the odds of airflow obstruction and decreased the odds of above average FEV1. Alternately, increasing chitotriosidase decreased the odds of abnormal FEV1/FVC and increased the odds of FEV1≥107%. Serum YKL-40 was not associated with FEV1/FVC or FEV1 in this cohort. Conclusions Increased serum chitotriosidase reduces the odds of developing obstruction after WTC-particulate matter exposure and is associated with recovery of lung function. Alternately, elevated IgE is a risk factor for airflow obstruction and progressive lung function decline. PMID:23744081

  7. A Diverse Range of Bacterial and Eukaryotic Chitinases Hydrolyzes the LacNAc (Galβ1–4GlcNAc) and LacdiNAc (GalNAcβ1–4GlcNAc) Motifs Found on Vertebrate and Insect Cells*

    PubMed Central

    Frederiksen, Rikki F.; Yoshimura, Yayoi; Storgaard, Birgit G.; Paspaliari, Dafni K.; Petersen, Bent O.; Chen, Kowa; Larsen, Tanja; Duus, Jens Ø.; Ingmer, Hanne; Bovin, Nicolai V.; Westerlind, Ulrika; Blixt, Ola; Palcic, Monica M.; Leisner, Jørgen J.

    2015-01-01

    There is emerging evidence that chitinases have additional functions beyond degrading environmental chitin, such as involvement in innate and acquired immune responses, tissue remodeling, fibrosis, and serving as virulence factors of bacterial pathogens. We have recently shown that both the human chitotriosidase and a chitinase from Salmonella enterica serovar Typhimurium hydrolyze LacNAc from Galβ1–4GlcNAcβ-tetramethylrhodamine (LacNAc-TMR (Galβ1–4GlcNAcβ(CH2)8CONH(CH2)2NHCO-TMR)), a fluorescently labeled model substrate for glycans found in mammals. In this study we have examined the binding affinities of the Salmonella chitinase by carbohydrate microarray screening and found that it binds to a range of compounds, including five that contain LacNAc structures. We have further examined the hydrolytic specificity of this enzyme and chitinases from Sodalis glossinidius and Polysphondylium pallidum, which are phylogenetically related to the Salmonella chitinase, as well as unrelated chitinases from Listeria monocytogenes using the fluorescently labeled substrate analogs LacdiNAc-TMR (GalNAcβ1–4GlcNAcβ-TMR), LacNAc-TMR, and LacNAcβ1–6LacNAcβ-TMR. We found that all chitinases examined hydrolyzed LacdiNAc from the TMR aglycone to various degrees, whereas they were less active toward LacNAc-TMR conjugates. LacdiNAc is found in the mammalian glycome and is a common motif in invertebrate glycans. This substrate specificity was evident for chitinases of different phylogenetic origins. Three of the chitinases also hydrolyzed the β1–6 bond in LacNAcβ1–6LacNAcβ-TMR, an activity that is of potential importance in relation to mammalian glycans. The enzymatic affinities for these mammalian-like structures suggest additional functional roles of chitinases beyond chitin hydrolysis. PMID:25561735

  8. Strong aphicidal activity of GlcNAc(β1→4)Glc disaccharides: synthesis, physiological effects, and chitinase inhibition.

    PubMed

    Dussouy, Christophe; Bultel, Laurent; Saguez, Julien; Cherqui, Anas; Khelifa, Mounia; Grand, Eric; Giordanengo, Philippe; Kovensky, José

    2012-08-06

    The synthesis of four GlcNAc(β1→4)Glc disaccharides containing 2-O-acetyl and/or 6-sulfate groups was performed in high yields with total 1,2-trans stereoselectivity. These disaccharides were evaluated as candidates for insect chitinase inhibition and aphicidal activity. All the compounds prepared displayed physiological effects on M. persicae aphids; however, the inhibition of chitinases of different sources (bacteria, fungus, and aphid) followed different patterns according to subtle structural characteristics.

  9. Substrate-binding specificity of chitinase and chitosanase as revealed by active-site architecture analysis.

    PubMed

    Liu, Shijia; Shao, Shangjin; Li, Linlin; Cheng, Zhi; Tian, Li; Gao, Peiji; Wang, Lushan

    2015-12-11

    Chitinases and chitosanases, referred to as chitinolytic enzymes, are two important categories of glycoside hydrolases (GH) that play a key role in degrading chitin and chitosan, two naturally abundant polysaccharides. Here, we investigate the active site architecture of the major chitosanase (GH8, GH46) and chitinase families (GH18, GH19). Both charged (Glu, His, Arg, Asp) and aromatic amino acids (Tyr, Trp, Phe) are observed with higher frequency within chitinolytic active sites as compared to elsewhere in the enzyme structure, indicating significant roles related to enzyme function. Hydrogen bonds between chitinolytic enzymes and the substrate C2 functional groups, i.e. amino groups and N-acetyl groups, drive substrate recognition, while non-specific CH-π interactions between aromatic residues and substrate mainly contribute to tighter binding and enhanced processivity evident in GH8 and GH18 enzymes. For different families of chitinolytic enzymes, the number, type, and position of substrate atoms bound in the active site vary, resulting in different substrate-binding specificities. The data presented here explain the synergistic action of multiple enzyme families at a molecular level and provide a more reasonable method for functional annotation, which can be further applied toward the practical engineering of chitinases and chitosanases.

  10. Bacterial community composition and chitinase gene diversity of vermicompost with antifungal activity.

    PubMed

    Yasir, Muhammad; Aslam, Zubair; Kim, Seon Won; Lee, Seon-Woo; Jeon, Che Ok; Chung, Young Ryun

    2009-10-01

    Bacterial communities and chitinase gene diversity of vermicompost (VC) were investigated to clarify the influence of earthworms on the inhibition of plant pathogenic fungi in VC. The spore germination of Fusarium moniliforme was reduced in VC aqueous extracts prepared from paper sludge and dairy sludge (fresh sludge, FS). The bacterial communities were examined by culture-dependent and -independent analyses. Unique clones selected from 16S rRNA libraries of FS and VC on the basis of restriction fragment length polymorphism (RFLP) fell into the major lineages of the domain bacteria Proteobacteria, Bacteroidetes, Verrucomicrobia, Actinobacteria and Firmicutes. Among culture isolates, Actinobacteria dominated in VC, while almost equal numbers of Actinobacteria and Proteobacteria were present in FS. Analysis of chitinolytic isolates and chitinase gene diversity revealed that chitinolytic bacterial communities were enriched in VC. Populations of bacteria that inhibited plant fungal pathogens were higher in VC than in FS and particularly chitinolytic isolates were most active against the target fungi.

  11. The mycorrhizal fungus Amanita muscaria induces chitinase activity in roots and in suspension-cultured cells of its host Picea abies.

    PubMed

    Sauter, M; Hager, A

    1989-08-01

    A cell-wall fraction of the mycorrhizal fungus Amanita muscaria increased the chitinase activity in suspension-cultured cells of spruce (Picea abies (L.) Karst.) which is a frequent host of Amanita muscaria in nature. Chitinase activity was also increased in roots of spruce trees upon incubation with the fungal elicitor. Non-induced levels of chitinase activity in spruce were higher in suspension cells than in roots whereas the elicitorinduced increase of chitinase activity was higher in roots. Treatment of cells with hormones (auxins and cytokinin) resulted in a severalfold depression of enzyme activity. However, the chitinase activity of hormone-treated as well as hormone-free cells showed an elicitor-induced increase. Suspension cells of spruce secreted a large amount of enzyme into the medium. It is postulated that chitinases released from the host cells in an ectomycorrhizal system partly degrade the fungal cell walls, thus possibly facilitating the exchange of metabolites between the symbionts.

  12. Two chitinase-like proteins abundantly accumulated in latex of mulberry show insecticidal activity

    PubMed Central

    2010-01-01

    Background Plant latex is the cytoplasm of highly specialized cells known as laticifers, and is thought to have a critical role in defense against herbivorous insects. Proteins abundantly accumulated in latex might therefore be involved in the defense system. Results We purified latex abundant protein a and b (LA-a and LA-b) from mulberry (Morus sp.) and analyzed their properties. LA-a and LA-b have molecular masses of approximately 50 and 46 kDa, respectively, and are abundant in the soluble fraction of latex. Western blotting analysis suggested that they share sequence similarity with each other. The sequences of LA-a and LA-b, as determined by Edman degradation, showed chitin-binding domains of plant chitinases at the N termini. These proteins showed small but significant chitinase and chitosanase activities. Lectin RCA120 indicated that, unlike common plant chitinases, LA-a and LA-b are glycosylated. LA-a and LA-b showed insecticidal activities when fed to larvae of the model insect Drosophila melanogaster. Conclusions Our results suggest that the two LA proteins have a crucial role in defense against herbivorous insects, possibly by hydrolyzing their chitin. PMID:20109180

  13. Differential effect of purified spruce chitinases and beta-1,3-glucanases on the activity of elicitors from ectomycorrhizal fungi.

    PubMed

    Salzer, P; Hübner, B; Sirrenberg, A; Hager, A

    1997-07-01

    Two chitinases (EC 3.2.1.14) and two beta-1,3-glucanases (EC 3.2.1.39) were purified from the culture medium of spruce (Picea abines [L.] Karst.) cells to study their role in modifying elicitors, cell walls, growth, and hyphal morphology of ectomycorrhizal fungi. The 36-kD class I chitinase (isoelectric point [pl] 8.0) and the 28-kD chitinase (pl 8.7) decreased the activity of elicitor preparations from Hebeloma crustuliniforme (Bull. ex Fries.) Quél., Amanita muscaria (L.) Pers., and Suillus variegatus (Sw.: Fr.) O.K., as demonstrated by using the elicitor-induced extracellular alkalinization in spruce cells as a test system. In addition, chitinases released monomeric products from the walls of these ectomycorrhizal fungi. The beta-1,3-glucanases (35 kD, pl 3.7 and 3.9), in contrast, had little influence on the activity of the fungal elicitors and released only from walls of A. muscaria some polymeric products. Furthermore, chitinases alone and in combination with beta-1,3-glucanases had no effect on the growth and morphology of the hyphae. Thus, it is suggested that apoplastic chitinases in the root cortex destroy elicitors from the ectomycorrhizal fungi without damaging the fungus. By this mechanism the host plant could attenuate the elicitor signal and adjust its own defense reactions to a level allowing symbiotic interaction.

  14. Characterization and antifungal activity of gazyumaru (Ficus microcarpa) latex chitinases: both the chitin-binding and the antifungal activities of class I chitinase are reinforced with increasing ionic strength.

    PubMed

    Taira, Toki; Ohdomari, Atsuko; Nakama, Naoya; Shimoji, Makiko; Ishihara, Masanobu

    2005-04-01

    Three chitinases, designated gazyumaru latex chitinase (GLx Chi)-A, -B, and -C, were purified from the latex of gazyumaru (Ficus microcarpa). GLx Chi-A,-B, and -C are an acidic class III (33 kDa, pI 4.0), a basic class I (32 kDa, pI 9.3), and a basic class II chitinase (27 kDa, pI > 10) respectively. GLx Chi-A did not exhibit any antifungal activity. At low ionic strength, GLx Chi-C exhibited strong antifungal activity, to a similar extent as GLx Chi-B. The antifungal activity of GLx Chi-C became weaker with increasing ionic strength, whereas that of GLx Chi-B became slightly stronger. GLx Chi-B and -C bound to the fungal cell-walls at low ionic strength, and then GLx Chi-C was dissociated from them by an escalation of ionic strength, but this was not the case for GLx Chi-B. The chitin-binding activity of GLx Chi-B was enhanced by increasing ionic strength. These results suggest that the chitin-binding domain of basic class I chitinase binds to the chitin in fungal cell walls by hydrophobic interaction and assists the antifungal action of the chitinase.

  15. Refolding of β-Stranded Class I Chitinases of Hippophae rhamnoides Enhances the Antifreeze Activity during Cold Acclimation

    PubMed Central

    Gupta, Ravi; Deswal, Renu

    2014-01-01

    Class I chitinases hydrolyse the β-1,4-linkage of chitin and also acquire antifreeze activity in some of the overwintering plants during cold stress. Two chitinases, HrCHT1a of 31 kDa and HrCHT1b of 34 kDa, were purified from cold acclimated and non-acclimated seabuckthorn seedlings using chitin affinity chromatography. 2-D gels of HrCHT1a and HrCHT1b showed single spots with pIs 7.0 and 4.6 respectively. N-terminal sequence of HrCHT1b matched with the class I chitinase of rice and antifreeze proteins while HrCHT1a could not be sequenced as it was N-terminally blocked. Unlike previous reports, where antifreeze activity of chitinase was cold inducible, our results showed that antifreeze activity is constitutive property of class I chitinase as both HrCHT1a and HrCHT1b isolated even from non-acclimated seedlings, exhibited antifreeze activity. Interestingly, HrCHT1a and HrCHT1b purified from cold acclimated seedlings, exhibited 4 and 2 times higher antifreeze activities than those purified from non-acclimated seedlings, suggesting that antifreeze activity increased during cold acclimation. HrCHT1b exhibited 23–33% higher hydrolytic activity and 2–4 times lower antifreeze activity than HrCHT1a did. HrCHT1b was found to be a glycoprotein; however, its antifreeze activity was independent of glycosylation as even deglycosylated HrCHT1b exhibited antifreeze activity. Circular dichroism (CD) analysis showed that both these chitinases were rich in unusual β-stranded conformation (36–43%) and the content of β-strand increased (∼11%) during cold acclimation. Surprisingly, calcium decreased both the activities of HrCHT1b while in case of HrCHT1a, a decrease in the hydrolytic activity and enhancement in its antifreeze activity was observed. CD results showed that addition of calcium also increased the β-stranded conformation of HrCHT1a and HrCHT1b. This is the first report, which shows that antifreeze activity is constitutive property of class I chitinase and cold

  16. Differential enzymatic activity of common haplotypic versions of the human acidic Mammalian chitinase protein.

    PubMed

    Seibold, Max A; Reese, Tiffany A; Choudhry, Shweta; Salam, Muhammad T; Beckman, Kenny; Eng, Celeste; Atakilit, Amha; Meade, Kelley; Lenoir, Michael; Watson, H Geoffrey; Thyne, Shannon; Kumar, Rajesh; Weiss, Kevin B; Grammer, Leslie C; Avila, Pedro; Schleimer, Robert P; Fahy, John V; Rodriguez-Santana, Jose; Rodriguez-Cintron, William; Boot, Rolf G; Sheppard, Dean; Gilliland, Frank D; Locksley, Richard M; Burchard, Esteban G

    2009-07-17

    Mouse models have shown the importance of acidic mammalian chitinase activity in settings of chitin exposure and allergic inflammation. However, little is known regarding genetic regulation of AMCase enzymatic activity in human allergic diseases. Resequencing the AMCase gene exons we identified 8 non-synonymous single nucleotide polymorphisms including three novel variants (A290G, G296A, G339T) near the gene area coding for the enzyme active site, all in linkage disequilibrium. AMCase protein isoforms, encoded by two gene-wide haplotypes, and differentiated by these three single nucleotide polymorphisms, were recombinantly expressed and purified. Biochemical analysis revealed the isoform encoded by the variant haplotype displayed a distinct pH profile exhibiting greater retention of chitinase activity at acidic and basic pH values. Determination of absolute kinetic activity found the variant isoform encoded by the variant haplotype was 4-, 2.5-, and 10-fold more active than the wild type AMCase isoform at pH 2.2, 4.6, and 7.0, respectively. Modeling of the AMCase isoforms revealed positional changes in amino acids critical for both pH specificity and substrate binding. Genetic association analyses of AMCase haplotypes for asthma revealed significant protective associations between the variant haplotype in several asthma cohorts. The structural, kinetic, and genetic data regarding the AMCase isoforms are consistent with the Th2-priming effects of environmental chitin and a role for AMCase in negatively regulating this stimulus.

  17. Chitinase activity in the epidermis of the fiddler crab, Uca pugilator, as an in vivo screen for molt-interfering xenobiotics.

    PubMed

    Zou, Enmin; Bonvillain, Ryan

    2004-12-01

    We describe an in vivo screening assay that uses epidermal chitinase activity as the endpoint following a 7-day exposure of Uca pugilator to test chemicals. Chitinase, a chitinolytic enzyme, is the end product of endocrine cascades of a multi-hormonal system for control of crustacean molting. Wherever a molt-interfering agent adversely impacts the Y-organ-ecdysteroid receptor axis, the effect should be manifested by the activity of chitinase in the epidermis. Therefore, epidermal chitinase activity is an ideal endpoint for molt-interfering effects of xenobiotics. The validity of epidermal chitinase activity being used for such a purpose is supported by our finding that two injections of 20-hydroxyecdysone at 25 microg/g live weight induced a twofold increase in chitinase activity in the epidermis of U. pugilator. A total of nine chemicals were screened for molting hormone and anti-molting activities. o,p'-DDT was found to significantly inhibit epidermal chitinase activity while kepone and methoxychlor exhibited a tendency of inhibition of enzymatic activity. None of the remaining six chemicals, namely, p,p'-dichlorodiphenyltrichloroethane (p,p'-DDT), atrazine, tributyltin (TBT), methoprene, dieldrin and permethrin, had an effect on epidermal chitinase activity.

  18. Lysozyme- and chitinase activity in latex bearing plants of genus Euphorbia--A contribution to plant defense mechanism.

    PubMed

    Sytwala, Sonja; Günther, Florian; Melzig, Matthias F

    2015-10-01

    Occurrence of latices in plants is widespread, there are 40 families of plants characterized to establish lactiferous structures. Latices exhibit a constitutive part of plant defense due to the stickiness. The appearance of proteins incorporated in latices is well characterized, and hydrolytic active proteins are considerable. A lot of plants constitute so-called pathogenesis-related (PR) proteins, to overcome stressful conditions. In our investigation we are focused on latex bearing plants of Euphorbiaceae Juss., and investigated the appearance of chitinase- and lysozyme activity in particular. The present outcomes represent a comprehensive study, relating to the occurrence of lysozyme and chitinase activity of genus Euphorbia at the first time. 110 different species of genus Euphorbia L. were tested, and the appearance of chitinase and lysozyme were determined in different quantities. The appearance itself, and the physicochemical properties of latices indicate an efficient interaction for plant defense against pathogen attack.

  19. Purification and characterization of chitinase from Alcaligenes faecalis AU02 by utilizing marine wastes and its antioxidant activity.

    PubMed

    Annamalai, Neelamegam; Veeramuthu Rajeswari, Mayavan; Vijayalakshmi, Shanmugam; Balasubramanian, Thangavel

    2011-12-01

    Marine waste is an abundant renewable source for the recovery of several value added metabolites with potential industrial applications. This study describes the production of chitinase on marine waste, with the subsequent use of the same marine waste for the extraction of antioxidants. A chitinase-producing bacterium isolated from seafood effluent was identified as Alcaligenes faecalis AU02. Optimal chitinase production was obtained in culture conditions of 37°C for 72 h in 100 ml medium containing 1% shrimp and crab shell powder (1:1) (w/v), 0.1% K(2)HPO(4), and 0.05% MgSO(4)·7H(2)O. The molecular weight of chitinase was determined by SDS-PAGE to be 36 kDa. The optimum pH, temperature, pH stability, and thermal stability of chitinase were about 8, 37°C, 5-12, and 40-80°C, respectively. The antioxidant activity of A. faecalis AU02 culture supernatant was determined through scavenging ability on 1,1-diphenyl-2-picrylhydrazyl (DPPH) as 84%, and the antioxidant compound was characterized by TLC and its FT-IR spectrum. The present study proposed that marine wastes can be utilized to generate a high-value-added product and that pharmacological studies can extend its use to the field of medicine.

  20. Entomotoxicity, protease and chitinase activity of Bacillus thuringiensis fermented wastewater sludge with a high solids content.

    PubMed

    Brar, Satinder K; Verma, M; Tyagi, R D; Valéro, J R; Surampalli, R Y

    2009-10-01

    This study investigated the production of biopesticides, protease and chitinase activity by Bacillus thuringiensis grown in raw wastewater sludge at high solids concentration (30 g/L). The rheology of wastewater sludge was modified with addition of Tween-80 (0.2% v/v). This addition resulted in 1.6 and 1.3-fold increase in cell and spore count, respectively. The maximum specific growth rate (micro(max)) augmented from 0.17 to 0.22 h(-1) and entomotoxicity (Tx) increased by 29.7%. Meanwhile, volumetric mass transfer coefficient (k(L)a) showed marked variations during fermentation, and oxygen uptake rate (OUR) increased 2-fold. The proteolytic activity increased while chitinase decreased for Tween amended wastewater sludge, but the entomotoxicity increased. The specific entomotoxicity followed power law when plotted against spore concentration and the relation between Tx and protease activity was linear. The viscosity varied and volume percent of particles increased in Tween-80 amended wastewater sludge and particle size (D(50)) decreased at the end of fermentation. Thus, there was an increase in entomotoxicity at higher suspended solids (30 g/L) as Tween addition improved rheology (viscosity, particle size, surface tension); enhanced maximum growth rate and OUR.

  1. A rapid test for chitinase activity that uses 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide.

    PubMed Central

    O'Brien, M; Colwell, R R

    1987-01-01

    A total of 101 strains of bacteria from environmental and clinical sources, most of which were gram negative, were tested for chitobiase activity by using a filter paper spot test with 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide as the substrate. The results were compared with those obtained by a conventional plate method for chitinase activity by using colloidal chitin as the substrate. There was excellent agreement in the results for both methods. The filter paper spot test with 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide has the advantages of being rapid, simple to perform, and inexpensive. This method should be adaptable to a wider range of microorganisms, particularly those with unusual growth requirements. PMID:3662513

  2. The chitinase C gene PsChiC from Pseudomonas sp. and its synergistic effects on larvicidal activity

    PubMed Central

    Zhong, Wanfang; Ding, Shaojun; Guo, Huifang

    2015-01-01

    Pseudomonas sp. strain TXG6-1, a chitinolytic gram-negative bacterium, was isolated from a vegetable field in Taixing city, Jiangsu Province, China. In this study, a Pseudomonas chitinase C gene (PsChiC) was isolated from the chromosomal DNA of this bacterium using a pair of specific primers. The PsChiC gene consisted of an open reading frame of 1443 nucleotides and encoded 480 amino acid residues with a calculated molecular mass of 51.66 kDa. The deduced PsChiC amino acid sequence lacked a signal sequence and consisted of a glycoside hydrolase family 18 catalytic domain responsible for chitinase activity, a fibronectin type III-like domain (FLD) and a C-terminal chitin-binding domain (ChBD). The amino acid sequence of PsChiCshowed high sequence homology (> 95%) with chitinase C from Serratia marcescens. SDS-PAGE showed that the molecular mass of chitinase PsChiC was 52 kDa. Chitinase assays revealed that the chitobiosidase and endochitinase activities of PsChiCwere 51.6- and 84.1-fold higher than those of pET30a, respectively. Although PsChiC showed little insecticidal activity towards Spodoptera litura larvae, an insecticidal assay indicated that PsChiC increased the insecticidal toxicity of SpltNPV by 1.78-fold at 192 h and hastened death. These results suggest that PsChiC from Pseudomonas sp. could be useful in improving the pathogenicity of baculoviruses. PMID:26500441

  3. Mutational analysis of amino acid residues involved in catalytic activity of a family 18 chitinase from tulip bulbs.

    PubMed

    Suzukawa, Keisuke; Yamagami, Takeshi; Ohnuma, Takayuki; Hirakawa, Hideki; Kuhara, Satoru; Aso, Yoichi; Ishiguro, Masatsune

    2003-02-01

    We expressed chitinase-1 (TBC-1) from tulip bulbs (Tulipa bakeri) in E. coli cells and used site-directed mutagenesis to identify amino acid residues essential for catalytic activity. Mutations at Glu-125 and Trp-251 completely abolished enzyme activity, and activity decreased with mutations at Asp-123 and Trp-172 when glycolchitin was the substrate. Activity changed with the mutations of Trp-251 to one of several amino acids with side-chains of little hydrophobicity, suggesting that hydrophobic interaction of Trp-251 is important for the activity. Molecular dynamics (MD) simulation analysis with hevamine as the model compound showed that the distance between Asp-123 and Glu-125 was extended by mutation of Trp-251. Kinetic studies of Trp-251-mutated chitinases confirmed these various phenomena. The results suggested that Glu-125 and Trp-251 are essential for enzyme activity and that Trp-251 had a direct role in ligand binding.

  4. Loss and Gain of Human Acidic Mammalian Chitinase Activity by Nonsynonymous SNPs

    PubMed Central

    Okawa, Kazuaki; Ohno, Misa; Kashimura, Akinori; Kimura, Masahiro; Kobayashi, Yuki; Sakaguchi, Masayoshi; Sugahara, Yasusato; Kamaya, Minori; Kino, Yoshihiro; Bauer, Peter O.; Oyama, Fumitaka

    2016-01-01

    Acidic mammalian chitinase (AMCase) is implicated in asthma, allergic inflammation, and food processing. Little is known about genetic and evolutional regulation of chitinolytic activity of AMCase. Here, we relate human AMCase polymorphisms to the mouse AMCase, and show that the highly active variants encoded by nonsynonymous single-nucleotide polymorphisms (nsSNPs) are consistent with the mouse AMCase sequence. The chitinolytic activity of the recombinant human AMCase was significantly lower than that of the mouse counterpart. By creating mouse-human chimeric AMCase protein we found that the presence of the N-terminal region of human AMCase containing conserved active site residues reduced the enzymatic activity of the molecule. We were able to significantly increase the activity of human AMCase by amino acid substitutions encoded by nsSNPs (N45, D47, and R61) with those conserved in the mouse homologue (D45, N47, and M61). For abolition of the mouse AMCase activity, introduction of M61R mutation was sufficient. M61 is conserved in most of primates other than human and orangutan as well as in other mammals. Orangutan has I61 substitution, which also markedly reduced the activity of the mouse AMCase, indicating that the M61 is a crucial residue for the chitinolytic activity. Altogether, our data suggest that human AMCase has lost its chitinolytic activity by integration of nsSNPs during evolution and that the enzyme can be reactivated by introducing amino acids conserved in the mouse counterpart. PMID:27702777

  5. Inbreeding Alters Activities of the Stress-Related Enzymes Chitinases and β-1,3-Glucanases

    PubMed Central

    Leimu, Roosa; Kloss, Lena; Fischer, Markus

    2012-01-01

    Pathogenesis-related proteins, chitinases (CHT) and β-1,3-glucanases (GLU), are stress proteins up-regulated as response to extrinsic environmental stress in plants. It is unknown whether these PR proteins are also influenced by inbreeding, which has been suggested to constitute intrinsic genetic stress, and which is also known to affect the ability of plants to cope with environmental stress. We investigated activities of CHT and GLU in response to inbreeding in plants from 13 Ragged Robin (Lychnis flos-cuculi) populations. We also studied whether activities of these enzymes were associated with levels of herbivore damage and pathogen infection in the populations from which the plants originated. We found an increase in pathogenesis-related protein activity in inbred plants from five out of the 13 investigated populations, which suggests that these proteins may play a role in how plants respond to intrinsic genetic stress brought about by inbreeding in some populations depending on the allele frequencies of loci affecting the expression of CHT and the past levels of inbreeding. More importantly, we found that CHT activities were higher in plants from populations with higher levels of herbivore or pathogen damage, but inbreeding reduced CHT activity in these populations disrupting the increased activities of this resistance-related enzyme in populations where high resistance is beneficial. These results provide novel information on the effects of plant inbreeding on plant–enemy interactions on a biochemical level. PMID:22879940

  6. Chitin recognition via chitotriosidase promotes pathologic type-2 helper T cell responses to cryptococcal infection.

    PubMed

    Wiesner, Darin L; Specht, Charles A; Lee, Chrono K; Smith, Kyle D; Mukaremera, Liliane; Lee, S Thera; Lee, Chun G; Elias, Jack A; Nielsen, Judith N; Boulware, David R; Bohjanen, Paul R; Jenkins, Marc K; Levitz, Stuart M; Nielsen, Kirsten

    2015-03-01

    Pulmonary mycoses are often associated with type-2 helper T (Th2) cell responses. However, mechanisms of Th2 cell accumulation are multifactorial and incompletely known. To investigate Th2 cell responses to pulmonary fungal infection, we developed a peptide-MHCII tetramer to track antigen-specific CD4+ T cells produced in response to infection with the fungal pathogen Cryptococcus neoformans. We noted massive accruement of pathologic cryptococcal antigen-specific Th2 cells in the lungs following infection that was coordinated by lung-resident CD11b+ IRF4-dependent conventional dendritic cells. Other researchers have demonstrated that this dendritic cell subset is also capable of priming protective Th17 cell responses to another pulmonary fungal infection, Aspergillus fumigatus. Thus, higher order detection of specific features of fungal infection by these dendritic cells must direct Th2 cell lineage commitment. Since chitin-containing parasites commonly elicit Th2 responses, we hypothesized that recognition of fungal chitin is an important determinant of Th2 cell-mediated mycosis. Using C. neoformans mutants or purified chitin, we found that chitin abundance impacted Th2 cell accumulation and disease. Importantly, we determined Th2 cell induction depended on cleavage of chitin via the mammalian chitinase, chitotriosidase, an enzyme that was also prevalent in humans experiencing overt cryptococcosis. The data presented herein offers a new perspective on fungal disease susceptibility, whereby chitin recognition via chitotriosidase leads to the initiation of harmful Th2 cell differentiation by CD11b+ conventional dendritic cells in response to pulmonary fungal infection.

  7. Chitotriosidase - a putative biomarker for sporadic amyotrophic lateral sclerosis

    PubMed Central

    2013-01-01

    Background Potential biomarkers to aid diagnosis and therapy need to be identified for Amyotrophic Lateral Sclerosis, a progressive motor neuronal degenerative disorder. The present study was designed to identify the factor(s) which are differentially expressed in the cerebrospinal fluid (CSF) of patients with sporadic amyotrophic lateral sclerosis (SALS; ALS-CSF), and could be associated with the pathogenesis of this disease. Results Quantitative mass spectrometry of ALS-CSF and control-CSF (from orthopaedic surgical patients undergoing spinal anaesthesia) samples showed upregulation of 31 proteins in the ALS-CSF, amongst which a ten-fold increase in the levels of chitotriosidase-1 (CHIT-1) was seen compared to the controls. A seventeen-fold increase in the CHIT-1 levels was detected by ELISA, while a ten-fold elevated enzyme activity was also observed. Both these results confirmed the finding of LC-MS/MS. CHIT-1 was found to be expressed by the Iba-1 immunopositive microglia. Conclusion Elevated CHIT-1 levels in the ALS-CSF suggest a definitive role for the enzyme in the disease pathogenesis. Its synthesis and release from microglia into the CSF may be an aligned event of neurodegeneration. Thus, high levels of CHIT-1 signify enhanced microglial activity which may exacerbate the process of neurodegeneration. In view of the multifold increase observed in ALS-CSF, it can serve as a potential CSF biomarker for the diagnosis of SALS. PMID:24295388

  8. Antifungal activity of chitinases from Trichoderma aureoviride DY-59 and Rhizopus microsporus VS-9.

    PubMed

    Nguyen, Nam Van; Kim, Young-Ju; Oh, Kyung-Taek; Jung, Woo-Jin; Park, Ro-Dong

    2008-01-01

    Two chitinolytic fungal strains, Trichoderma aureoviride DY-59 and Rhizopus microsporus VS-9, were isolated from soil samples of Korea and Vietnam, respectively. DY-59 and VS-9 crude chitinases secreted by these fungi in the 0.5% swollen chitin culture medium had an optimal pH of 4 and the optimal temperatures of 40 degrees C and 60 degrees C, respectively. Enzymatic hydrolysis products from crab swollen chitin were N-acetyl-beta-D-glucosamine (GlcNAc) by DY-59 chitinase, and GlcNAc and N, N'-diacetylchitobiose (GlcNAc)2 by VS-9 chitinases. The chitinases degraded the cell wall of Fusarium solani hyphae to produce oligosaccharides, among which GlcNAc, (GlcNAc)2, and pentamer (GlcNAc)5 were identified by high-pressure liquid chromatography. DY-59 and VS-9 chitinases inhibited F. solani microconidial germination by more than 70% and 60% at final protein concentrations of 5 and 27 microg mL(-1), respectively, at 30 degrees C for 20 h treatment.

  9. Activity, stability and folding analysis of the chitinase from Entamoeba histolytica.

    PubMed

    Muñoz, Patricia L A; Minchaca, Alexis Z; Mares, Rosa E; Ramos, Marco A

    2016-02-01

    Human amebiasis, caused by the parasitic protozoan Entamoeba histolytica, remains as a significant public health issue in developing countries. The life cycle of the parasite compromises two main stages, trophozoite and cyst, linked by two major events: encystation and excystation. Interestingly, the cyst stage has a chitin wall that helps the parasite to withstand harsh environmental conditions. Since the amebic chitinase, EhCHT1, has been recognized as a key player in both encystation and excystation, it is plausible to consider that specific inhibition could arrest the life cycle of the parasite and, thus, stop the infection. However, to selectively target EhCHT1 it is important to recognize its unique biochemical features to have the ability to control its cellular function. Hence, to gain further insights into the structure-function relationship, we conducted an experimental approach to examine the effects of pH, temperature, and denaturant concentration on the enzymatic activity and protein stability. Additionally, dependence on in vivo oxidative folding was further studied using a bacterial model. Our results attest the potential of EhCHT1 as a target for the design and development of new or improved anti-amebic therapeutics. Likewise, the potential of the oxidoreductase EhPDI, involved in oxidative folding of amebic proteins, was also confirmed.

  10. Chitinase-like proteins with antifungal activity from emperor banana fruits.

    PubMed

    Ho, Vincent S M; Ng, Tzi Bun

    2007-01-01

    Two 30-kDa proteins with N-terminal sequence homology to chitinases have been isolated from fruits of the emperor banana by using a protocol that involved (NH(4))(2)SO(4) precipitation, affinity chromatography on Affi-gel blue gel, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono S and gel filtration by FPLC on Superdex 75. The proteins were adsorbed on Affi-gel blue gel and Mono S. They both inhibited mycelial growth in Fusarium oxysporum but not in Mycosphaerella arachidicola. The chitinase-like protein more strongly bound on Mono S was obtained with a slightly lower yield and exhibited a higher antifungal potency toward F. oxysporum when compared with the less strongly bound chitinase-like protein.

  11. Enrichment of chitinolytic microorganisms: isolation and characterization of a chitinase exhibiting antifungal activity against phytopathogenic fungi from a novel Streptomyces strain.

    PubMed

    Hoster, Frank; Schmitz, Jessica E; Daniel, Rolf

    2005-01-01

    Thirteen different chitin-degrading bacteria were isolated from soil and sediment samples. Five of these strains (SGE2, SGE4, SSL3, MG1, and MG3) exhibited antifungal activity against phytopathogenic fungi. Analyses of the 16S rRNA genes and the substrate spectra revealed that the isolates belong to the genera Bacillus or Streptomyces. The closest relatives were Bacillus chitinolyticus (SGE2, SGE4, and SSL3), B. ehimensis (MG1), and Streptomyces griseus (MG3). The chitinases present in the culture supernatants of the five isolates revealed optimal activity between 45 degrees C and 50 degrees C and at pH values of 4 (SSL3), 5 (SGE2 and MG1), 6 (SGE4), and 5-7 (MG3). The crude chitinase preparations of all five strains possessed antifungal activity. The chitinase of MG3 (ChiIS) was studied further, since the crude enzyme conferred strong growth suppression of all fungi tested and was very active over the entire pH range tested. The chiIS gene was cloned and the gene product was purified. The deduced protein consisted of 303 amino acids with a predicted molecular mass of 31,836 Da. Sequence analysis revealed that ChiIS of MG3 is similar to chitinases of Streptomyces species, which belong to family 19 of glycosyl hydrolases. Purified ChiIS showed remarkable antifungal activity and stability.

  12. Molecular Docking and Site-directed Mutagenesis of a Bacillus thuringiensis Chitinase to Improve Chitinolytic, Synergistic Lepidopteran-larvicidal and Nematicidal Activities

    PubMed Central

    Ni, Hong; Zeng, Siquan; Qin, Xu; Sun, Xiaowen; Zhang, Shan; Zhao, Xiuyun; Yu, Ziniu; Li, Lin

    2015-01-01

    Bacterial chitinases are useful in the biocontrol of agriculturally important pests and fungal pathogens. However, the utility of naturally occurring bacterial chitinases is often limited by their low enzyme activity. In this study, we constructed mutants of a Bacillus thuringiensis chitinase with enhanced activity based on homology modeling, molecular docking, and the site-directed mutagenesis of target residues to modify spatial positions, steric hindrances, or hydrophilicity/hydrophobicity. We first identified a gene from B. thuringiensis YBT-9602 that encodes a chitinase (Chi9602) belonging to glycosyl hydrolase family 18 with conserved substrate-binding and substrate-catalytic motifs. We constructed a structural model of a truncated version of Chi9602 (Chi960235-459) containing the substrate-binding domain using the homologous 1ITX protein of Bacillus circulans as the template. We performed molecular docking analysis of Chi960235-459 using di-N-acetyl-D-glucosamine as the ligand. We then selected 10 residues of interest from the docking area for the site-directed mutagenesis experiments and expression in Escherichia coli. Assays of the chitinolytic activity of the purified chitinases revealed that the three mutants exhibited increased chitinolytic activity. The ChiW50A mutant exhibited a greater than 60 % increase in chitinolytic activity, with similar pH, temperature and metal ion requirements, compared to wild-type Chi9602. Furthermore, ChiW50A exhibited pest-controlling activity and antifungal activity. Remarkable synergistic effects of this mutant with B. thuringiensis spore-crystal preparations against Helicoverpa armigera and Caenorhabditis elegans larvae and obvious activity against several plant-pathogenic fungi were observed. PMID:25678849

  13. Molecular docking and site-directed mutagenesis of a Bacillus thuringiensis chitinase to improve chitinolytic, synergistic lepidopteran-larvicidal and nematicidal activities.

    PubMed

    Ni, Hong; Zeng, Siquan; Qin, Xu; Sun, Xiaowen; Zhang, Shan; Zhao, Xiuyun; Yu, Ziniu; Li, Lin

    2015-01-01

    Bacterial chitinases are useful in the biocontrol of agriculturally important pests and fungal pathogens. However, the utility of naturally occurring bacterial chitinases is often limited by their low enzyme activity. In this study, we constructed mutants of a Bacillus thuringiensis chitinase with enhanced activity based on homology modeling, molecular docking, and the site-directed mutagenesis of target residues to modify spatial positions, steric hindrances, or hydrophilicity/hydrophobicity. We first identified a gene from B. thuringiensis YBT-9602 that encodes a chitinase (Chi9602) belonging to glycosyl hydrolase family 18 with conserved substrate-binding and substrate-catalytic motifs. We constructed a structural model of a truncated version of Chi9602 (Chi9602(35-459)) containing the substrate-binding domain using the homologous 1ITX protein of Bacillus circulans as the template. We performed molecular docking analysis of Chi9602(35-459) using di-N-acetyl-D-glucosamine as the ligand. We then selected 10 residues of interest from the docking area for the site-directed mutagenesis experiments and expression in Escherichia coli. Assays of the chitinolytic activity of the purified chitinases revealed that the three mutants exhibited increased chitinolytic activity. The ChiW50A mutant exhibited a greater than 60 % increase in chitinolytic activity, with similar pH, temperature and metal ion requirements, compared to wild-type Chi9602. Furthermore, ChiW50A exhibited pest-controlling activity and antifungal activity. Remarkable synergistic effects of this mutant with B. thuringiensis spore-crystal preparations against Helicoverpa armigera and Caenorhabditis elegans larvae and obvious activity against several plant-pathogenic fungi were observed.

  14. Chitinases: An update

    PubMed Central

    Hamid, Rifat; Khan, Minhaj A.; Ahmad, Mahboob; Ahmad, Malik Mobeen; Abdin, Malik Zainul; Musarrat, Javed; Javed, Saleem

    2013-01-01

    Chitin, the second most abundant polysaccharide in nature after cellulose, is found in the exoskeleton of insects, fungi, yeast, and algae, and in the internal structures of other vertebrates. Chitinases are enzymes that degrade chitin. Chitinases contribute to the generation of carbon and nitrogen in the ecosystem. Chitin and chitinolytic enzymes are gaining importance for their biotechnological applications, especially the chitinases exploited in agriculture fields to control pathogens. Chitinases have a use in human health care, especially in human diseases like asthma. Chitinases have wide-ranging applications including the preparation of pharmaceutically important chitooligosaccharides and N-acetyl D glucosamine, preparation of single-cell protein, isolation of protoplasts from fungi and yeast, control of pathogenic fungi, treatment of chitinous waste, mosquito control and morphogenesis, etc. In this review, the various types of chitinases and the chitinases found in different organisms such as bacteria, plants, fungi, and mammals are discussed. PMID:23559820

  15. Purification and characterization of a highly thermostable chitinase from the stomach of the red scorpionfish Scorpaena scrofa with bioinsecticidal activity toward cowpea weevil Callosobruchus maculatus (Coleoptera: Bruchidae).

    PubMed

    Laribi-Habchi, Hassiba; Dziril, Maya; Badis, Abdelmalek; Mouhoub, Samia; Mameri, Nabil

    2012-01-01

    This present study is the first attempt to report on the purification and characterization of a chitinase from the stomach of the red scorpionfish Scorpaena scrofa. A 50-kDa chitinase (SsChi50) was purified to homogeneity, and matrix assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) analysis showed that SsChi50 was a monomer with a molecular mass of 50,103 Da. The 25 N-terminal residues of SsChi50 displayed high homology with family-18 chitinases. Optimal activity was obtained at pH 5.0 at 80 °C. SsChi50 was stable at pH and temperature ranges of 3.0 to 7.0 and 70 to 90 °C for 48 and 4 h respectively. Among the inhibitors and metals tested, p-chloromercuribenzoic acid, N-ethylmaleimide, Hg(2+), and Hg(+) completely inhibited enzyme activity. Chitinase activity was high on colloidal chitin, glycol chitin, glycol chitosane, chitotriose, and chitooligosaccharide. Chitinase activity towards synthetic substrates in the order of p-NP-(GlcNAc)(n) (n = 2-4) was p-NP-(GlcNAc)(2) > p-NP-(GlcNAc)(4) > p-NP-(GlcNAc)(3). Our results suggest that the SsChi50 enzyme preferentially hydrolyzed the second glycosidic link from the non-reducing end of (GlcNAc)(n). This enzyme obeyed Michaelis-Menten kinetics, the K(m) and k(cat) values being 0.412 mg, colloidal chitin mL(-1) and 5.33 s(-1) respectively. An in vivo bioinsecticidal assay was developed for SsChi50 against Callosobruchus maculatus adults. The enzyme showed bioinsecticidal activity toward Callosobruchus maculatus, indicating the possibility of using it in biotechnological strategies for insect management for stored cowpea seeds.

  16. A single amino acid substitution in a chitinase of the legume Medicago truncatula is sufficient to gain Nod-factor hydrolase activity

    PubMed Central

    Zhang, Lan-Yue; Cai, Jie; Li, Ru-Jie; Liu, Wei; Wagner, Christian; Wong, Kam-Bo; Xie, Zhi-Ping; Staehelin, Christian

    2016-01-01

    The symbiotic interaction between nitrogen-fixing rhizobia and legumes depends on lipo-chitooligosaccharidic Nod-factors (NFs). The NF hydrolase MtNFH1 of Medicago truncatula is a symbiotic enzyme that hydrolytically inactivates NFs with a C16 : 2 acyl chain produced by the microsymbiont Sinorhizobium meliloti 1021. MtNFH1 is related to class V chitinases (glycoside hydrolase family 18) but lacks chitinase activity. Here, we investigated the substrate specificity of MtNFH1-related proteins. MtCHIT5a and MtCHIT5b of M. truncatula as well as LjCHIT5 of Lotus japonicus showed chitinase activity, suggesting a role in plant defence. The enzymes failed to hydrolyse NFs from S. meliloti. NFs from Rhizobium leguminosarum with a C18 : 4 acyl moiety were neither hydrolysed by these chitinases nor by MtNFH1. Construction of chimeric proteins and further amino acid replacements in MtCHIT5b were performed to identify chitinase variants that gained the ability to hydrolyse NFs. A single serine-to-proline substitution was sufficient to convert MtCHIT5b into an NF-cleaving enzyme. MtNFH1 with the corresponding proline-to-serine substitution failed to hydrolyse NFs. These results are in agreement with a substrate-enzyme model that predicts NF cleavage when the C16 : 2 moiety is placed into a distinct fatty acid-binding cleft. Our findings support the view that MtNFH1 evolved from the ancestral MtCHIT5b by gene duplication and subsequent symbiosis-related neofunctionalization. PMID:27383628

  17. Production in Pichia pastoris, antifungal activity and crystal structure of a class I chitinase from cowpea (Vigna unguiculata): Insights into sugar binding mode and hydrolytic action.

    PubMed

    Landim, Patrícia G Castro; Correia, Tuana O; Silva, Fredy D A; Nepomuceno, Denise R; Costa, Helen P S; Pereira, Humberto M; Lobo, Marina D P; Moreno, Frederico B M B; Brandão-Neto, José; Medeiros, Suelen C; Vasconcelos, Ilka M; Oliveira, José T A; Sousa, Bruno L; Barroso-Neto, Ito L; Freire, Valder N; Carvalho, Cristina P S; Monteiro-Moreira, Ana C O; Grangeiro, Thalles B

    2017-04-01

    A cowpea class I chitinase (VuChiI) was expressed in the methylotrophic yeast P. pastoris. The recombinant protein was secreted into the culture medium and purified by affinity chromatography on a chitin matrix. The purified chitinase migrated on SDS-polyacrylamide gel electrophoresis as two closely-related bands with apparent molecular masses of 34 and 37 kDa. The identity of these bands as VuChiI was demonstrated by mass spectrometry analysis of tryptic peptides and N-terminal amino acid sequencing. The recombinant chitinase was able to hydrolyze colloidal chitin but did not exhibit enzymatic activity toward synthetic substrates. The highest hydrolytic activity of the cowpea chitinase toward colloidal chitin was observed at pH 5.0. Furthermore, most VuChiI activity (approximately 92%) was retained after heating to 50 °C for 30 min, whereas treatment with 5 mM Cu(2+) caused a reduction of 67% in the enzyme's chitinolytic activity. The recombinant protein had antifungal activity as revealed by its ability to inhibit the spore germination and mycelial growth of Penicillium herquei. The three-dimensional structure of VuChiI was resolved at a resolution of 1.55 Å by molecular replacement. The refined model had 245 amino acid residues and 381 water molecules, and the final R-factor and Rfree values were 14.78 and 17.22%, respectively. The catalytic domain of VuChiI adopts an α-helix-rich fold, stabilized by 3 disulfide bridges and possessing a wide catalytic cleft. Analysis of the crystallographic model and molecular docking calculations using chito-oligosaccharides provided evidences about the VuChiI residues involved in sugar binding and catalysis, and a possible mechanism of antifungal action is suggested.

  18. A hevein-like protein and a class I chitinase with antifungal activity from leaves of the paper mulberry.

    PubMed

    Zhao, Ming; Ma, Yan; Pan, Ying-Hong; Zhang, Chun-Hua; Yuan, Wen-Xia

    2011-08-01

    Paper mulberry (Broussonetia papyrifera, syn. Morus papyrifera L.) is a Chinese traditional medicine and its low-molecular-weight extracts are reported to have antifungal activity. In this study, two proteins (PMAPI and PMAPII) with activity against Trichoderma viride were obtained from paper mulberry leaves with a fast protein liquid chromatography (FPLC) unit. The purification protocol employed (NH(4))(2)SO(4) precipitation, ion-exchange chromatography and hydrophobic-interaction chromatography on FPLC. Molecular masses were 18,798 Da for PMAPI, and 31,178 Da for PMAPII determined by Matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Peptide mapping fingerprint analysis showed that PMAPI has no peptides similar to PMAPII. N-terminal amino acid sequencing revealed that PMAPI is a hevein-like protein, and PMAPII is a class I chitinase. They both had a half-maximal inhibitory concentration (IC50) of 0.1 µg/µL against T. viride. This is the first report of high-molecular-weight extracts with antifungal activity from paper mulberry.

  19. Crystallization and preliminary X-ray diffraction analysis of an active-site mutant of `loopless' family GH19 chitinase from Bryum coronatum in a complex with chitotetraose.

    PubMed

    Ohnuma, Takayuki; Umemoto, Naoyuki; Taira, Toki; Fukamizo, Tamo; Numata, Tomoyuki

    2013-12-01

    The catalytic mechanism of family GH19 chitinases is not well understood owing to insufficient information regarding the three-dimensional structures of enzyme-substrate complexes. Here, the crystallization and preliminary X-ray diffraction analysis of a selenomethionine-labelled active-site mutant of `loopless' family GH19 chitinase from the moss Bryum coronatum in complex with chitotetraose, (GlcNAc)4, are reported. The crystals were grown using the vapour-diffusion method. They diffracted to 1.58 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 74.5, b = 58.4, c = 48.1 Å, β = 115.6°. The asymmetric unit of the crystals is expected to contain one protein molecule, with a Matthews coefficient of 2.08 Å(3) Da(-1) and a solvent content of 41%.

  20. Computational analysis of difenoconazole interaction with soil chitinases

    NASA Astrophysics Data System (ADS)

    Vlǎdoiu, D. L.; Filimon, M. N.; Ostafe, V.; Isvoran, A.

    2015-01-01

    This study focusses on the investigation of the potential binding of the fungicide difenoconazole to soil chitinases using a computational approach. Computational characterization of the substrate binding sites of Serratia marcescens and Bacillus cereus chitinases using Fpocket tool reflects the role of hydrophobic residues for the substrate binding and the high local hydrophobic density of both sites. Molecular docking study reveals that difenoconazole is able to bind to Serratia marcescens and Bacillus cereus chitinases active sites, the binding energies being comparable.

  1. Flavonoid Interaction with a Chitinase from Grape Berry Skin: Protein Identification and Modulation of the Enzymatic Activity.

    PubMed

    Filippi, Antonio; Petrussa, Elisa; Rajcevic, Uros; Čurin Šerbec, Vladka; Passamonti, Sabina; Renzone, Giovanni; Scaloni, Andrea; Zancani, Marco; Vianello, Angelo; Braidot, Enrico

    2016-09-28

    In the present study, an antibody raised against a peptide sequence of rat bilitranslocase (anti-peptide Ab) was tested on microsomal proteins obtained from red grape berry skin. Previously, this antibody had demonstrated to recognize plant membrane proteins associated with flavonoid binding and transport. Immuno-proteomic assays identified a number of proteins reacting with this particular antibody, suggesting that the flavonoid binding and interaction may be extended not only to carriers of these molecules, but also to enzymes with very different functions. One of these proteins is a pathogenesis-related (PR) class IV chitinase, whose in vitro chitinolytic activity was modulated by two of the most representative flavonoids of grape, quercetin and catechin, as assessed by both spectrophotometric and fluorimetric assays in grape microsomes and commercial enzyme preparations. The effect of these flavonoids on the catalysis and its kinetic parameters was also evaluated, evidencing that they determine a hormetic dose-dependent response. These results highlight the importance of flavonoids not only as antioxidants or antimicrobial effectors, but also as modulators of plant growth and stress response. Implications of the present suggestion are here discussed in the light of environment and pesticide-reduction concerns.

  2. A high-throughput matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry-based assay of chitinase activity.

    PubMed

    Price, Neil P J; Naumann, Todd A

    2011-04-01

    A high-throughput matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) assay is described for determination of chitolytic enzyme activity. The assay uses unmodified chitin oligosaccharide substrates and is readily achievable on a microliter scale (2μl of total volume containing 2μg of substrate and 1ng of protein). The speed and sensitivity of the assay make it potentially well suited for the high-throughput screening of chitinase inhibitors. The mass spectrum is acquired in approximately 2min, as opposed to typically 30-40min for a single run with a high-performance liquid chromatography (HPLC)-based assay. By using the multiple-place MALDI MS targets, we estimate that 100 assays could be run in approximately 2-3h without needing to remove the target from the instrument. In addition, because the substrate and product chitomers are visualized simultaneously in the TOF spectrum, this gives immediate information about the cleavage site and mechanism of the enzyme under study. The assay was used to monitor the purification and transgenic expression of plant class IV chitinases. By performing the assay with chitomer substrates and C-glycoside chitomer analogs, the enzyme mechanism of the class IV chitinases is described for the first time.

  3. Chitinases: in agriculture and human healthcare.

    PubMed

    Nagpure, Anand; Choudhary, Bharti; Gupta, Rajinder K

    2014-09-01

    Biological control of phytopathogenic fungi and insects continues to inspire the research and development of environmentally friendly bioactive alternatives. Potentially lytic enzymes, chitinases can act as a biocontrol agent against agriculturally important fungi and insects. The cell wall in fungi and protective covers, i.e. cuticle in insects shares a key structural polymer, chitin, a β-1,4-linked N-acetylglucosamine polymer. Therefore, it is advantageous to develop a common biocontrol agent against both of these groups. As chitin is absent in plants and mammals, targeting its metabolism will signify an eco-friendly strategy for the control of agriculturally important fungi and insects but is innocuous to mammals, plants, beneficial insects and other organisms. In addition, development of chitinase transgenic plant varieties probably holds the most promising method for augmenting agricultural crop protection and productivity, when properly integrated into traditional systems. Recently, human proteins with chitinase activity and chitinase-like proteins were identified and established as biomarkers for human diseases. This review covers the recent advances of chitinases as a biocontrol agent and its various applications including preparation of medically important chitooligosaccharides, bioconversion of chitin as well as in implementing chitinases as diagnostic and prognostic markers for numerous diseases and the prospect of their future utilization.

  4. Genetic analysis of the chitinase system of Serratia marcescens 2170.

    PubMed Central

    Watanabe, T; Kimura, K; Sumiya, T; Nikaidou, N; Suzuki, K; Suzuki, M; Taiyoji, M; Ferrer, S; Regue, M

    1997-01-01

    To carry out a genetic analysis of the degradation and utilization of chitin by Serratia marcescens 2170, various Tn5 insertion mutants with characteristic defects in chitinase production were isolated and partially characterized. Prior to the isolation of the mutants, proteins secreted into culture medium in the presence of chitin were analyzed. Four chitinases, A, B, C1, and C2, among other proteins, were detected in the culture supernatant of S. marcescens 2170. All four chitinases and a 21-kDa protein (CBP21) lacking chitinase activity showed chitin binding activity. Cloning and sequencing analysis of the genes encoding chitinases A and B of strain 2170 revealed extensive similarities to those of other strains of S. marcescens described previously. Tn5 insertion mutagenesis of strain 2170 was carried out, and mutants which formed altered clearing zones of colloidal chitin were selected. The obtained mutants were divided into five classes as follows: mutants with (i) no clearing zones, (ii) fuzzy clearing zones, (iii) large clearing zones, (iv) delayed clearing zones, and (v) small clearing zones. Preliminary characterization suggested that some of these mutants have defects in chitinase excretion, a negatively regulating mechanism of chitinase gene expression, an essential factor for chitinase gene expression, and a structural gene for a particular chitinase. These mutants could allow researchers to identify the genes involved in the degradation and utilization of chitin by S. marcescens 2170. PMID:9371460

  5. Potentiation of the synergistic activities of chitinases ChiA, ChiB and ChiC from Serratia marcescens CFFSUR-B2 by chitobiase (Chb) and chitin binding protein (CBP).

    PubMed

    Gutiérrez-Román, Martha Ingrid; Dunn, Michael F; Tinoco-Valencia, Raunel; Holguín-Meléndez, Francisco; Huerta-Palacios, Graciela; Guillén-Navarro, Karina

    2014-01-01

    With the goal of understanding the chitinolytic mechanism of the potential biological control strain Serratia marcescens CFFSUR-B2, genes encoding chitinases ChiA, ChiB and ChiC, chitobiase (Chb) and chitin binding protein (CBP) were cloned, the protein products overexpressed in Escherichia coli as 6His-Sumo fusion proteins and purified by affinity chromatography. Following affinity tag removal, the chitinolytic activity of the recombinant proteins was evaluated individually and in combination using colloidal chitin as substrate. ChiB and ChiC were highly active while ChiA was inactive. Reactions containing both ChiB and ChiC showed significantly increased N-acetylglucosamine trimer and dimer formation, but decreased monomer formation, compared to reactions with either enzyme alone. This suggests that while both ChiB and ChiC have a general affinity for the same substrate, they attack different sites and together degrade chitin more efficiently than either enzyme separately. Chb and CBP in combination with ChiB and ChiC (individually or together) increased their chitinase activity. We report for the first time the potentiating effect of Chb on the activity of the chitinases and the synergistic activity of a mixture of all five proteins (the three chitinases, Chb and CBP). These results contribute to our understanding of the mechanism of action of the chitinases produced by strain CFFSUR-B2 and provide a molecular basis for its high potential as a biocontrol agent against fungal pathogens.

  6. Characterization of a Grape Class IV Chitinase

    PubMed Central

    2015-01-01

    A chitinase was purified from Vitis vinifera Manzoni Bianco grape juice and characterized. On the basis of proteomic analysis of tryptic peptides, a significant match identified the enzyme as a type IV grape chitinase previously found in juices of other V. vinifera varieties. The optimal pH and temperature for activity toward colloidal chitin were found to be 6 and 30 °C, respectively. The enzyme was found to hydrolyze chitin and oligomers of N-acetylglucosamine, generating N,N′-diacetylchitobiose and N-acetylglucosamine as products, but was inactive toward N,N′-diacetylchitobiose. The enzyme exhibited both endo- and exochitinase activities. Because yeast contains a small amount of chitin in the cell wall, the possibility of growth inhibition was tested. At a concentration and pH expected in ripe grapes, no inhibition of wine yeast growth by the chitinase was observed. PMID:24845689

  7. A High-Throughput MALDI-TOF Mass Spectrometry-Based Assay of Chitinase Activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A high-throughput MALDI-TOF mass spectrometric assay is described for assay of chitolytic enzyme activity. The assay uses unmodified chitin oligosaccharide substrates, and is readily achievable on a microliter scale (2 µL total volume, containing 2 µg of substrate and 1 ng of protein). The speed a...

  8. Cloning, expression, and characterization of an antifungal chitinase from Leucaena leucocephala de Wit.

    PubMed

    Kaomek, Mana; Mizuno, Kouichi; Fujimura, Tatsuhito; Sriyotha, Poonsook; Cairns, James R Ketudat

    2003-04-01

    Chitinase cDNAs from Leucaena leucocephala seedlings were cloned by PCR amplification with degenerate primers based on conserved class I chitinase sequences and cDNA library screening. Two closely related chitinase cDNAs were sequenced and inferred to encode precursor proteins of 323 (KB1) and 326 (KB2) amino acids. Expression of the KB2 chitinase from a pET32a plasmid in Origami (DE3) Escherichia coli produced high chitinase activity in the cell lysate. The recombinant thioredoxin fusion protein was purified and cleaved to yield a 32-kDa chitinase. The recombinant chitinase hydrolyzed colloidal chitin with endochitinase-type activity. It also inhibited growth of 13 of the 14 fungal strains tested.

  9. Plant Chitinases and Their Roles in Resistance to Fungal Diseases

    PubMed Central

    Punja, Zamir K.; Zhang, Ye-Yan

    1993-01-01

    Chitinases are enzymes that hydrolyze the N-acetylglucosamine polymer chitin, and they occur in diverse plant tissues over a broad range of crop and noncrop species. The enzymes may be expressed constitutively at low levels but are dramatically enhanced by numerous abiotic agents (ethylene, salicylic acid, salt solutions, ozone, UV light) and by biotic factors (fungi, bacteria, viruses, viroids, fungal cell wall components, and oligosaccharides). Different classes of plant chitinases are distinguishable by molecular, biochemical, and physicochemical criteria. Thus, plant chitinases may differ in substrate-binding characteristics, localization within the cell, and specific activities. Because chitin is a structural component of the cell wall of many phytopathogenic fungi, extensive research has been conducted to determine whether plant chitinases have a role in defense against fungal diseases. Plant chitinases have different degrees of antifungal activity to several fungi in vitro. In vivo, although rapid accumulation and high levels of chitinases (together with numerous other pathogenesis-related proteins) occur in resistant tissues expressing a hypersensitive reaction, high levels also can occur in susceptible tissues. Expression of cloned chitinase genes in transgenic plants has provided further evidence for their role in plant defense. The level of protection observed in these plants is variable and may be influenced by the specific activity of the enzyme, its localization and concentration within the cell, the characteristics of the fungal pathogen, and the nature of the host-pathogen interaction. The expression of chitinase in combination with one or several different antifungal proteins should have a greater effect on reducing disease development, given the complexities of fungal-plant cell interactions and resistance responses in plants. The effects of plant chitinases on nematode development in vitro and in vivo are worthy of investigation. PMID:19279806

  10. Structural and functional characterization of a small chitin-active lytic polysaccharide monooxygenase domain of a multi-modular chitinase from Jonesia denitrificans.

    PubMed

    Mekasha, Sophanit; Forsberg, Zarah; Dalhus, Bjørn; Bacik, John-Paul; Choudhary, Swati; Schmidt-Dannert, Claudia; Vaaje-Kolstad, Gustav; Eijsink, Vincent G H

    2016-01-01

    Lytic polysaccharide monooxygenases (LPMOs) boost enzymatic depolymerization of recalcitrant polysaccharides, such as chitin and cellulose. We have studied a chitin-active LPMO domain (JdLPMO10A) that is considerably smaller (15.5 kDa) than all structurally characterized LPMOs so far and that is part of a modular protein containing a GH18 chitinase. The 1.55 Å resolution structure revealed deletions of interacting loops that protrude from the core β-sandwich scaffold in larger LPMO10s. Despite these deletions, the enzyme is active on alpha- and beta-chitin, and the chitin-binding surface previously described for larger LPMOs is fully conserved. JdLPMO10A may represent a minimal scaffold needed to catalyse the powerful LPMO reaction.

  11. Changes in glycosylation of human blood plasma chitotriosidase in patients with type 2 diabetes.

    PubMed

    Żurawska-Płaksej, Ewa; Kratz, Ewa Maria; Ferens-Sieczkowska, Mirosława; Knapik-Kordecka, Maria; Piwowar, Agnieszka

    2016-02-01

    Human blood plasma chitotriosidase (CHIT1) is a glycoprotein with chitinolytic activity with not fully elucidated biological function. Its increased level is observed in type 2 diabetes mellitus (T2DM) and is associated with development of diabetic complications. The CHIT1 glycosylation profile and degree is still poorly studied and never investigated in T2DM. Therefore the aim of the present study was to examine the association between glycosylation profile and degree and diabetes with accompanying nephropathy. In blood plasma of 28 patients with T2DM and 11 healthy subjects the CHIT1 concentration and specific activity were examined. The profile and degree of CHIT1 glycosylation were determined by lectin-ELISA using lectins specific to O-glycans (Jacalin, MPL, VVL) and sialo-specific SNA and MAA. We revealed that both concentration and specific activity of CHIT1 significantly increased in T2DM, especially in nephropathy with elevated albuminuria. The relative reactivities with lectins, except Jacalin, decreased progressively with T2DM occurrence and albuminuria progression. The most significant differences were observed between control vs. albuminuric group (Micro and Macro). It is also possible that the observed differences in immunoblotting pattern in molecular masses of CHIT1 bands between T2DM patients and healthy subjects may be caused by the differences in degree of CHIT1 glycosylation. The analysis of CHIT1 glycosylation status and the determination of CHIT1 concentration together with its enzymatic activity in blood plasma might constitute additional valuable diagnosis tools for the evaluation the T2DM patients with accompanying nephropathy. Extension of the lectin panel specific to O-glycans occurs useful for the further research using microarray formats, which are expected to accelerate “lectin-based glycan profiling” of glycoproteins.

  12. Cloning, Expression and 3D Structure Prediction of Chitinase from Chitinolyticbacter meiyuanensis SYBC-H1

    PubMed Central

    Hao, Zhikui; Wu, Hangui; Yang, Meiling; Chen, Jianjun; Xi, Limin; Zhao, Weijie; Yu, Jialin; Liu, Jiayang; Liao, Xiangru; Huang, Qingguo

    2016-01-01

    Two CHI genes from Chitinolyticbacter meiyuanensis SYBC-H1 encoding chitinases were identified and their protein 3D structures were predicted. According to the amino acid sequence alignment, CHI1 gene encoding 166 aa had a structural domain similar to the GH18 type II chitinase, and CHI2 gene encoding 383 aa had the same catalytic domain as the glycoside hydrolase family 19 chitinase. In this study, CHI2 chitinase were expressed in Escherichia coli BL21 cells, and this protein was purified by ammonium sulfate precipitation, DEAE-cellulose, and Sephadex G-100 chromatography. Optimal activity of CHI2 chitinase occurred at a temperature of 40 °C and a pH of 6.5. The presence of metal ions Fe3+, Fe2+, and Zn2+ inhibited CHI2 chitinase activity, while Na+ and K+ promoted its activity. Furthermore, the presence of EGTA, EDTA, and β-mercaptoethanol significantly increased the stability of CHI2 chitinase. The CHI2 chitinase was active with p-NP-GlcNAc, with the Km and Vm values of 23.0 µmol/L and 9.1 mM/min at a temperature of 37 °C, respectively. Additionally, the CHI2 chitinase was characterized as an N-acetyl glucosaminidase based on the hydrolysate from chitin. Overall, our results demonstrated CHI2 chitinase with remarkable biochemical properties is suitable for bioconversion of chitin waste. PMID:27240345

  13. [Chitinases in bioengeneering research].

    PubMed

    Shakhbazov, A V; Kartel', N A

    2008-08-01

    Techniques of introduction of foreign genes into the plant genome have been intensely developed in order to directionally improve properties of crops. One of the key directions in plant bioengineering is searching for and analyzing promising genes, in particular, to construct genotypes with high resistance to pathogens and pests. In this review, the use for this purpose of transgenes coding for chitinase family enzymes is considered. Many of these transgenes have proved to be efficient factors for elevating plant resistance to pathogenic fungi.

  14. Expressing a fusion protein with protease and chitinase activities increases the virulence of the insect pathogen Beauveria bassiana.

    PubMed

    Fang, Weiguo; Feng, Jin; Fan, Yanhua; Zhang, Yongjun; Bidochka, Michael J; Leger, Raymond J St; Pei, Yan

    2009-10-01

    Entomopathogenic fungi, such as Beauveria bassiana and Metarhizium anisopliae are being developed as alternatives to chemical insecticides. They infect insects by direct penetration of the cuticle using a combination of physical pressure and extracellular hydrolytic enzymes such as proteases and chitinases. Previously we found that overexpression of a subtilisin-like protease (Pr1A) or a chitinase (Bbchit1) resulted in increased virulence of M. anisopliae and B. bassiana, respectively. In this study, we found that a mixture of the B. bassiana Pr1A homolog (CDEP1) and Bbchit1 degraded insect cuticle in vitro more efficiently than either CDEP1 or Bbchit1 alone. Based on this we produced three plasmid constructs; (1) Bbchit1, (2) CDEP1, and (3) a fusion gene of Bbchit1 linked to CDEP1 each under the control of the constitutive gpd promoter from Aspergillus nidulans. B. bassiana transformants secreting the fusion protein (CDEP1:Bbchit1) penetrated the cuticle significantly faster than the wild type or transformants overexpressing either Bbchit1 or CDEP1. Compared to the wild type, the transformant overexpressing CDEP1 showed a 12.5% reduction in LT(50), without a reduction in LC(50). The LT(50) of the transformant expressing CDEP1:Bbchit1 was reduced by 24.9%. Strikingly, expression of CDEP1:Bbchit1 resulted in a 60.5% reduction in LC(50), more than twice the reduction obtained by overexpression of Bbchit1 (28.5%). This work represents a significant step towards the development of hypervirulent insect pathogens for effective pest control.

  15. Characterization and identification of chitinase producing Streptomyces venezuelae P10.

    PubMed

    Mukherjee, G; Sen, S K

    2004-05-01

    In an attempt to isolate chitinase producers from soil, a streptomycete strain was found potent using natural chitin as the substrate. Chitinolytic activity was tested directly on agar plates, also with crude enzyme. Chitinase assay showed that the isolate could produce 0.8 U/ml of the enzyme. The morphological, cultural, physiological and biochemical characters of the isolate P10 were studied, and identified as Streptomyces venezuelae P10.

  16. High prevalence of chitotriosidase deficiency in Peruvian Amerindians exposed to chitin-bearing food and enteroparasites

    PubMed Central

    Manno, N.; Sherratt, S.; Boaretto, F.; Coico, F. Mejìa; Camus, C. Espinoza; Campos, C. Jara; Musumeci, S.; Battisti, A.; Quinnell, R.J.; León, J. Mostacero; Vazza, G.; Mostacciuolo, M.L.; Paoletti, M.G.; Falcone, F.H.

    2014-01-01

    The human genome encodes a gene for an enzymatically active chitinase (CHIT1) located in a single copy on Chromosome 1, which is highly expressed by activated macrophages and in other cells of the innate immune response. Several dysfunctional mutations are known in CHIT1, including a 24-bp duplication in Exon 10 causing catalytic deficiency. This duplication is a common variant conserved in many human populations, except in West and South Africans. Thus it has been proposed that human migration out of Africa and the consequent reduction of exposure to chitin from environmental factors may have enabled the conservation of dysfunctional mutations in human chitinases. Our data obtained from 85 indigenous Amerindians from Peru, representative of populations characterized by high prevalence of chitin-bearing enteroparasites and intense entomophagy, reveal a very high frequency of the 24-bp duplication (47.06%), and of other single nucleotide polymorphisms which are known to partially affect enzymatic activity (G102S: 42.7% and A442G/V: 25.5%). Our finding is in line with a founder effect, but appears to confute our previous hypothesis of a protective role against parasite infection and sustains the discussion on the redundancy of chitinolytic function. PMID:25256524

  17. Function of a recombinant Chitinase derived from a virulent Aeromonas hydrophila isolated from diseased channel catfish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A chitinase was identified in extracellular products of a virulent Aeromonas hydrophila isolated from diseased channel catfish (Ictalurus punctatus). Bioactive recombinant chitinase (rChi-Ah) was produced in Escherichia coli. Purified rChi-Ah had optimal activity at temperature of 42°C and pH 6.5. T...

  18. Functional characterization of chitinase-3 reveals involvement of chitinases in early embryo immunity in zebrafish.

    PubMed

    Teng, Zinan; Sun, Chen; Liu, Shousheng; Wang, Hongmiao; Zhang, Shicui

    2014-10-01

    The function and mechanism of chitinases in early embryonic development remain largely unknown. We show here that recombinant chitinase-3 (rChi3) is able to hydrolyze the artificial chitin substrate, 4-methylumbelliferyl-β-D-N,N',N″-triacetylchitotrioside, and to bind to and inhibit the growth of the fungus Candida albicans, implicating that Chi3 plays a dual function in innate immunity and chitin-bearing food digestion in zebrafish. This is further corroborated by the expression profile of Chi3 in the liver and gut, which are both immune- and digestion-relevant organs. Compared with rChi3, rChi3-CD lacking CBD still retains partial capacity to bind to C. albicans, but its enzymatic and antifungal activities are significantly reduced. By contrast, rChi3-E140N with the putative catalytic residue E140 mutated shows little affinity to chitin, and its enzymatic and antifungal activities are nearly completely lost. These suggest that both enzymatic and antifungal activities of Chi3 are dependent on the presence of CBD and E140. We also clearly demonstrate that in zebrafish, both the embryo extract and the developing embryo display antifungal activity against C. albicans, and all the findings point to chitinase-3 (Chi3) being a newly-identified factor involved in the antifungal activity. Taken together, a dual function in both innate immunity and food digestion in embryo is proposed for zebrafish Chi3. It also provides a new angle to understand the immune role of chitinases in early embryonic development of animals.

  19. A novel strain of Brevibacillus laterosporus produces chitinases that contribute to its biocontrol potential.

    PubMed

    Prasanna, Lakshmi; Eijsink, Vincent G H; Meadow, Richard; Gåseidnes, Sigrid

    2013-02-01

    A novel strain exhibiting entomopathogenic and chitinolytic activity was isolated from mangrove marsh soil in India. The isolate was identified as Brevibacillus laterosporus by phenotypic characterization and 16S rRNA sequencing and designated Lak1210. When grown in the presence of colloidal chitin as the sole carbon source, the isolate produced extracellular chitinases. Chitinase activity was inhibited by allosamidin indicating that the enzymes belong to the family 18 chitinases. The chitinases were purified by ammonium sulfate precipitation followed by chitin affinity chromatography yielding chitinases and chitinase fragments with 90, 75, 70, 55, 45, and 25 kDa masses. Mass spectrometric analyses of tryptic fragments showed that these fragments belong to two distinct chitinases that are almost identical to two putative chitinases, a 89.6-kDa four-domain chitodextrinase and a 69.4-kDa two-domain enzyme called ChiA1, that are encoded on the recently sequenced genome of B. laterosporus LMG15441. The chitinase mixture showed two pH optima, at 6.0 and 8.0, and an optimum temperature of 70 °C. The enzymes exhibited antifungal activity against the phytopathogenic fungus Fusarium equiseti. Insect toxicity bioassays with larvae of diamondback moths (Plutella xylostella), showed that addition of chitinases reduced the time to reach 50 % mortality upon infection with non-induced B. laterosporus from 3.3 to 2.1 days. This study provides evidence for the presence of inducible, extracellular chitinolytic enzymes in B. laterosporus that contribute to the strain's antifungal activity and insecticidal activity.

  20. BjMYB1, a transcription factor implicated in plant defence through activating BjCHI1 chitinase expression by binding to a W-box-like element

    PubMed Central

    Gao, Ying; Jia, Shuangwei; Wang, Chunlian; Wang, Fujun; Wang, Fajun; Zhao, Kaijun

    2016-01-01

    We previously identified the W-box-like-4 (Wbl-4) element (GTAGTGACTCAT), one of six Wbl elements in the BjC-P promoter of the unusual chitinase gene BjCHI1 from Brassica juncea, as the core element responsive to fungal infection. Here, we report the isolation and characterization of the cognate transcription factor interacting with the Wbl-4 element. Using Wbl-4 as a target, we performed yeast one-hybrid screening of a B. juncea cDNA library and isolated an R2R3-MYB transcription factor designated as BjMYB1. BjMYB1 was localized in the nucleus of plant cells. EMSA assays confirmed that BjMYB1 binds to the Wbl-4 element. Transiently expressed BjMYB1 up-regulated the activity of the BjC-P promoter through its binding to the Wbl-4 element in tobacco (Nicotiana benthamiana) leaves. In B. juncea, BjMYB1 displayed a similar induced expression pattern as that of BjCHI1 upon infection by the fungus Botrytis cinerea. Moreover, heterogeneous overexpression of BjMYB1 significantly elevated the resistance of transgenic Arabidopsis thaliana to the fungus B. cinerea. These results suggest that BjMYB1 is potentially involved in host defence against fungal attack through activating the expression of BjCHI1 by binding to the Wbl-4 element in the BjC-P promoter. This finding demonstrates a novel DNA target of plant MYB transcription factors. PMID:27353280

  1. Malaria parasite chitinase and penetration of the mosquito peritrophic membrane.

    PubMed Central

    Huber, M; Cabib, E; Miller, L H

    1991-01-01

    Malaria parasites (ookinetes) appear to digest the peritrophic membrane in the mosquito midgut during penetration. Previous studies demonstrated that lectins specific for N-acetylglucosamine bind to the peritrophic membrane and proposed that the membrane contains chitin [Rudin, W. & Hecker, H. (1989) Parasitol. Res. 75, 268-279]. In the present study, we show that the peritrophic membrane is digested by Serratia marcescens chitinase (EC 3.2.1.14), leading to the release of N-acetylglucosamine and fragmentation of the membrane. We also report the presence of a malaria parasite chitinase that digests 4-methylumbelliferyl chitotriose. The enzyme is not detectable until 15 hr after zygote formation, the time required for maturation of the parasite from a zygote to an ookinete, the invasive form of the parasite. At 20 hr, the enzyme begins to appear in the culture supernatant. The chitinase extracted from the parasite and found in the culture supernatant consists of a major band and two minor bands of activity on native polyacrylamide gel electrophoresis. The presence of chitin in the peritrophic membrane, the disruption of the peritrophic membrane during invasion, and the presence of chitinase in ookinetes suggest that the chitinase in ookinetes is used in the penetration of the peritrophic membrane. Images PMID:2011589

  2. Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen suppressive soil

    SciTech Connect

    Hjort, K.; Bergstrom, M.; Adesina, M.F.; Jansson, J.K.; Smalla, K.; Sjoling, S.

    2009-09-01

    Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal-restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal-restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF{sup 103} of the isolate, Streptomyces mutomycini and/or Streptomyces clavifer. There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.

  3. Chitinase from Autographa californica multiple nucleopolyhedrovirus: rapid purification from Sf-9 medium and mode of action.

    PubMed

    Fukamizo, Tamo; Sato, Hirokazu; Mizuhara, Mamiko; Ohnuma, Takayuki; Gotoh, Takeshi; Hiwatashi, Kazuyuki; Takahashi, Saori

    2011-01-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) chitinase is involved in the final liquefaction of infected host larvae. We purified the chitinase rapidly to homogeneity from Sf-9 cells infected with AcMNPV by a simple procedure using a pepstatin-aminohexyl-Sepharose column. In past studies, a recombinant AcMNPV chitinase was found to exhibit both exo- and endo-chitinase activities by analysis using artificial substrates with a fluorescent probe. In this study, however, we obtained more accurate information on the mode of action of the chitinase by HPLC analysis of the enzymatic products using natural oligosaccharide and polysaccharide substrates. The AcMNPV chitinase hydrolyzed the second β-1,4 glycosidic linkage from the non-reducing end of the chitin oligosaccharide substrates [(GlcNAc)(n), n=4, 5, and 6], producing the β-anomer of (GlcNAc)₂. The mode of action was similar to that of Serratia marcescens chitinase A (SmChiA), the amino acid sequence of which is 60.5% homologous to that of the AcMNPV enzyme. The enzyme also hydrolyzed solid β-chitin, producing only (GlcNAc)₂. The AcMNPV chitinase processively hydrolyzes solid β-chitin in a manner similar to SmChiA. The processive mechanism of the enzyme appears to be advantageous in liquefaction of infected host larvae.

  4. Expression of a novel chitinase by the fungal endophyte in Poa ampla.

    PubMed

    Li, Huaijun Michael; Sullivan, Ray; Moy, Melinda; Kobayashi, Donald Y; Belanger, Faith C

    2004-01-01

    Many wild and cultivated cool-season grass species are naturally infected with fungal endophytes of the genera Neotyphodium and Epichloë. These associations generally are considered mutualistic with the plants benefiting from reduced herbivory and the fungi benefiting from nutrients supplied by the plants. The fungi secrete proteins that might have a role in the interspecies symbiosis. In the interaction between Poa ampla Merr. and the endophyte Neotyphodium sp., a fungal chitinase was detected in the apoplastic protein fraction. The chitinase was also the major protein secreted in culture. Sequence analysis of the chitinase revealed it has a low level of amino acid sequence identity to other fungal chitinases and one of the conserved active site residues is altered. DNA gel-blot analysis indicated the chitinase was encoded by a single gene. Expression of similar chitinases also was detected in endophyte-infected tall fescue (Festuca arundinacea Schreb.), perennial ryegrass (Lolium perenne L.) and Chewings fescue (Festuca rubra L. subsp. fallax [Thuill] Nyman). This is the first report of an endophyte chitinase expressed in the infected host grass. As a secreted hydrolytic enzyme, the chitinase might have roles in the nutrition, growth or defense of the endophyte.

  5. Cloning and functional analysis of a novel chitinase gene Trchi1 from Trichothecium roseum.

    PubMed

    Xian, Hongquan; Li, Jiarui; Zhang, Liqing; Li, Duochuan

    2012-10-01

    Chitinases produced by mycoparasites play an important role in disease control in plants. To explore the functions of chitinases in Trichothecium roseum, we cloned a new chitinase gene named Trchi1 from T. roseum by RT (reverse transcription)-PCR techniques. The T. roseum gene, Trchi1, contains an 1278-bp ORF that shares 76 % similarity with chitinase from Bionectria ochroleuca (ABV57861 3G6L_A). A plant expression vector, containing the Trchi1 gene driven by the CaMV35S promoter, was constructed and transformed into tobacco via Agrobacterium tumefaciens. Southern blot analysis showed that Trchi1 was integrated into the tobacco genome. Total chitinase activity in Trchi1-transgenic tobacco leaves was enhanced 2.2- to 5.8- times with respect to non-transgenic leaves. Transgenic tobacco plants transformed with the Trchi1 gene had increased resistance to Alternaria alternata and Colletotrichum nicotianae.

  6. Enhanced expression of chitinase during the autolysis of mushroom in Coprinellus congregatus.

    PubMed

    Lim, Hyangsoon; Choi, Hyoung T

    2009-04-01

    Fungal cell walls consist of various glucans and chitin. An inky cap, Coprinellus congregates, produced mushrooms at 25 degrees C in a regime of 15 h light/9 h dark, and then the mushroom was autolyzed rapidly to generate black liquid droplets where no cell wall was detected by microscopy. A chitinase cDNA from the matured mushroom cells of C. congregates that consisted of 1,541 nucleotides was successfully cloned using the rapid amplification of cDNA ends (RACE)-PCR technique. Its deduced 441 amino acid sequence had the conserved catalytic domain as in other fungal chitinase family 18. Chitinase activity was higher at the matured mushroom stage than primordial and young mushroom stage. When the expression of the cloned chitinase was examined by real-time PCR using the chitinase-specific primers, it was increased more than twice to 20 times during the autolytic process of mushroom than young mushroom or primordial stages, respectively.

  7. Colloid chitin azure is a dispersible, low-cost substrate for chitinase measurements in a sensitive, fast, reproducible assay.

    PubMed

    Shen, Chia-Rui; Chen, Yu-Sheng; Yang, Ching-Jen; Chen, Jeen-Kuan; Liu, Chao-Lin

    2010-02-01

    Chitin and its derivatives are widely used as biomedical materials because of their versatility and biocompatibility. Chitinases are enzymes that produce chito-oligosaccharides from chitin. The assay of chitinase activity is difficult because few appropriate substrates are available. In this study, the authors developed an efficient and low-cost chitinase assay using colloidal chitin azure. The assay feasibility is evaluated and compared with traditional assays employing colloidal chitin and chitin azure. The authors found that the optimum pH for determining chitinase activity using colloid chitin azure was pH 5 or 8. The method was sensitive, and the assay was complete within 30 min. When the assay was used to measure chitinase activities produced by 2 strains of chitinolytic bacteria, BCTS (an Escherichia coli BL21 [DE3] expressing a secretory recombinant chitinase) and AS1 (a chitinolytic bacterium with low levels of chitinase), it was shown that cultivation in Bushnell-Haas selection medium caused AS1 to secrete a higher level of chitinase than was secreted when the bacterium grew in other media. In summary, colloid chitin azure is a sensitive, feasible, reproducible, and low-cost substrate for the assay of chitinase activity.

  8. Secreted major Venus flytrap chitinase enables digestion of Arthropod prey.

    PubMed

    Paszota, Paulina; Escalante-Perez, Maria; Thomsen, Line R; Risør, Michael W; Dembski, Alicja; Sanglas, Laura; Nielsen, Tania A; Karring, Henrik; Thøgersen, Ida B; Hedrich, Rainer; Enghild, Jan J; Kreuzer, Ines; Sanggaard, Kristian W

    2014-02-01

    Predation plays a major role in energy and nutrient flow in the biological food chain. Plant carnivory has attracted much interest since Darwin's time, but many fundamental properties of the carnivorous lifestyle are largely unexplored. In particular, the chain of events leading from prey perception to its digestive utilization remains to be elucidated. One of the first steps after the capture of animal prey, i.e. the enzymatic breakup of the insects' chitin-based shell, is reflected by considerable chitinase activity in the secreted digestive fluid in the carnivorous plant Venus flytrap. This study addresses the molecular nature, function, and regulation of the underlying enzyme, VF chitinase-I. Using mass spectrometry based de novo sequencing, VF chitinase-I was identified in the secreted fluid. As anticipated for one of the most prominent proteins in the flytrap's "green stomach" during prey digestion, transcription of VF chitinase-I is restricted to glands and enhanced by secretion-inducing stimuli. In their natural habitat, Venus flytrap is exposed to high temperatures. We expressed and purified recombinant VF chitinase-I and show that the enzyme exhibits the hallmark properties expected from an enzyme active in the hot and acidic digestive fluid of Dionaea muscipula. Structural modeling revealed a relative compact globular form of VF chitinase-I, which might contribute to its overall stability and resistance to proteolysis. These peculiar characteristics could well serve industrial purposes, especially because of the ability to hydrolyze both soluble and crystalline chitin substrates including the commercially important cleavage of α-chitin.

  9. Human gastric juice contains chitinase that can degrade chitin.

    PubMed

    Paoletti, Maurizio G; Norberto, Lorenzo; Damini, Roberta; Musumeci, Salvatore

    2007-01-01

    Chitin digestion by humans has generally been questioned or denied. Only recently chitinases have been found in several human tissues and their role has been associated with defense against parasite infections and to some allergic conditions. In this pilot study we tested the gastric juices of 25 Italian subjects on the artificial substrates 4-methylumbelliferyl-beta-D-N,N',diacetylchitobiose or/and fluorescein isothiocyanate (FITC) chitin to demonstrate the presence of a chitinase activity. Since this chitinase activity was demonstrated at acidic pH, it is currently referred to acidic mammalian chitinase (AMCase). AMCase activity was present in gastric juices of twenty of 25 Italian patients in a range of activity from 0.21 to 36.27 nmol/ml/h and from 8,881 to 1,254,782 fluorescence emission (CPS), according to the used methods. In the remaining five of 25 gastric juices, AMCase activity was almost absent in both assay methods. An allosamidine inhibition test and the measurement at different pH values confirmed that this activity was characteristic of AMCase. The absence of activity in 20% of the gastric juices may be a consequence of virtual absence of chitinous food in the Western diet.

  10. CT gene modulate differential expression of chitinase gene under variant habitats in Vibrio cholerae

    PubMed Central

    Verma, Yogendra Kumar; Verma, Mahendra Kumar

    2013-01-01

    Objective To investigate the interrelation of cholera toxin gene (CT gene) in expression of chitinase gene under different pH conditions among pathogenic and Non-pathogenic strains of Vibrio cholera (V. cholera). Methods The chitinase assay well diffusion method and calorimetric chitinase assay were performed. Further, time depended chitinase activity among pathogenic and nonpathogenic strain was evaluated with control as Escherichia coli. The expressed protein in variant environment was purified by cascade of chromatographic techniques. The partially purified protein was analyzed by SDS-PAGE in both the strain of V. cholera. Results The results have shown differential expression of chitinase gene among vibrio in time depended chitinase activity, purification of expressed protein and SDS-PAGE analysis. Conclusions From the current study, two conclusions came in picture, habitat is prime factor that regulation of chitin gene expression among many bacterial strains, second, moreover among the vibrio pathogenic strains (CT+) expression of chitinase gene is more precisely regulated by CT gene rather than external environments while in non-pathogenic strain ( CT-) completely absent.

  11. Chitinases Are Essential for Cell Separation in Ustilago maydis

    PubMed Central

    Langner, Thorsten; Öztürk, Merve; Hartmann, Sarah; Cord-Landwehr, Stefan; Moerschbacher, Bruno; Walton, Jonathan D.

    2015-01-01

    Chitin is an essential component of the fungal cell wall, providing rigidity and stability. Its degradation is mediated by chitinases and supposedly ensures the dynamic plasticity of the cell wall during growth and morphogenesis. Hence, chitinases should be particularly important for fungi with dramatic morphological changes, such as Ustilago maydis. This smut fungus switches from yeast to filamentous growth for plant infection, proliferates as a mycelium in planta, and forms teliospores for spreading. Here, we investigate the contribution of its four chitinolytic enzymes to the different morphological changes during the complete life cycle in a comprehensive study of deletion strains combined with biochemical and cell biological approaches. Interestingly, two chitinases act redundantly in cell separation during yeast growth. They mediate the degradation of remnant chitin in the fragmentation zone between mother and daughter cell. In contrast, even the complete lack of chitinolytic activity does not affect formation of the infectious filament, infection, biotrophic growth, or teliospore germination. Thus, unexpectedly we can exclude a major role for chitinolytic enzymes in morphogenesis or pathogenicity of U. maydis. Nevertheless, redundant activity of even two chitinases is essential for cell separation during saprophytic growth, possibly to improve nutrient access or spreading of yeast cells by wind or rain. PMID:25934689

  12. Biochemistry of plant class IV chitinases and fungal chitinase-modifying proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant class IV chitinases have 2 domains, a small (3 kDa) amino-terminal domain with homology to carbohydrate binding peptides, and a larger (25 kDa) catalytic domain. The biological function of these chitinases is not known. But it is known that some pathogenic fungi secrete chitinase modifying pro...

  13. In silico identification of novel chitinase-like proteins in the silkworm, Bombyx mori, genome.

    PubMed

    Pan, Ye; Lü, Peng; Wang, Yong; Yin, Lijing; Ma, Hexiang; Ma, Guohong; Chen, Keping; He, Yuanqing

    2012-01-01

    In insects, chitinases participate in the periodic shedding of old exoskeletons and the turnover of peritrophic membranes. Chitinase family members have been identified in dozens of species, including Tribolium castaneum, Drosophila melanogaster, and Anopheles gambiae. In this study, nine chitinases and three hypothetical chitinases have been identified in Bombyx mori L. (Lepidoptera: Bombycidae) through genome-wide searching. Phylogenetic analyses revealed that seven of them belong to the seven chitinase groups, respectively. BmCht25 and BmCht26 could not be grouped into the known chitinase groups, and might belong to two new groups of the chitinase family. BmCht10, BmCht25, and BmIDGF have glutamate amino acid substitutions in the active catalytic domain. Only BmCht5 and BmCht10 contain CBD domain and PEST sequences (rich in proline, glutamic acid, serine, and threonine). BmCht5 and BmCht26 are located on chromosome 7, and others (BmCht6, BmCht7, BmCht10, BmCht11, BmCht20, BmIDGF) are located on separate chromosomes of Bombyx mori, respectively. The present study provides important background information for future studies using Bombyx mori as a model organism for insect development and virus and host interaction.

  14. Partial biochemical characterization of crude extract extracellular chitinase enzyme from Bacillus subtilis B 298

    NASA Astrophysics Data System (ADS)

    Lestari, P.; Prihatiningsih, N.; Djatmiko, H. A.

    2017-02-01

    Extraction and characterization of extracellular chitinase from Bacillus subtilis B 298 have been done. Growth curve determination of B. subtilis B 298, production curve determination of crude extract chitinase from B. subtilis B 298, and partial biochemical characterization of crude extract chitinase have been achieved in this study. Optimum growth of B. subtilis B 298 was achieved at logarithmic phase within 9 hours incubation time, so it was used as inoculum for enzyme production. According to production curve of the enzyme, it was known that incubation time which gave the highest chitinase activity of 15 hours with activity of 6.937 U/mL respectively. Effect of various temperatures on chitinase activity showed that optimum activity was achieved at 40°C with an activity of 5.764 U/mL respectively. Meanwhile, the optimum pH for chitinase activity was achieved at pH of 5.0 with an activity of 6.813 U/mL respectively. This enzyme was then classified as metalloenzyme due to the decline of the activity by EDTA addition. All divalent cations tested acted as inhibitors.

  15. Chitinases from Bacteria to Human: Properties, Applications, and Future Perspectives

    PubMed Central

    Rathore, Abhishek Singh; Gupta, Rinkoo D.

    2015-01-01

    Chitin is the second most plenteous polysaccharide in nature after cellulose, present in cell walls of several fungi, exoskeletons of insects, and crustacean shells. Chitin does not accumulate in the environment due to presence of bacterial chitinases, despite its abundance. These enzymes are able to degrade chitin present in the cell walls of fungi as well as the exoskeletons of insect. They have shown being the potential agents for biological control of the plant diseases caused by various pathogenic fungi and insect pests and thus can be used as an alternative to chemical pesticides. There has been steady increase in demand of chitin derivatives, obtained by action of chitinases on chitin polymer for various industrial, clinical, and pharmaceutical purposes. Hence, this review focuses on properties and applications of chitinases starting from bacteria, followed by fungi, insects, plants, and vertebrates. Designing of chitinase by applying directed laboratory evolution and rational approaches for improved catalytic activity for cost-effective field applications has also been explored. PMID:26664744

  16. Identification of a chitinase modifying protein from Fusarium verticillioides: truncation of a host resistance protein by a fungalysin metalloprotease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chitinase modifying proteins (cmps) are proteases, secreted by fungal pathogens, which truncate the plant class IV chitinases ChitA and ChitB during maize ear rot. Cmp activity has been characterized for Bipolaris zeicola and Stenocarpella maydis, but the identities of the proteases are not known. H...

  17. Degradation of chitin and chitosan by a recombinant chitinase derived from a virulent Aeromonas hydrophila isolated from diseased channel catfish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A chitinase was identified in extracellular products of a virulent Aeromonas hydrophila isolated from diseased channel catfish (Ictalurus punctatus). Bioactive recombinant chitinase (rChi-Ah) was produced in Escherichia coli. Purified rChi-Ah had optimal activity at temperature of 42°C and pH 6.5. T...

  18. Recent development of two chitinase inhibitors, Argifin and Argadin, produced by soil microorganisms

    PubMed Central

    Hirose, Tomoyasu; Sunazuka, Toshiaki; Ōmura, Satoshi

    2010-01-01

    Chitin, the second most abundant polysaccharide in nature, occurs in fungi, some algae and many invertebrates, including insects. Thus, chitin synthesis and degradation could represent specific targets for fungicides and insecticides. Chitinases hydrolyze chitin into oligomers of N-acetyl-d-glucosamine at key points in the life cycles of organisms, consequently, chitinase inhibitors have become subject of increasing interest. This review covers the development of two chitinase inhibitors of natural origin, Argifin and Argadin, isolated from the cultured broth of microorganisms in our laboratory. In particular, the practical total synthesis of these natural products, the synthesis of lead compounds via computer-aided rational molecular design, and discovery methods that generate only highly-active compounds using a kinetic target(chitinase)-guided synthesis approach (termed in situ click chemistry) are described. PMID:20154467

  19. Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens

    PubMed Central

    Jones, Jonathan D. G.; Grady, Karen L.; Suslow, Trevor V.; Bedbrook, John R.

    1986-01-01

    Analysis of clones isolated from a cosmid DNA library indicates that the Serratia marcescens chromosome contains at least two genes, chiA and chiB, which encode distinct secreted forms of the enzyme chitinase. These genes have been characterized by inspection of chitinase activity and secreted proteins in Escherichia coli strains containing subclones of these cosmids. The two chitinase genes show no detectable homology to each other. DNA sequence analysis of one of the genes predicts an amino acid sequence with an N-terminal signal peptide typical of genes encoding secreted bacterial proteins. This gene was mutagenized by cloning a neomycin phosphotransferase gene within its coding region, and the insertion mutation was recombined into the parental S. marcescens strain. The resulting chiA mutant transconjugant showed reduced chitinase production, reduced inhibition of fungal spore germination and reduced biological control of a fungal plant pathogen. ImagesFig. 2. PMID:16453672

  20. Chitinase production by Bacillus thuringiensis and Bacillus licheniformis: their potential in antifungal biocontrol.

    PubMed

    Gomaa, Eman Zakaria

    2012-02-01

    Thirty bacterial strains were isolated from the rhizosphere of plants collected from Egypt and screened for production of chitinase enzymes. Bacillus thuringiensis NM101-19 and Bacillus licheniformis NM120-17 had the highest chitinolytic activities amongst those investigated. The production of chitinase by B. thuringiensis and B. licheniformis was optimized using colloidal chitin medium amended with 1.5% colloidal chitin, with casein as a nitrogen source, at 30°C after five days of incubation. An enhancement of chitinase production by the two species was observed by addition of sugar substances and dried fungal mats to the colloidal chitin media. The optimal conditions for chitinase activity by B. thuringiensis and B. licheniformis were at 40°C, pH 7.0 and pH 8.0, respectively. Na(+), Mg(2+), Cu(2+), and Ca(2+) caused enhancement of enzyme activities whereas they were markedly inhibited by Zn(2+), Hg(2+), and Ag(+). In vitro, B. thuringiensis and B. licheniformis chitinases had potential for cell wall lysis of many phytopathogenic fungi tested. The addition of B. thuringiensis chitinase was more effective than that of B. licheniformis in increasing the germination of soybean seeds infected with various phytopathogenic fungi.

  1. Cloning and expression analysis of the chitinase gene Ifu-chit2 from Isaria fumosorosea

    PubMed Central

    Meng, Huimin; Wang, Zhangxun; Meng, Xiangyun; Xie, Ling; Huang, Bo

    2015-01-01

    Entomopathogenic fungi can produce a series of chitinases, some of which function synergistically with proteases and other hydrolytic enzymes to degrade the insect cuticle. In the present study, the chitinase gene Ifu-chit2 from Isaria fumosorosea was investigated. The Ifu-chit2 gene is 1,435-bp long, interrupted by three short introns, and encodes a predicted protein of 423 amino acids with a 22 residue signal peptide. The predicted Ifu-Chit2 protein is highly homologous to Beauveria bassiana chitinase Bbchit2 and belongs to the glycohydrolase family 18. Ifu-Chit2 was expressed in Escherichia coli to verify chitinase activity, and the recombinant enzyme exhibited activity with a colloidal chitin substrate. Furthermore, the expression profiles of Ifu-chit2 were analyzed at different induction times under in vivo conditions. Quantitative real-time PCR analysis revealed that Ifu-chit2 expression peaked at two days post-induction. The expression of chitinase Ifu-chit2 in vivo suggests that the chitinase may play a role in the early stage of pathogenesis. PMID:26500443

  2. Functional specialization among insect chitinase family genes revealed by RNA interference.

    PubMed

    Zhu, Qingsong; Arakane, Yasuyuki; Beeman, Richard W; Kramer, Karl J; Muthukrishnan, Subbaratnam

    2008-05-06

    The biological functions of individual members of the large family of chitinase-like proteins from the red flour beetle, Tribolium castaneum (Tc), were examined by using gene-specific RNAi. One chitinase, TcCHT5, was found to be required for pupal-adult molting only. A lethal phenotype was observed when the transcript level of TcCHT5 was down-regulated by injection of TcCHT5-specific dsRNA into larvae. The larvae had metamorphosed into pupae and then to pharate adults but did not complete adult eclosion. Specific knockdown of transcripts for another chitinase, TcCHT10, which has multiple catalytic domains, prevented embryo hatch, larval molting, pupation, and adult metamorphosis, indicating a vital role for TcCHT10 during each of these processes. A third chitinase-like protein, TcCHT7, was required for abdominal contraction and wing/elytra extension immediately after pupation but was dispensable for larval-larval molting, pupation, and adult eclosion. The wing/elytra abnormalities found in TcCHT7-silenced pupae were also manifest in the ensuing adults. A fourth chitinase-like protein, TcIDGF4, exhibited no chitinolytic activity but contributed to adult eclosion. No phenotypic effects were observed after knockdown of transcripts for several other chitinase-like proteins, including imaginal disk growth factor IDGF2. These data indicate functional specialization among insect chitinase family genes, primarily during the molting process, and provide a biological rationale for the presence of a large assortment of chitinase-like proteins.

  3. Cloning and characterization of a small family 19 chitinase from moss (Bryum coronatum).

    PubMed

    Taira, Toki; Mahoe, Yoko; Kawamoto, Noriko; Onaga, Shoko; Iwasaki, Hironori; Ohnuma, Takayuki; Fukamizo, Tamo

    2011-05-01

    Chitinase-A (BcChi-A) was purified from a moss, Bryum coronatum, by several steps of column chromatography. The purified BcChi-A was found to be a molecular mass of 25 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of 3.5. A cDNA encoding BcChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1012 nucleotides and encoded an open reading frame of 228 amino acid residues. The predicted mature BcChi-A consists of 205 amino acid residues and has a molecular weight of 22,654. Sequence analysis indicated that BcChi-A is glycoside hydrolase family-19 (GH19) chitinase lacking loops I, II, IV and V, and a C-terminal loop, which are present in the catalytic domain of plant class I and II chitinases. BcChi-A is a compact chitinase that has the fewest loop regions of the GH19 chitinases. Enzymatic experiments using chitooligosaccharides showed that BcChi-A has higher activity toward shorter substrates than class II enzymes. This characteristic is likely due to the loss of the loop regions that are located at the end of the substrate-binding cleft and would be involved in substrate binding of class II enzymes. This is the first report of a chitinase from mosses, nonvascular plants.

  4. Does 24bp Duplication of Human CHIT1 Gene (Chitotriosidase1) Predispose to Filarial Chyluria? A Case-Control Study

    PubMed Central

    Pant, Shriya; Agarwal, Jyotsna; Gangwar, Pravin K; Waseem, Mohammad; Gupta, Prashant; Sankhwar, Satya N; Purkait, Bimalesh

    2016-01-01

    Introduction Chyluria which is endemic in many parts of the world is mainly caused by Wuchereria bancrofti. CHIT1 (chitotriosidase) is produced by macrophages and plays an important role in the defense against chitin containing pathogen such as filarial parasite. Variation in the coding region with 24 bp duplication allele results in reduced CHIT1 activity that enhance the survival of parasite which may play a role in the occurrence of disease. Aim To examine the role of 24bp duplication of CHIT1 gene in patients of filarial chyluria (FC). Materials and Methods A case-control study was carried out where 155 confirmed FC patients and equal number of age-, sex- and residence-matched controls without any symptoms or signs of lymphatic filariasis, confirmed by negative immunochromatographic card test (ICT) and IgG/IgM combo rapid antibody test, from a hospital-based population were enrolled. Filarial aetiology was confirmed on the basis of DEC-provocative test (Giemsa staining), ICT and IgG/IgM- antifiarial antibody test. The patients positive by either of these tests were enrolled as FC cases. 24bp duplication in CHIT1 gene in FC was detected by the product size 99bp of amplified gene using polymerase chain reaction. Results The mean ages of patients and controls were 38.25±12.09 and 35.45±12.53 years, respectively while male: female ratio was 2.4:1. The mean duration of illness in chyluria patients was 62.81±60.83 months and mean number of episodes was 2.54±1.11. Homozygous wild type, heterozygous and homozygous mutant frequencies were 10.3%, 81.3% and 8.4% in FC patients and 18.7%, 75.5%, and 5.8% in controls, respectively. The 24bp duplication in CHIT1 gene showed a significant association in Heterozygous (HT) genotype with Odd Ratio (OR) of 1.95, 95% Confidence Interval (CI) (1.01-3.77); p=0.04. However, the homozygous mutant genotype (TT) was found to be non-significant with OR of 2.61, 95% CI (0.91-7.45); p=0.07. The combination of both HT+TT was also found

  5. Characterization of an antifungal and cryoprotective class I chitinase from table grape berries (Vitis vinifera cv. Cardinal).

    PubMed

    Fernandez-Caballero, Carlos; Romero, Irene; Goñi, Oscar; Escribano, M Isabel; Merodio, Carmen; Sanchez-Ballesta, M Teresa

    2009-10-14

    Gene expression of a class I chitinase (Vcchit1b) in the skin of table grapes was analyzed as a molecular marker for changes induced at low temperature and also to study the effect of high CO(2) levels modulating transcript levels at 0 degrees C. An active recombinant VcCHIT1b was overexpressed in Escherichia coli, and as the protein was produced as insoluble inclusion bodies, it was solubilized and refolded. The purified recombinant chitinase showed an optimum pH of 6.0 and a temperature of 50 degrees C, retaining activity at 0 and -10 degrees C. Purified chitinase exerted in vitro antifungal activity against Botrytis cinerea. Furthermore, recombinant chitinase was able to cryoprotect lactate dehydrogenase against freeze/thaw inactivation. However, the recombinant VcCHIT1b did not show any antifreeze activity when the thermal hysteresis activity was measured using differential scanning calorimetry.

  6. Effect of chitinase on resistance to fungal pathogens in sea buckthorn, Hippophae rhamnoides, and cloning of Class I and III chitinase genes.

    PubMed

    Sun, Yan-Lin; Hong, Soon-Kwan

    2012-08-01

    Sea buckthorn (Hippophae rhamnoides L.) is naturally distributed from Asia to Europe. It has been widely planted as an ornamental shrub and is rich in nutritional and medicinal compounds. Fungal pathogens that cause diseases such as dried-shrink disease are threats to the production of this plant. In this study, we isolated the dried-shrink disease pathogen from bark and total chitinase protein from leaves of infected plants. The results of the Oxford Cup experiment suggested that chitinase protein inhibited the growth of this pathogen. To improve pathogen resistance, we cloned chitinase Class I and III genes in H. rhamnoides, designated Hrchi1 and Hrchi3. The full-length cDNA of the open reading frame region of Hrchi1 contained 903 bp encoding 300 amino acids and Hrchi3 contained 894 bp encoding 297 amino acids. Active domain analysis, protein types, and secondary and 3D structures were predicted using online software.

  7. Purification and characterization of an antifungal chitinase from Citrobacter freundii str. nov. haritD11.

    PubMed

    Meruvu, Haritha; Donthireddy, Sri Rami Reddy

    2014-01-01

    The purpose of the research was to study the purification and partial characterization of antifungal alkaline chitinase from a newly isolated Citrobacter freundii haritD11. The enzyme was purified in a three-step procedure involving ammonium sulfate precipitation, dialysis, and Sephadex G-100 gel filtration chromatography. The enzyme was shown to have a relative high molecular weight of 64 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and was purified 7.3-fold with a yield of 18.8 %. It was most active at 35 °C, pH 8.0, with colloid chitin as substrate and was very stable at alkaline pH contradicting the characteristic that most of the bacterial chitinases are active at acidic pH. Further, the purified chitinase exhibited remarkable antifungal activity against pathogenic fungi Aspergillus flavus MTCC 2798 and Aspergillus niger MTCC 9652 showing diametric inhibition zones of 27 mm and 21 mm, respectively.

  8. Coexpression of chitinase and the cry11Aa1 toxin genes in Bacillus thuringiensis serovar israelensis.

    PubMed

    Sirichotpakorn, N; Rongnoparut, P; Choosang, K; Panbangred, W

    2001-10-01

    At the spore stage, a cloned chitinase gene was coexpressed with the regulatory gene p19 and the toxin gene cry11Aa1 in the hosts Bacillus thuringiensis serovar israelensis strains 4Q2-72 and c4Q2-72. The chitinase gene was derived from a high-chitinase producer, Bacillus licheniformis TP-1. Two transcriptional fusion plasmids between the p19 or p19-cry11Aa1 genes and the promoterless chitinase gene were constructed. In transcription order, the p16-19CHI construct contained the p19 gene together with the chitinase gene only while the p16-1968CHI construct contained p19 together with the toxin gene cry11Aa1 and the chitinase gene. The inserted sequences were regulated by a spore-specific promoter located upstream of p19. The recombinant chitinase of all transformed B. thuringiensis serovar israelensis strains was initially synthesized at low level at about 9 h of growth when a portion of the cells started to sporulate. It increased thereafter and reached maximum levels of 5.5, 4.9, and 4.7 mU/ml at 48 h, for strain 4Q2-72 transformed with p16-19CHI and p16-1968CHI and strain c4Q2-72 transformed with p16-19CHI, respectively. This activity was approximately 2 times higher than the maximum activity (2.7 mU/ml) of the parental strain, B. licheniformis TP-1. Although crude chitinase alone from B. thuringiensis serovar israelensis c4Q2-72 (p16-19CHI) at 4.5 mU/ml caused 40% mortality in second instar Aedes aegypti larvae, transformants containing the chitinase alone or in combination with cry11Aa1 resulted in lower toxicity to A. aegypti larvae than the untransformed 4Q2-72 host. For example the LC(50) for the transformed 4Q2-72 harboring the chitinase gene only (p16-19CHI) was 5.6 x 10(4) +/- 0.7 x 10(4) cells, 40 times higher than that of the untransformed host at 1.4 x 10(3) +/- 0.19 x 10(3). The lower toxicity correlated with poor sporulation in the transformants (i.e., 35 times lower than that in the untransformed host). However, the transformed 4Q2-72 strain

  9. ArabidopsisChitinases: a Genomic Survey.

    PubMed

    Passarinho, Paul A; de Vries, Sacco C

    2002-01-01

    Plant chitinases (EC 3.2.1.14) belong to relatively large gene families subdivided in classes that suggest class-specific functions. They are commonly induced upon the attack of pathogens and by various sources of stress, which led to associating them with plant defense in general. However, it is becoming apparent that most of them display several functions during the plant life cycle, including taking part in developmental processes such as pollination and embryo development. The number of chitinases combined with their multiple functions has been an obstacle to a better understanding of their role in plants. It is therefore important to identify and inventory all chitinase genes of a plant species to be able to dissect their function and understand the relations between the different classes. Complete sequencing of the Arabidopsis genome has made this task feasible and we present here a survey of all putative chitinase-encoding genes accompanied by a detailed analysis of their sequence. Based on their characteristics and on studies on other plant chitinases, we propose an overview of their possible functions as well as modified annotations for some of them.

  10. Tissue distribution, synthesis stage, and ethylene induction of pineapple (Ananas comosus) chitinases.

    PubMed

    Taira, Toki; Toma, Noriko; Ichi, Marika; Takeuchi, Makoto; Ishihara, Masanobu

    2005-04-01

    We examined the tissue distribution, synthesis stage, and ethylene induction of three types of pineapple chitinase using chitinase activity gel and immunoblot analysis. Type A (acidic class III) exists in all tissues, while type B (weakly basic class I, which has strong antifungal activity) and type C (acidic class I) are localized mainly in the leaf and stem. In a pericarp, type A exists at all stages during fruit development, while type B and type C exist only at the early stage. Synthesis of type A is induced by ethylene, while that of types B and C is not affected by it. These results suggest that the physiological roles of these three types of chitinase in pineapple are different.

  11. Characterization of two Listeria innocua chitinases of different sizes that were expressed in Escherichia coli.

    PubMed

    Honda, Shotaro; Wakita, Satoshi; Sugahara, Yasusato; Kawakita, Masao; Oyama, Fumitaka; Sakaguchi, Masayoshi

    2016-09-01

    Two putative chitinase genes, lin0153 and lin1996, from the nonpathogenic bacterium Listeria innocua were expressed in Escherichia coli, and the gene products were characterized. The genes were close homologs of chitinases from the pathogenic bacterium Listeria monocytogenes, in which chitinases and chitin-binding proteins play important roles in pathogenesis in mice-infection models. The purified recombinant enzymes that are different in size, LinChi78 (lin0153 product) and LinChi35 (lin1996 product)-with molecular masses of 82 and 38 kDa, including vector-derived additional sequences, respectively-exhibited optimum catalytic activity under neutral and acidic conditions at 50 °C, respectively, and were stable over broad pH (4-11) and temperature (4-40 °C) ranges. LinChi35 displayed higher k cat and K M values for 4-nitrophenyl N,N-diacetyl-β-D-chitobioside [4NP-(GlcNAc)2] than LinChi78. Both enzymes produced primarily dimers from colloidal chitin as a substrate. However, LinChi78 and LinChi35 could hydrolyze oligomeric substrates in a processive exo- and nonprocessive endo-manner, respectively, and showed different reactivity toward oligomeric substrates. Both enzymes could bind chitin beads but were different in their binding ability toward crystalline α-chitin and cellulose. The structure-function relationships of these chitinases are discussed in reference to other bacterial chitinases.

  12. Trichoderma asperellum Chi42 Genes Encode Chitinase

    PubMed Central

    Quang, Hoang Tan; Hung, Nguyen Bao; Huy, Nguyen Duc; Phuong, Truong Thi Bich; Ha, Tran Thi Thu

    2011-01-01

    Four Trichoderma strains (CH2, SH16, PQ34, and TN42) were isolated from soil samples collected from Quang Tri and Thua Thien Hue provinces in Vietnam. The strains exhibited high chitinolytic secretion. Strain PQ34 formed the largest zone of chitinase-mediated clearance (> 4 cm in diameter) in agar containing 1% (w/v) colloidal chitin. Analysis of the internal transcribed spacer regions of these strains indicated that they were Trichoderma asperellum. The molecular weights of the chitinases were approximately 42 kDa. Chitinase genes (chi42) of T. asperellum strains TN42, CH2, SH16, and PQ34 were 98~99% homologous to the ech42 gene of T. harzianum CB-Pin-01 (accession No. DQ166036). The deduced amino acid sequences of both T. asperellum strains SH16 and TN42 shared 100% similarity. PMID:22783101

  13. Crystal structures and inhibitor binding properties of plant class V chitinases: the cycad enzyme exhibits unique structural and functional features.

    PubMed

    Umemoto, Naoyuki; Kanda, Yuka; Ohnuma, Takayuki; Osawa, Takuo; Numata, Tomoyuki; Sakuda, Shohei; Taira, Toki; Fukamizo, Tamo

    2015-04-01

    A class V (glycoside hydrolase family 18) chitinase from the cycad Cycas revoluta (CrChiA) is a plant chitinase that has been reported to possess efficient transglycosylation (TG) activity. We solved the crystal structure of CrChiA, and compared it with those of class V chitinases from Nicotiana tabacum (NtChiV) and Arabidopsis thaliana (AtChiC), which do not efficiently catalyze the TG reaction. All three chitinases had a similar (α/β)8 barrel fold with an (α + β) insertion domain. In the acceptor binding site (+1, +2 and +3) of CrChiA, the Trp168 side chain was found to stack face-to-face with the +3 sugar. However, this interaction was not found in the identical regions of NtChiV and AtChiC. In the DxDxE motif, which is essential for catalysis, the carboxyl group of the middle Asp (Asp117) was always oriented toward the catalytic acid Glu119 in CrChiA, whereas the corresponding Asp in NtChiV and AtChiC was oriented toward the first Asp. These structural features of CrChiA appear to be responsible for the efficient TG activity. When binding of the inhibitor allosamidin was evaluated using isothermal titration calorimetry, the changes in binding free energy of the three chitinases were found to be similar to each other, i.e. between -9.5 and -9.8 kcal mol(-1) . However, solvation and conformational entropy changes in CrChiA were markedly different from those in NtChiV and AtChiC, but similar to those of chitinase A from Serratia marcescens (SmChiA), which also exhibits significant TG activity. These results provide insight into the molecular mechanism underlying the TG reaction and the molecular evolution from bacterial chitinases to plant class V chitinases.

  14. Chitinase but N-acetyl-β-D-glucosaminidase production correlates to the biomass decline in Penicillium and Aspergillus species.

    PubMed

    Pusztahelyi, Tünde; Pócsi, István

    2014-06-01

    Hydrolytic enzyme production is typical of the autolysis in filamentous fungi; however, less attention has been given to the physiological role of the enzymes. Here, the aim was to investigate the possible relation of the chitinolytic enzymes to the changes in the biomass in some filamentous fungi of high importance for pharmaceutical or food industry. In Penicillium and Aspergillus filamentous fungi, which showed different characteristics in submerged cultures, the growth and biomass decline rates were calculated and correlated to the chitinase and N-acetyl-β-D-glucosaminidase enzyme productions. Correlation was found between the biomass decrease rate and the chitinase level at the stationary growth phase; while chitinase production covariates negatively with N-acetyl-β-D-glucosaminidase activities. The chitinase production and the intensive autolysis hindered the production of N-acetyl-β-D-glucosaminidase and, therefore, could hinder the cell death in the cultures.

  15. Insectivorous Bats Digest Chitin in the Stomach Using Acidic Mammalian Chitinase

    PubMed Central

    Strobel, Sara; Roswag, Anna; Becker, Nina I.; Trenczek, Tina E.; Encarnação, Jorge A.

    2013-01-01

    The gastrointestinal tract of animals is adapted to their primary source of food to optimize resource use and energy intake. Temperate bat species mainly feed on arthropods. These contain the energy-rich carbohydrate chitin, which is indigestible for the endogenous enzymes of a typical mammalian gastrointestinal tract. However, the gastrointestinal tract of bat species should be adapted to their diet and be able to digest chitin. We hypothesized that (i) European vespertilionid bat species have the digestive enzyme chitinase and that (ii) the chitinolytic activity is located in the intestine, as has been found for North American bat species. The gastrointestinal tracts of seven bat species (Pipistrellus pipistrellus, Plecotus auritus, Myotis bechsteinii, Myotis nattereri, Myotis daubentonii, Myotis myotis, and Nyctalus leisleri) were tested for chitinolytic activity by diffusion assay. Gastrointestinal tracts of P. pipistrellus, P. auritus, M. nattereri, M. myotis, and N. leisleri were examined for acidic mammalian chitinase by western blot analysis. Tissue sections of the gastrointestinal tract of P. pipistrellus were immunohistochemically analyzed to locate the acidic mammalian chitinase. Chitinolytic activity was detected in the stomachs of all bat species. Western blot analysis confirmed the acidic mammalian chitinase in stomach samples. Immunohistochemistry of the P. pipistrellus gastrointestinal tract indicated that acidic mammalian chitinase is located in the stomach chief cells at the base of the gastric glands. In conclusion, European vespertilionid bat species have acidic mammalian chitinase that is produced in the gastric glands of the stomach. Therefore, the gastrointestinal tracts of insectivorous bat species evolved an enzymatic adaptation to their diet. PMID:24019876

  16. Isolation, purification, crystallization and preliminary crystallographic studies of chitinase from tamarind (Tamarindus indica) seeds

    PubMed Central

    Patil, Dipak N.; Datta, Manali; Chaudhary, Anshul; Tomar, Shailly; Kumar Sharma, Ashwani; Kumar, Pravindra

    2009-01-01

    A protein with chitinase activity has been isolated and purified from tamarind (Tamarindus indica) seeds. N-terminal amino-acid sequence analysis of this protein confirmed it to be an ∼34 kDa endochitinase which belongs to the acidic class III chitinase family. The protein was crystallized by the vapour-diffusion method using PEG 4000. The crystals belonged to the tetragonal space group P41, with two molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.6 Å. PMID:19342775

  17. Polyglycine hydrolases secreted by Pleosporineae fungi that target the linker region of plant class IV chitinases.

    PubMed

    Naumann, Todd A; Wicklow, Donald T; Price, Neil P J

    2014-06-01

    Cmps (chitinase-modifying proteins) are fungal proteases that truncate plant class IV chitinases by cleaving near their N-termini. We previously described Fv-cmp, a fungalysin protease that cleaves a conserved glycine-cysteine bond within the hevein domain. In the present paper we describe a new type of cmp, polyglycine hydrolases, as proteases that selectively cleave glycine-glycine peptide bonds within the polyglycine linker of plant class IV chitinases. Polyglycine hydrolases were purified from Cochliobolus carbonum (syn. Bipolaris zeicola; Bz-cmp) and Epicoccum sorghi (syn. Phoma sorghina; Es-cmp) and were shown to cleave three different maize class IV chitinase substrates. The proteolytic cleavage sites were assessed by SDS/PAGE and MALDI-TOF-MS and indicated the cleavage of multiple peptide bonds within the polyglycine linker regions. Site-directed mutagenesis was used to produce mutants of maize ChitB chitinase in which two serine residues in its linker were systematically modified to glycine. Serine to glycine changes in the ChitB linker resulted in higher susceptibility to truncation by Bz-cmp and altered substrate specificity for Bz-cmp and Es-cmp, such that different glycine-glycine peptide bonds were cleaved. Removal of the hevein domain led to loss of Es-cmp activity, indicating that interactions outside of the active site are important for recognition. Our findings demonstrate that plant class IV chitinases with polyglycine linkers are targeted for truncation by selective polyglycine hydrolases that are secreted by plant pathogenic fungi. This novel proteolysis of polyglycine motifs is previously unreported, but the specificity is similar to that of bacterial lysostaphin proteases, which cleave pentaglycine cross-links from peptidoglycan.

  18. A molt-associated chitinase cDNA from the spruce budworm, Choristoneura fumiferana.

    PubMed

    Zheng, Y; Zheng, S; Cheng, X; Ladd, T; Lingohr, E J; Krell, P J; Arif, B M; Retnakaran, A; Feng, Q

    2002-12-01

    Chitinase (CfChitinase) cDNA from the spruce budworm, Choristoneura fumiferana, was cloned using reverse transcription PCR and cDNA library screening. The CfChitinase cDNA was determined to be 2856 nucleotides long with the longest open reading frame made up of 1671 nucleotides that encoded a protein that was 557 amino acid long with a predicted molecular mass of 62 kDa. The deduced amino acid sequence showed 76-79% identity with other lepidopteran chitinases. Northern blots revealed that transcripts of CfChitinase appeared prior to each molt and peaked on the day of ecdysis from the second instar to the pupal stage but disappeared immediately after the molt. No transcripts could be detected in the early first instar prior to the spinning of the hibernaculum or in the diapausing second instars or during the intermolt periods of the other instars. Western blot analysis revealed that the protein appeared 12 h prior to ecdysis and disappeared 12 h after ecdysis from the sixth instar to pupal stage. The 20-hydroxyecdysone analog, tebufenozide (RH5992), induced expression of CfChitinase in the early stage of the sixth instar and caused a precocious and incomplete molt into an extra larval stage. During the sixth instar to the pupal molt, transcripts could be detected only in the epidermis and fat bodies, but not in the midgut. Western blots showed that the protein was present in the epidermis and midgut, but not in the fat bodies. The recombinant protein expressed in Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) showed high levels of chitinolytic activity with an optimal pH range 6-9. Glycosylation appeared to be necessary for the chitinolytic activity and secretion of the recombinant protein.

  19. Unexpected effects of chitinases on the peach-potato aphid (Myzus persicae Sulzer) when delivered via transgenic potato plants (Solanum tuberosum Linné) and in vitro.

    PubMed

    Saguez, Julien; Hainez, Romaric; Cherqui, Anas; Van Wuytswinkel, Olivier; Jeanpierre, Haude; Lebon, Gaël; Noiraud, Nathalie; Beaujean, Antony; Jouanin, Lise; Laberche, Jean-Claude; Vincent, Charles; Giordanengo, Philippe

    2005-02-01

    With the aim of producing insect-resistant potato plants, internode explants of Solanum tuberosum L. cv. Désirée were transformed with an Agrobacterium strain C58pMP90 containing an insect (Phaedon cochleariae: Coleoptera, Chrysomelidae) chitinase gene and the neomycin phosphotransferase (nptII) gene as selectable marker, both under the control of the viral CaMV 35S promoter. Three transformed potato lines (CH3, CH5 and CH25) exhibiting the highest chitinolytic activities were selected for feeding experiments with the peach-potato aphid, Myzus persicae (Sulzer), under controlled photoperiod and temperature conditions. Aphids fed on transgenic potato plants showed a reduced pre-reproductive period and an enhanced daily fecundity. Transgenic potato lines did not affect nymphal mortality, but improved several biological parameters related to aphid population's growth. Artificial diets were used to provide active (1, 10, 100 and 500 microg ml(-1)) and inactive (500 microg ml(-1)) bacterial (Serratia marcescens) chitinase to M. persicae. These compounds increased nymph survival at all active chitinase doses when compared to the control diet, while inactive chitinase did not. Although the pre-reproductive period was slightly shortened and the daily fecundity slightly higher, active and inactive chitinase provided as food led a reduction from 1 to 1.5 day population's doubling time. Therefore chitinase activity was responsible for the probiotic effects on aphids. Our results question the relevance of a chitinase-based strategy in the context of potato culture protection.

  20. Phylogeny of chitinases and its implications for estimating horizontal gene transfer from chitinase-transgenic silver birch (Betula pendula).

    PubMed

    Lohtander, Katileena; Pasonen, Hanna-Leena; Aalto, Markku K; Palva, Tapio; Pappinen, Ari; Rikkinen, Jouko

    2008-01-01

    Chitinases are hydrolytic enzymes that have been employed in biotechnology in attempts to increase plants' resistance against fungal pathogens. Genetically modified plants have given rise to concerns of the spreading of transgenes into the environment through vertical or horizontal gene transfer (HGT). In this study, chitinase-like sequences from silver birch (Betula pendula) EST-libraries were identified and their phylogenetic relationships to other chitinases were studied. Phylogenetic analyses were used to estimate the frequency of historical gene transfer events of chitinase genes between plants and other organisms, and the usefulness of phylogenetic analyses as a source of information for the risk assessment of transgenic silver birch carrying a sugar beet chitinase IV gene was evaluated. Thirteen partial chitinase-like sequences, with an approximate length of 600 bp, were obtained from the EST-libraries. The sequences belonged to five chitinase classes. Some bacterial chitinases from Streptomyces and Burkholderia, as well as a chitinase from an oomycete, Phytophthora infestans, grouped together with the class IV chitinases of plants, supporting the hypothesis that some class IV chitinases in bacteria have evolved from eukaryotic chitinases via horizontal gene transfer. According to our analyses, HGT of a chitinase IV gene from eukaryotes to bacteria has presumably occurred only once. Based on this, the likelihood for the HGT of chitinase IV gene from transgenic birch to other organisms is extremely low. However, as risk is a function of both the likelihood and consequences of an event, the effects of rare HGT event(s) will finally determine the level of the risk.

  1. An ammonium sulfate sensitive chitinase from Streptomyces sp. CS501.

    PubMed

    Rahman, Md Arifur; Choi, Yun Hee; Pradeep, G C; Yoo, Jin Cheol

    2014-12-01

    A chitinase from Streptomyces sp. CS501 was isolated from the Korean soil sample, purified by single-step chromatography, and biochemically characterized. The extracellular chitinase (Ch501) was purified to 4.60 fold with yield of 28.74 % using Sepharose Cl-6B column. The molecular mass of Ch501 was approximately 43 kDa as estimated by SDS-PAGE and zymography. The enzyme (Ch501) was found to be stable over a broad pH range (5.0-10.0) and temperature (up to 50 °C), and have an optimum temperature of 60 °C. N-terminal sequence of Ch501 was AAYDDAAAAA. Intriguingly, Ch501 was highly sensitive to ammonium sulfate but it's completely suppressed activity was recovered after desalting out. TLC analysis of Ch501 showed the production of N-acetyl D-glucosamine (GlcNAc) and Diacetylchitobiose (GlcNAc)2, as a principal hydrolyzed product. Ch501 shows antifungal activity against Fusarium solani and Aspergillus brasiliensis, which can be used for the biological control of fungus. As has been simple in purification, stable in a broad range of pH, ability to produce oligosaccharides, and antifungal activity showed that Ch501 has potential applications in industries as for chitooligosaccharides production used as prebiotics and/or for the biological control of plant pathogens in agriculture.

  2. Recognition of Corn Defense Chitinases by Fungal Polyglycine Hydrolases.

    PubMed

    Naumann, Todd A; Bakota, Erica L; Price, Neil P J

    2017-04-06

    Polyglycine hydrolases (PGH)s are secreted fungal endoproteases that cleave peptide bonds in the polyglycine interdomain linker of ChitA chitinase, an antifungal protein from domesticated corn (Zea mays ssp. mays). These target-specific endoproteases are unusual because they do not cut a specific peptide bond but select one of many Gly-Gly bonds within the polyglycine region. Some Gly-Gly bonds are cleaved frequently while others are never cleaved. Moreover, we have previously shown that PGHs from different fungal pathogens prefer to cleave different Gly-Gly peptide bonds. It is not understood how PGHs selectively cleave the ChitA linker, especially because its polyglycine structure lacks peptide sidechains. To gain insights into this process we synthesized several peptide analogs of ChitA to evaluate them as potential substrates and inhibitors of Es-cmp, a PGH from the plant pathogenic fungus Epicoccum sorghi. Our results showed that part of the PGH recognition site for substrate chitinases is adjacent to the polyglycine linker on the carboxy side. More specifically, four amino acid residues were implicated, each spaced four residues apart on an alpha helix. Moreover, analogous peptides with selective Gly->sarcosine (N-methylglycine) mutations or a specific Ser->Thr mutation retained inhibitor activity but were no longer cleaved by PGH. Additonally, our findings suggest that peptide analogs of ChitA that inhibit PGH activity could be used to strengthen plant defenses. This article is protected by copyright. All rights reserved.

  3. A broad pH range and processive chitinase from a metagenome library

    PubMed Central

    Thimoteo, S.S.; Glogauer, A.; Faoro, H.; de Souza, E.M.; Huergo, L.F.; Moerschbacher, B.M.; Pedrosa, F.O.

    2017-01-01

    Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked β(1-4) present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N′-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin. PMID:28076454

  4. A broad pH range and processive chitinase from a metagenome library.

    PubMed

    Thimoteo, S S; Glogauer, A; Faoro, H; de Souza, E M; Huergo, L F; Moerschbacher, B M; Pedrosa, F O

    2017-01-05

    Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked β(1-4) present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N'-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.

  5. Identification and Characterization of a Chitinase Antigen from Pseudomonas aeruginosa Strain 385

    PubMed Central

    Thompson, Suzanne E.; Smith, Mark; Wilkinson, Mark C.; Peek, Keith

    2001-01-01

    A chitinase antigen has been identified in Pseudomonas aeruginosa strain 385 using sera from animals immunized with a whole-cell vaccine. The majority of the activity was shown to be in the cytoplasm, with some activity in the membrane fraction. The chitinase was not secreted into the culture medium. Purification of the enzyme was achieved by exploiting its binding to crab shell chitin. The purified enzyme had a molecular mass of 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 5.2. NH2-terminal amino acid sequencing revealed two sequences of M(I/L)RID and (Q/M/V)AREDAAAAM that gave an exact match to sequences in a translated putative open reading frame from the P. aeruginosa genome. The chitinase was active against chitin azure, ethylene glycol chitin, and colloidal chitin. It did not display any lysozyme activity. Using synthetic 4-methylumbelliferyl chitin substrates, it was shown to be an endochitinase. The Km and kcat for 4-nitrophenyl-β-d-N,N′-diacetylchitobiose were 4.28 mM and 1.7 s−1 respectively, and for 4-nitrophenyl-β-d-N,N′,N′′-triacetylchitotriose, they were 0.48 mM and 0.16 s−1 respectively. The pH optimum was determined to be pH 6.75, and 90% activity was maintained over the pH range 6.5 to 7.1. The enzyme was stable over the pH range 5 to 10 for 3 h and to temperatures up to 50°C for 30 min. The chitinase bound strongly to chitin, chitin azure, colloidal chitin, lichenan, and cellulose but poorly to chitosan, xylan, and heparin. It is suggested that the chitinase functions primarily as a chitobiosidase, removing chitobiose from the nonreducing ends of chitin and chitin oligosaccharides. PMID:11525997

  6. Extracellular chitinases of fluorescent pseudomonads antifungal to Fusarium oxysporum f. sp. dianthi causing carnation wilt.

    PubMed

    Ajit, Naosekpam Singh; Verma, Rajni; Shanmugam, V

    2006-04-01

    Vascular wilt of carnation caused by Fusarium oxysporum f. sp. dianthi (Prill. & Delacr.) W. C. Synder & H.N. Hans inflicts substantial yield and quality loss to the crop. Mycolytic enzymes such as chitinases are antifungal and contribute significantly to the antagonistic activity of fluorescent pseudomonads belonging to plant-growth-promoting rhizobacteria. Fluorescent pseudomonads antagonistic to the vascular wilt pathogen were studied for their ability to grow and produce chitinases on different substrates. Bacterial cells grown on chitin-containing media showed enhanced growth and enzyme production with increased anti-fungal activity against the pathogen. Furthermore, the cell-free bacterial culture filtrate from chitin-containing media also significantly inhibited the mycelial growth. Both the strains and their cell-free culture filtrate from chitin-amended media showed the formation of lytic zones on chitin agar, indicating chitinolytic ability. Extracellular proteins of highly antagonistic bacterial strain were isolated from cell-free extracts of media amended with chitin and fungal cell wall. These cell-free conditioned media contained one to seven polypeptides. Western blot analysis revealed two isoforms of chitinase with molecular masses of 43 and 18.5 kDa. Further plate assay for mycelial growth inhibition showed the 43-kDa protein to be antifungal. The foregoing studies clearly established the significance of chitinases in the antagonism of fluorescent pseudomonads, showing avenues for possible exploitation in carnation wilt management.

  7. Expression, purification, crystallization and preliminary crystallographic analysis of chitinase A from Vibrio carchariae

    SciTech Connect

    Songsiriritthigul, Chomphunuch; Yuvaniyama, Jirundon; Robinson, Robert C.; Vongsuwan, Archara; Prinz, Heino; Suginta, Wipa

    2005-10-01

    This article describes the high-level expression, purification and crystallization as well as preliminary X-ray diffraction study of a family 18 chitinase, chitinase A from V. carchariae. Chitinase A of Vibrio carchariae was expressed in Escherichia coli M15 host cells as a 575-amino-acid fragment with full enzymatic activity using the pQE60 expression vector. The yield of the highly purified recombinant protein was approximately 70 mg per litre of bacterial culture. The molecular mass of the expressed protein was determined by HPLC/ESI–MS to be 63 770, including the hexahistidine tag. Crystals of recombinant chitinase A were grown to a suitable size for X-ray structure analysis in a precipitant containing 10%(v/v) PEG 400, 0.1 M sodium acetate pH 4.6 and 0.125 M CaCl{sub 2}. The crystals belonged to the tetragonal space group P422, with two molecules per asymmetric unit and unit-cell parameters a = b = 127.64, c = 171.42 Å. A complete diffraction data set was collected to 2.14 Å resolution using a Rigaku/MSC R-AXIS IV{sup ++} detector system mounted on an RU-H3R rotating-anode X-ray generator.

  8. Molecular modelling, dynamics simulation and characterization of antifungal chitinase from Sechium edule.

    PubMed

    Bhattacharjee, Bipasha; Pathaw, Neeta; Chrungoo, Nikhil K; Bhattacharjee, Atanu

    2017-03-30

    Chitinases are varied sized proteins which have the ability to degrade chitin and are present in a huge range of organisms like fungi, yeasts, arthropods, humans etc. and have been getting increased attention due to their biocontrol properties. In silico analysis sheds light on the extensive properties of this plant protein. In this paper, a particular antifungal protein Chitinase sourced from Sechium edule from East Khasi Hills, Meghalaya was characterized using an array of bioinformatics tools. The modelled protein showed conserved domains characteristic to glycosyl hydrolase, family 18 superfamily. Likewise, a part of the conserved domain area fits in with xylanase inhibitor Xip-1 and the class ΙΙΙ plant chitinases, for example, concanavalin B, hevamine, which have a GH18 area. The modelled wild type protein exhibited secondary characteristics comprising of 48.8% helix, 62.2% sheets and 13.8% turns, displaying an aliphatic index of 80.53 and instability index of 48.88 inferring upon the fact that the protein is relatively unstable without its appropriate environment. The paper functions as the first attempt to portray molecular dynamics simulation of Chitinase from Sechium edule reinforced by modelling and thorough characteristic analysis of the protein by employing parameters like Ramachandran Plot, Chou and Fasman Secondary Structure prediction, ProtParam etc. Further approaches like protein engineering and activity analysis suggested.

  9. The role of enzyme distortion in the single displacement mechanism of family 19 chitinases

    PubMed Central

    Brameld, Ken A.; Goddard, William A.

    1998-01-01

    By using molecular dynamics simulations, we have examined the binding of a hexaNAG substrate and two potential hydrolysis intermediates (an oxazoline ion and an oxocarbenium ion) to a family 19 barley chitinase. We find the hexaNAG substrate binds with all sugars in a chair conformation, unlike the family 18 chitinase which causes substrate distortion. Glu 67 is in a position to protonate the anomeric oxygen linking sugar residues D and E whereas Asn 199 serves to hydrogen bond with the C2′ N-acetyl group of sugar D, thus preventing the formation of an oxazoline ion intermediate. In addition, Glu 89 is part of a flexible loop region allowing a conformational change to occur within the active site to bring the oxocarbenium ion intermediate and Glu 89 closer by 4–5 Å. A hydrolysis product with inversion of the anomeric configuration occurs because of nucleophilic attack by a water molecule that is coordinated by Glu 89 and Ser 120. Issues important for the design of inhibitors specific to family 19 chitinases over family 18 chitinases also are discussed. PMID:9539727

  10. Stimulatory effects of chitinase on growth and immune defense of orange-spotted grouper (Epinephelus coioides).

    PubMed

    Zhang, Yanhong; Feng, Shaozhen; Chen, Jun; Qin, Chaobin; Lin, Haoran; Li, Wensheng

    2012-05-01

    Chitinase, belonging to either family 18 or family 19 of the glycosylhydrolases, hydrolyze chitin into oligosaccharides. In the present study, the cDNA fragment encoding orange-spotted grouper (Epinephelus coioides) chitinase1 was subcloned into pPIC3.5K vector and expressed in Pichia pastoris GS115. The results showed that a band with the size of about 53 kDa could be detected by SDS-PAGE and Western blot. The recombinant protein of grouper chitinase1 (rgChi1) was added into the fish diet containing shrimp shell chitin for feeding experiment lasting 8 weeks. The weight of orange-spotted grouper, fed with diets containing rgChi1 at 0, 5, 10 and 20 μg/g was calculated on the 2nd, 4th, 6th and 8th weeks, and difference in growth rates was first observed in the 6th week of the feeding period and it kept until the end of the feeding experiment. At the end of 8 weeks feeding trial, the percent weight gain (PWG), growth rate (GR) and specific growth rate (SGR) of fish fed with 10 and 20 μg rgChi1/g feed were significantly higher compared to the control group. The neuropeptide Y (NPY), growth-hormone-releasing hormone (GHRH), growth-hormone (GH), interleukin-1beta (IL-1β), cyclooxygenase-2 (COX-2), superoxide dismutase (SOD) (Cu/Zn) and SOD (Mn) mRNA expression of fish fed with diet containing 10 μg/g or/and 20 μg/g rgChi1 were obviously higher than the control group. The lysozyme (LZM) and total SOD activity of fish fed with diet containing rgChi1 at 10 and 20 μg/g were significantly higher than that of the control. The aspartate aminotransferase (AST)/glutamic oxalacetic transaminases (GOT) activity in 20 μg/g group decreased compared to the control group. These results indicated that the grouper chitinase1 was successfully produced using the P. pastoris expression system and the recombinant protein had obvious effects on growth and immune defense. The mRNA expression and protein secretion of grouper chitinase1 and chitinase2 were significantly stimulated in

  11. Enzymatic characterization of the Plasmodium vivax chitinase, a potential malaria transmission-blocking target

    PubMed Central

    Takeo, Satoru; Hisamori, Daisuke; Matsuda, Shusaku; Vinetz, Joseph; Sattabongkot, Jetsumon; Tsuboi, Takafumi

    2009-01-01

    The chitinase (EC 3.2.1.14) of the human malaria parasite Plasmodium falciparum, PfCHT1, has been validated as a malaria transmission-blocking vaccine (TBV). The present study aimed to delineate functional characteristics of the P. vivax chitinase PvCHT1, whose primary structure differs from that of PfCHT1 by having proenzyme and chitin-binding domains. The recombinant protein rPvCHT1 expressed with a wheat germ cell-free system hydrolyzed 4-methylumbelliferone (4MU) derivatives of chitin oligosaccharides (β-1,4-poly-N-acetyl glucosamine (GlcNAc)). An anti-rPvCHT1 polyclonal antiserum reacted with in vitro-obtained P. vivax ookinetes in anterior cytoplasm, showing uneven patchy distribution. Enzymatic activity of rPvCHT1 shared the exclusive endochitinase property with parallelly expressed rPfCHT1 as demonstrated by a marked substrate preference for 4MU-GlcNAc3 compared to shorter GlcNAc substrates. While rPvCHT1 was found to be sensitive to the general family-18 chitinase inhibitor, allosamidin, its pH (maximal in neutral environment) and temperature (max. at ~25 °C) activity profiles and sensitivity to allosamidin (IC50=6 μM) were different from rPfCHT1. The results in this first report of functional rPvCHT1 synthesis indicate that the P. vivax chitinase is enzymatically close to long form Plasmodium chitinases represented by P. gallinaceum PgCHT1. PMID:19427918

  12. Carbohydrate-binding motif in Chitinase 3-like 1 (CHI3L1/YKL-40) specifically activates Akt signaling pathway in colonic epithelial cells

    PubMed Central

    Chen, Chun-Chuan; Llado, Victoria; Eurich, Katrin; Tran, Hoa T.; Mizoguchi, Emiko

    2011-01-01

    Host-microbial interactions play a key role during the development of colitis. We have previously shown that chinase 3-like 1 (CHI3L1) is an inducible molecule overexpressed in colonic epithelial cells (CECs) under inflammatory conditions. In this study, we found that chitin-binding motif (CBM) of CHI3L1 is specifically associated with the CHI3L1-mediated activation of the Akt-signaling in CEC by transfecting the CBM-mutant CHI3L1 vectors in SW480 CECs. Downstream, CHI3L1 enhanced the secretion of IL-8 and TNFα in a dose-dependent manner. We previously show that 325 through 339 amino-acids in CBM are crucial for the biological function of CHI3L1. Here we demonstrated that 325th–339th residues of CBM in CHI3L1 is a critical region for the activation of Akt, IL-8 production, and for a specific cellular localization of CHI3L1. In conclusion, CBM region of CHI3L1 is critical in activating Akt signaling in CECs, and the activation may be associated with the development of chronic colitis. PMID:21546314

  13. Structural Investigation of a Novel N-Acetyl Glucosamine Binding Chi-Lectin Which Reveals Evolutionary Relationship with Class III Chitinases

    PubMed Central

    Patil, Dipak N.; Datta, Manali; Dev, Aditya; Dhindwal, Sonali; Singh, Nirpendra; Dasauni, Pushpanjali; Kundu, Suman; Sharma, Ashwani K.; Tomar, Shailly; Kumar, Pravindra

    2013-01-01

    The glycosyl hydrolase 18 (GH18) family consists of active chitinases as well as chitinase like lectins/proteins (CLPs). The CLPs share significant sequence and structural similarities with active chitinases, however, do not display chitinase activity. Some of these proteins are reported to have specific functions and carbohydrate binding property. In the present study, we report a novel chitinase like lectin (TCLL) from Tamarindus indica. The crystal structures of native TCLL and its complex with N-acetyl glucosamine were determined. Similar to the other CLPs of the GH18 members, TCLL lacks chitinase activity due to mutations of key active site residues. Comparison of TCLL with chitinases and other chitin binding CLPs shows that TCLL has substitution of some chitin binding site residues and more open binding cleft due to major differences in the loop region. Interestingly, the biochemical studies suggest that TCLL is an N-acetyl glucosamine specific chi-lectin, which is further confirmed by the complex structure of TCLL with N-acetyl glucosamine complex. TCLL has two distinct N-acetyl glucosamine binding sites S1 and S2 that contain similar polar residues, although interaction pattern with N-acetyl glucosamine varies extensively among them. Moreover, TCLL structure depicts that how plants utilize existing structural scaffolds ingenuously to attain new functions. To date, this is the first structural investigation of a chi-lectin from plants that explore novel carbohydrate binding sites other than chitin binding groove observed in GH18 family members. Consequently, TCLL structure confers evidence for evolutionary link of lectins with chitinases. PMID:23717482

  14. Chitinase modifying proteins from phylogenetically distinct lineages of Brassica pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chitinase modifying proteins (CMPs) are secreted fungal proteases that truncate specific plant class IV chitinases by cleaving peptide bonds in their amino termini. We recently identified a CMP from the Zea mays (maize) pathogen Fusarium verticillioides and found that it is a member of the fungalysi...

  15. Family GH19 plant class IV chitinase from Zea mays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize ChitA chitinase is composed of a small, hevein-like domain attached to a carboxy-terminal chitinase domain. During fungal ear rot, the hevein-like domain is cleaved by secreted fungal proteases to produce truncated forms of ChitA. Here we report a structural and biochemical characterization of...

  16. Modification of recombinant maize ChitA chitinase by fungal chitinase-modifying proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In commercial maize, there are at least two different alleles of the chiA gene that encode alloforms of ChitA chitinase, a protein that is abundant in developing seed. Both known alloforms are modified by Bz-cmp, a protein secreted by the fungal pathogen Bipolaris zeicola. One alloform (ChitA-B73) i...

  17. Induced production of chitinase to enhance entomotoxicity of Bacillus thuringiensis employing starch industry wastewater as a substrate.

    PubMed

    Vu, Khanh Dang; Yan, S; Tyagi, R D; Valéro, J R; Surampalli, R Y

    2009-11-01

    Induced production of chitinase during bioconversion of starch industry wastewater (SIW) to Bacillus thuringiensis var. kurstaki HD-1 (Btk) based biopesticides was studied in shake flask as well as in computer-controlled fermentors. SIW was fortified with different concentrations (0%; 0.05%; 0.1%; 0.2%; 0.3% w/v) of colloidal chitin and its consequences were ascertained in terms of Btk growth (total cell count and viable spore count), chitinase, protease and amylase activities and entomotoxicity. At optimum concentration of 0.2% w/v colloidal chitin, the entomotoxicity of fermented broth and suspended pellet was enhanced from 12.4x10(9) (without chitin) to 14.4x10(9) SBU/L and from 18.2x10(9) (without chitin) to 25.1x10(9) SBU/L, respectively. Further, experiments were conducted for Btk growth in a computer-controlled 15 L bioreactor using SIW as a raw material with (0.2% w/v chitin, to induce chitinase) and without fortification of colloidal chitin. It was found that the total cell count, spore count, delta-endotoxin concentration (alkaline solubilised insecticidal crystal proteins), amylase and protease activities were reduced whereas the entomotoxicity and chitinase activity was increased with chitin fortification. The chitinase activity attained a maximum value at 24 h (15 mU/ml) and entomotoxicity of suspended pellet reached highest (26.7x10(9) SBU/L) at 36 h of fermentation with chitin supplementation of SIW. In control (without chitin), the highest value of entomotoxicity of suspended pellet (20.5x10(9) SBU/L) reached at 48 h of fermentation. A quantitative synergistic action of delta-endotoxin concentration, spore concentration and chitinase activity on the entomotoxicity against spruce budworm larvae was observed.

  18. Development associated profiling of chitinase and microRNA of Helicoverpa armigera identified chitinase repressive microRNA.

    PubMed

    Agrawal, Neema; Sachdev, Bindiya; Rodrigues, Janneth; Sree, K Sowjanya; Bhatnagar, Raj K

    2013-01-01

    Expression of chitinase is developmentally regulated in insects in consonance with their molting process. During the larval-larval metamorphosis in Helicoverpa armigera, chitinase gene expression varies from high to negligible. In the five-day metamorphic course of fifth-instar larvae, chitinase transcript is least abundant on third day and maximal on fifth day. MicroRNA library prepared from these highest and lowest chitinase-expressing larval stages resulted in isolation of several miRNAs. In silico analysis of sequenced miRNAs revealed three miRNAs having sequence similarity to 3'UTR of chitinase. Gene-targeted specific action of these miRNAs, was investigated by luciferase reporter having 3'UTR of chitinase. Only one of three miRNAs, miR-24, inhibited luciferase expression. Further, a day-wise in vivo quantification of miR-24 in fifth-instar larvae revealed a negative correlation with corresponding chitinase transcript abundance. The force-feeding of synthetic miR-24 induced significant morphological aberrations accompanied with arrest of molting. These miR-24 force-fed larvae revealed significantly reduced chitinase transcript abundance.

  19. Partial purification, characterization, and kinetic studies of a low-molecular-weight, alkali-tolerant chitinase enzyme from Bacillus subtilis JN032305, A potential biocontrol strain.

    PubMed

    Shivakumar, Srividya; Karmali, Anika Nayak; Ruhimbana, Charles

    2014-01-01

    A new alkalophilic low-molecular-mass chitinase of 14 kD from the potent biocontrol agent Bacillus subtilis JN032305 was partially purified and enzymology of the chitinase was studied. The enzyme showed optimal pH of 9.0 and temperature of 50°C. The enzyme was found stable during the 60-min incubation at 50 °C. The chitinase was inhibited by group specific agents like IAA, DAN, TLCK, and SDS and metal ions Mg(2+), Ca(2+), Fe(2+), Mn(2+), Ba(2+), and Hg(2+), whereas Zn(2+) did not show significant inhibitory effect against the chitinase. PMSF partially inhibited the enzyme. Substrates specificity tests indicated that the enzyme showed 75% of relative activity on glycol chitin, 58% on carboxymethylcellulose (CMC), 33% on chitin flakes, and 166% laminarin compared to that on colloidal chitin. The enzyme also hydrolyzed 4-methylumbelliferyl-N-acetyl-D-glucosaminide, indicating its chitobiase activity. The chitinase of this study has broad specificity, which could hydrolyze not only the glycosidic bond in GlcNAc-GlcNAc but also that of related carbohydrates with glycosidic linkages. The partially purified chitinase not only showed antifungal activity against Rhizoctonia solani and Colletotrichum gloeosporioides, two potent phytopathogens of chilli, but also increased the germination of chilli seeds when infected with the two potent phytopathogenic fungi.

  20. Characterization of Thermotolerant Chitinases Encoded by a Brevibacillus laterosporus Strain Isolated from a Suburban Wetland.

    PubMed

    Liu, Pulin; Cheng, Deyong; Miao, Lihong

    2015-12-04

    To isolate and characterize chitinases that can be applied with practical advantages, 57 isolates of chitin-degrading bacteria were isolated from the soil of a suburban wetland. 16S rRNA gene analysis revealed that the majority of these strains belonged to two genera, Paenibacillus and Brevibacillus. Taking thermostability into account, the chitinases (ChiA and ChiC) of a B. laterosporus strain were studied further. Ni-NTA affinity-purified ChiA and ChiC were optimally active at pH 7.0 and 6.0, respectively, and showed high temperature stability up to 55 °C. Kinetic analysis revealed that ChiC has a lower affinity and stronger catalytic activity toward colloidal chitin than ChiA. With their stability in a broad temperature range, ChiA and ChiC can be utilized for the industrial bioconversion of chitin wastes into biologically active products.

  1. Characterization of Thermotolerant Chitinases Encoded by a Brevibacillus laterosporus Strain Isolated from a Suburban Wetland

    PubMed Central

    Liu, Pulin; Cheng, Deyong; Miao, Lihong

    2015-01-01

    To isolate and characterize chitinases that can be applied with practical advantages, 57 isolates of chitin-degrading bacteria were isolated from the soil of a suburban wetland. 16S rRNA gene analysis revealed that the majority of these strains belonged to two genera, Paenibacillus and Brevibacillus. Taking thermostability into account, the chitinases (ChiA and ChiC) of a B. laterosporus strain were studied further. Ni-NTA affinity-purified ChiA and ChiC were optimally active at pH 7.0 and 6.0, respectively, and showed high temperature stability up to 55 °C. Kinetic analysis revealed that ChiC has a lower affinity and stronger catalytic activity toward colloidal chitin than ChiA. With their stability in a broad temperature range, ChiA and ChiC can be utilized for the industrial bioconversion of chitin wastes into biologically active products. PMID:26690223

  2. Optimization of nutrition factors on chitinase production from a newly isolated Chitiolyticbacter meiyuanensis SYBC-H1

    PubMed Central

    Hao, Zhikui; Cai, Yujie; Liao, Xiangru; Zhang, Xiaoli; Fang, Zhiyou; Zhang, Dabing

    2012-01-01

    The present study reports statistical medial optimization for chitinase production by a novel bacterial strain isolated from soil recently, which the name Chitinolyticbacter meiyuanensis SYBC-H1 is proposed. A sequential statistical methodology comprising of Plackett-Burman and response surface methodology (RSM) was applied to enhance the fermentative production of chitinase, in which inulin was firstly used as an effective carbon source. As a result, maximum chitinase activity of 5.17 U/mL was obtained in the optimized medium, which was 15.5-fold higher than that in the basal medium. The triplicate verification experiments were performed under the optimized nutrients levels which indicated that it well agreed with the predicted value. PMID:24031816

  3. Production of a Thermostable and Alkaline Chitinase by Bacillus thuringiensis subsp. kurstaki Strain HBK-51

    PubMed Central

    Kuzu, Secil Berna; Güvenmez, Hatice Korkmaz; Denizci, Aziz Akin

    2012-01-01

    This paper reports the isolation and identification of chitinase-producing Bacillus from chitin-containing wastes, production of a thermostable and alkaline chitinasese, and enzyme characterization. Bacillus thuringiensis subsp. kurstaki HBK-51 was isolated from soil and was identified. Chitinase was obtained from supernatant of B. thuringiensis HBK-51 strain and showed its optimum activity at 110°C and at pH 9.0. Following 3 hours of incubation period, the enzyme showed a high level of activity at 110°C (96% remaining activity) and between pH 9.0 and 12.0 (98% remaining activity). Considering these characteristics, the enzyme was described as hyperthermophile-thermostable and highly alkaline. Two bands of the enzyme weighing 50 and 125 kDa were obtained following 12% SDS-PAGE analyses. Among the metal ions and chemicals used, Ni2+ (32%), K+ (44%), and Cu2+ (56%) increased the enzyme activity while EDTA (7%), SDS (7%), Hg2+ (11%), and ethyl-acetimidate (20%) decreased the activity of the enzyme. Bacillus thuringiensis subsp. kurstaki HBK-51 is an important strain which can be used in several biotechnological applications as a chitinase producer. PMID:23304523

  4. Stomach Chitinase from Japanese Sardine Sardinops melanostictus: Purification, Characterization, and Molecular Cloning of Chitinase Isozymes with a Long Linker.

    PubMed

    Kawashima, Satoshi; Ikehata, Hiroki; Tada, Chihiro; Ogino, Tomohiro; Kakizaki, Hiromi; Ikeda, Mana; Fukushima, Hideto; Matsumiya, Masahiro

    2016-01-20

    Fish express two different chitinases, acidic fish chitinase-1 (AFCase-1) and acidic fish chitinase-2 (AFCase-2), in the stomach. AFCase-1 and AFCase-2 have different degradation patterns, as fish efficiently degrade chitin ingested as food. For a comparison with the enzymatic properties and the primary structures of chitinase isozymes obtained previously from the stomach of demersal fish, in this study, we purified chitinase isozymes from the stomach of Japanese sardine Sardinops melanostictus, a surface fish that feeds on plankton, characterized the properties of these isozymes, and cloned the cDNAs encoding chitinases. We also predicted 3D structure models using the primary structures of S. melanostictus stomach chitinases. Two chitinase isozymes, SmeChiA (45 kDa) and SmeChiB (56 kDa), were purified from the stomach of S. melanostictus. Moreover, two cDNAs, SmeChi-1 encoding SmeChiA, and SmeChi-2 encoding SmeChiB were cloned. The linker regions of the deduced amino acid sequences of SmeChi-1 and SmeChi-2 (SmeChi-1 and SmeChi-2) are the longest among the fish stomach chitinases. In the cleavage pattern groups toward short substrates and the phylogenetic tree analysis, SmeChi-1 and SmeChi-2 were classified into AFCase-1 and AFCase-2, respectively. SmeChi-1 and SmeChi-2 had catalytic domains that consisted of a TIM-barrel (β/α)₈-fold structure and a deep substrate-binding cleft. This is the first study showing the 3D structure models of fish stomach chitinases.

  5. Molecular cloning, expression and biochemical characterisation of a cold-adapted novel recombinant chitinase from Glaciozyma antarctica PI12

    PubMed Central

    2011-01-01

    Background Cold-adapted enzymes are proteins produced by psychrophilic organisms that display a high catalytic efficiency at extremely low temperatures. Chitin consists of the insoluble homopolysaccharide β-(1, 4)-linked N-acetylglucosamine, which is the second most abundant biopolymer found in nature. Chitinases (EC 3.2.1.14) play an important role in chitin recycling in nature. Biodegradation of chitin by the action of cold-adapted chitinases offers significant advantages in industrial applications such as the treatment of chitin-rich waste at low temperatures, the biocontrol of phytopathogens in cold environments and the biocontrol of microbial spoilage of refrigerated food. Results A gene encoding a cold-adapted chitinase (CHI II) from Glaciozyma antarctica PI12 was isolated using Rapid Amplification of cDNA Ends (RACE) and RT-PCR techniques. The isolated gene was successfully expressed in the Pichia pastoris expression system. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 1,215 bp, which encodes a 404 amino acid protein. The recombinant chitinase was secreted into the medium when induced with 1% methanol in BMMY medium at 25°C. The purified recombinant chitinase exhibited two bands, corresponding to the non-glycosylated and glycosylated proteins, by SDS-PAGE with molecular masses of approximately 39 and 50 kDa, respectively. The enzyme displayed an acidic pH characteristic with an optimum pH at 4.0 and an optimum temperature at 15°C. The enzyme was stable between pH 3.0-4.5 and was able to retain its activity from 5 to 25°C. The presence of K+, Mn2+ and Co2+ ions increased the enzyme activity up to 20%. Analysis of the insoluble substrates showed that the purified recombinant chitinase had a strong affinity towards colloidal chitin and little effect on glycol chitosan. CHI II recombinant chitinase exhibited higher Vmax and Kcat values toward colloidal chitin than other substrates at low temperatures. Conclusion By

  6. Posttranslational processing of a new class of hydroxyproline-containing proteins. Prolyl hydroxylation and C-terminal cleavage of tobacco (Nicotiana tabacum) vacuolar chitinase.

    PubMed

    Sticher, L; Hofsteenge, J; Neuhaus, J M; Boller, T; Meins, F

    1993-04-01

    The fungicidal class I chitinases (EC 3.2.1.14) are believed to be important in defending plants against microbial pathogens. The vacuolar isoforms of tobacco (Nicotiana tabacum), chitinases A and B, are the first examples of a new type of hydroxyproline-containing protein with intracellular location, enzymic activity, and a small number of hydroxyprolyl residues restricted to a single, short peptide sequence. We have investigated the posttranslational processing and intracellular transport of transgene-encoded chitinase A in callus cultures of Nicotiana tabacum L. cv Havana 425 and leaves of Nicotiana sylvestris Spegazzini and Comes. Pulse-chase experiments and cell fractionation show that chitinase A is processed in two distinct steps. In the first step, the nascent protein undergoes an increase in apparent M(r) of approximately 1500 detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experiments with the inhibitor of prolyl hydroxylation, alpha,alpha'-dipyridyl, and pulse-chase labeling of cells expressing recombinant forms of chitinase A indicate that the anomalous increase in M(r) is due to hydroxylation of prolyl residues. This step occurs in the endomembrane system before sorting for secretion and vacuolar transport and does not appear to be required for correct targeting of chitinase A to the vacuole. The second step is a proteolytic cleavage. Sequencing of tryptic peptides of the mature proteins indicates that during processing essentially all molecules of chitinase A and B lose a C-terminal heptapeptide, which has been shown to be a vacuolar targeting signal. This appears to occur primarily in the endomembrane system late in intracellular transport. A model for the posttranslational modification of chitinase A is proposed.

  7. Chitinase from a novel strain of Serratia marcescens JPP1 for biocontrol of aflatoxin: molecular characterization and production optimization using response surface methodology.

    PubMed

    Wang, Kai; Yan, Pei-sheng; Cao, Li-xin

    2014-01-01

    Chitinase is one of the most important mycolytic enzymes with industrial significance, and produced by a number of organisms. A chitinase producing isolate Serratia marcescens JPP1 was obtained from peanut hulls in Jiangsu Province, China, and exhibited antagonistic activity against aflatoxins. In this study, we describe the optimization of medium composition with increased production of chitinase for the selected bacteria using statistical methods: Plackett-Burman design was applied to find the key ingredients, and central composite design of response surface methodology was used to optimize the levels of key ingredients for the best yield of chitinase. Maximum chitinase production was predicted to be 23.09 U/mL for a 2.1-fold increase in medium containing 12.70 g/L colloidal chitin, 7.34 g/L glucose, 5.00 g/L peptone, 1.32 g/L (NH4)2SO4, 0.7 g/L K2HPO4, and 0.5 g/L MgSO4 · 7H2O. Polymerase chain reaction (PCR) amplification of the JPP1 chitinase gene was performed and obtained a 1,789 bp nucleotide sequence; its open reading frame encoded a protein of 499 amino acids named as ChiBjp.

  8. Chitinase from a Novel Strain of Serratia marcescens JPP1 for Biocontrol of Aflatoxin: Molecular Characterization and Production Optimization Using Response Surface Methodology

    PubMed Central

    Wang, Kai; Yan, Pei-sheng; Cao, Li-xin

    2014-01-01

    Chitinase is one of the most important mycolytic enzymes with industrial significance, and produced by a number of organisms. A chitinase producing isolate Serratia marcescens JPP1 was obtained from peanut hulls in Jiangsu Province, China, and exhibited antagonistic activity against aflatoxins. In this study, we describe the optimization of medium composition with increased production of chitinase for the selected bacteria using statistical methods: Plackett-Burman design was applied to find the key ingredients, and central composite design of response surface methodology was used to optimize the levels of key ingredients for the best yield of chitinase. Maximum chitinase production was predicted to be 23.09 U/mL for a 2.1-fold increase in medium containing 12.70 g/L colloidal chitin, 7.34 g/L glucose, 5.00 g/L peptone, 1.32 g/L (NH4)2SO4, 0.7 g/L K2HPO4, and 0.5 g/L MgSO4·7H2O. Polymerase chain reaction (PCR) amplification of the JPP1 chitinase gene was performed and obtained a 1,789 bp nucleotide sequence; its open reading frame encoded a protein of 499 amino acids named as ChiBjp. PMID:24812619

  9. Cloning of the gene Lecanicillium psalliotae chitinase Lpchi1 and identification of its potential role in the biocontrol of root-knot nematode Meloidogyne incognita.

    PubMed

    Gan, Zhongwei; Yang, Jinkui; Tao, Nan; Liang, Lianming; Mi, Qili; Li, Juan; Zhang, Ke-Qin

    2007-10-01

    The nematophagous fungus Lecanicillium psalliotae (syn. Verticillium psalliotae) is a well-known biocontrol agent. In this study, a chitinase gene Lpchi1 was isolated for the first time from L. psalliotae using degenerate primers and DNA-walking technique. The cloned gene Lpchi1 encoding 423 amino acid residues shares a high degree of homology with other pathogenicity-related chitinases from entomopathogenic and mycoparasitic fungi. The complementary DNA sequence of the mature chitinase was amplified via reverse transcription polymerase chain reaction and expressed well in Pichia pastoris GS115. Through gel filtration, the recombinant chitinase was purified as a protein of ca. 45 kDa with an optimal activity at pH 7.0 and 37.6 degrees C. The purified chitinase LPCHI1 was found degrading chitinous components of eggs of the root-knot nematode Meloidogyne incognita and significantly influence its development. Moreover, our results also demonstrate that the protease Ver112 and the chitinase LPCHI1 from the same fungus interacted on the egg infection.

  10. Structure and disulfide bonding pattern of the hevein-like peptide domains from plant class IV chitinases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Corn (Zea mays) and Arabidopsis (Arabidopsis thaliana) produce GH family 19 plant class IV chitinases. These chitinases contain two domains: a small N-terminal hevein region, and a C-terminal chitinase. Numerous structures of GH19 chitinase domains have been reported, including the chitinase domain ...

  11. Bacterial chitinases and chitin-binding proteins as virulence factors.

    PubMed

    Frederiksen, Rikki F; Paspaliari, Dafni K; Larsen, Tanja; Storgaard, Birgit G; Larsen, Marianne H; Ingmer, Hanne; Palcic, Monica M; Leisner, Jørgen J

    2013-05-01

    Bacterial chitinases (EC 3.2.1.14) and chitin-binding proteins (CBPs) play a fundamental role in the degradation of the ubiquitous biopolymer chitin, and the degradation products serve as an important nutrient source for marine- and soil-dwelling bacteria. However, it has recently become clear that representatives of both Gram-positive and Gram-negative bacterial pathogens encode chitinases and CBPs that support infection of non-chitinous mammalian hosts. This review addresses this biological role of bacterial chitinases and CBPs in terms of substrate specificities, regulation, secretion and involvement in cellular and animal infection.

  12. Expression of a chitinase gene from Metarhizium anisopliae in tobacco plants confers resistance against Rhizoctonia solani.

    PubMed

    Kern, Marcelo Fernando; Maraschin, Simone de Faria; Vom Endt, Débora; Schrank, Augusto; Vainstein, Marilene Henning; Pasquali, Giancarlo

    2010-04-01

    The chit1 gene from the entomopathogenic fungus Metarhizium anisopliae, encoding the endochitinase CHIT42, was placed under the control of the CaMV 35S promoter, and the resulting construct was transferred to tobacco. Seventeen kanamycin-resistant transgenic lines were recovered, and the presence of the transgene was confirmed by polymerase chain reactions and Southern blot hybridization. The number of chit1 copies was determined to be varying from one to four. Copy number had observable effects neither on plant growth nor development. Substantial heterogeneity concerning production of the recombinant chitinase, and both general and specific chitinolytic activities were detected in leaf extracts from primary transformants. The highest chitinase activities were found in plants harboring two copies of chit1 inserts at different loci. Progeny derived from self-pollination of the primary transgenics revealed a stable inheritance pattern, with transgene segregation following a mendelian dihybrid ratio. Two selected plants expressing high levels of CHIT42 were consistently resistant to the soilborne pathogen Rhizoctonia solani, suggesting a direct relationship between enzyme activity and reduction of foliar area affected by fungal lesions. To date, this is the first report of resistance to fungal attack in plants mediated by a recombinant chitinase from an entomopathogenic and acaricide fungus.

  13. Molecular cloning of chitinase 33 (chit33) gene from Trichoderma atroviride

    PubMed Central

    Matroudi, S.; Zamani, M.R.; Motallebi, M.

    2008-01-01

    In this study Trichoderma atroviride was selected as over producer of chitinase enzyme among 30 different isolates of Trichoderma sp. on the basis of chitinase specific activity. From this isolate the genomic and cDNA clones encoding chit33 have been isolated and sequenced. Comparison of genomic and cDNA sequences for defining gene structure indicates that this gene contains three short introns and also an open reading frame coding for a protein of 321 amino acids. The deduced amino acid sequence includes a 19 aa putative signal peptide. Homology between this sequence and other reported Trichoderma Chit33 proteins are discussed. The coding sequence of chit33 gene was cloned in pEt26b(+) expression vector and expressed in E. coli. PMID:24031242

  14. Isolation and characterization of a chitinase gene from entomopathogenic fungus Verticillium lecanii

    PubMed Central

    Zhu, Yanping; Pan, Jieru; Qiu, Junzhi; Guan, Xiong

    2008-01-01

    Entomopathogenic fungus Verticillium lecanii is a promising whitefly and aphid control agent. Chitinases secreted by this insect pathogen have considerable importance in the biological control of some insect pests. An endochitinase gene Vlchit1 from the fungus was cloned and overexpressed in Escherichia coli. The Vlchit1 gene not only contains an open reading frame (ORF) which encodes a protein of 423 amino acids (aa), but also is interrupted by three short introns. Vlchit1 protein showed that the chitinase Vlchit1 has a (a/b)8 TIM barrel structure. Overexpression test and Enzymatic activity assay indicated that the Vlchit1 is a functional enzyme that can hydrolyze the chitin substrate, so the Vlchit1 gene can service as a useful gene source for genetic manipulation leading to strain improvement of entomopathogenic fungi or constructing new transgenic plants with resistance to various fungal and insects pests. PMID:24031223

  15. Expression of the chitinase gene from Trichoderma aureoviride in Saccharomyces cerevisiae.

    PubMed

    Jinzhu, Song; Qian, Yang; Beidong, Liu; Dianfu, Chen

    2005-11-01

    Chitinase gene ech42 was obtained from Trichoderma aureoviride M and amplified by PCR. The isolated DNA of ech42 was then sequenced. The results showed that the open reading frame of ech42 was 1,447 bp long, encoding 421 amino acids. Three introns were found in the sequence. The cloning vector pMD18-T and an E. coli DH5alpha host were used to yield clones as E. coli DH5alpha/ech42. The ech42 gene was integrated into the genomic DNA of pYES2 by insertion into a single site for recombination, yielding the recombinant pYES2/ech42. Chitinase expressed by pYES2/ech42 was induced by galactose (maximal activity 0.50 units ml(-1)) and was produced in fermentation liquid cultured for 36 h.

  16. Polyglycine hydrolases: fungal b-lactamase-like endoproteases that cleave polyglycine regions within plant class IV chitinases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyglycine hydrolases are secreted fungal proteases that cleave glycine-glycine peptide bonds in the inter-domain linker region of specific plant defense chitinases. Previously, we reported the catalytic activity of polyglycine hydrolases from the phytopathogens Epicoccum sorghi (Es-cmp) and Cochli...

  17. Molecular cloning, characterization, and expression of a chitinase from the entomopathogenic fungus Paecilomyces javanicus.

    PubMed

    Chen, Chien-Cheng; Kumar, H G Ashok; Kumar, Senthil; Tzean, Shean-Shong; Yeh, Kai-Wun

    2007-07-01

    Paecilomyces javanicus is an entomopathogenic fungus of coleopteran and lepidopteran insects. Here we report on cloning, characterization, and expression patterns of a chitinase from P. javanicus. A strong chitinase activity was detected in P. javanicus cultures added to chitin. The full-length cDNA, designated PjChi-1, was cloned from mycelia by using both degenerate primer/reverse transcription polymerase chain reaction (RT-PCR) amplification and 5'-/3'-RACE extension. The 1.18-kb cDNA gene contains a 1035-bp open reading frame and encodes a 345-amino acid polypeptide with a deduced molecular mass of 37 kDa. A conserved motif for chitinase activity -F82DGIDIDWE90- was present in deduced amino acid sequence. Both RT-PCR and Northern analysis revealed that the expression of the PjChi gene was constitutive at low level, but enhanced to high level when chitin was the substrate. Fungal inhibitory assay showed that PjChi-1 inhibited the growth of phytopathogenic fungi such as Sclerotium rolfsii, Colletotrichum gloeosporioides, Aspergillus nidulans, and Rhizoctonia solani.

  18. Purification and characterization of chitinase showing antifungal and biodegradation properties obtained from Streptomyces anulatus CS242.

    PubMed

    Mander, Poonam; Cho, Seung Sik; Choi, Yun Hee; Panthi, Sandesh; Choi, Yoon Seok; Kim, Hwan Mook; Yoo, Jin Cheol

    2016-07-01

    In an effort to identify a microbial enzyme that can be useful as a fungicide and biodegradation agent of chitinous wastes, a chitinase (Chi242) was purified from the culture supernatant of Streptomyces anulatus CS242 utilizing powder of shrimp shell wastes as a sole carbon source. It was purified employing ammonium sulfate precipitation and gel permeation chromatography techniques. The molecular weight of the purified chitinase was ~38 kDa by SDS-PAGE. The N-terminal amino acid sequence (A-P-G-A-P-G-T-G-A-L) showed close similarity to those of other Streptomyes chitinases. The purified enzyme displayed optimal activity at pH 6.0 and 50 °C respectively. It showed substantial thermal stability for 2 h at 30-60 °C, and exhibited broad pH stability in the range 5.0-13.0 for 48 h at 4 °C. Scanning electron microscopy confirmed the ability of this enzyme to adsorb onto solid shrimp bio-waste and to degrade chitin microfibers. Chi242 could proficiently convert colloidal chitin to N-acetyl glucosamine (GlcNAc) and N-acetyl chitobiose (GlcNAc)2 signifying that this enzyme is suitable for bioconversion of chitin waste. In addition, it exerted an effective antifungal activity towards fungal pathogen signifying its role as a biocontrol agent. Thus, a single microbial cell of Streptomyces anulatus CS242 justified its dual role.

  19. Two cold-induced family 19 glycosyl hydrolases from cherimoya (Annona cherimola) fruit: an antifungal chitinase and a cold-adapted chitinase.

    PubMed

    Goñi, Oscar; Sanchez-Ballesta, María T; Merodio, Carmen; Escribano, María I

    2013-11-01

    Two cold-induced chitinases were isolated and purified from the mesocarp cherimoyas (Annona cherimola Mill.) and they were characterised as acidic endochitinases with a Mr of 24.79 and 47.77kDa (AChi24 and AChi48, respectively), both family 19 glycosyl hydrolases. These purified chitinases differed significantly in their biochemical and biophysical properties. While both enzymes had similar optimal acidic pH values, AChi24 was enzymatically active and stable at alkaline pH values, as well as displaying an optimal temperature of 45°C and moderate thermostability. Kinetic studies revealed a great catalytic efficiency of AChi24 for oligomeric and polymeric substrates. Conversely, AChi48 hydrolysis showed positive co-operativity that was associated to a mixture of different functional oligomeric states through weak transient protein interactions. The rise in the AChi48 kcat at increasing enzyme concentrations provided evidence of its oligomerisation. AChi48 chitinase was active and stable in a broad acidic pH range, and while it was relatively labile as temperatures increased, with an optimal temperature of 35°C, it retained about 50% of its maximal activity from 5 to 50°C. Thermodynamic characterisation reflected the high kcat of AChi48 and the remarkably lower ΔH(‡), ΔS(‡) and ΔG(‡) values at 5°C compared to AChi24, indicating that the hydrolytic activity of AChi48 was less thermodependent. In vitro functional studies revealed that AChi24 had a strong antifungal defence potential against Botrytis cinerea, whereas they displayed no cryoprotective or antifreeze activity. Hence, based on biochemical, thermodynamic and functional data, this study demonstrates that two acidic endochitinases are induced at low temperatures in a subtropical fruit, and that one of them acts in an oligomeric cold-adapted manner.

  20. Identification, Phylogeny, and Transcript of Chitinase Family Genes in Sugarcane

    PubMed Central

    Su, Yachun; Xu, Liping; Wang, Shanshan; Wang, Zhuqing; Yang, Yuting; Chen, Yun; Que, Youxiong

    2015-01-01

    Chitinases are pathogensis-related proteins, which play an important role in plant defense mechanisms. The role of the sugarcane chitinase family genes remains unclear due to the highly heterozygous and aneuploidy chromosome genetic background of sugarcane. Ten differentially expressed chitinase genes (belonging to class I~VII) were obtained from RNA-seq analysis of both incompatible and compatible sugarcane genotypes during Sporisorium scitamineum challenge. Their structural properties and expression patterns were analyzed. Seven chitinases (ScChiI1, ScChiI2, ScChiI3, ScChiIII1, ScChiIII2, ScChiIV1 and ScChiVI1) showed more positive with early response and maintained increased transcripts in the incompatible interaction than those in the compatible one. Three (ScChiII1, ScChiV1 and ScChiVII1) seemed to have no significant difference in expression patterns between incompatible and compatible interactions. The ten chitinases were expressed differentially in response to hormone treatment as well as having distinct tissue specificity. ScChiI1, ScChiIV1 and ScChiVII1 were induced by various abiotic stresses (NaCl, CuCl2, PEG and 4 °C) and their involvement in plant immunity was demonstrated by over-expression in Nicotiana benthamiana. The results suggest that sugarcane chitinase family exhibit differential responses to biotic and abiotic stress, providing new insights into their function. PMID:26035173

  1. Chitinase Expression Due to Reduction in Fusaric Acid Level in an Antagonistic Trichoderma harzianum S17TH.

    PubMed

    Sharma, Vivek; Bhandari, Pamita; Singh, Bikram; Bhatacharya, Amita; Shanmugam, Veerubommu

    2013-06-01

    To study the effect of reduction in phytotoxin level on fungal chitinases, antagonistic Trichoderma spp. were screened for their ability to reduce the level of fusaric acid (FA), the phytotoxin produced by Fusarium spp. A T. harzianum isolate S17TH was able to tolerate high levels of FA (up to 500 ppm) but was unable to reduce the toxin to a significant level (non-toxic) added to minimal synthetic broth (MSB). However, the isolate was able to reduce 400 ppm FA in the liquid medium after 7 days to a non-toxic level and displayed similar level of antagonism over the control (without FA). In studies of the effect of the reduction in FA (400 ppm) level on chitinase gene expression in PCR assays, nag1 was significantly repressed but ech42 expression was only slightly repressed. Chitinase activity was either reduced or absent in the extracellular proteins of MSB supplemented with 400 ppm FA, which could be attributed to the effect of residual FA or its breakdown products through unknown mechanisms. Selection of S17TH as a toxin insensitive isolate that could commensurate the negative effect on chitinase activity makes it a potential antagonist against Fusarium spp.

  2. Expression and efficient secretion of a functional chitinase from Chromobacterium violaceum in Escherichia coli

    PubMed Central

    2013-01-01

    Background Chromobacterium violaceum is a free-living β-proteobacterium found in tropical and subtropical regions. The genomic sequencing of C. violaceum ATCC 12472 has revealed many genes that underpin its adaptability to diverse ecosystems. Moreover, C. violaceum genes with potential applications in industry, medicine and agriculture have also been identified, such as those encoding chitinases. However, none of the chitinase genes of the ATCC 12472 strain have been subjected to experimental validation. Chitinases (EC 3.2.1.14) hydrolyze the β-(1,4) linkages in chitin, an abundant biopolymer found in arthropods, mollusks and fungi. These enzymes are of great biotechnological interest as potential biocontrol agents against pests and pathogens. This work aimed to experimentally validate one of the chitinases from C. violaceum. Results The open reading frame (ORF) CV2935 of C. violaceum ATCC 12472 encodes a protein (439 residues) that is composed of a signal peptide, a chitin-binding domain, a linker region, and a C-terminal catalytic domain belonging to family 18 of the glycoside hydrolases. The ORF was amplified by PCR and cloned into the expression vector pET303/CT-His. High levels of chitinolytic activity were detected in the cell-free culture supernatant of E. coli BL21(DE3) cells harboring the recombinant plasmid and induced with IPTG. The secreted recombinant protein was purified by affinity chromatography on a chitin matrix and showed an apparent molecular mass of 43.8 kDa, as estimated by denaturing polyacrylamide gel electrophoresis. N-terminal sequencing confirmed the proper removal of the native signal peptide during the secretion of the recombinant product. The enzyme was able to hydrolyze colloidal chitin and the synthetic substrates p-nitrophenyl-β-D-N,N’-diacetylchitobiose and p-nitrophenyl-β-D-N,N’,N”-triacetylchitotriose. The optimum pH for its activity was 5.0, and the enzyme retained ~32% of its activity when heated to 60°C for 30

  3. Isolation and identification of chitinolytic bacteria of pohara river of South East Sulawesi and the optimization production of chitinase enzyme

    NASA Astrophysics Data System (ADS)

    Halimahtussadiyah, R.; Natsir, Muh.; Kurniawati, Desy; Utamy, Sukma Puspita

    2017-03-01

    Isolation and identification of chitinolytic bacteria from pohara river and optimation of chitinase enzyme production has been conducted. The aims of the study were isolation, characterize and optimaze of chitinase enzyme production. This study was carried out in three stages; isolation and selection of chitinolytic bacteria, characterization and identification of selected bacteria; optimization of the production of the enzyme (substrate concentration, temperature, and pH), and the determination of growth curve of T3 isolate. The chitinase activity assay was carried out using Schales method. The results of the screening obtained 6 isolates of potential bacteria of chitinolytic. The T3 isolate then was selected for the enzyme production, because it had the highest chitinolytic index of 22.31 mm. The morphological and biochemical observation showed that T3 isolate as a group of bacteria Aerobacter with Gram-negative nature, and shaped bacillus. The optimum condition for chitinase enzyme production was in chitin substrat concentration 0.06%, temperature of 30°C, and pH of 6.

  4. Class I β-1,3-Glucanase and Chitinase Are Expressed in the Micropylar Endosperm of Tomato Seeds Prior to Radicle Emergence1

    PubMed Central

    Wu, Chun-Ta; Leubner-Metzger, Gerhard; Meins, Frederick; Bradford, Kent J.

    2001-01-01

    β-1,3-Glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) mRNAs, proteins, and enzyme activities were expressed specifically in the micropylar tissues of imbibed tomato (Lycopersicon esculentum Mill.) seeds prior to radicle emergence. RNA hybridization and immunoblotting demonstrated that both enzymes were class I basic isoforms. β-1,3-Glucanase was expressed exclusively in the endosperm cap tissue, whereas chitinase localized to both endosperm cap and radicle tip tissues. β-1,3-Glucanase and chitinase appeared in the micropylar tissues of gibberellin-deficient gib-1 tomato seeds only when supplied with gibberellin. Accumulation of β-1,3-glucanase mRNA, protein and enzyme activity was reduced by 100 μM abscisic acid, which delayed or prevented radicle emergence but not endosperm cap weakening. In contrast, expression of chitinase mRNA, protein, and enzyme activity was not affected by abscisic acid. Neither of these enzymes significantly hydrolyzed isolated tomato endosperm cap cell walls. Although both β-1,3-glucanase and chitinase were expressed in tomato endosperm cap tissue prior to radicle emergence, we found no evidence that they were directly involved in cell wall modification or tissue weakening. Possible functions of these hydrolases during tomato seed germination are discussed. PMID:11457981

  5. Effects of sugar beet chitinase IV on root-associated fungal community of transgenic silver birch in a field trial.

    PubMed

    Pasonen, Hanna-Leena; Lu, Jinrong; Niskanen, Anna-Maija; Seppänen, Sanna-Kaisa; Rytkönen, Anna; Raunio, Janne; Pappinen, Ari; Kasanen, Risto; Timonen, Sari

    2009-10-01

    Heterogenous chitinases have been introduced in many plant species with the aim to increase the resistance of plants to fungal diseases. We studied the effects of the heterologous expression of sugar beet chitinase IV on the intensity of ectomycorrhizal (ECM) colonization and the structure of fungal communities in the field trial of 15 transgenic and 8 wild-type silver birch (Betula pendula Roth) genotypes. Fungal sequences were separated in denaturing gradient gel electrophoresis and identified by sequencing the ITS1 region to reveal the operational taxonomic units. ECM colonization was less intense in 7 out of 15 transgenic lines than in the corresponding non-transgenic control plants, but the slight decrease in overall ECM colonization in transgenic lines could not be related to sugar beet chitinase IV expression or total endochitinase activity. One transgenic line showing fairly weak sugar beet chitinase IV expression without significantly increased total endochitinase activity differed significantly from the non-transgenic controls in the structure of fungal community. Five sequences belonging to three different fungal genera (Hebeloma, Inocybe, Laccaria) were indicative of wild-type genotypes, and one sequence (Lactarius) indicated one transgenic line. In cluster analysis, the non-transgenic control grouped together with the transgenic lines indicating that genotype was a more important factor determining the structure of fungal communities than the transgenic status of the plants. With the tested birch lines, no clear evidence for the effect of the heterologous expression of sugar beet chitinase IV on ECM colonization or the structure of fungal community was found.

  6. Isolation and characterization of chitinase-producing Bacillus and Paenibacillus strains from salted and fermented shrimp, Acetes japonicus.

    PubMed

    Han, Kook-Il; Patnaik, Bharat Bhusan; Kim, Yong Hyun; Kwon, Hyun-Jung; Han, Yeon Soo; Han, Man-Deuk

    2014-04-01

    Chitinases catalyze the conversion of chitin and are produced by a wide range of bacteria. The biological applications of these enzymes have been exploited in food and pharmaceutical industries. We isolated 2 halophilic chitinase-producing novel strains of bacteria-SCH-1 and SCH-2 from Saeu-jeot, a traditional Korean salted and fermented food made with shrimp (Acetes japonicus). The isolated strains- SCH-1 and SCH-2 were Gram-positive, rod-shaped, endospore-forming facultative anaerobes, with strain SCH-2 showing peritrichous flagella. Molecular characterization of the 16S rRNA gene identified the strains SCH-1 and SCH-2 as Bacillus sp. and Paenibacillus sp. respectively. Basic Local Alignment Search Tool and subsequent phylogenetic analysis of strain SCH-1 showed an identity of 97.83% with Bacillus cereus ATCC 14579 (NR_074540), whereas strain SCH-2 showed an identity of 99.16% with Paenibacillus lautus JCM 9073 (NR_040882). Furthermore, the SCH-1 strain could use glucose, N-acetyl glucosamine, esculin, and maltose as carbon source substrates. Cellular fatty acid analysis showed that iso-C15:0 and anteiso-C15:0 are the major acids in strain SCH-1 and SCH-2, respectively. The SCH-1 strain showed a higher chitinase activity at 15.71 unit/mg protein compared with SCH-2 strain. Chitinase isozymes of Bacillus sp. SCH-1was expressed as 2 bands having sizes of 41 and 50 kDa, and as 4 bands with sizes of 30, 37, 45.7, and 50 kDa in Paenibacillus sp. SCH-2. The rich chitinase activity with the isozyme profiles of the isolated Bacillus and Paenibacillus strains provide advancement in the study of fermentation and may play putative functions in the chitin bioconversion of sea crustacean foods.

  7. Cloning of a Serratia marcescens Gene Encoding Chitinase

    PubMed Central

    Fuchs, R. L.; McPherson, S. A.; Drahos, D. J.

    1986-01-01

    Serratia marcescens, a chitinase-producing microorganism, was shown to produce five unique chitinolytic proteins with subunit molecular masses of 21, 36, 48, 52, and 57 kilodaltons. A cosmid library of S. marcescens DNA was constructed in the broad-host-range cosmid pLAFR1 and screened in Escherichia coli for clones capable of degrading chitin. A total of four independent clones (22- to 27-kilobase inserts) were isolated, characterized by restriction endonuclease digestion, and shown to share a common 9.5-kilobase EcoR1 fragment apparently encoding the same 57-kilodalton chitinase, the most abundant chitinase produced by S. marcescens. Chitinase expression from these constructs in both E. coli and Pseudomonas fluorescens 701E1 is apparently driven by an S. marcescens promoter. The significantly higher chitinase levels produced in E. coli relative to those in P. fluorescens 701E1 suggest that E. coli may recognize this promoter sequence more efficiently than P. fluorescens. Images PMID:16347012

  8. Chitin and chitinase: Role in pathogenicity, allergenicity and health.

    PubMed

    Patel, Seema; Goyal, Arun

    2017-04-01

    Chitin, a polysaccharide with particular abundance in fungi, nematodes and arthropods is immunogenic. It acts as a threat to other organisms, to tackle which they have been endowed with chitinase enzyme. Even if this enzyme is not present in all organisms, they possess proteins having chitin-binding domain(s) (ChtBD). Many lethal viruses like Ebola, and HCV (Hepatitis C virus) have these domains to manipulate their carriers and target organisms. In keeping with the basic rule of survival, the self-origin (own body component) chitins and chitinases are protective, but that of non-self origin (from other organisms) are detrimental to health. The exogenous chitins and chitinases provoke human innate immunity to generate a deluge of inflammatory cytokines, which injure organs (leading to asthma, atopic dermatitis etc.), and in persistent situations lead to death (multiple sclerosis, systemic lupus erythromatosus (SLE), cancer, etc.). Unfortunately, chitin-chitinase-stimulated hypersensitivity is a common cause of occupational allergy. On the other hand, chitin, and its deacetylated derivative chitosan are increasingly proving useful in pharmaceutical, agriculture, and biocontrol applications. This critical review discusses the complex nexus of chitin and chitinase and assesses both their pathogenic as well as utilitarian aspects.

  9. Co-evolution of chitinases from maize and other cereals with secreted proteases from Pleosporineae fungi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant class IV chitinases are composed of a carboxy-terminal chitinase domain that is attached, through a linker sequence, to a small amino-terminal domain that can be thought of as a structured peptide. While both the peptide-like domain and the chitinase domain share sequence homology throughout m...

  10. Truncation of class IV chitinases from Arabidopsis by secreted fungal proteases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant class IV chitinases have a small, amino-terminal chitin binding domain and a larger chitinase domain. Previous work on Zea mays chitinases ChitA and ChitB showed that their chitin binding domains bind insoluble chitin, that their catalytic domains degrade short, soluble forms of chitin, and th...

  11. Novel extracellular chitinases rapidly and specifically induced by general bacterial elicitors and suppressed by virulent bacteria as a marker of early basal resistance in tobacco.

    PubMed

    Ott, Péter G; Varga, Gabriella J; Szatmári, Agnes; Bozsó, Zoltan; Klement, Eva; Medzihradszky, Katalin F; Besenyei, Eszter; Czelleng, Arnold; Klement, Zoltán

    2006-02-01

    Early basal resistance (EBR, formerly known as early induced resistance) is triggered by general bacterial elicitors. EBR has been suggested to inhibit or retard expression of the type III secretion system of pathogenic bacteria and may also prevent nonpathogenic bacteria from colonizing the plant tissue. The quickness of EBR here plays a crucial role, compensating for a low bactericidal efficacy. This inhibitory activity should take place in the cell wall, as bacteria do not enter living plant cells. We found several soluble proteins in the intercellular fluid of tobacco leaf parenchyma that coincided with EBR under different environmental (light and temperature) conditions known to affect EBR. The two most prominent proteins proved to be novel chitinases (EC 3.2.1.14) that were transcriptionally induced before and during EBR development. Their expression in the apoplast was fast and not stress-regulated as opposed to many pathogenesis-related proteins. Nonpathogenic, saprophytic, and avirulent bacteria all induced EBR and the chitinases. Studies using these chitinases as EBR markers revealed that the virulent Pseudomonas syringae pv. tabaci, being sensitive to EBR, must suppress it while suppressing the chitinases. EBR, the chitinases, as well as their suppression are quantitatively related, implying a delicate balance determining the outcome of an infection.

  12. Identification of the chitinase genes from the diamondback moth, Plutella xylostella.

    PubMed

    Liao, Z H; Kuo, T C; Kao, C H; Chou, T M; Kao, Y H; Huang, R N

    2016-12-01

    Chitinases have an indispensable function in chitin metabolism and are well characterized in numerous insect species. Although the diamondback moth (DBM) Plutella xylostella, which has a high reproductive potential, short generation time, and characteristic adaptation to adverse environments, has become one of the most serious pests of cruciferous plants worldwide, the information on the chitinases of the moth is presently limited. In the present study, using degenerated polymerase chain reaction (PCR) and rapid amplification of cDNA ends-PCR strategies, four chitinase genes of P. xylostella were cloned, and an exhaustive search was conducted for chitinase-like sequences from the P. xylostella genome and transcriptomic database. Based on the domain analysis of the deduced amino acid sequences and the phylogenetic analysis of the catalytic domain sequences, we identified 15 chitinase genes from P. xylostella. Two of the gut-specific chitinases did not cluster with any of the known phylogenetic groups of chitinases and might be in a new group of the chitinase family. Moreover, in our study, group VIII chitinase was not identified. The structures, classifications and expression patterns of the chitinases of P. xylostella were further delineated, and with this information, further investigations on the functions of chitinase genes in DBM could be facilitated.

  13. Design and synthesis of 4'-O-alkyl-chitobiosyl-4-methylumbelliferone as human chitinase fluorogenic substrates.

    PubMed

    Duivenvoorden, Boudewijn A; Ghauharali, Karen; Scheij, Saskia; Boot, Rolf G; Aerts, Johannes M F G; van der Marel, Gijsbert A; Overkleeft, Herman S; Codée, Jeroen D C

    2014-11-18

    The synthesis of three fluorogenic chitobiosyl derivatives, modified at the non-reducing 4'-OH with, either a methyl, an isopropyl or a cyclohexylmethyl substituent, is described. The 4'-capped 4-methylumbelliferyl chitobiosides are hydrolysed by the human chitinase CHIT1 following Michaelis-Menten kinetics and in contrast to unmodified chitobiosyl-4-methylumbelliferone do not undergo transglycosylation. The compounds are also relatively poor hexosaminidase substrates and thus provide useful alternatives to 4'-deoxychitobiosyl-4-methylumbelliferone, previously reported by us as fluorogenic substrate to monitor CHIT1 activity as a marker for Gaucher disease state.

  14. Pesticide tolerance of Paenibacillus sp. D1 and its chitinase.

    PubMed

    Singh, Anil Kumar; Ghodke, Indrajeet; Chhatpar, H S

    2009-01-01

    Excessive use of pesticides in agriculture has led to several problems pertaining to loss of soil fertility and environmental degradation. Biological control agents offer the best alternative to reduce use of toxic pesticides. Paenibacillus sp. D1 isolated from the effluent treatment plant of a seafood processing industry exhibited broad spectrum tolerance towards a number of pesticides at concentrations higher than recommended for field applications. The isolate showed enhanced growth and chitinase production in the presence of some protectant fungicides. None of the tested demethylase inhibitor (DMI) fungicides inhibited growth and chitinase production except triadimefon. The isolate was also tolerant to most commonly used insecticides belonging to the organophosphate, carbamate and cyclodiene organochloride classes. Chitinase of Paenibacillus sp. D1 was found to be more tolerant than the organism itself and was highly stable in the presence of pesticides at the temperature under field conditions in Gujarat, India, i.e. 40 degrees C. This was suggestive of its potential in integrated pest management (IPM) to significantly reduce the use of harmful chemicals. To our knowledge this is the first extensive study on pesticide tolerance of the Paenibacillus species and its chitinase.

  15. Recognition of corn defense chitinases by fungal polyglycine hydrolases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyglycine hydrolases (PGH)s are secreted fungal endoproteases that cleave peptide bonds in the polyglycine interdomain linker of ChitA chitinase, an antifungal protein from domesticated corn (Zea mays ssp. mays). These target-specific endoproteases are unusual because they do not cut a defined pep...

  16. Acidic Chitinase Limits Allergic Inflammation and Promotes Intestinal Nematode Expulsion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Acidic mammalian chitinase (AMCase) is stereotypically induced during mammalian immune responses to helminths and allergens—yet, its precise role in immunity and inflammation is unclear. Here we show that in the lung, genetic ablation of AMCase failed to diminish type 2 inflammation against helmint...

  17. Production of prodigiosin and chitinases by tropical Serratia marcescens strains with potential to control plant pathogens.

    PubMed

    Gutiérrez-Román, Martha Ingrid; Holguín-Meléndez, Francisco; Bello-Mendoza, Ricardo; Guillén-Navarro, Karina; Dunn, Michael F; Huerta-Palacios, Graciela

    2012-01-01

    The potential of three Serratia marcescens strains (CFFSUR-B2, CFFSUR-B3 and CFFSUR-B4) isolated from tropical regions in Mexico to inhibit the mycelial growth and conidial germination of Colletotrichum gloeosporioides, causal agent of fruit anthracnose, was evaluated. The ability of these strains to produce prodigiosin and chitinases when cultivated in oil seed-based media (peanut, sesame, soybean and castor bean) and in Luria-Bertani medium was determined. All of the strains exhibited similar fungal antagonistic activities and inhibited myceliar growth by more than 40% while inhibiting conidial germination by 81-89% (P = 0.01). The highest level of prodigiosin (40 μg/ml) was produced in the peanut-based medium while growth in soybean-based medium allowed the highest production of chitinases (56 units/ml), independent of the strain used. Strain CFFSUR-B2 grown in peanut medium was used to evaluate the effect of inoculum density and initial pH on metabolite production. The amount of prodigiosin produced increased with greater inoculum densities, with an initial density of 1 × 10(12) resulting in the highest production (60 μg/ml). Prodigiosin production was not affected by pH. The strains studied have the advantage of being adapted to tropical climates and are able to produce chitinases in the absence of chitin induction in vitro. These characteristics suggest their potential as biocontrol agents for fungal pathogens in tropical regions of the world.

  18. Purification and characterisation of an acidic and antifungal chitinase produced by a Streptomyces sp.

    PubMed

    Karthik, Narayanan; Binod, Parameswaran; Pandey, Ashok

    2015-01-01

    An extremely acidic extracellular chitinase produced by a Streptomyces sp. was purified 12.44-fold by ammonium sulphate precipitation, ion-exchange chromatography and gel-permeation chromatography and further characterised. The molecular mass of the enzyme was estimated to be about 40 kDa by SDS-PAGE. The optimum pH and temperature of the purified enzyme were pH 2 and 6, and 50 °C respectively. The enzyme showed high stability in the acidic pH range of 2-6 and temperature stability of up to 50 °C. Additionally, the effect of some cations and other chemical compounds on the chitinase activity was studied. The activity of the enzyme was considerably retained under salinity conditions of up to 3%. The Km and Vmax values of the enzyme were determined to be 6.74 mg mL(-1) and 61.3 U mg(-1) respectively using colloidal chitin. This enzyme exhibited antifungal activity against phytopathogens revealing a potential biocontrol application in agriculture.

  19. Colloidal chitin stained with Remazol Brilliant Blue R, a useful substrate to select chitinolytic microorganisms and to evaluate chitinases.

    PubMed

    Gómez Ramírez, M; Rojas Avelizapa, L I; Rojas Avelizapa, N G; Cruz Camarillo, R

    2004-02-01

    A simple and sensitive method based on the use of colloidal chitin stained with Remazol Brilliant Blue R (RBB) is proposed to evaluate chitinase activity. If this colloidal-stained substrate is included as a carbon source in a liquid medium, this technique allows the selection or the comparison of chitinolytic microorganisms. The colloidal substrate is proportionally solubilized and the dye released is spectrophotometrically quantified at 595 nm. The procedures used for the staining and fixing of RBB in the colloidal chitin, and a comparison with the commercial substrate chitin-azure, are presented. The influence of several physicochemical and enzymatic parameters on the release of dyes is also shown. Both stained substrates were used for studying the effect of pH, substrate concentration, temperature and time on the chitinase reaction of Bacillus thuringiensis Bt-107.

  20. Isolation and characterization of chitinase isoforms from the bulbs of four species of the genus Tulipa.

    PubMed

    Yamagami, T; Taira, T; Aso, Y; Ishiguro, M

    1998-03-01

    Six chitinase isoforms, designated TBC-1 to TBC-6, were purified to homogenity from the bulbs of four species (Tulipa bakeri, T. tarda, T. turkestanica, and T. praestans) of the genus Tulipa by CM-cellulose column chromatography, Butyl-Toyopearl 650M hydrophobic column chromatography, gel filtration on Sephadex G-75, and Mono-S fast protein liquid chromatography (FPLC). The chitinases had molecular weights of 30,000 and isoelectric points of 5.2 to 6.1. These chitinases were found to proteins with similar amino acid compositions and N-terminal sequences. The tulip chitinases all had two half-cystine residues, one more than gladiolus bulb class IIIb chitinase, but many fewer than chitinases of plant class I (15-17 Cys residues/mol), II (5-8 Cys residues/mol), or III (6 Cys residues/mol). The N-terminal sequences of tulip chitinases were similar to the sequence of the gladiolus chitinase, but did not resemble sequence of any class of plant chitinase. The optimal pH of these chitinases toward glycolchitin was pH 5. TBC-1 hydrolyzed (GlcNAc)6 into (GlcNAc)2, (GlcNAc)3, and (GlcNAc)4, and hydrolyzed (GlcNAc)5 into (GlcNAc)2 and (GlcNAc)3.

  1. Functional analyses of chitinases in the moss Physcomitrella patens: chitin oligosaccharide-induced gene expression and enzymatic characterization.

    PubMed

    Kobaru, Saki; Tanaka, Ryusuke; Taira, Toki; Uchiumi, Toshiki

    2016-12-01

    Plant chitinases play diverse roles including defense against pathogenic fungi. Using reverse-transcription quantitative PCR analysis, we found that six chitinase (PpChi) genes and two genes for chitin elicitor receptor kinases (PpCERKs) are expressed at considerable levels in the moss Physcomitrella patens subsp. patens. The expressed PpChis belonged to glycoside hydrolase family 19 (class I: PpChi-Ia and -Ib; class II: PpChi-IIa and -IIc; and class IV: PpChi-IV) and to glycoside hydrolase family 18 (class V: PpChi-Vb). Treatment with chitin tetramer or hexamer increased the expression of class I and IV PpChi genes and decreased that of class II PpChi genes. Recombinant PpChi-Ia, PpChi-IV, and PpChi-Vb were characterized. PpChi-IV exhibited higher activity against chitin tetramer and pentamer than PpChi-Ia did. PpChi-Vb showed transglycosylation activity and PpChi-Ia inhibited fungal growth. These results suggest that chitinases of different classes play different roles in defense mechanism of moss plant against fungal pathogens.

  2. Characterization of an Acidic Chitinase from Seeds of Black Soybean (Glycine max (L) Merr Tainan No. 3)

    PubMed Central

    Chang, Ya-Min; Chen, Li-Chun; Wang, Hsin-Yi; Chiang, Chui-Liang; Chang, Chen-Tien; Chung, Yun-Chin

    2014-01-01

    Using 4-methylumbelliferyl-β-D-N,N′,N″-triacetylchitotrioside (4-MU-GlcNAc3) as a substrate, an acidic chitinase was purified from seeds of black soybean (Glycine max Tainan no. 3) by ammonium sulfate fractionation and three successive steps of column chromatography. The purified chitinase was a monomeric enzyme with molecular mass of 20.1 kDa and isoelectric point of 4.34. The enzyme catalyzed the hydrolysis of synthetic substrates p-nitrophenyl N-acetyl chitooligosaccharides with chain length from 3 to 5 (GlcNAcn, n = 3-5), and pNp-GlcNAc4 was the most degradable substrate. Using pNp-GlcNAc4 as a substrate, the optimal pH for the enzyme reaction was 4.0; kinetic parameters Km and kcat were 245 µM and 10.31 min−1, respectively. This enzyme also showed activity toward CM-chitin-RBV, a polymer form of chitin, and N-acetyl chitooligosaccharides, an oligomer form of chitin. The smallest oligomer substrate was an N-acetylglucosamine tetramer. These results suggested that this enzyme was an endo-splitting chitinase with short substrate cleavage activity and useful for biotechnological applications, in particular for the production of N-acetyl chitooligosaccharides. PMID:25437446

  3. Characterization of an acidic chitinase from seeds of black soybean (Glycine max (L) Merr Tainan No. 3).

    PubMed

    Chang, Ya-Min; Chen, Li-Chun; Wang, Hsin-Yi; Chiang, Chui-Liang; Chang, Chen-Tien; Chung, Yun-Chin

    2014-01-01

    Using 4-methylumbelliferyl-β-D-N,N',N″-triacetylchitotrioside (4-MU-GlcNAc3) as a substrate, an acidic chitinase was purified from seeds of black soybean (Glycine max Tainan no. 3) by ammonium sulfate fractionation and three successive steps of column chromatography. The purified chitinase was a monomeric enzyme with molecular mass of 20.1 kDa and isoelectric point of 4.34. The enzyme catalyzed the hydrolysis of synthetic substrates p-nitrophenyl N-acetyl chitooligosaccharides with chain length from 3 to 5 (GlcNAcn, n = 3-5), and pNp-GlcNAc4 was the most degradable substrate. Using pNp-GlcNAc4 as a substrate, the optimal pH for the enzyme reaction was 4.0; kinetic parameters Km and kcat were 245 µM and 10.31 min-1, respectively. This enzyme also showed activity toward CM-chitin-RBV, a polymer form of chitin, and N-acetyl chitooligosaccharides, an oligomer form of chitin. The smallest oligomer substrate was an N-acetylglucosamine tetramer. These results suggested that this enzyme was an endo-splitting chitinase with short substrate cleavage activity and useful for biotechnological applications, in particular for the production of N-acetyl chitooligosaccharides.

  4. Acidic mammalian chitinase is a proteases-resistant glycosidase in mouse digestive system

    PubMed Central

    Ohno, Misa; Kimura, Masahiro; Miyazaki, Haruko; Okawa, Kazuaki; Onuki, Riho; Nemoto, Chiyuki; Tabata, Eri; Wakita, Satoshi; Kashimura, Akinori; Sakaguchi, Masayoshi; Sugahara, Yasusato; Nukina, Nobuyuki; Bauer, Peter O.; Oyama, Fumitaka

    2016-01-01

    Chitinases are enzymes that hydrolyze chitin, a polymer of β-1, 4-linked N-acetyl-D-glucosamine (GlcNAc). Chitin has long been considered as a source of dietary fiber that is not digested in the mammalian digestive system. Here, we provide evidence that acidic mammalian chitinase (AMCase) can function as a major digestive enzyme that constitutively degrades chitin substrates and produces (GlcNAc)2 fragments in the mouse gastrointestinal environment. AMCase was resistant to endogenous pepsin C digestion and remained active in the mouse stomach extract at pH 2.0. The AMCase mRNA levels were much higher than those of four major gastric proteins and two housekeeping genes and comparable to the level of pepsinogen C in the mouse stomach tissues. Furthermore, AMCase was expressed in the gastric pepsinogen-synthesizing chief cells. The enzyme was also stable and active in the presence of trypsin and chymotrypsin at pH 7.6, where pepsin C was completely degraded. Mouse AMCase degraded polymeric colloidal and crystalline chitin substrates in the gastrointestinal environments in presence of the proteolytic enzymes. Thus, AMCase can function as a protease-resistant major glycosidase under the conditions of stomach and intestine and degrade chitin substrates to produce (GlcNAc)2, a source of carbon, nitrogen and energy. PMID:27883045

  5. Thermostable chitinase from Cohnella sp. A01: isolation and product optimization.

    PubMed

    Aliabadi, Nasrin; Aminzadeh, Saeed; Karkhane, Ali Asghar; Haghbeen, Kamahldin

    Twelve bacterial strains isolated from shrimp farming ponds were screened for their growth activity on chitin as the sole carbon source. The highly chitinolytic bacterial strain was detected by qualitative cup plate assay and tentatively identified to be Cohnella sp. A01 based on 16S rDNA sequencing and by matching the key morphological, physiological, and biochemical characteristics. The cultivation of Cohnella sp. A01 in the suitable liquid medium resulted in the production of high levels of enzyme. The colloidal chitin, peptone, and K2HPO4 represented the best carbon, nitrogen, and phosphorus sources, respectively. Enzyme production by Cohnella sp. A01 was optimized by the Taguchi method. Our results demonstrated that inoculation amount and temperature of incubation were the most significant factors influencing chitinase production. From the tested values, the best pH/temperature was obtained at pH 5 and 70°C, with Km and Vmax values of chitinase to be 5.6mg/mL and 0.87μmol/min, respectively. Ag(+), Co(2+), iodoacetamide, and iodoacetic acid inhibited the enzyme activity, whereas Mn(2+), Cu(2+), Tweens (20 and 80), Triton X-100, and EDTA increased the same. In addition, the study of the morphological alteration of chitin treated by enzyme by SEM revealed cracks and pores on the chitin surface, indicating a potential application of this enzyme in several industries.

  6. Structural and functional definition of the human chitinase chitin-binding domain.

    PubMed

    Tjoelker, L W; Gosting, L; Frey, S; Hunter, C L; Trong, H L; Steiner, B; Brammer, H; Gray, P W

    2000-01-07

    Mammalian chitinase, a chitinolytic enzyme expressed by macrophages, has been detected in atherosclerotic plaques and is elevated in blood and tissues of guinea pigs infected with Aspergillus. Its normal physiological function is unknown. To understand how the enzyme interacts with its substrate, we have characterized the chitin-binding domain. The C-terminal 49 amino acids make up the minimal sequence required for chitin binding activity. The absence of this domain does not affect the ability of the enzyme to hydrolyze the soluble substrate, triacetylchitotriose, but abolishes hydrolysis of insoluble chitin. Within the minimal chitin-binding domain are six cysteines; mutation of any one of these to serine results in complete loss of chitin binding activity. Analysis of purified recombinant chitin-binding domain revealed the presence of three disulfide linkages. The recombinant domain binds specifically to chitin but does not bind chitosan, cellulose, xylan, beta-1, 3-glucan, beta-1,3-1,4-glucan, or mannan. Fluorescently tagged chitin-binding domain was used to demonstrate chitin-specific binding to Saccharomyces cerevisiae, Candida albicans, Mucor rouxii, and Neurospora crassa. These experiments define structural features of the minimal domain of human chitinase required for both specifically binding to and hydrolyzing insoluble chitin and demonstrate relevant binding within the context of the fungal cell wall.

  7. Overexpression and characterization of thermostable chitinase from Bacillus atrophaeus SC081 in Escherichia coli.

    PubMed

    Cho, Eun Kyung; Choi, In Soon; Choi, Young Ju

    2011-03-01

    The chitinase-producing strain SC081 was isolated from Korean traditional soy sauce and identified as Bacillus atrophaeus based on a phylogenetic analysis of the 16S rDNA sequence and a phenotypic analysis. A gene encoding chitinase from B. atrophaeus SC081 was cloned in Escherichia coli and was named SCChi-1 (GQ360078). The SCChi-1 nucleotide sequences were composed of 1788 base pairs and 596 amino acids, which were 92.6, 89.6, 89.3, and 78.9% identical to those of Bacillus subtilis (ABG57262), Bacillus pumilus (ABI15082), Bacillus amyloliquefaciens (ABO15008), and Bacillus licheniformis (ACF40833), respectively. A recombinant SCChi-1 containing a hexahistidine tag at the amino- terminus was constructed, overexpressed, and purified in E. coli to characterize SCChi-1. H(6)SCChi-1 revealed a hydrolytic band on zymograms containing 0.1% glycol chitin and showed the highest lytic activity on colloidal chitin and acidic chitosan. The optimal temperature and pH for chitinolytic activity were 50°C and pH 8.0, respectively.

  8. Characterization of Chitinase C from a Marine Bacterium, Alteromonas sp. Strain O-7, and Its Corresponding Gene and Domain Structure

    PubMed Central

    Tsujibo, Hiroshi; Orikoshi, Hideyuki; Shiotani, Kayoko; Hayashi, Miyuki; Umeda, Junko; Miyamoto, Katsushiro; Imada, Chiaki; Okami, Yoshiro; Inamori, Yoshihiko

    1998-01-01

    One of the chitinase genes of Alteromonas sp. strain O-7, the chitinase C-encoding gene (chiC), was cloned, and the nucleotide sequence was determined. An open reading frame coded for a protein of 430 amino acids with a predicted molecular mass of 46,680 Da. Alignment of the deduced amino acid sequence demonstrated that ChiC contained three functional domains, the N-terminal domain, a fibronectin type III-like domain, and a catalytic domain. The N-terminal domain (59 amino acids) was similar to that found in the C-terminal extension of ChiA (50 amino acids) of this strain and furthermore showed significant sequence homology to the regions found in several chitinases and cellulases. Thus, to evaluate the role of the domain, we constructed the hybrid gene that directs the synthesis of the fusion protein with glutathione S-transferase activity. Both the fusion protein and the N-terminal domain itself bound to chitin, indicating that the N-terminal domain of ChiC constitutes an independent chitin-binding domain. PMID:9464381

  9. Kinetic characterization of Aspergillus niger chitinase CfcI using a HPAEC-PAD method for native chitin oligosaccharides.

    PubMed

    van Munster, Jolanda M; Sanders, Peter; ten Kate, Geralt A; Dijkhuizen, Lubbert; van der Maarel, Marc J E C

    2015-04-30

    The abundant polymer chitin can be degraded by chitinases (EC 3.2.1.14) and β-N-acetyl-hexosaminidases (EC 3.2.1.52) to oligosaccharides and N-acetyl-glucosamine (GlcNAc) monomers. Kinetic characterization of these enzymes requires product quantification by an assay method with a low detection limit, preferably compatible with the use of native, non-labeled substrates. Here we report a quantitative HPAEC-PAD method that allows fast separation of chitin oligosaccharides (COS) ranging from (GlcNac)1-6 at detection limits of 1-3 pmol and a linear range of 5-250 pmol. Quantification under intra- and interday precision conditions was performed with 2.1-5.4% relative standard deviation (RSD) and 1.2-10.3% RSD, respectively. This method was successfully used for the determination of the kinetic parameters of the Aspergillus niger chitinase CfcI with native COS. CfcI was recently shown to release GlcNAc from the reducing end of COS, a new activity for fungal chitinases. A Carbohydrate Binding Module of family 18 (CBM18) is inserted in the CfcI catalytic domain. Site directed mutagenesis was used to assess the functionality of this CfcI-CBM18: four of its key amino acids were replaced by glycine residues, yielding CfcISYNF. Comparison of the kinetic parameters of CfcI and CfcISYNF confirmed that this CBM18 is functionally involved in catalysis.

  10. A modular family 19 chitinase found in the prokaryotic organism Streptomyces griseus HUT 6037.

    PubMed Central

    Ohno, T; Armand, S; Hata, T; Nikaidou, N; Henrissat, B; Mitsutomi, M; Watanabe, T

    1996-01-01

    The specificity of chitinase C-1 of Streptomyces griseus HUT 6037 for the hydrolysis of the beta-1,4-glycosidic linkages in partially acetylated chitosan is different from that of other microbial chitinases. In order to study the primary structure of this unique chitinase, the chiC gene specifying chitinase C-1 was cloned and its nucleotide sequence was determined. The gene encodes a polypeptide of 294 amino acids with a calculated size of 31.4 kDa. Comparison of the amino acid sequence of the deduced polypeptide with that of other proteins revealed a C-terminal catalytic domain displaying considerable sequence similarity to the catalytic domain of plant class I, II, and IV chitinases which form glycosyl hydrolase family 19. The N-terminal domain of the deduced polypeptide exhibits sequence similarity to substrate-binding domains of several microbial chitinases and cellulases but not to the chitin-binding domains of plant chitinases. The previously purified chitinase C-1 from S. griseus is suggested to be generated by proteolytic removal of the N-terminal chitin-binding domain and corresponds to the catalytic domain of the chitinase encoded by the chiC gene. High-performance liquid chromatography analysis of the hydrolysis products from N-acetyl chitotetraose revealed that chitinase C-1 catalyzes hydrolysis of the glycosidic bond with inversion of the anomeric configuration, in agreement with the previously reported inverting mechanism of plant class I chitinases. This is the first report of a family 19 chitinase found in an organism other than higher plants. PMID:8752320

  11. Preparation of nanoscale Bacillus thuringiensis chitinases using silica nanoparticles for nematicide delivery.

    PubMed

    Qin, Xu; Xiang, Xuemei; Sun, Xiaowen; Ni, Hong; Li, Lin

    2016-01-01

    A series of amino, carboxylic, and aldehydic surface-grafted silica nanoparticles (SNPs) was prepared based on SiO2 NYSi40 nanoparticles to develop an efficient, biocompatible, and cost-effective biopesticide delivery system. Bacillus thuringiensis chitinase (Chi9602) was immobilized onto SNP surface to prepare nanoscale chitinases (SNPCs) through electrostatic adsorption and covalent binding. The specimens were characterized by Fourier transform infrared, scanning electron microscopy, and zeta-potential analyses. The delivery capacity of the SNPs in Caenorhabditis elegans N2 was observed by immunofluorescence. Results demonstrated that amino-grafted SiO2 nanoparticles with Chi9602 electrostatically adsorbed onto their surface (SNPC2) exhibited a relatively high enzyme immobilization rate (80.2%) and the highest (94.1%) residual enzyme activity among all SNPCs. SNPC2 also showed wider pH tolerance and relatively higher thermostability and ultraviolet radiation resistance capacity than Chi9602. Bioassays further showed that SNPC2 synergistically enhanced the nematicidal effect of B. thuringiensis YBT-020 preparation against C. elegans, with a reduced LC50 of 8.35mg/mL and a shortened LT50 of 12.04h. Immunofluorescence assays showed that SNPC2 had considerable delivery capacity to carry a large protein into C. elegans. Therefore, SNP2 can serve as an efficient nanocarrier for the delivery of macromolecular proteic biopesticides or drugs, indicating potential agricultural or biotechnological applications.

  12. Purification and characterization of chitinase from Streptomyces violascens NRRL B2700.

    PubMed

    Gangwar, Mamta; Singh, Vineeta; Pandey, Asheesh Kumar; Tripathi, C K M; Mishra, B N

    2016-01-01

    Chitinase is one of the important enzymes as it is directly linked to Chitin that has wide applications in industrial, medical and commercial fields for its biocompatibility and biodegradability. Here, we report extracellular chitinase production by Streptomyces violascens NRRL B2700 under submerged fermentation condition. Chitinase production started after 10 h of incubation and reached to maximum level at 72 h of cultivation. Studies on the influence of additional carbon and nitrogen sources on chitinase production revealed that maltose, xylose, fructose, lactose, soybean meal and ammonium nitrate served as good carbon and nitrogen sources to enhance chitinase yield by 1.6 to 6 fold. Medium supplemented with 1% colloidal chitin produced high chitinase concentration (0.1714 U/mg). The enzyme chitinase was purified from the culture broth by 75% ammonium sulphate precipitation, DEAE-cellulose ion-exchange and sephadex G-100 gel filtration. The molecular mass of the purified chitinase was 65 kDa as estimated by SDS-PAGE. The apparent Michaelis constant (K(m)) and the maximum rate (V(max)) of the enzyme for colloidal chitin were 1.556 mg/mL and 2.680 μM/min/mg, respectively suggested high affinity towards-chitin. Possibly, it is the first report on production of chitinase from S. violascens NRRL B2700. The findings were encouraging, especially for cost effective production, and further warrants media and purification optimization studies for enhanced yield.

  13. A new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) affects Soybean Asian rust (Phakopsora pachyrhizi) spore germination

    PubMed Central

    2011-01-01

    Background Asian rust (Phakopsora pachyrhizi) is a common disease in Brazilian soybean fields and it is difficult to control. To identify a biochemical candidate with potential to combat this disease, a new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP) leaves was cloned into the pGAPZα-B vector for expression in Pichia pastoris. Results A cDNA encoding a chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP), was isolated from leaves. The amino acid sequence predicts a (β/α)8 topology common to Class III Chitinases (glycoside hydrolase family 18 proteins; GH18), and shares similarity with other GH18 members, although it lacks the glutamic acid residue essential for catalysis, which is replaced by glutamine. CaclXIP was expressed as a recombinant protein in Pichia pastoris. Enzymatic assay showed that purified recombinant CaclXIP had only residual chitinolytic activity. However, it inhibited xylanases from Acrophialophora nainiana by approx. 60% when present at 12:1 (w/w) enzyme:inhibitor ratio. Additionally, CaclXIP at 1.5 μg/μL inhibited the germination of spores of Phakopsora pachyrhizi by 45%. Conclusions Our data suggests that CaclXIP belongs to a class of naturally inactive chitinases that have evolved to act in plant cell defence as xylanase inhibitors. Its role on inhibiting germination of fungal spores makes it an eligible candidate gene for the control of Asian rust. PMID:21299880

  14. Enhanced degradation of α-chitin materials prepared from shrimp processing byproduct and production of N-acetyl-D-glucosamine by thermoactive chitinases from soil mesophilic fungi.

    PubMed

    Suresh, P V; Anil Kumar, P K

    2012-07-01

    Soil isolates of mesophilic Penicillium monoverticillium CFR 2, Aspergillus flavus CFR 10 and Fusarium oxysporum CFR 8 were cultivated in solid state fermentation (SSF) using wheat bran solid medium supplemented with α-chitin in order to produce chitinolytic enzyme. Under SSF cultivation, maximum enzymes (U/g IDS) production was 41.0 (endo-chitinase) and 195.4 (β-N-acetylhexosaminidase) by P. monoverticillium, 26.8 (endo-chitinase) and 222.1 (β-N-acetylhexosaminidase) by A. flavus and 13.3 (endo-chitinase) and 168.3 (β-N-acetylhexosaminidase) by F. oxysporum after 166 h of incubation. The crude endo-chitinase and β-N-acetylhexosaminidase derived from A. flavus and F. oxysporum revealed optimum temperature at 62 ± 1°C, but the enzymes from P. monoverticillium showed optimum temperature at 52 ± 1°C for maximum activity. Several fold increase in endo-chitinase and β-N-acetylhexosaminidase activities in the crude enzymes preparation was achieved after concentrating with polyethylene glycol. The concentrated crude chitinases from P. monoverticillium, A. flavus and F. oxysporum, respectively yielded 95.6, 96.6 and 96.1 mmol/l of N-acetyl-D: -glucosamine (GlcNAc) in 48 h of reaction from colloidal chitin. While, the crude enzyme preparations of P. monoverticillium, A. flavus and F. oxysporum produced 10.11, 6.85 and 10.7 mmol/l of GlcNAc respectively, in 48 h of reaction from crystalline α-chitin. HPLC analysis of colloidal chitin hydrolysates prepared with crude chitinases derived from P. monoverticillium, A. flavus and F. oxysporum revealed that the major reaction product was monomeric GlcNAc (~80%) and a small amount of (GlcNAc)(4) (~20%), indicating the potential of these enzymes for efficient production of GlcNAc from α-chitin.

  15. Antimicrobial peptide inhibition of fungalysin proteases that target plant type 19 Family IV defense chitinases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cereal crops and other plants produce secreted seed chitinases that reduce pathogenic infection, most likely by targeting the fungal chitinous cell wall. We have shown that corn (Zea mays) produces three GH family 19, plant class IV chitinases, that help in protecting the plant against Fusarium and ...

  16. Polyglycine hydrolases secreted by Pleosporineae fungi that target the linker region of plant class IV chitinases*

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chitinase modifying proteins (cmps) are fungal proteases that truncate plant class IV chitinases by cleaving near their amino termini. We previously described Fv-cmp, a fungalysin protease that cleaves a conserved glycine-cysteine bond within the hevein domain. Here we describe a new type of cmp—pol...

  17. Structural and functional analysis of chitinase gene family in wheat (Triticum aestivum).

    PubMed

    Mishra, A K; Pandey, Bharati; Tyagi, Chetna; Chakraborty, Ohika; Kumar, Amrender; Jain, A K

    2015-04-01

    Chitinases are the hydrolytic enzymes which protect plants against pathogen attack. However, the precise role of chitinases in disease resistance has not been explored in wheat. In the present study, in silico approach, including secondary structure analysis, detailed signature pattern study, cis-acting regulatory elements survey, evolutionary trends and three-dimensional molecular modeling was used for different chitinase classes of wheat (Triticum aestivum). Homology modeling of class I, II, IV and 3 chitinase proteins was performed using the template crystal structure. The model structures were further refined by molecular mechanics methods using different tools, such as Procheck, ProSA and Verify3D. Secondary structure studies revealed greater percentage of residues forming a helix conformation with specific signature pattern, similar to casein kinase II phosphorylation site, amidation site, N-myristoylation (N-MYR) site and protein kinase C phoshorylation site. The expression profile suggested that wheat chitinase gene was highly expressed in cell culture and callus. We found that wheat chitinases showed more functional similarity with rice and barley. The results provide insight into the evolution of the chitinase family, constituting a diverse array of pathogenesis-related proteins. The study also provides insight into the possible binding sites of chitinase proteins and may further enhance our knowledge of fungal resistance mechanism in plants.

  18. Purification, crystallization and preliminary X-ray crystallographic analysis of chitinase from Bacillus cereus NCTU2

    SciTech Connect

    Kuo, Chueh-Yuan; Wu, Yue-Jin; Hsieh, Yin-Cheng; Guan, Hong-Hsiang; Tsai, Huei-Ju; Lin, Yi-Hung; Huang, Yen-Chieh; Liu, Ming-Yih; Li, Yaw-Kuen; Chen, Chun-Jung

    2006-09-01

    The crystallization of B. cereus chitinase is reported. Chitinases (EC 3.2.1.14) are found in a broad range of organisms, including bacteria, fungi and higher plants, and play different roles depending on their origin. A chitinase from Bacillus cereus NCTU2 (ChiNCTU2) capable of hydrolyzing chitin as a carbon and nitrogen nutrient has been identified as a member of the family 18 glycoside hydrolases. ChiNCTU2 of molecular weight 36 kDa has been crystallized using the hanging-drop vapour-diffusion method. According to the diffraction of chitinase crystals at 1.10 Å resolution, the crystal belongs to space group P2{sub 1}, with unit-cell parameters a = 50.79, b = 48.79, c = 66.87 Å, β = 99.31°. Preliminary analysis indicates there is one chitinase molecule in the asymmetric unit, with a solvent content of 43.4%.

  19. Protein A-mouse acidic mammalian chitinase-V5-His expressed in periplasmic space of Escherichia coli possesses chitinase functions comparable to CHO-expressed protein.

    PubMed

    Kashimura, Akinori; Okawa, Kazuaki; Ishikawa, Kotarou; Kida, Yuta; Iwabuchi, Kokoro; Matsushima, Yudai; Sakaguchi, Masayoshi; Sugahara, Yasusato; Oyama, Fumitaka

    2013-01-01

    Acidic mammalian chitinase (AMCase) has been shown to be associated with asthma in mouse models, allergic inflammation and food processing. Here, we describe an E. coli-expression system that allows for the periplasmic production of active AMCase fused to Protein A at the N-terminus and V5 epitope and (His)6 tag (V5-His) at the C-terminus (Protein A-AMCase-V5-His) in E. coli. The mouse AMCase cDNA was cloned into the vector pEZZ18, which is an expression vector containing the Staphylococcus Protein A promoter, with the signal sequence and truncated form of Protein A for extracellular expression in E. coli. Most of the Protein A-AMCase-V5-His was present in the periplasmic space with chitinolytic activity, which was measured using a chromogenic substrate, 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside. The Protein A-AMCase-V5-His was purified from periplasmic fractions using an IgG Sepharose column followed by a Ni Sepharose chromatography. The recombinant protein showed a robust peak of activity with a maximum observed activity at pH 2.0, where an optimal temperature was 54°C. When this protein was preincubated between pH 1.0 and pH 11.0 on ice for 1 h, full chitinolytic activity was retained. This protein was also heat-stable till 54°C, both at pH 2.0 and 7.0. The chitinolytic activity of the recombinant AMCase against 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside was comparable to the CHO-expressed AMCase. Furthermore, the recombinant AMCase bound to chitin beads, cleaved colloidal chitin and released mainly N,N'-diacetylchitobiose fragments. Thus, the E. coli-expressed Protein A-mouse AMCase-V5-His fusion protein possesses chitinase functions comparable to the CHO-expressed AMCase. This recombinant protein can be used to elucidate detailed biomedical functions of the mouse AMCase.

  20. Genome Sequence, Comparative Analysis, and Evolutionary Insights into Chitinases of Entomopathogenic Fungus Hirsutella thompsonii

    PubMed Central

    Agrawal, Yamini; Khatri, Indu; Subramanian, Srikrishna; Shenoy, Belle Damodara

    2015-01-01

    Hirsutella thompsonii (Ht) is a fungal pathogen of acarines and the primary cause of epizootics among mites. The draft genomes of two isolates of Ht (MTCC 3556: Ht3, 34.6 Mb and MTCC 6686: Ht6, 34.7 Mb) are presented and compared with the genomes of Beauveria bassiana (Bb) ARSEF 2860 and Ophiocordyceps sinensis (Os) CO18. Comparative analysis of carbohydrate active enzymes, pathogen–host interaction genes, metabolism-associated genes, and genes involved in biosynthesis of secondary metabolites in the four genomes was carried out. Reduction in gene family sizes in Ht3 and Os as compared with Ht6 and Bb is observed. Analysis of the mating type genes in Ht reveals the presence of MAT idiomorphs which is suggestive of cryptic sexual traits in Ht. We further identify and classify putative chitinases that may function as virulence factors in fungal entomopathogens due to their role in degradation of arthropod cuticle. PMID:25716828

  1. Enhanced resistance to Sclerotinia sclerotiorum in Brassica napus by co-expression of defensin and chimeric chitinase genes.

    PubMed

    Zarinpanjeh, Nasim; Motallebi, Mostafa; Zamani, Mohammad Reza; Ziaei, Mahboobeh

    2016-11-01

    Sclerotinia stem rot caused by Sclerotinia sclerotiorum is one of the major fungal diseases of Brassica napus L. To develop resistance against this fungal disease, the defensin gene from Raphanus sativus and chimeric chit42 from Trichoderma atroviride with a C-terminal fused chitin-binding domain from Serratia marcescens were co-expressed in canola via Agrobacterium-mediated transformation. Twenty transformants were confirmed to carry the two transgenes as detected by polymerase chain reaction (PCR), with 4.8 % transformation efficiency. The chitinase activity of PCR-positive transgenic plants were measured in the presence of colloidal chitin, and five transgenic lines showing the highest chitinase activity were selected for checking the copy number of the transgenes through Southern blot hybridisation. Two plants carried a single copy of the transgenes, while the remainder carried either two or three copies of the transgenes. The antifungal activity of two transgenic lines that carried a single copy of the transgenes (T4 and T10) was studied by a radial diffusion assay. It was observed that the constitutive expression of these transgenes in the T4 and T10 transgenic lines suppressed the growth of S. sclerotiorum by 49 % and 47 %, respectively. The two transgenic lines were then let to self-pollinate to produce the T2 generation. Greenhouse bioassays were performed on the transgenic T2 young leaves by challenging with S. sclerotiorum and the results revealed that the expression of defensin and chimeric chitinase from a heterologous source in canola demonstrated enhanced resistance against sclerotinia stem rot disease.

  2. Detection, characterization and evolution of internal repeats in Chitinases of known 3-D structure.

    PubMed

    Sivaji, Manigandan; Sadasivam, Vinoth; Narayanasamy, Jayabalan; Samuel, Selvaraj; Fan, Chuanzhu

    2014-01-01

    Chitinase proteins have evolved and diversified almost in all organisms ranging from prokaryotes to eukaryotes. During evolution, internal repeats may appear in amino acid sequences of proteins which alter the structural and functional features. Here we deciphered the internal repeats from Chitinase and characterized the structural similarities between them. Out of 24 diverse Chitinase sequences selected, six sequences (2CJL, 2DSK, 2XVP, 2Z37, 3EBV and 3HBE) did not contain any internal repeats of amino acid sequences. Ten sequences contained repeats of length <50, and the remaining 8 sequences contained repeat length between 50 and 100 residues. Two Chitinase sequences, 1ITX and 3SIM, were found to be structurally similar when analyzed using secondary structure of Chitinase from secondary and 3-Dimensional structure database of Protein Data Bank. Internal repeats of 3N17 and 1O6I were also involved in the ligand-binding site of those Chitinase proteins, respectively. Our analyses enhance our understanding towards the identification of structural characteristics of internal repeats in Chitinase proteins.

  3. Purification and characterization of two bifunctional chitinases/lysozymes extracellularly produced by Pseudomonas aeruginosa K-187 in a shrimp and crab shell powder medium.

    PubMed Central

    Wang, S L; Chang, W T

    1997-01-01

    Two extracellular chitinases (FI and FII) were purified from the culture supernatant of Pseudomonas aeruginosa K-187. The molecular weights of FI and FII were 30,000 and 32,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 60,000 and 30,000, respectively, by gel filtration. The pIs for FI and FII were 5.2 and 4.8, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of FI were pH 8, 50 degrees C, pH 6 to 9, and 50 degrees C; those of FII were pH 7, 40 degrees C, pH 5 to 10, and 60 degrees C. The activities of both enzymes were activated by Cu2+; strongly inhibited by Mn2+, Mg2+, and Zn2+; and completely inhibited by glutathione, dithiothreitol, and 2-mercaptoethanol. Both chitinases showed lysozyme activity. The purified enzymes had antibacterial and cell lysis activities with many kinds of bacteria. This is the first report of a bifunctional chitinase/lysozyme from a prokaryote. PMID:9023918

  4. Purification, characterization, and molecular cloning of an extracellular chitinase from Bacillus licheniformis stain LHH100 isolated from wastewater samples in Algeria.

    PubMed

    Laribi-Habchi, Hassiba; Bouanane-Darenfed, Amel; Drouiche, Nadjib; Pauss, André; Mameri, Nabil

    2015-01-01

    An extracellular chitinase (ChiA-65) was produced and purified from a newly isolated Bacillus licheniformis LHH100. Pure protein was obtained after heat treatment and ammonium sulphate precipitation followed by Sephacryl S-200 chromatography. Based on matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis, the purified enzyme is a monomer with a molecular mass of 65,195.13 Da. The sequence of the 27 N-terminal residues of the mature ChiA-65 showed high homology with family-18 chitinases. Optimal activity was achieved at pH 4 and 75 °C. Among the inhibitors and metals tested, p-chloromercuribenzoic acid, N-ethylmaleimide, Hg(2+), and Hg(+) completely inhibited enzyme activity. Chitinase activity was high on colloidal chitin, glycol chitin, glycol chitosane, chitotriose, and chitooligosaccharide. Chitinase activity towards synthetic substrates in the order of p-NP-(GlcNAc)n (n = 2-4) was p-NP-(GlcNAc)2 > p-NP-(GlcNAc)4 > p-NP-(GlcNAc)3. Our results suggest that ChiA-65 preferentially hydrolyzed the second glycosidic link from the non-reducing end of (GlcNAc)n. ChiA-65 obeyed Michaelis-Menten kinetics, the Km and kcat values being 0.385 mg, colloidal chitin/ml and 5000 s(-1), respectively. The chiA-65 gene encoding ChiA-65 was cloned in Escherichia coli and its sequence was determined. Above all, ChiA-65 exhibited remarkable biochemical properties suggesting that this enzyme is suitable for bioconversion of chitin waste.

  5. Heterogonous expression and characterization of a plant class IV chitinase from the pitcher of the carnivorous plant Nepenthes alata.

    PubMed

    Ishisaki, Kana; Honda, Yuji; Taniguchi, Hajime; Hatano, Naoya; Hamada, Tatsuro

    2012-03-01

    A class IV chitinase belonging to the glycoside hydrolase 19 family from Nepenthes alata (NaCHIT1) was expressed in Escherichia coli. The enzyme exhibited weak activity toward polymeric substrates and significant activity toward (GlcNAc)(n) [β-1,4-linked oligosaccharide of GlcNAc with a polymerization degree of n (n = 4-6)]. The enzyme hydrolyzed the third and fourth glycosidic linkages from the non-reducing end of (GlcNAc)(6). The pH optimum of the enzymatic reaction was 5.5 at 37°C. The optimal temperature for activity was 60°C in 50 mM sodium acetate buffer (pH 5.5). The anomeric form of the products indicated that it was an inverting enzyme. The k(cat)/K(m) of the (GlcNAc)(n) hydrolysis increased with an increase in the degree of polymerization. Amino acid sequence alignment analysis between NaCHIT1 and a class IV chitinase from a Picea abies (Norway spruce) suggested that the deletion of four loops likely led the enzyme to optimize the (GlcNAc)(n) hydrolytic reaction rather than the hydrolysis of polymeric substrates.

  6. Temporal, spatial and induced expression of chitinase in the spruce budworm, Choristoneura fumiferana.

    PubMed

    Zheng, Y-P; Retnakaran, A; Krell, P J; Arif, B M; Primavera, M; Feng, Q-L

    2003-03-01

    Temporal, spatial and induced expression of Choristoneura fumiferana chitinase (CfChitinase) was studied using immunohistochemistry and Western blots. CfChitinase was detected in the integument, the midgut peritrophic membrane, the cuticular lining of the trachea, the spiracle, and salivary glands. The enzyme was expressed as larvae were preparing to molt from one instar to the next. The spatial and temporal expression patterns are consistent with its function in degrading chitin during the molting process. The 20-hydroxyecdysone agonist, tebufenozide (RH5992), induced the expression of the CfChitinase gene in the early stage of the sixth-instar larvae and the enzyme was detected in the epidermis and molting fluid 24 h post treatment.

  7. Antifungal chitinase against human pathogenic yeasts from Coprinellus congregatus.

    PubMed

    Yoo, Yeeun; Choi, Hyoung T

    2014-05-01

    The inky cap, Coprinellus congregatus, produces mushrooms which become autolyzed rapidly to generate black liquid droplets, in which no cell wall is detected by microscopy. A chitinase (Chi2) which is synthesized during the autolytic phase of C. congregatus inhibits the growths of Candida albicans and Cryptococcus neoformans up to 10% at the concentration of 10 μg/ml, about 50% at concentration of 20 μg/ml, and up to 95% at the concentration of 70 μg/ml. Upon treatment these yeast cells are observed to be severely deformed, with the formation of large holes in the cell wall. The two yeast species show no growth inhibition at the concentration of 5 μg/ml, which means the minimum inhibitory concentrations for both yeast species are 10 μg/ml under these experimental conditions.

  8. Field performance of chitinase transgenic silver birches (Betula pendula): resistance to fungal diseases.

    PubMed

    Pasonen, H-L; Seppänen, S-K; Degefu, Y; Rytkönen, A; von Weissenberg, K; Pappinen, A

    2004-08-01

    A field trial of 15 transgenic birch lines expressing a sugar beet chitinase IV gene and the corresponding controls was established in southern Finland to study the effects of the level of sugar beet chitinase IV expression on birch resistance to fungal diseases. The symptoms caused by natural infections of two fungal pathogens, Pyrenopeziza betulicola (leaf spot disease) and Melampsoridium betulinum (birch rust), were analysed in the field during a period of 3 years. The lines that had shown a high level of sugar beet chitinase IV mRNA accumulation in the greenhouse also showed high sugar beet chitinase IV expression after 3 years in the field. The level of sugar beet chitinase IV expression did not significantly improve the resistance of transgenic birches to leaf spot disease. Instead, some transgenic lines were significantly more susceptible to leaf spot than the controls. The level of sugar beet chitinase IV expression did have an improving effect on most parameters of birch rust; the groups of lines showing high or intermediate transgene expression were more resistant to birch rust than those showing low expression. This result indicates that the tested transformation may provide a tool for increasing the resistance of silver birch to birch rust.

  9. Purification and characterisation of a 31-kDa chitinase from the Myzus Persicae aphid: a target for hemiptera biocontrol.

    PubMed

    Francis, Frédéric; Saguez, Julien; Cherqui, Anas; Vandermoten, Sophie; Vincent, Charles; Versali, Marie-France; Dommès, Jacques; De Pauw, Edwin; Giordanengo, Philippe; Haubruge, Eric

    2012-03-01

    Hydrolytic enzymes involved in chitin degradation are important to allow moulting during insect development. Chitinases are interesting targets to disturb growth and develop alternative strategies to control insect pests. In this work, a chitinase from the aphid Myzus persicae was purified with a 36-fold purification rate in a three step procedure by ammonium sulphate fractionation, anion-exchange chromatography on a DEAE column and on an affinity Concanavalin A column. The purified chitinase purity assessed by 1D and 2D SDS-PAGE revealed a single band and three spots at 31 kDa, respectively. Chitinases were found to have high homologies with Concanavalins A and B, two chitinase-related proteins, a fungal endochitinase and an aphid acetylhydrolase by peptide identification by Maldi-Tof-Tof. The efficiency of two potent chitinase inhibitors, namely allosamidin and psammaplin A, was tested and showed significant rate of enzymatic inhibition.

  10. Production of N-Acetyl-d-glucosamine from Mycelial Waste by a Combination of Bacterial Chitinases and an Insect N-Acetyl-d-glucosaminidase.

    PubMed

    Zhu, Weixing; Wang, Di; Liu, Tian; Yang, Qing

    2016-09-07

    N-Acetyl-d-glucosamine (GlcNAc) has great potential to be used as a food additive and medicine. The enzymatic degradation of chitin-containing biomass for producing GlcNAc is an eco-friendly approach but suffers from a high cost. The economical efficiency can be improved by both optimizing the member and ratio of the chitinolytic enzymes and using new inexpensive substrates. To address this, a novel combination of bacterial and insect chitinolytic enzymes was developed in this study to efficiently produce GlcNAc from the mycelia of Asperillus niger, a fermentation waste. This enzyme combination contained three bacterial chitinases (chitinase A from Serratia marcescens (SmChiA), SmChiB, SmChiC) and one insect N-acetyl-d-glucosaminidase from Ostrinia furnacalis (OfHex1) in a ratio of 39.1% of SmChiA, 26.7% of SmChiB, 32.9% of SmChiC, and 1.3% of OfHex1. A yield of 6.3 mM (1.4 mg/mL) GlcNAc with a purity of 95% can be obtained from 10 mg/mL mycelial powder in 24 h. The enzyme combination reported here exhibited 5.8-fold higher hydrolytic activity over the commercial chitinase preparation derived from Streptomyces griseus.

  11. An acidic class III chitinase in sugar beet: induction by Cercospora beticola, characterization, and expression in transgenic tobacco plants.

    PubMed

    Nielsen, K K; Mikkelsen, J D; Kragh, K M; Bojsen, K

    1993-01-01

    An acidic chitinase (SE) was found to accumulate in leaves of sugar beet (Beta vulgaris) during infection with Cercospora beticola. Two isoforms, SE1 and SE2, with MW of 29 kDa and pI of approximately 3.0 were purified to homogeneity. SE2 is an endochitinase that also exhibits exochitinase activity, i.e., it is capable of hydrolyzing chito-oligosaccharides, including chitobiose, into N-acetyl-glucosamine. Partial amino acid sequence data for SE2 were used to obtain a cDNA clone by polymerase chain reaction. The clone was used to isolate a cDNA clone encoding SE2. The deduced amino acid sequence for SE2 is 58-67% identical to the class III chitinases from cucumber, Arabidopsis, and tobacco. A transient induction of SE2 mRNA during the early stages of infection with C. beticola is much stronger in tolerant plants than in susceptible plants. Transgenic tobacco (Nicotiana benthamiana) plants constitutively accumulate SE2 protein in the intercellular space of their leaves. In a preliminary infection experiment, the transgenic plants did not show increase in resistance against C. nicotianae.

  12. PcchiB1, encoding a class V chitinase, is affected by PcVelA and PcLaeA, and is responsible for cell wall integrity in Penicillium chrysogenum.

    PubMed

    Kamerewerd, Jens; Zadra, Ivo; Kürnsteiner, Hubert; Kück, Ulrich

    2011-11-01

    Penicillin production in Penicillium chrysogenum is controlled by PcVelA and PcLaeA, two components of the regulatory velvet-like complex. Comparative microarray analysis with mutants lacking PcVelA or PcLaeA revealed a set of 62 common genes affected by the loss of both components. A downregulated gene in both knockout strains is PcchiB1, potentially encoding a class V chitinase. Under nutrient-depleted conditions, transcript levels of PcchiB1 are strongly upregulated, and the gene product contributes to more than 50 % of extracellular chitinase activity. Functional characterization by generating PcchiB1-disruption strains revealed that PcChiB1 is responsible for cell wall integrity and pellet formation in P. chrysogenum. Further, fluorescence microscopy with a DsRed-labelled chitinase suggests a cell wall association of the protein. An unexpected phenotype occurred when knockout strains were grown on media containing N-acetylglucosamine as the sole C and N source, where, in contrast to the recipient, a penicillin producer strain, the mutants and an ancestral strain show distinct mycelial growth. We discuss the relevance of this class V chitinase for morphology in an industrially important fungus.

  13. Involvement of the MAPK and PI3K pathways in chitinase 3-like 1-regulated hyperoxia-induced airway epithelial cell death

    SciTech Connect

    Kim, Mi Na; Lee, Kyung Eun; Hong, Jung Yeon; Heo, Won Il; Kim, Kyung Won; Kim, Kyu Earn; Sohn, Myung Hyun

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer Hyperoxia induces apoptosis and chitinase 3-like 1 expression in human airway epithelial cells. Black-Right-Pointing-Pointer Presence of chitinase 3-like 1 affects airway epithelial cell death after hyperoxic exposure. Black-Right-Pointing-Pointer Silencing chitinase 3-like 1 manipulate the phosphorylation of ERK, p38 and Akt. -- Abstract: Background: Exposure to 100% oxygen causes hyperoxic acute lung injury characterized by cell death and injury of alveolar epithelial cells. Recently, the role of chitinase 3-like 1 (CHI3L1), a member of the glycosyl hydrolase 18 family that lacks chitinase activity, in oxidative stress was demonstrated in murine models. High levels of serum CHI3L1 have been associated with various diseases of the lung, such as asthma, chronic obstructive pulmonary disease, and cancer. However, the role of CHI3L1 in human airway epithelial cells undergoing oxidative stress remains unknown. In addition, the signaling pathways associated with CHI3L1 in this process are poorly understood. Purpose: In this study, we demonstrate the role of CHI3L1, along with the MAPK and PI3K signaling pathways, in hyperoxia-exposed airway epithelial cells. Method: The human airway epithelial cell line, BEAS-2B, was exposed to >95% oxygen (hyperoxia) for up to 72 h. Hyperoxia-induced cell death was determined by assessing cell viability, Annexin-V FITC staining, caspase-3 and -7 expression, and electron microscopy. CHI3L1 knockdown and overexpression studies were conducted in BEAS-2B cells to examine the role of CHI3L1 in hyperoxia-induced apoptosis. Activation of the MAPK and PI3K pathways was also investigated to determine the role of these signaling cascades in this process. Results: Hyperoxia exposure increased CHI3L1 expression and apoptosis in a time-dependent manner. CHI3L1 knockdown protected cells from hyperoxia-induced apoptosis. In contrast, CHI3L1 overexpression promoted cell death after hyperoxia exposure. Finally

  14. Time-Dependent Increase of Chitinase1 in APP/PS1 Double Transgenic Mice.

    PubMed

    Xiao, Qian; Shi, Rui; Yang, Wenxiu; Zou, Yan; Du, Yinshi; Zhang, Man; Yu, Weihua; Lü, Yang

    2016-07-01

    It is reported that chitinase1 increases in Alzheimer's disease (AD). However, the alteration of chitinase1 in the progress of AD is still unclear. Thus, we designed the present study to detect chitinase1 level in different stages of APP/PS1 double transgenic mice. Experimental models were APP/PS1 double transgenic mice with 4, 12 and 22 months. Cognitive function was detected by Morris water maze test in APP/PS1 mice as well as controls. ELISA and the quantitative RT-PCR were used to detect chitinase1 level in different groups. The study displayed that expression of chitinase1 gradually increased in a time-dependent manner in APP/PS1 mice, while there were no statistical differences among the wild-type mice in varies ages. Moreover, chitnase1 increased significantly in APP/PS1 mice aged 12 and 22 months compared with the age matched wild-type group, respectively. However, no difference of chitnase1 was found between 4 months-old APP/PS1 mice and wild-type mice. Comparing with the age matched wild type group, the consequences of mRNA on the increase in chitnase1 is in accordance with protein in APP/PS1 mice. Furthermore, Morris water maze showed that 4 months-old APP/PS1 mice have normal spatial learning and impaired spatial memory; both spatial learning and spatial memory in 12 and 22 months-old APP/PS1 mice were declined. Time-dependent increase of chitnase1 in APP/PS1 double transgenic mice indicates that the level of chitinase1 is associated with decline of cognition. Therefore, chitinase1 might be a biomarker of disease progression in AD.

  15. Chitinase 3-like 1: prognostic biomarker in clinically isolated syndromes.

    PubMed

    Cantó, Ester; Tintoré, Mar; Villar, Luisa M; Costa, Carme; Nurtdinov, Ramil; Álvarez-Cermeño, José C; Arrambide, Georgina; Reverter, Ferran; Deisenhammer, Florian; Hegen, Harald; Khademi, Mohsen; Olsson, Tomas; Tumani, Hayrettin; Rodríguez-Martín, Eulalia; Piehl, Fredrik; Bartos, Ales; Zimova, Denisa; Kotoucova, Jolana; Kuhle, Jens; Kappos, Ludwig; García-Merino, Juan Antonio; Sánchez, Antonio José; Saiz, Albert; Blanco, Yolanda; Hintzen, Rogier; Jafari, Naghmeh; Brassat, David; Lauda, Florian; Roesler, Romy; Rejdak, Konrad; Papuc, Ewa; de Andrés, Clara; Rauch, Stefan; Khalil, Michael; Enzinger, Christian; Galimberti, Daniela; Scarpini, Elio; Teunissen, Charlotte; Sánchez, Alex; Rovira, Alex; Montalban, Xavier; Comabella, Manuel

    2015-04-01

    Chitinase 3-like 1 (CHI3L1) has been proposed as a biomarker associated with the conversion to clinically definite multiple sclerosis in patients with clinically isolated syndromes, based on the finding of increased cerebrospinal fluid CHI3L1 levels in clinically isolated syndrome patients who later converted to multiple sclerosis compared to those who remained as clinically isolated syndrome. Here, we aimed to validate CHI3L1 as a prognostic biomarker in a large cohort of patients with clinically isolated syndrome. This is a longitudinal cohort study of clinically isolated syndrome patients with clinical, magnetic resonance imaging, and cerebrospinal fluid data prospectively acquired. A total of 813 cerebrospinal fluid samples from patients with clinically isolated syndrome were recruited from 15 European multiple sclerosis centres. Cerebrospinal fluid CHI3L1 levels were measured by enzyme-linked immunosorbent assay. Multivariable Cox regression models were used to investigate the association between cerebrospinal fluid CHI3L1 levels and time to conversion to multiple sclerosis and time to reach Expanded Disability Status Scale 3.0. CHI3L1 levels were higher in patients who converted to clinically definite multiple sclerosis compared to patients who continued as clinically isolated syndrome (P = 8.1 × 10(-11)). In the Cox regression analysis, CHI3L1 levels were a risk factor for conversion to multiple sclerosis (hazard ratio = 1.7; P = 1.1 × 10(-5) using Poser criteria; hazard ratio = 1.6; P = 3.7 × 10(-6) for McDonald criteria) independent of other covariates such as brain magnetic resonance imaging abnormalities and presence of cerebrospinal fluid oligoclonal bands, and were the only significant independent risk factor associated with the development of disability (hazard ratio = 3.8; P = 2.5 × 10(-8)). High CHI3L1 levels were associated with shorter time to multiple sclerosis (P = 3.2 × 10(-9) using Poser criteria; P = 5.6 × 10(-11) for McDonald criteria

  16. Chitin binding proteins act synergistically with chitinases in Serratia proteamaculans 568.

    PubMed

    Purushotham, Pallinti; Arun, P V Parvati Sai; Prakash, Jogadhenu S S; Podile, Appa Rao

    2012-01-01

    Genome sequence of Serratia proteamaculans 568 revealed the presence of three family 33 chitin binding proteins (CBPs). The three Sp CBPs (Sp CBP21, Sp CBP28 and Sp CBP50) were heterologously expressed and purified. Sp CBP21 and Sp CBP50 showed binding preference to β-chitin, while Sp CBP28 did not bind to chitin and cellulose substrates. Both Sp CBP21 and Sp CBP50 were synergistic with four chitinases from S. proteamaculans 568 (Sp ChiA, Sp ChiB, Sp ChiC and Sp ChiD) in degradation of α- and β-chitin, especially in the presence of external electron donor (reduced glutathione). Sp ChiD benefited most from Sp CBP21 or Sp CBP50 on α-chitin, while Sp ChiB and Sp ChiD had major advantage with these Sp CBPs on β-chitin. Dose responsive studies indicated that both the Sp CBPs exhibit synergism ≥ 0.2 µM. The addition of both Sp CBP21 and Sp CBP50 in different ratios to a synergistic mixture did not significantly increase the activity. Highly conserved polar residues, important in binding and activity of CBP21 from S. marcescens (Sm CBP21), were present in Sp CBP21 and Sp CBP50, while Sp CBP28 had only one such polar residue. The inability of Sp CBP28 to bind to the test substrates could be attributed to the absence of important polar residues.

  17. Sequence analysis and gene expression of putative oil palm chitinase and chitinase-like proteins in response to colonization of Ganoderma boninense and Trichoderma harzianum.

    PubMed

    Yeoh, K-A; Othman, A; Meon, S; Abdullah, F; Ho, C-L

    2013-01-01

    Chitinases are glycosyl hydrolases that cleave the β-1,4-glycosidic linkages between N-acetylglucosamine residues in chitin which is a major component of fungal cell wall. Plant chitinases hydrolyze fungal chitin to chitin oligosaccharides that serve as elicitors of plant defense system against fungal pathogens. However, plants synthesize many chitinase isozymes and some of them are not pathogenesis-related. In this study, three full-length cDNA sequences encoding a putative chitinase (EgChit3-1) and two chitinase-like proteins (EgChit1-1 and EgChit5-1) have been cloned from oil palm (Elaeis guineensis) by polymerase chain reaction (PCR). The abundance of these transcripts in the roots and leaves of oil palm seedlings treated with Ganoderma boninense (a fungal pathogen) or Trichoderma harzianum (an avirulent symbiont), and a combination of both fungi at 3, 6 and 12 weeks post infection were profiled by real time quantitative reverse-transcription (qRT)-PCR. Our findings showed that the gene expression of EgChit3-1 increased significantly in the roots of oil palm seedlings treated with either G. boninense or T. harzianum and a combination of both; whereas the gene expression of EgChit1-1 in the treated roots of oil palm seedlings was not significantly higher compared to those of the untreated oil palm roots. The gene expression of EgChit5-1 was only higher in the roots of oil palm seedlings treated with T. harzianum compared to those of the untreated oil palm roots. In addition, the gene expression of EgChit1-1 and EgChit3-1 showed a significantly higher gene expression in the leaf samples of oil palm seedlings treated with either G. boninense or T. harzianum.

  18. Acidic chitinase primes the protective immune response to gastrointestinal nematodes.

    PubMed

    Vannella, Kevin M; Ramalingam, Thirumalai R; Hart, Kevin M; de Queiroz Prado, Rafael; Sciurba, Joshua; Barron, Luke; Borthwick, Lee A; Smith, Allen D; Mentink-Kane, Margaret; White, Sandra; Thompson, Robert W; Cheever, Allen W; Bock, Kevin; Moore, Ian; Fitz, Lori J; Urban, Joseph F; Wynn, Thomas A

    2016-05-01

    Acidic mammalian chitinase (AMCase) is known to be induced by allergens and helminths, yet its role in immunity is unclear. Using AMCase-deficient mice, we show that AMCase deficiency reduced the number of group 2 innate lymphoid cells during allergen challenge but was not required for establishment of type 2 inflammation in the lung in response to allergens or helminths. In contrast, AMCase-deficient mice showed a profound defect in type 2 immunity following infection with the chitin-containing gastrointestinal nematodes Nippostrongylus brasiliensis and Heligmosomoides polygyrus bakeri. The impaired immunity was associated with reduced mucus production and decreased intestinal expression of the signature type 2 response genes Il13, Chil3, Retnlb, and Clca1. CD103(+) dendritic cells, which regulate T cell homing, were also reduced in mesenteric lymph nodes of infected AMCase-deficient mice. Thus, AMCase functions as a critical initiator of protective type 2 responses to intestinal nematodes but is largely dispensable for allergic responses in the lung.

  19. Molecular characterization of stress resistance-related chitinase genes of Brassica rapa.

    PubMed

    Ahmed, Nasar Uddin; Park, Jong-In; Jung, Hee-Jeong; Kang, Kwon-Kyoo; Hur, Yoonkang; Lim, Yong-Pyo; Nou, Ill-Sup

    2012-09-01

    Brassica is an important vegetable group worldwide that is impacted by biotic and abiotic stresses. Molecular biology techniques offer the most efficient approach to address these concerns. Inducible plant defense responses include the production of pathogenesis-related (PR) proteins, and chitinases are very important PR proteins. We collected 30 chitinase like genes, three from our full-length cDNA library of Brassica rapa cv. Osome and 27 from Brassica databases. Sequence analysis and comparison study confirmed that they were all class I-V and VII chitinase genes. These genes also showed a high degree of homology with other biotic stress resistance-related plant chitinases. An organ-specific expression of these genes was observed and among these, seven genes showed significant responses after infection with Fusarium oxysporum f.sp. conglutinans in cabbage and sixteen genes showed responsive expression after abiotic stress treatments in Chinese cabbage. BrCLP1, 8, 10, 17 and 18 responded commonly after biotic and abiotic stress treatments indicating their higher potentials. Taken together, the results presented herein suggest that these chitinase genes may be useful resources in the development of stress resistant Brassica.

  20. Crystal structure of class III chitinase from pomegranate provides the insight into its metal storage capacity.

    PubMed

    Masuda, Taro; Zhao, Guanghua; Mikami, Bunzo

    2015-01-01

    Chitinase hydrolyzes the β-1,4-glycosidic bond in chitin. In higher plants, this enzyme has been regarded as a pathogenesis-related protein. Recently, we identified a class III chitinase, which functions as a calcium storage protein in pomegranate (Punica granatum) seed (PSC, pomegranate seed chitinase). Here, we solved a crystal structure of PSC at 1.6 Å resolution. Although its overall structure, including the structure of catalytic site and non-proline cis-peptides, was closely similar to those of other class III chitinases, PSC had some unique structural characteristics. First, there were some metal-binding sites with coordinated water molecules on the surface of PSC. Second, many unconserved aspartate residues were present in the PSC sequence which rendered the surface of PSC negatively charged. This acidic electrostatic property is in contrast to that of hevamine, well-characterized plant class III chitinase, which has rather a positively charged surface. Thus, the crystal structure provides a clue for metal association property of PSC.

  1. Abundance of truncated and full-length ChitA and ChitB chitinases in healthy and diseased maize tissues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chitinase modifying proteins, cmps, are secreted fungal proteases that combat plant defenses by truncating plant class IV chitinases. We initially discovered that ChitA and ChitB, two plant class IV chitinases that are abundant in developing and mature kernels of corn, are truncated by cmps during e...

  2. Creation of Customized Bioactivity within a 14-Membered Macrolide Scaffold: Design, Synthesis, and Biological Evaluation Using a Family-18 Chitinase.

    PubMed

    Sugawara, Akihiro; Maita, Nobuo; Gouda, Hiroaki; Yamamoto, Tsuyoshi; Hirose, Tomoyasu; Kimura, Saori; Saito, Yoshifumi; Nakano, Hayato; Kasai, Takako; Nakano, Hirofumi; Shiomi, Kazuro; Hirono, Shuichi; Watanabe, Takeshi; Taniguchi, Hisaaki; Omura, Satoshi; Sunazuka, Toshiaki

    2015-06-25

    Argifin, a 17-membered pentapeptide, inhibits chitinase. As argifin has properties that render it unsuitable as a drug development candidate, we devised a mechanism to create the structural component of argifin that bestows the chitinase inhibition and introduce it into a 14-membered macrolide scaffold. Here we describe (1) the designed macrolide, which exhibits ∼200-fold more potent chitinase inhibition than argifin, (2) the binding modes of the macrolide with Serratia marcescens chitinase B, and (3) the computed analysis explaining the reason for derivatives displaying increased inhibition compared to argifin, the macrolide aglycone displaying inhibition in a nanomolar range. This promises a class of chitinase inhibitors with novel skeletons, providing innovative insight for drug design and the use of macrolides as adaptable, flexible templates for use in drug discovery research and development.

  3. Production, purification and application of extracellular chitinase from Cellulosimicrobium cellulans 191

    PubMed Central

    Fleuri, Luciana F.; Kawaguti, Haroldo Y.; Sato, Hélia H.

    2009-01-01

    This study concerned the production, purification and application of extracellular chitinase from Cellulosimicrobium cellulans strain 191. In shaken flasks the maximum yield of chitinase was 6.9 U/mL after 72 h of cultivation at 25°C and 200 rpm. In a 5 L fermenter with 1.5 vvm aeration, the highest yield obtained was 4.19 U/mL after 168 h of fermentation at 25°C and 200 rpm, and using 3 vvm, it was 4.38 U/mL after 144 h of fermentation. The chitinase (61 KDa) was purified about 6.65 times by Sepharose CL 4B 200 gel filtration with a yield of 46.61%. The purified enzyme was able to lyse the cell walls of some fungi and to form protoplasts. PMID:24031407

  4. The chitinase-like protein YKL-40 increases mucin5AC production in human bronchial epithelial cells

    SciTech Connect

    Liu, Chunyi; Li, Qi; Zhou, Xiangdong; Kolosov, Victor P.; Perelman, Juliy M.

    2013-11-01

    Mucus overproduction is an important feature in patients with chronic inflammatory airway diseases. However, the regulatory mechanisms that mediate excessive mucin production remain elusive. Recently, the level of YKL-40, a chitinase-like protein, has been found to be significantly increased in chronic inflammatory airway diseases and has been shown to be associated with the severity of these diseases. In this study, we sought to explore the effect of YKL-40 on mucin5AC (MUC5AC) production in chronic inflammatory airway diseases and the potential signaling pathways involved in this process. We found that elevated YKL-40 levels increased the mRNA and protein expression of MUC5AC in a dose- and time-dependent manner, in association with the phosphorylation of extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB), reflecting their activation. These responses were significantly suppressed by the knockdown of protease-activating receptor 2 (PAR2) with specific small interfering RNA or the inhibitors of ERK and NF-κB. YKL-40-induced MUC5AC overproduction was also effectively attenuated by the inhibitor of focal adhesion kinase (FAK). Taken together, these results imply that YKL-40 can stimulate excessive MUC5AC production through PAR2- and FAK-mediated mechanisms. - Highlights: • MUC5AC is the major secreted mucin in chronic inflammatory airway diseases. • YKL-40 is a prototype of the chitinase-like protein in mammals. • YKL-40 is an active player in chronic inflammatory airway diseases. • YKL-40 can increase MUC5AC production via PAR2-mediated pathway. • FAK is another candidate to mediate YKL-40-induced MUC5AC overexpression.

  5. Bacterial chitin binding proteins show differential substrate binding and synergy with chitinases.

    PubMed

    Manjeet, Kaur; Purushotham, Pallinti; Neeraja, Chilukoti; Podile, Appa Rao

    2013-08-25

    Glycosyl hydrolase (GH) family 18 chitinases (Chi) and family 33 chitin binding proteins (CBPs) from Bacillus thuringiensis serovar kurstaki (BtChi and BtCBP), B. licheniformis DSM13 (BliChi and BliCBP) and Serratia proteamaculans 568 (SpChiB and SpCBP21) were used to study the efficiency and synergistic action of BtChi, BliChi and SpChiB individually with BtCBP, BliCBP or SpCBP21. Chitinase assay revealed that only BtChi and SpChiB showed synergism in hydrolysis of chitin, while there was no increase in products generated by BliChi, in the presence of the three above mentioned CBPs. This suggests that some (specific) CBPs are able to exert a synergistic effect on (specific) chitinases. A mutant of BliChi, designated as BliGH, was constructed by deleting the C-terminal fibronectin III (FnIII) and carbohydrate binding module 5 (CBM5) to assess the contribution of FnIII and CBM5 domains in the synergistic interactions of GH18 chitinases with CBPs. Chitinase assay with BliGH revealed that the accessory domains play a major role in making BliChi an efficient enzyme. We studied binding of BtCBP and BliCBP to α- and β-chitin. The BtCBP, BliCBP or SpCBP21 did not act synergistically with chitinases in hydrolysis of the chitin, interspersed with other polymers, present in fungal cell walls.

  6. Identification and expression analysis of chitinase genes related to biotic stress resistance in Brassica.

    PubMed

    Ahmed, Nasar Uddin; Park, Jong-In; Seo, Mi-Suk; Kumar, Thamilarasan Senthil; Lee, In-Ho; Park, Beom-Seok; Nou, Ill-Sup

    2012-04-01

    Brassica is a very important vegetable group because of its contribution to human nutrition and consequent economic benefits. However, biotic stress is a major concern for these crops and molecular biology techniques offer the most efficient of approaches to address this concern. Chitinase is an important biotic stress resistance-related gene. We identified three genes designated as Brassica chitinase like protein (BrCLP1), BrCLP2 and BrCLP3 from a full-length cDNA library of Brassica rapa cv. Osome. Sequence analysis of these genes confirmed that BrCLP1 was a class IV chitinase, and BrCLP2 and BrCLP3 were class VII chitinases. Also, these genes showed a high degree of homology with other biotic stress resistance-related plant chitinases. In expression analysis, organ-specific expression of all three genes was high except BrCLP1 in all the organs tested and BrCLP2 showed the highest expression compared to the other genes in flower buds. All these genes also showed expression during all developmental growth stages of Chinese cabbage. In addition, BrCLP1 was up-regulated with certain time of infection by Pectobacterium carotovorum subsp. carotovorum in Chinese cabbage plants during microarray expression analysis. On the other hand, expression of BrCLP2 and BrCLP3 were increased after 6 h post inoculation (hpi) but decreased from 12 hpi. All these data suggest that these three chitinase genes may be involved in plant resistance against biotic stresses.

  7. A glycosynthase derived from an inverting GH19 chitinase from the moss Bryum coronatum.

    PubMed

    Ohnuma, Takayuki; Fukuda, Tatsuya; Dozen, Satoshi; Honda, Yuji; Kitaoka, Motomitsu; Fukamizo, Tamo

    2012-06-15

    BcChi-A, a GH19 chitinase from the moss Bryum coronatum, is an endo-acting enzyme that hydrolyses the glycosidic bonds of chitin, (GlcNAc)(n) [a β-1,4-linked polysaccharide of GlcNAc (N-acetylglucosamine) with a polymerization degree of n], through an inverting mechanism. When the wild-type enzyme was incubated with α-(GlcNAc)2-F [α-(GlcNAc)(2) fluoride] in the absence or presence of (GlcNAc)(2), (GlcNAc)(2) and hydrogen fluoride were found to be produced through the Hehre resynthesis-hydrolysis mechanism. To convert BcChi-A into a glycosynthase, we employed the strategy reported by Honda et al. [(2006) J. Biol. Chem. 281, 1426-1431; (2008) Glycobiology 18, 325-330] of mutating Ser(102), which holds a nucleophilic water molecule, and Glu(70), which acts as a catalytic base, producing S102A, S102C, S102D, S102G, S102H, S102T, E70G and E70Q. In all of the mutated enzymes, except S102T, hydrolytic activity towards (GlcNAc)(6) was not detected under the conditions we used. Among the inactive BcChi-A mutants, S102A, S102C, S102G and E70G were found to successfully synthesize (GlcNAc)(4) as a major product from α-(GlcNAc)(2)-F in the presence of (GlcNAc)(2). The S102A mutant showed the greatest glycosynthase activity owing to its enhanced F(-) releasing activity and its suppressed hydrolytic activity. This is the first report on a glycosynthase that employs amino sugar fluoride as a donor substrate.

  8. Heterologous expression and characterization of two chitinase 5 enzymes from the migratory locust Locusta migratoria.

    PubMed

    Li, Ying-Long; Song, Hui-Fang; Zhang, Xue-Yao; Li, Da-Qi; Zhang, Ting-Ting; Ma, En-Bo; Zhang, Jian-Zhen

    2016-06-01

    Insect chitinases are involved in degradation of chitin from the exoskeleton or peritrophic metrix of midgut. In Locusta migratoria, two duplicated Cht5s (LmCht5-1 and LmCht5-2) have been shown to have distinct molecular characteristics and biological roles. To explore the protein properties of the two LmCht5s, we heterologously expressed both enzymes using baculovirus expression system in SF9 cells, and characterized kinetic and carbohydrate-binding properties of purified enzymes. LmCht5-1 and LmCht5-2 exhibited similar pH and temperature optimums. LmCht5-1 has lower Km value for the oligomeric substrate (4MU-(GlcNAc)3 ), and higher Km value for the longer substrate (CM-Chitin-RBV) compared with LmCht5-2. A comparison of amino acids and homology modeling of catalytic domain presented similar TIM barrel structures and differentiated amino acids between two proteins. LmCht5-1 has a chitin-binding domain (CBD) tightly bound to colloidal chitin, but LmCht5-2 does not have a CBD for binding to colloidal chitin. Our results suggested both LmCht5-1 and LmCht5-2, which have the critical glutamate residue in region II of catalytic domain, exhibited chitinolytic activity cleaving both polymeric and oligomeric substrates. LmCht5-1 had relatively higher activity against the oligomeric substrate, 4MU-(GlcNAc)3 , whereas LmCht5-2 exhibited higher activity toward the longer substrate, CM-Chitin-RBV. These findings are helpful for further research to clarify their different roles in insect growth and development.

  9. Chitinases biosynthesis by immobilized Aeromonas hydrophila SBK1 by prawn shells valorization and application of enzyme cocktail for fungal protoplast preparation.

    PubMed

    Halder, Suman Kumar; Maity, Chiranjit; Jana, Arijit; Ghosh, Kuntal; Das, Arpan; Paul, Tanmay; Das Mohapatra, Pradeep Kumar; Pati, Bikas Ranjan; Mondal, Keshab Chandra

    2014-02-01

    Production and optimization of β-N-acetyl glucosaminidase and chitinase by Ca-alginate immobilized Aeromonas hydrophila SBK1 was carried out using prawn shell as cost-effective substrate. Beads prepared with 5.0% Na-alginate (containing 2.0% colloidal chitin) and 1.0 M CaCl2 showed considerable beads integrity and supported maximum production of chitinolytic enzymes. Bead diameter, 3 mm; temperature, 35°C; pH 7.0; agitation, 90 rpm were found ideal for the maximum production of the enzymes. The fermentation and thermodynamic indices revealed the feasibility of immobilized cells over free cells for enzymes production. Reasonable amount of chitosaccharides (degree of polymerization; 1-6) accumulated in the production media which have paramount antioxidant activity. Scale up experiment was successfully carried out in 5 L fermentor. In immobilized state, the chitosaccharides yield and antioxidant activity increased about 44.76% and 22.22%, whereas specific productivity of β-N-acetyl glucosaminidase and chitinase increased by 22.86% and 33.37% over free state. The cell entrapped beads can be reused upto ten cycles without marked loss of its biocatalytic efficiency. High level of protoplast of Aspergillus niger was generated by treating mycelia with 10 U/ml of crude chitinase after 4 h at pH 7.0 and in the temperature 35-40°C, and 67% of the protoplasts were found to be regenerated.

  10. Domain organization and phylogenetic analysis of the chitinase-like family of proteins in three species of insects.

    PubMed

    Zhu, Qingsong; Arakane, Yasuyuki; Banerjee, Debarshi; Beeman, Richard W; Kramer, Karl J; Muthukrishnan, Subbaratnam

    2008-04-01

    A bioinformatics-based investigation of three insect species with completed genome sequences has revealed that insect chitinase-like proteins (glycosylhydrolase family 18) are encoded by a rather large and diverse group of genes. We identified 16, 16 and 13 putative chitinase-like genes in the genomic databases of the red flour beetle, Tribolium castaneum, the fruit fly, Drosophila melanogaster, and the malaria mosquito, Anopheles gambiae, respectively. Chitinase-like proteins encoded by this gene family were classified into five groups based on phylogenetic analyses. Group I chitinases are secreted proteins that are the most abundant such enzymes in molting fluid and/or integument, and represent the prototype enzyme of the family, with a single copy each of the catalytic domain and chitin-binding domain (ChBD) connected by an S/T-rich linker polypeptide. Group II chitinases are unusually larger-sized secreted proteins that contain multiple catalytic domains and ChBDs. Group III chitinases contain two catalytic domains and are predicted to be membrane-anchored proteins. Group IV chitinases are the most divergent. They usually lack a ChBD and/or an S/T-rich linker domain, and are known or predicted to be secreted proteins found in gut or fat body. Group V proteins include the putative chitinase-like imaginal disc growth factors (IDGFs). In each of the three insect genomes, multiple genes encode group IV and group V chitinase-like proteins. In contrast, groups I-III are each represented by only a singe gene in each species.

  11. Effect of Bombyx mori chitinase against Japanese pine sawyer (Monochamus alternatus) adults as a biopesticide.

    PubMed

    Kabir, Khondkar Ehteshamul; Sugimoto, Hiroyuki; Tado, Hiroyuki; Endo, Katsuhiko; Yamanaka, Akira; Tanaka, Shuhei; Koga, Daizo

    2006-01-01

    Bombyx mori chitinase (Bm-CHI), with a molecular mass of 75 kDa, was investigated on the possibility that it can serve as a biocontrol agent against the adult Japanese pine sawyer (JPS), Monochamus alternatus (Coleoptera: Cerambycidae). Oral ingestion of purified chitinase at concentrations of 3 microM (11.25 microg/50 microl) and 0.3 micoM (1.125 microg/50 microl) caused high mortality in JPS, a significant decrease in bark consumption, and, only in high concentration, a slight reduction of body weight. Fluorescence assays indicated that peritrophic membrane (PM) chitin is degraded by the action of orally ingested Bm-CHI at 3 microM concentration only. Scanning electron micrographs clearly indicated that the beetles that ingested Bm-CHI of the same high concentration had their PM perforated and disrupted, but ultrastructural studies showed that the ingested chitinase did not affect the midgut epithelium. These findings open up the possibility of using insect chitinase as a biopesticidal enzyme. It should have agronomic potential for insect control.

  12. Differential aphicidal effects of chitinase inhibitors on the polyphagous homopteran Myzus persicae (Sulzer).

    PubMed

    Saguez, Julien; Dubois, Françoise; Vincent, Charles; Laberche, Jean-Claude; Sangwan-Norreel, Brigitte S; Giordanengo, Philippe

    2006-12-01

    Four chitinase inhibitors, cyclo-(Proline-Tyrosine), cyclo-(Histidine-Proline), allosamidin and psammaplin A, were selected for in vitro feeding experiments with the peach-potato aphid, Myzus persicae (Sulzer), under controlled photoperiod and temperature conditions. Artificial diets were used to provide chitinase inhibitors at 10, 50 and 100 microg mL(-1) to M. persicae. Except for cyclo-(Proline-Tyrosine), which did not modify aphid demographic parameters, chitinase inhibitors induced differential aphicidal effects on M. persicae. At all doses, cyclo-(Histidine-Proline) induced significant effects affecting daily fecundity, intrinsic rate of natural increase (r(m)) and doubling time of population. When compared with the control diet, allosamidin decreased nymph survival and daily fecundity, increasing the doubling time of population from 1 to 1.5 days. Psammaplin A was the most toxic inhibitor when delivered via artificial diet, as it induced the death of all aphids reared at 50 and 100 microg mL(-1). The results demonstrate the potential use of chitinase inhibitors as aphid management tools.

  13. Draft Genome Sequence of Marine-Derived Aeromonas caviae CHZ306, a Potential Chitinase Producer Strain

    PubMed Central

    Zimpel, Cristina Kraemer; Guimaraes, Ana Marcia Sa; Pessoa, Adalberto; Rivera, Irma Nelly Gutierrez

    2016-01-01

    We report here a draft genome sequence of Aeromonas caviae CHZ306, a marine-derived bacterium with the ability to hydrolyze chitin and express high levels of chitinases. The assembly resulted in 65 scaffolds with approximately 4.78 Mb. Genomic analysis revealed different genes encoding chitin-degrading enzymes that can be used for chitin derivative production. PMID:27856589

  14. Draft Genome Sequence of Marine-Derived Aeromonas caviae CHZ306, a Potential Chitinase Producer Strain.

    PubMed

    Cardozo, Flávio Augusto; Zimpel, Cristina Kraemer; Guimaraes, Ana Marcia Sa; Pessoa, Adalberto; Rivera, Irma Nelly Gutierrez

    2016-11-17

    We report here a draft genome sequence of Aeromonas caviae CHZ306, a marine-derived bacterium with the ability to hydrolyze chitin and express high levels of chitinases. The assembly resulted in 65 scaffolds with approximately 4.78 Mb. Genomic analysis revealed different genes encoding chitin-degrading enzymes that can be used for chitin derivative production.

  15. Maize Seed Chitinase is Modified by a Protein Secreted by Bipolaris zeicola

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plants contain defense mechanisms that prevent infection by most fungi. Some specialized fungi have the ability to overcome plant defenses. The Zea mays (maize) seed chitinase ChitA has been previously reported as an antifungal protein. Here we report that ChitA is converted to a modified form by...

  16. Use of Metarhizium anisopliae Chitinase Genes for Genotyping and Virulence Characterization

    PubMed Central

    Niassy, Saliou; Subramanian, Sevgan; Ekesi, Sunday; Bargul, Joel L.; Villinger, Jandouwe; Maniania, Nguya K.

    2013-01-01

    Virulence is the primary factor used for selection of entomopathogenic fungi (EPF) for development as biopesticides. To understand the genetic mechanisms underlying differences in virulence of fungal isolates on various arthropod pests, we compared the chitinase genes, chi2 and chi4, of 8 isolates of Metarhizium anisopliae. The clustering of the isolates showed various groups depending on their virulence. However, the analysis of their chitinase DNA sequences chi2 and chi4 did not reveal major divergences. Although their protein translates have been implicated in fungal virulence, the predicted protein structure of chi2 was identical for all isolates. Despite the critical role of chitin digestion in fungal infection, we conclude that chi2 and chi4 genes cannot serve as molecular markers to characterize observed variations in virulence among M. anisopliae isolates as previously suggested. Nevertheless, processes controlling the efficient upregulation of chitinase expression might be responsible for different virulence characteristics. Further studies using comparative “in vitro” chitin digestion techniques would be more appropriate to compare the quality and the quantity of chitinase production between fungal isolates. PMID:23936804

  17. Cells from the skin of patients with systemic sclerosis secrete chitinase 3-like protein 1

    PubMed Central

    Ho, Yuen Yee; Baron, Murray; Recklies, Anneliese D.; Roughley, Peter J.; Mort, John S.

    2014-01-01

    Background The chitinase-like protein, Chi3L1, is associated with increased fibrotic activity as well as inflammatory processes. The capacity of skin cells from systemic sclerosis (SSc) patients to produce Chi3L1, and the stimulation of its synthesis by cytokines or growth factors known to be associated with SSc, was investigated. Methods Cells were isolated from forearm and/or abdomen skin biopsies taken from SSc patients and normal individuals and stimulated with cytokines and growth factors to assess Chi3L1 expression. Chi3L1-expressing cells were characterized by immunohistochemical staining. Results Chi3L1 was not secreted by skin cells from normal individuals nor was its synthesis induced by any of the cytokines or growth factors investigated. In contrast, Chi3L1 secretion was induced by OSM or IL-1 in cells from all forearm biopsies of SSc patients, and endogenous secretion in the absence of cytokines was detected in several specimens. Patients with Chi3L1-producing cells at both the arm and abdomen had a disease duration of less than 3 years. Endogenous Chi3L1 production was not a property of the major fibroblast population nor of myofibroblasts, but rather was related to the presence of stem-like cells not present in normal skin. Other cells, however, contributed to the upregulation of Chi3L1 by OSM. Conclusions The emergence of cells primed to respond to OSM with increased Chi3L1 production appears to be associated with pathological processes active in SSc. General significance The presence of progenitor cells expressing the chilectin Chi3L1 in SSc skin appears to play a role in the initiation of the disease process. PMID:26675476

  18. ScChi, Encoding an Acidic Class III Chitinase of Sugarcane, Confers Positive Responses to Biotic and Abiotic Stresses in Sugarcane

    PubMed Central

    Su, Yachun; Xu, Liping; Fu, Zhiwei; Yang, Yuting; Guo, Jinlong; Wang, Shanshan; Que, Youxiong

    2014-01-01

    Chitinases (EC 3.2.2.14), expressed during the plant-pathogen interaction, are associated with plant defense against pathogens. In the present study, a positive correlation between chitinase activity and sugarcane smut resistance was found. ScChi (GenBank accession no. KF664180), a Class III chitinase gene, encoded a 31.37 kDa polypeptide, was cloned and identified. Subcellular localization revealed ScChi targeting to the nucleus, cytoplasm and the plasma membrane. Real-time quantitative PCR (RT-qPCR) results showed that ScChi was highly expressed in leaf and stem epidermal tissues. The ScChi transcript was both higher and maintained longer in the resistance cultivar during challenge with Sporisorium scitamineum. The ScChi also showed an obvious induction of transcription after treatment with SA (salicylic acid), H2O2, MeJA (methyl jasmonate), ABA (abscisic acid), NaCl, CuCl2, PEG (polyethylene glycol) and low temperature (4 °C). The expression levels of ScChi and six immunity associated marker genes were upregulated by the transient overexpression of ScChi. Besides, histochemical assay of Nicotiana benthamiana leaves overexpressing pCAMBIA 1301-ScChi exhibited deep DAB (3,3′-diaminobenzidinesolution) staining color and high conductivity, indicating the high level of H2O2 accumulation. These results suggest a close relationship between the expression of ScChi and plant immunity. In conclusion, the positive responses of ScChi to the biotic and abiotic stimuli reveal that this gene is a stress-related gene of sugarcane. PMID:24552874

  19. Human cartilage chitinase 3-like protein 2: cloning, expression, and production of polyclonal and monoclonal antibodies for osteoarthritis detection and identification of potential binding partners.

    PubMed

    Ranok, Araya; Khunkaewla, Panida; Suginta, Wipa

    2013-10-01

    Human cartilage chitinase 3-like protein 2 (CHI3L2 or YKL-39) is a member of family-18 glycosyl hydrolases that lacks chitinase activity. YKL-39 is known as a potential marker for the activation of chondrocytes and the progression of osteoarthritis. In this study, we cloned and expressed a functional form of human YKL-39 in the bacterial system. The Escherichia coli expressed YKL-30 was used as immugen for production of anti YKL-39 polyclonal and monoclonal antibodies. Both antibody types were highly selective, reacting only with YKL-39. Isotype mapping identified two hybridoma clones (so called clones 6H11 and 8H3) to be IgM isotype. Dot blot assay showed that the monoclonal antibody was strongly active with the synovial fluid of an osteoarthritis patient, human monocyte, and T lymphocyte cell lines. Database search for protein binding partners gave high hits with several glycoproteins that play particular roles in cartilage tissue scaffolding, connective tissue formation, and cell-cell interactions. In conclusion, anti YKL-39 polyclonal and monoclonal antibodies were raised and tested to be suitable for immunological applications, such as the investigation of the YKL-39 regulating pathway and the development of an immunosensing tool for sensitive detection of cartilage tissue destruction.

  20. Two chitinase 5 genes from Locusta migratoria: molecular characteristics and functional differentiation.

    PubMed

    Li, Daqi; Zhang, Jianqin; Wang, Yan; Liu, Xiaojian; Ma, Enbo; Sun, Yi; Li, Sheng; Zhu, Kun Yan; Zhang, Jianzhen

    2015-03-01

    The duplication of chitinase 5 (Cht5) into two to five different genes has been reported only in mosquito species to date. Here, we report the duplication of Cht5 genes (LmCht5-1 and LmCht5-2) in the migratory locust (Locusta migratoria). Both LmCht5-1 (505 aa) and LmCht5-2 (492 aa) possess a signal peptide and a catalytic domain with four conserved motifs, but only LmCht5-1 contains a chitin-binding domain. Structural and phylogenetic analyses suggest that LmCht5-1 is orthologous to other insect Cht5 genes, whereas LmCht5-2 might be newly duplicated. Both LmCht5 genes were expressed in all tested tissues with LmCht5-1 highly expressed in hindgut and LmCht5-2 highly expressed in integument, foregut, hindgut and fat bodies. From the fourth-instar nymphs to the adults, LmCht5-1 and LmCht5-2 showed similar developmental expression patterns with transcript peaks prior to each nymphal molting, suggesting that their expression levels are similarly regulated. Treatment with 20-hydroxyecdysone (20E; the most active molting hormone) and reducing expression of EcR (ecdysone receptor gene) by RNAi increased and decreased expression of both LmCht5 genes, respectively, indicating that both genes are responsive to 20E. Although transcript level of LmCht5-2 is generally 10-fold higher than that of LmCht5-1, RNAi-mediated suppression of LmCht5-1 transcript led to severe molting defects and lethality, but such effects were not seen with RNAi of LmCht5-2, suggesting that the newly duplicated LmCht5-2 is not essential for development and survivorship of the locust.

  1. Autolysis and aging of Penicillium chrysogenum cultures under carbon starvation: Chitinase production and antifungal effect of allosamidin.

    PubMed

    Sámi, László; Pusztahelyi, Tünde; Emri, Tamás; Varecza, Zoltán; Fekete, Andrea; Grallert, Agnes; Karanyi, Zsolt; Kiss, László; Pócsi, István

    2001-08-01

    In carbon-depleted cultures of Penicillium chrysogenum, age-related chitinases were shown to play a crucial role in both autolysis and fragmentation as indicated by in vivo enzyme inhibition experiments using allosamidin. This pseudotrisaccharide even hindered significantly the outgrowth of new hyphal tips from the surviving yeastlike fragments after glucose supplementation. The antifungal effect of allosamidin on autolyzing P. chrysogenum mycelia was fungistatic rather than fungicidal. In growing hyphae, membrane-bound microsomal chitinase zymogen(s) were detected, which may be indicative of some compartmentalization of these hydrolases. Later, during autolysis, no zymogenic chitinase was detected in any enzyme fraction studied, including microsomes. These observations may explain the different sensitivity of growing and autolyzing mycelia to allosamidin. Chitinases taking part in the age-related fragmentation of hyphae and the outgrowth of surviving hyphal fragments seem to be potent targets for future antifungal drug research.

  2. Human YKL39 (chitinase 3-like protein 2), an osteoarthritis-associated gene, enhances proliferation and type II collagen expression in ATDC5 cells

    SciTech Connect

    Miyatake, Kazumasa; Tsuji, Kunikazu; Yamaga, Mika; Yamada, Jun; Matsukura, Yu; Abula, Kahaer; Sekiya, Ichiro; Muneta, Takeshi

    2013-02-01

    Highlights: ► hYKL-39 expression is increased in osteoarthritic articular chondrocytes. ► To examine the molecular functions of hYKL-39 in chondrocytes, we overexpressed hYKL-39 in chondrocytic ATDC5 cells. ► hYKL-39 enhanced proliferation and colony formation in ATDC5 cells. ► hYKL-39 increased type II collagen expression in ATDC5 cells treated with chondrogenic medium. -- Abstract: Human YKL39 (chitinase 3-like protein 2/CHI3L2) is a secreted 39 kDa protein produced by articular chondrocytes and synoviocytes. Recent studies showed that hYKL-39 expression is increased in osteoarthritic articular chondrocytes suggesting the involvement of hYKL-39 in the progression of osteoarthritis (OA). However little is known regarding the molecular function of hYKL-39 in joint homeostasis. Sequence analyses indicated that hYKL-39 has significant identity with the human chitotorisidase family molecules, although it is considered that hYKL-39 has no enzymatic activity since it lacks putative chitinase catalytic motif. In this study, to examine the molecular function of hYKL-39 in chondrocytes, we overexpressed hYKL-39 in ATDC5 cells. Here we report that hYKL-39 enhances colony forming activity, cell proliferation, and type II collagen expression in these cells. These data suggest that hYKL-39 is a novel growth and differentiation factor involved in cartilage homeostasis.

  3. Sinorhizobium meliloti-induced chitinase gene expression in Medicago truncatula ecotype R108-1: a comparison between symbiosis-specific class V and defence-related class IV chitinases.

    PubMed

    Salzer, Peter; Feddermann, Nadja; Wiemken, Andres; Boller, Thomas; Staehelin, Christian

    2004-08-01

    The Medicago truncatula (Gaertn.) ecotypes Jemalong A17 and R108-1 differ in Sinorhizobium meliloti-induced chitinase gene expression. The pathogen-inducible class IV chitinase gene, Mtchit 4, was strongly induced during nodule formation of the ecotype Jemalong A17 with the S. meliloti wild-type strain 1021. In the ecotype R108-1, the S. meliloti wild types Sm1021 and Sm41 did not induce Mtchit 4 expression. On the other hand, expression of the putative class V chitinase gene, Mtchit 5, was found in roots of M. truncatula cv. R108-1 nodulated with either of the rhizobial strains. Mtchit 5 expression was specific for interactions with rhizobia. It was not induced in response to fungal pathogen attack, and not induced in roots colonized with arbuscular mycorrhizal (AM) fungi. Elevated Mtchit 5 gene expression was first detectable in roots forming nodule primordia. In contrast to Mtchit 4, expression of Mtchit 5 was stimulated by purified Nod factors. Conversely, Mtchit 4 expression was strongly elevated in nodules formed with the K-antigen-deficient mutant PP699. Expression levels of Mtchit 5 were similarly increased in nodules formed with PP699 and its parental wild-type strain Sm41. Phylogenetic analysis of the deduced amino acid sequences of Mtchit 5 (calculated molecular weight = 41,810 Da, isoelectric point pH 7.7) and Mtchit 4 (calculated molecular weight 30,527 Da, isoelectric point pH 4.9) revealed that the putative Mtchit 5 chitinase forms a separate clade within class V chitinases of plants, whereas the Mtchit 4 chitinase clusters with pathogen-induced class IV chitinases from other plants. These findings demonstrate that: (i) Rhizobium-induced chitinase gene expression in M. truncatula occurs in a plant ecotype-specific manner, (ii) Mtchit 5 is a putative chitinase gene that is specifically induced by rhizobia, and (iii) rhizobia-specific and defence-related chitinase genes are differentially influenced by rhizobial Nod factors and K antigens.

  4. Enhanced resistance to blister blight in transgenic tea (Camellia sinensis [L.] O. Kuntze) by overexpression of class I chitinase gene from potato (Solanum tuberosum).

    PubMed

    Singh, H Ranjit; Deka, Manab; Das, Sudripta

    2015-07-01

    Tea is the second most consumed beverage in the world. A crop loss of up to 43 % has been reported due to blister blight disease of tea caused by a fungus, Exobasidium vexans. Thus, it directly affects the tea industry qualitatively and quantitatively. Solanum tuberosum class I chitinase gene (AF153195) is a plant pathogenesis-related gene. It was introduced into tea genome via Agrobacterium-mediated transformation with hygromycin phosphotransferase (hpt) gene conferring hygromycin resistance as plant selectable marker. A total of 41 hygromycin resistant plantlets were obtained, and PCR analysis established 12 plantlets confirming about the stable integration of transgene in the plant genome. Real-time PCR detected transgene expression in four transgenic plantlets (T28, C57, C9, and T31). Resistance to biotrophic fungal pathogen, E. vexans, was tested by detached leaf infection assay of greenhouse acclimated plantlets. An inhibitory activity against the fungal pathogen was evident from the detached leaves from the transformants compared with the control. Fungal lesion formed on control plantlet whereas the transgenic plantlets showed resistance to inoculated fungal pathogen by the formation of hypersensitivity reaction area. This result suggests that constitutive expression of the potato class I chitinase gene can be exploited to improve resistance to fungal pathogen, E. vexans, in economical perennial plantation crop like tea.

  5. Molecular characterization of plantain class i chitinase gene and its expression in response to infection by Gloeosporium musarum Cke and Massee and other abiotic stimuli.

    PubMed

    Fan, Jianming; Wang, Hongbin; Feng, Dongru; Liu, Bin; Liu, Haiyan; Wang, Jinfa

    2007-11-01

    We have cloned a chitinase cDNA (MpChi-1) from plantain (Musa paradisiacal L) using rapid amplification of cDNA ends (RACE) according to a sequence fragment which we had cloned using the suppression subtractive hybridization (SSH) technique. The MpChi-1 encodes a protein of 326 amino acids and belongs to acidic chitinase class Ib subfamily. MpChi-1 shares high identity with rice endochitinase (XP_468714) and different each other only at three residues. Homology modelling indicated these three substitutions would not change the configuration of the activity site of the enzyme. We have expressed recombinant MpChi-1 and purified by ammonium sulphate precipitation and preparative reversed phase HPLC. The recombinant protein could hydrolyse chitin and inhibit the growth of the Gloeosporium musarum Cke and Massee in vitro. Northern blot revealed that the MpChi-1 transcripts rapidly after inoculation with G. musarum and maximum mRNA accumulation reached at 48 h. Jasmonic acid (JA) and salicylic acid (SA) could induce MpChi-1 expression, while mechanical wounding, silver nitrate and osmotic stress stimulated only a slight accumulation of MpChi-1 transcripts. Abscisic acid (ABA) could induce MpChi-1 transcript. These results suggest the MpChi-1 plays important role in the events of the hypersensitive reaction (HR).

  6. Chitinase-like gene family in the brown planthopper, Nilaparvata lugens.

    PubMed

    Xi, Y; Pan, P-L; Ye, Y-X; Yu, B; Xu, H-J; Zhang, C-X

    2015-02-01

    Chitinases are important enzymes required for chitin degradation and reconstruction in insects. Based on a bioinformatics investigation, we identified 12 genes encoding putative chitinase-like proteins, including 10 chitinases (Cht), one imaginal disc growth factor (IDGF) and one endo-β-N-acetylglucosaminidase (ENGase) in the genome of the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae). These 12 genes were clustered into nine different groups, with 11 in glycoside hydrolase family 18 groups (groups I-VIII) and one in the ENGase group. Developmental and tissue-specific expression pattern analysis revealed that the transcript levels of eight genes peaked periodically during moulting and were mainly expressed in the integument, except NlCht2, NlCht4, NlIDGF and NlENGase. NlCht2, NlIDGF and NlENGase were expressed at all stages with slight periodical changes and mainly expressed in the female reproductive organs in adults, whereas NlCht4 was highly expressed only at the adult stage in the male reproductive organs. Lethal phenotypes were observed in insects challenged by double-stranded RNAs for NlCht1, NlCht5, NlCht7, NlCht9 and NlCht10 during moulting, suggesting their significant roles in old cuticle degradation. NlCht1 was the most sensitive gene, inducing 50% mortality even at 0.01 ng per insect. Our results illustrate the structural and functional differences of chitinase-like family genes and provide potential targets for RNA interference-based rice planthopper management.

  7. Identification of a chitinase-producing bacterium C4 and histopathologic study on locusts.

    PubMed

    Yong, Tao; Zhangfu, Long; Jing, Xie; Hong, Jin; Hongyan, Ran; Ke, Tao; Shaorong, Ge; Kun, Liu; Shigui, Liu

    2005-02-01

    In order to develop the potential of chitinase-producing micro-organisms as biocontrol agents for insect pests, five chitinase-producing bacterial strains (C1, C2, C3, C4 and C5) previously isolated from soil samples were chosen to infect grassland locusts. The data showed that the mortality rate of locusts fed with strain C4 was significantly higher than that of other groups, and its pathogenicity was confirmed by Koch's law. Midgut tissues of locusts infected with C4 were examined with a light microscope. Apparent histopathologic changes in midgut cells partly explained the pathogenesis of locusts. Therefore, strain C4 was considered to be a potential biocontrol agent. To determine the taxonomic position of C4, physiological and biochemical characteristics were determined and molecular identification was performed. The 16S rDNA gene of C4 was amplified, cloned and sequenced. Comparative sequence analysis demonstrated that C4 corresponded to the genera Sanguibacter, Oerskovia and Cellulomonas. On the basis of phenotypic characterization and sequence similarity analysis, strain C4 was more closely related to the genus Sanguibacter. This chitinase-producing strain C4, which closely corresponds to the species of the genus Sanguibacter and is pathogenic to locusts, is here reported for the first time.

  8. Crystal structures of Vibrio harveyi chitinase A complexed with chitooligosaccharides: implications for the catalytic mechanism.

    PubMed

    Songsiriritthigul, Chomphunuch; Pantoom, Supansa; Aguda, Adeleke H; Robinson, Robert C; Suginta, Wipa

    2008-06-01

    This research describes four X-ray structures of Vibrio harveyi chitinase A and its catalytically inactive mutant (E315M) in the presence and absence of substrates. The overall structure of chitinase A is that of a typical family-18 glycosyl hydrolase comprising three distinct domains: (i) the amino-terminal chitin-binding domain; (ii) the main catalytic (alpha/beta)(8) TIM-barrel domain; and (iii) the small (alpha+beta) insertion domain. The catalytic cleft of chitinase A has a long, deep groove, which contains six chitooligosaccharide ring-binding subsites (-4)(-3)(-2)(-1)(+1)(+2). The binding cleft of the ligand-free E315M is partially blocked by the C-terminal (His)(6)-tag. Structures of E315M-chitooligosaccharide complexes display a linear conformation of pentaNAG, but a bent conformation of hexaNAG. Analysis of the final 2F(o)-F(c) omit map of E315M-NAG6 reveals the existence of the linear conformation of the hexaNAG at a lower occupancy with respect to the bent conformation. These crystallographic data provide evidence that the interacting sugars undergo conformational changes prior to hydrolysis by the wild-type enzyme.

  9. Go Fly a Chitin: The Mystery of Chitin and Chitinases in Vertebrate Tissues.

    PubMed

    Stern, Robert

    2017-01-01

    A controversy arose decades ago whether the DG42 gene product expressed during frog embryogenesis synthesized hyaluronan or chitin. Both sets of investigators were correct. It is now possible to understand how prescient those findings were. Synthesis of a seven to nine chitin sugar chain fragment is required before hyaluronan synthesis begins. Thus, DG42 indeed synthesizes both hyaluronan and chitin. Hyaluronan turns over rapidly in vertebrate tissues, but chitin oligomers are difficult to degrade. They accumulate and can cause pathology. Chitin is a simple beta-linked repeating sugar homopolymer found prominently in the building block structures of fungi, molluscs, arthropods, and other forms of invertebrate life. It is a highly resistant insoluble material requiring chitin synthases for production and chitinases for degradation. Mysteriously, chitins and chitinases also occur in vertebrate tissues, while it had previously been assumed that no chitins were contained therein. That assumption is now challenged based on recent biochemical evidence. Chitin does accumulate in many tissues, but may be particularly toxic to neurons. Its accumulation in the brain may account for the cognitive decline found in patients with Alzheimer's disease. The DG42 observations together with the participation of chitins and chitinases in several human diseases, among which in addition to Alzheimer's disease include Gaucher's disease, asthma, and aspects of abnormal immune recognition justify a reexamination of these topics. The purpose of this review is to summarize data in order to place chitins and their attendant enzymes in a rational framework in an attempt to create a cohesive story.

  10. Optimization of Chitinase Production by Bacillus pumilus Using Plackett-Burman Design and Response Surface Methodology

    PubMed Central

    Tasharrofi, Noshin; Adrangi, Sina; Fazeli, Mehdi; Rastegar, Hossein; Khoshayand, Mohammad Reza; Faramarzi, Mohammad Ali

    2011-01-01

    A soil bacterium capable of degrading chitin on chitin agar plates was isolated and identified as Bacillus pumilus isolate U5 on the basis of 16S rDNA sequence analysis. In order to optimize culture conditions for chitinase production by this bacterium, a two step approach was employed. First, the effects of several medium components were studied using the Plackett-Burman design. Among various components tested, chitin and yeast extract showed positive effect on enzyme production while MgSO4 and FeSO4 had negative effect. However, the linear model proved to be insufficient for determining the optimum levels for these components due to a highly significant curvature effect. In the second step, Box-Behnken response surface methodology was used to determine the optimum values. It was noticed that a quadratic polynomial equation fitted he experimental data appropriately. The optimum concentrations for chitin, yeast extract, MgSO4 and FeSO4 were found to be 4.76, 0.439, 0.0055 and 0.019 g/L, respectively, with a predicted value of chitinase production of 97.67 U/100 mL. Using this statistically optimized medium, the practical chitinase production reached 96.1 U/100 mL. PMID:24250411

  11. A class V chitinase from Arabidopsis thaliana: gene responses, enzymatic properties, and crystallographic analysis.

    PubMed

    Ohnuma, Takayuki; Numata, Tomoyuki; Osawa, Takuo; Mizuhara, Mamiko; Lampela, Outi; Juffer, André H; Skriver, Karen; Fukamizo, Tamo

    2011-07-01

    Expression of a class V chitinase gene (At4g19810, AtChiC) in Arabidopsis thaliana was examined by quantitative real-time PCR and by analyzing microarray data available at Genevestigator. The gene expression was induced by the plant stress-related hormones abscisic acid (ABA) and jasmonic acid (JA) and by the stress resulting from the elicitor flagellin, NaCl, and osmosis. The recombinant AtChiC protein was produced in E. coli, purified, and characterized with respect to the structure and function. The recombinant AtChiC hydrolyzed N-acetylglucosamine oligomers producing dimers from the non-reducing end of the substrates. The crystal structure of AtChiC was determined by the molecular replacement method at 2.0 Å resolution. AtChiC was found to adopt an (β/α)(8) fold with a small insertion domain composed of an α-helix and a five-stranded β-sheet. From docking simulation of AtChiC with pentameric substrate, the amino acid residues responsible for substrate binding were found to be well conserved when compared with those of the class V chitinase from Nicotiana tabacum (NtChiV). All of the structural and functional properties of AtChiC are quite similar to those obtained for NtChiV, and seem to be common to class V chitinases from higher plants.

  12. Biochemical characterization of a recombinant plant class III chitinase from the pitcher of the carnivorous plant Nepenthes alata.

    PubMed

    Ishisaki, Kana; Arai, Sachiko; Hamada, Tatsuro; Honda, Yuji

    2012-11-01

    A class III chitinase belonging to the GH18 family from Nepenthes alata (NaCHIT3) was expressed in Escherichia coli. The enzyme exhibited hydrolytic activity toward colloidal chitin, ethylene glycol chitin, and (GlcNAc)(n) (n=5 and 6). The enzyme hydrolyzed the fourth glycosidic linkage from the non-reducing end of (GlcNAc)(6). The anomeric form of the products indicated it was a retaining enzyme. The colloidal chitin hydrolytic reaction displayed high activity between pH 3.9 and 6.9, but the pH optimum of the (GlcNAc)(6) hydrolytic reaction was 3.9 at 37 °C. The optimal temperature for activity was 65 °C in 50 mM sodium acetate buffer (pH 3.9). The pH optima of NaCHIT3 and NaCHIT1 might be related to their roles in chitin degradation in the pitcher fluid.

  13. Molecular, Structural and Immunological Characterization of Der p 18, a Chitinase-Like House Dust Mite Allergen

    PubMed Central

    Resch, Yvonne; Blatt, Katharina; Malkus, Ursula; Fercher, Christian; Swoboda, Ines; Focke-Tejkl, Margit; Chen, Kuan-Wei; Seiberler, Susanne; Mittermann, Irene; Lupinek, Christian; Rodriguez-Dominguez, Azahara; Zieglmayer, Petra; Zieglmayer, René; Keller, Walter; Krzyzanek, Vladislav; Valent, Peter; Valenta, Rudolf; Vrtala, Susanne

    2016-01-01

    Background The house dust mite (HDM) allergen Der p 18 belongs to the glycoside hydrolase family 18 chitinases. The relevance of Der p 18 for house dust mite allergic patients has only been partly investigated. Objective To perform a detailed characterization of Der p 18 on a molecular, structural and immunological level. Methods Der p 18 was expressed in E. coli, purified to homogeneity, tested for chitin-binding activity and its secondary structure was analyzed by circular dichroism. Der p 18-specific IgG antibodies were produced in rabbits to localize the allergen in mites using immunogold electron microscopy and to search for cross-reactive allergens in other allergen sources (i.e. mites, crustacea, mollusca and insects). IgE reactivity of rDer p 18 was tested with sera from clinically well characterized HDM-allergic patients (n = 98) and its allergenic activity was analyzed in basophil activation experiments. Results Recombinant Der p 18 was expressed and purified as a folded, biologically active protein. It shows weak chitin-binding activity and partial cross-reactivity with Der f 18 from D. farinae but not with proteins from the other tested allergen sources. The allergen was mainly localized in the peritrophic matrix of the HDM gut and to a lower extent in fecal pellets. Der p 18 reacted with IgE from 10% of mite allergic patients from Austria and showed allergenic activity when tested for basophil activation in Der p 18-sensitized patients. Conclusion Der p 18 is a rather genus-specific minor allergen with weak chitin-binding activity but exhibits allergenic activity and therefore should be included in diagnostic test panels for HDM allergy. PMID:27548813

  14. Development of insect resistant maize plants expressing a chitinase gene from the cotton leaf worm, Spodoptera littoralis.

    PubMed

    Osman, Gamal H; Assem, Shireen K; Alreedy, Rasha M; El-Ghareeb, Doaa K; Basry, Mahmoud A; Rastogi, Anshu; Kalaji, Hazem M

    2015-12-14

    Due to the importance of chitinolytic enzymes for insect, nematode and fungal growth, they are receiving attention concerning their development as biopesticides or chemical defense proteins in transgenic plants and as microbial biocontrol agents. Targeting chitin associated with the extracellular matrices or cell wall by insect chitinases may be an effective approach for controlling pest insects and pathogenic fungi. The ability of chitinases to attack and digest chitin in the peritrophic matrix or exoskeleton raises the possibility to use them as insect control method. In this study, an insect chitinase cDNA from cotton leaf worm (Spodoptera littoralis) has been synthesized. Transgenic maize plant system was used to improve its tolerance against insects. Insect chitinase transcripts and proteins were expressed in transgenic maize plants. The functional integrity and expression of chitinase in progenies of the transgenic plants were confirmed by insect bioassays. The bioassays using transgenic corn plants against corn borer (Sesamia cretica) revealed that ~50% of the insects reared on transgenic corn plants died, suggesting that transgenic maize plants have enhanced resistance against S. cretica.

  15. Antifungal performance of extracellular chitinases and culture supernatants of Streptomyces galilaeus CFFSUR-B12 against Mycosphaerella fijiensis Morelet.

    PubMed

    Castillo, Benjamín Moreno; Dunn, Michael F; Navarro, Karina Guillén; Meléndez, Francisco Holguín; Ortiz, Magdalena Hernández; Guevara, Sergio Encarnación; Palacios, Graciela Huerta

    2016-03-01

    The tropical and mycoparasite strain Streptomyces galilaeus CFFSUR-B12 was evaluated as an antagonist of Mycosphaerella fijiensis Morelet, causal agent of the Black Sigatoka Disease (BSD) of banana. On zymograms of CFFSUR-B12 culture supernatants, we detected four chitinases of approximately 32 kDa (Chi32), 20 kDa (Chi20), and two with masses well over 170 kDa (ChiU) that showed little migration during denaturing electrophoresis at different concentrations of polyacrylamide. The thymol-sulphuric acid assay showed that the ChiU were glycosylated chitinases. Moreover, matrix assisted laser desorption ionization time-of-flight MS analysis revealed that the ChiU are the same protein and identical to a family 18 chitinase from Streptomyces sp. S4 (gi|498328075). Chi32 was similar to an extracellular protein from Streptomyces albus J1074 (gi|478687481) and Chi20 was non-significantly similar to chitinases from five different strains of Streptomyces (P > 0.05). Subsequently, Chi32 and Chi20 were partially purified by anion exchange and hydrophobic interaction chromatography and tested against M. fijiensis. Chitinases failed to inhibit ascospore germination, but inhibited up to 35 and 62% of germ tube elongation and mycelial growth, respectively. We found that crude culture supernatant and living cells of S. galilaeus CFFSUR-B12 were the most effective in inhibiting M. fijiensis and are potential biocontrol agents of BSD.

  16. Development of insect resistant maize plants expressing a chitinase gene from the cotton leaf worm, Spodoptera littoralis

    PubMed Central

    Osman, Gamal H.; Assem, Shireen K.; Alreedy, Rasha M.; El-Ghareeb, Doaa K.; Basry, Mahmoud A.; Rastogi, Anshu; Kalaji, Hazem M.

    2015-01-01

    Due to the importance of chitinolytic enzymes for insect, nematode and fungal growth, they are receiving attention concerning their development as biopesticides or chemical defense proteins in transgenic plants and as microbial biocontrol agents. Targeting chitin associated with the extracellular matrices or cell wall by insect chitinases may be an effective approach for controlling pest insects and pathogenic fungi. The ability of chitinases to attack and digest chitin in the peritrophic matrix or exoskeleton raises the possibility to use them as insect control method. In this study, an insect chitinase cDNA from cotton leaf worm (Spodoptera littoralis) has been synthesized. Transgenic maize plant system was used to improve its tolerance against insects. Insect chitinase transcripts and proteins were expressed in transgenic maize plants. The functional integrity and expression of chitinase in progenies of the transgenic plants were confirmed by insect bioassays. The bioassays using transgenic corn plants against corn borer (Sesamia cretica) revealed that ~50% of the insects reared on transgenic corn plants died, suggesting that transgenic maize plants have enhanced resistance against S. cretica. PMID:26658494

  17. Cloning and identification of Fv-cmp, a protease from Fusarium verticillioides that truncates Zea mays and Arabidopsis thaliana class IV chitinases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chitinase modifying proteins (cmps) are proteases, secreted by fungal pathogens, that were originally identified as proteins that truncate class IV chitinases of maize during ear rot. Cmps from Bipolaris zeicola and Stenocarpella maydis have been characterized, but the identities of the proteases h...

  18. Chitinase is stored and secreted from the inner body of microfilariae and has a role in exsheathment in the parasitic nematode Brugia malayi

    PubMed Central

    Wu, Yang; Preston, Gillian; Bianco, Albert E.

    2008-01-01

    Chitinase expression in microfilariae of the parasitic nematode Brugia malayi (B. malayi, Bm) is coincidental with the onset of their infectivity to mosquitoes. An antibody raised to Onchocerca volvulus (O. volvulus, Ov) infective-stage larval chitinase (Ov-CHI-1) was specifically reactive against B. malayi microfilarial chitinase and was used to study the localization of chitinase in B. malayi during microfilarial development and transmission to the insect vector. Immuno-electron microscopy (IEM) was used to demonstrate that the chitinase was confined to the inner body of the microfilariae and furthermore that chitinase was only present in sheathed microfilarial species, although the inner body is present in all species. Observation using the IEM implicates two distinct routes of chitinase secretion from the inner body, via either the pharyngeal thread, or during transmission of the microfilariae to the vector, contained in vesicle-like structures. Many morphological studies have described the structure of the inner body, but no function has been assigned to it as of yet. Although it has been commented that the cells surrounding the inner body and pharyngeal thread are those destined to become the intestine and pharynx and that the inner body represents a store of material. Our studies suggest that chitinase is one such product stored in the inner body and that it is secreted during the exsheathment of the microfilaria in the mosquito. PMID:18611418

  19. High-capacity calcium-binding chitinase III from pomegranate seeds (Punica granatum Linn.) is located in amyloplasts.

    PubMed

    Lv, Chenyan; Masuda, Taro; Yang, Haixia; Sun, Lei; Zhao, Guanghua

    2011-12-01

    We have recently identified a new class III chitinase from pomegranate seeds (PSC). Interestingly, this new chitinase naturally binds calcium ions with high capacity and low affinity, suggesting that PSC is a Ca-storage protein. Analysis of the amino acid sequence showed that this enzyme is rich in acidic amino acid residues, especially Asp, which are responsible for calcium binding. Different from other known chitinases, PSC is located in the stroma of amyloplasts in pomegranate seeds. Transmission electron microscopy (TEM) analysis indicated that the embryonic cells of pomegranate seeds are rich in calcium ions, most of which are distributed in the stroma and the starch granule of the amyloplasts, consistent with the above idea that PSC is involved in calcium storage, a newly non-defensive function.

  20. Crystal Structure of Chitinase ChiW from Paenibacillus sp. str. FPU-7 Reveals a Novel Type of Bacterial Cell-Surface-Expressed Multi-Modular Enzyme Machinery

    PubMed Central

    Itoh, Takafumi; Hibi, Takao; Suzuki, Fumiko; Sugimoto, Ikumi; Fujiwara, Akihiro; Inaka, Koji; Tanaka, Hiroaki; Ohta, Kazunori; Fujii, Yutaka; Taketo, Akira; Kimoto, Hisashi

    2016-01-01

    The Gram-positive bacterium Paenibacillus sp. str. FPU-7 effectively hydrolyzes chitin by using a number of chitinases. A unique chitinase with two catalytic domains, ChiW, is expressed on the cell surface of this bacterium and has high activity towards various chitins, even crystalline chitin. Here, the crystal structure of ChiW at 2.1 Å resolution is presented and describes how the enzyme degrades chitin on the bacterial cell surface. The crystal structure revealed a unique multi-modular architecture composed of six domains to function efficiently on the cell surface: a right-handed β-helix domain (carbohydrate-binding module family 54, CBM-54), a Gly-Ser-rich loop, 1st immunoglobulin-like (Ig-like) fold domain, 1st β/α-barrel catalytic domain (glycoside hydrolase family 18, GH-18), 2nd Ig-like fold domain and 2nd β/α-barrel catalytic domain (GH-18). The structure of the CBM-54, flexibly linked to the catalytic region of ChiW, is described here for the first time. It is similar to those of carbohydrate lyases but displayed no detectable carbohydrate degradation activities. The CBM-54 of ChiW bound to cell wall polysaccharides, such as chin, chitosan, β-1,3-glucan, xylan and cellulose. The structural and biochemical data obtained here also indicated that the enzyme has deep and short active site clefts with endo-acting character. The affinity of CBM-54 towards cell wall polysaccharides and the degradation pattern of the catalytic domains may help to efficiently decompose the cell wall chitin through the contact surface. Furthermore, we clarify that other Gram-positive bacteria possess similar cell-surface-expressed multi-modular enzymes for cell wall polysaccharide degradation. PMID:27907169

  1. Cloning, sequencing, and expression of the gene encoding Clostridium paraputrificum chitinase ChiB and analysis of the functions of novel cadherin-like domains and a chitin-binding domain.

    PubMed Central

    Morimoto, K; Karita, S; Kimura, T; Sakka, K; Ohmiya, K

    1997-01-01

    The Clostridium paraputrificum chiB gene, encoding chitinase B (ChiB), consists of an open reading frame of 2,493 nucleotides and encodes 831 amino acids with a deduced molecular weight of 90,020. The deduced ChiB is a modular enzyme composed of a family 18 catalytic domain responsible for chitinase activity, two reiterated domains of unknown function, and a chitin-binding domain (CBD). The reiterated domains are similar to the repeating units of cadherin proteins but not to fibronectin type III domains, and therefore they are referred to as cadherin-like domains. ChiB was purified from the periplasm fraction of Escherichia coli harboring the chiB gene. The molecular weight of the purified ChiB (87,000) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, was in good agreement with the value (86,578) calculated from the deduced amino acid sequence excluding the signal peptide. ChiB was active toward chitin from crab shells, colloidal chitin, glycol chitin, and 4-methylumbelliferyl beta-D-N,N'-diacetylchitobioside [4-MU-(GlcNAc)2]. The pH and temperature optima of the enzyme were 6.0 and 45 degrees C, respectively. The Km and Vmax values for 4-MU-(GlcNAc)2 were estimated to be 6.3 microM and 46 micromol/min/mg, respectively. SDS-PAGE, zymogram, and Western blot analyses using antiserum raised against purified ChiB suggested that ChiB was one of the major chitinase species in the culture supernatant of C. paraputrificum. Deletion analysis showed clearly that the CBD of ChiB plays an important role in hydrolysis of native chitin but not processed chitin such as colloidal chitin. PMID:9393694

  2. Biochemical characterization of chitinase 2 expressed during the autolytic phase of the inky cap, Coprinellus congregatus.

    PubMed

    Kang, Yuri; Kim, Hyewon; Choi, Hyoung T

    2013-04-01

    Fungal cell walls consist of various glucans and chitin. The inky cap, Coprinellus congregatus, produces mushrooms at 25°C in a regime of 15 h light/9 h dark, and then the mushroom is autolyzed rapidly to generate black liquid droplets in which no cell walls are detected by microscopy. Chitinase cDNA from the mature mushroom tissues of C. congregatus, which consisted of 1,622 nucleotides (chi2), was successfully cloned using the rapid amplification of cDNA ends polymerase chain reaction technique. The deduced 498 amino acid sequence of Chi2 had a conserved catalytic domain as in other fungal chitinase family 18 enzymes. The Chi2 enzyme was purified from the Pichia pastoris expression system, and its estimated molecular weight was 68 kDa. The optimum pH and temperature of Chi2 was pH 4.0 and 35°C, respectively when 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside was used as the substrate. The K m value and V max for the substrate A, 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside, was 0.175 mM and 0.16 OD min(-1)unit(-1), respectively.

  3. Characterization of cis-acting elements residing in the chitinase promoter of Bacillus pumilus SG2.

    PubMed

    Heravi, K Morabbi; Shali, A; Naghibzadeh, N; Ahmadian, G

    2014-05-01

    Bacillus pumilus SG2 is a chitinolytic bacterium that produces two chitinases, namely ChiS and ChiL. The chiS and chiL genes are consecutively expressed under a common promoter. Regulation of the chiS and chiL genes is under the control of carbon catabolite repression (CCR) in B. pumilus. This study aimed to investigate the cis-acting elements of the chitinase promoter. For this purpose, we transferred the chiS gene along with its specific promoter to Bacillus subtilis as a host. Primer extension analysis revealed two transcription start sites located 287 and 65 bp upstream of the chiS start codon. The distal promoter was highly compatible with the consensus sequence of the σ(A)-type promoters in B. subtilis, whereas the proximal promoter sequence showed less similarity to the σ(A)-type consensus sequence. A catabolite responsive element (cre), which is required for CCR in Bacillus species, was found to be 136 to 123 bp upstream of the chiS start codon. Interestingly, this cre site was located upstream of the -35 of the proximal promoter and downstream of the distal promoter. Deletion of this cre site sequence rendered the chiS expression constitutive.

  4. Role of Chitinase 3-Like-1 in Interleukin-18-Induced Pulmonary Type 1, Type 2, and Type 17 Inflammation; Alveolar Destruction; and Airway Fibrosis in the Murine Lung.

    PubMed

    Kang, Min-Jong; Yoon, Chang Min; Nam, Milang; Kim, Do-Hyun; Choi, Je-Min; Lee, Chun Geun; Elias, Jack A

    2015-12-01

    Chitinase 3-like 1 (Chi3l1), which is also called YKL-40 in humans and BRP-39 in mice, is the prototypic chitinase-like protein. Recent studies have highlighted its impressive ability to regulate the nature of tissue inflammation and the magnitude of tissue injury and fibroproliferative repair. This can be appreciated in studies that highlight its induction after cigarette smoke exposure, during which it inhibits alveolar destruction and the genesis of pulmonary emphysema. IL-18 is also known to be induced and activated by cigarette smoke, and, in murine models, the IL-18 pathway has been shown to be necessary and sufficient to generate chronic obstructive pulmonary disease-like inflammation, fibrosis, and tissue destruction. However, the relationship between Chi3l1 and IL-18 has not been defined. To address this issue we characterized the expression of Chi3l1/BRP-39 in control and lung-targeted IL-18 transgenic mice. We also characterized the effects of transgenic IL-18 in mice with wild-type and null Chi3l1 loci. The former studies demonstrated that IL-18 is a potent stimulator of Chi3l1/BRP-39 and that this stimulation is mediated via IFN-γ-, IL-13-, and IL-17A-dependent mechanisms. The latter studies demonstrated that, in the absence of Chi3l1/BRP-39, IL-18 induced type 2 and type 17 inflammation and fibrotic airway remodeling were significantly ameliorated, whereas type 1 inflammation, emphysematous alveolar destruction, and the expression of cytotoxic T lymphocyte perforin, granzyme, and retinoic acid early transcript 1 expression were enhanced. These studies demonstrate that IL-18 is a potent stimulator of Chi3l1 and that Chi3l1 is an important mediator of IL-18-induced inflammatory, fibrotic, alveolar remodeling, and cytotoxic responses.

  5. High-yield production of a chitinase from Aeromonas veronii B565 as a potential feed supplement for warm-water aquaculture.

    PubMed

    Zhang, Yuting; Zhou, Zhigang; Liu, Yuchun; Cao, Yanan; He, Suxu; Huo, Fengmin; Qin, Chubin; Yao, Bin; Ringø, Einar

    2014-02-01

    Chitin, present in crustacean shells, insects, and fungi, is the second most plentiful natural organic fiber after wood. To effectively use chitin in a cost-saving and environmentally friendly way in aquaculture, crustacean shells (e.g., shrimp-shell meal) are supplemented into aquafeed after degradation by chemical methods. Herein, we describe a chitinase from Aeromonas veronii B565, designated ChiB565, which potently degrades shrimp-shell chitin and resists proteolysis. We isolated recombinant ChiB565 of the expected molecular mass in large yield from Pichia pastoris. ChiB565 is optimally active at pH 5.0 and 50 °C and stable between pH 4.5 and 9.0 at 50 °C and below. Compared with the commercial chitinase C-6137, which cannot degrade shrimp-shell chitin, ChiB565 hydrolyzes shrimp-shell chitin in addition to colloidal chitin, powdered chitin, and β-1,3-1,4-glucan. The optimal enzyme concentration and reaction time for in vitro degradation of 0.1 g of powdered shrimp shell are 30 U of ChiB565 and 3 h, respectively. A synergistic protein-release effect occurred when ChiB565 and trypsin were incubated in vitro with shrimp shells. Tilapia were fed an experimental diet containing 5% (w/w) shrimp bran and 16.2 U/kg ChiB565, which significantly improved growth and feed conversion compared with a control diet lacking ChiB565. Dietary ChiB565 enhanced nitrogen digestibility and downregulated intestinal IL-1β expression. The immunologically relevant protective effects of dietary ChiB565 were also observed for 2 to 3 days following exposure to pathogenic Aeromonas hydrophila.

  6. Application of DNA Bar Codes for Screening of Industrially Important Fungi: the Haplotype of Trichoderma harzianum Sensu Stricto Indicates Superior Chitinase Formation▿

    PubMed Central

    Nagy, Viviana; Seidl, Verena; Szakacs, George; Komoń-Zelazowska, Monika; Kubicek, Christian P.; Druzhinina, Irina S.

    2007-01-01

    Selection of suitable strains for biotechnological purposes is frequently a random process supported by high-throughput methods. Using chitinase production by Hypocrea lixii/Trichoderma harzianum as a model, we tested whether fungal strains with superior enzyme formation may be diagnosed by DNA bar codes. We analyzed sequences of two phylogenetic marker loci, internal transcribed spacer 1 (ITS1) and ITS2 of the rRNA-encoding gene cluster and the large intron of the elongation factor 1-alpha gene, tef1, from 50 isolates of H. lixii/T. harzianum, which were also tested to determine their ability to produce chitinases in solid-state fermentation (SSF). Statistically supported superior chitinase production was obtained for strains carrying one of the observed ITS1 and ITS2 and tef1 alleles corresponding to an allele of T. harzianum type strain CBS 226.95. A tef1-based DNA bar code tool, TrichoCHIT, for rapid identification of these strains was developed. The geographic origin of the strains was irrelevant for chitinase production. The improved chitinase production by strains containing this haplotype was not due to better growth on N-acetyl-β-d-glucosamine or glucosamine. Isoenzyme electrophoresis showed that neither the isoenzyme profile of N-acetyl-β-glucosaminidases or the endochitinases nor the intensity of staining of individual chitinase bands correlated with total chitinase in the culture filtrate. The superior chitinase producers did not exhibit similarly increased cellulase formation. Biolog Phenotype MicroArray analysis identified lack of N-acetyl-β-d-mannosamine utilization as a specific trait of strains with the chitinase-overproducing haplotype. This observation was used to develop a plate screening assay for rapid microbiological identification of the strains. The data illustrate that desired industrial properties may be an attribute of certain populations within a species, and screening procedures should thus include a balanced mixture of all

  7. Crystal structures of chitin binding domains of chitinase from Thermococcus kodakarensis KOD1.

    PubMed

    Hanazono, Yuya; Takeda, Kazuki; Niwa, Satomi; Hibi, Masahito; Takahashi, Naoya; Kanai, Tamotsu; Atomi, Haruyuki; Miki, Kunio

    2016-01-01

    Chitinase from T. kodakarensis (TkChiA) catalyzes the hydrolysis of chitin. The enzyme consists of two catalytic and three binding domains (ChBD1, ChBD2 and ChBD3). ChBD2 and ChBD3 can bind to not only chitin but also cellulose. In both domains, the intervals of the side chains of the three tryptophan residues, which are located on the molecular surface, correspond to twice the length of the lattice of the chitin. A binding model with crystalline chitin implies that the tryptophan residues and a glutamate residue interact with the hexose ring by CH-π interactions and the amide group by a hydrogen bond, respectively.

  8. Backbone chemical shifts assignments, secondary structure, and ligand binding of a family GH-19 chitinase from moss, Bryum coronatum.

    PubMed

    Shinya, Shoko; Nagata, Takuya; Ohnuma, Takayuki; Taira, Toki; Nishimura, Shigenori; Fukamizo, Tamo

    2012-10-01

    Family GH19 chitinases have been recognized as important in the plant defense against fungal pathogens. However, their substrate-recognition mechanism is still unknown. We report here the first resonance assignment of NMR spectrum of a GH19 chitinase from moss, Bryum coronatum (BcChi-A). The backbone signals were nearly completely assigned, and the secondary structure was estimated based on the chemical shift values. The addition of the chitin dimer to the enzyme solution perturbed the chemical shifts of HSQC resonances of the amino acid residues forming the putative substrate-binding cleft. Further NMR analysis of the ligand binding to BcChi-A will improve understanding of the substrate-recognition mechanism of GH-19 enzymes.

  9. Molecular Analysis of Atypical Family 18 Chitinase from Fujian Oyster Crassostrea angulata and Its Physiological Role in the Digestive System.

    PubMed

    Yang, Bingye; Zhang, Mingming; Li, Lingling; Pu, Fei; You, Weiwei; Ke, Caihuan

    2015-01-01

    Chitinolytic enzymes have an important physiological significance in immune and digestive systems in plants and animals, but chitinase has not been identified as having a role in the digestive system in molluscan. In our study, a novel chitinase homologue, named Ca-Chit, has been cloned and characterized as the oyster Crassostrea angulate. The 3998bp full-length cDNA of Ca-Chit consisted of 23bp 5-UTR, 3288 ORF and 688bp 3-UTR. The deduced amino acids sequence shares homologue with the chitinase of family 18. The molecular weight of the protein was predicted to be 119.389 kDa, with a pI of 6.74. The Ca-Chit protein was a modular enzyme composed of a glycosyl hydrolase family 18 domain, threonine-rich region profile and a putative membrane anchor domain. Gene expression profiles monitored by quantitative RT-PCR in different adult tissues showed that the mRNA of Ca-Chit expressed markedly higher visceral mass than any other tissues. The results of the whole mount in-situ hybridization displayed that Ca-Chit starts to express the visceral mass of D-veliger larvae and then the digestive gland forms a crystalline structure during larval development. Furthermore, the adult oysters challenged by starvation indicated that the Ca-Chit expression would be regulated by feed. All the observations made suggest that Ca-Chit plays an important role in the digestive system of the oyster, Crassostrea angulate.

  10. Expression of Rice Chitinase Gene in Genetically Engineered Tomato Confers Enhanced Resistance to Fusarium Wilt and Early Blight

    PubMed Central

    Jabeen, Nyla; Chaudhary, Zubeda; Gulfraz, Muhammad; Rashid, Hamid; Mirza, Bushra

    2015-01-01

    This is the first study reporting the evaluation of transgenic lines of tomato harboring rice chitinase (RCG3) gene for resistance to two important fungal pathogens Fusarium oxysporum f. sp. lycopersici (Fol) causing fusarium wilt and Alternaria solani causing early blight (EB). In this study, three transgenic lines TL1, TL2 and TL3 of tomato Solanum lycopersicum Mill. cv. Riogrande genetically engineered with rice chitinase (RCG 3) gene and their R1 progeny was tested for resistance to Fol by root dip method and A. solani by detached leaf assay. All the R0 transgenic lines were highly resistant to these fungal pathogens compared to non-transgenic control plants. The pattern of segregation of three independent transformant for Fol and A. solani was also studied. Mendelian segregation was observed in transgenic lines 2 and 3 while it was not observed in transgenic line 1. It was concluded that introduction of chitinase gene in susceptible cultivar of tomato not only enhanced the resistance but was stably inherited in transgenic lines 2 and 3. PMID:26361473

  11. Genome-wide analysis of chitinase genes and their varied functions in larval moult, pupation and eclosion in the rice striped stem borer, Chilo suppressalis.

    PubMed

    Su, C; Tu, G; Huang, S; Yang, Q; Shahzad, M F; Li, F

    2016-08-01

    Some insect chitinases are required to degrade chitin and ensure successful metamorphosis. Although chitinase genes have been well characterized in several model insects, no reports exist for the rice striped stem borer, Chilo suppressalis, a highly destructive pest that causes huge yield losses in rice production. Here, we conducted a genome-level analysis of chitinase genes in C. suppressalis. After amplification of full-length transcripts with rapid amplification of cDNA ends, we identified 12 chitinase genes in C. suppressalis. All these genes had the conserved domains and motifs of glycoside hydrolase family 18 and grouped phylogenetically into five subgroups. C. suppressalis chitinase 1 (CsCht1) was highly expressed in late pupae, whereas CsCht3 was abundant in early pupae. Both CsCht2 and CsCht4 were highly expressed in larvae. CsCht2 was abundant specifically in the third-instar larvae and CsCht4 showed periodic high expression in 2- to 5-day-old larvae in each instar. Tissue specific expression analysis indicated that CsCht1 and CsCht3 were highly expressed in epidermis whereas CsCht2 and CsCht4 were specifically abundant in the midgut. Knockdown of CsCht1 resulted in adults with curled wings, indicating that CsCht1 might have an important role in wing expansion. Silencing of CsCht2 or CsCht4 arrested moulting, suggesting essential roles in larval development. When the expression of CsCht3 was interfered, defects in pupation occurred. Overall, we provide here the first catalogue of chitinase genes in the rice striped stem borer and have elucidated the functions of four chitinases in metamorphosis.

  12. Molecular cloning of class III chitinase gene from Avicennia marina and its expression analysis in response to cadmium and lead stress.

    PubMed

    Wang, Li-Ying; Wang, You-Shao; Zhang, Jing-Ping; Gu, Ji-Dong

    2015-10-01

    Mangrove species have high tolerance to heavy metal pollution. Chitinases have been widely reported as defense proteins in response to heavy metal stress in terrestrial plants. In this study, a full-length cDNA sequence encoding an acidic and basic class III chitinase (AmCHI III) was cloned by using RT-PCR and RACE methods in Avicennia marina. AmCHI III mRNA expression in leaf of A. marina were investigated under Cd, Pb stresses on using real-time quantitative PCR. The deduced AmCHI III protein consists of 302 amino acids, including a signal putative peptide region, and a catalytic domain. Homology modeling of the catalytic domain revealed a typical molecular structure of class III plant chitinases. Results further demonstrated that the regulation of AmCHI III mRNA expression in leaves was strongly dependent on Cd, Pb stresses. AmCHI III mRNA expressions were significantly increased in response to Cd, Pb, and peaked at 7 days Cd-exposure, 7 days Pb-exposure, respectively. AmCHI III mRNA expression exhibited more sensitive to Pb stress than Cd stress. This work was the first time cloing chitinase from A. marina, and it brought evidence on chitinase gene involving in heavy metals (Cd(2+) and Pb(2+)) resistance or detoxification in plants. Further studies including the promoter and upstream regulation, gene over-expression and the response of mangrove chitinases to other stresses will shed more light on the role of chitinase in mangrove plants.

  13. Purification and characterization of a 56 kDa chitinase isozyme (PaChiB) from the stomach of the silver croaker, Pennahia argentatus.

    PubMed

    Ikeda, Mana; Miyauchi, Kouji; Matsumiya, Masahiro

    2012-01-01

    A 56 kDa chitinase isozyme (PaChiB) was purified from the stomach of the silver croaker Pennahia argentatus. The optimum pH and pH stability of PaChiB were observed in an acidic pH range. When N-acetylchitooligosaccharides ((GlcNAc)n, n=2 -6) were used as substrates, PaChiB degraded (GlcNAc)4 -6 and produced (GlcNAc)2,3. It degraded (GlcNAc)5 to produce (GlcNAc)2 (23.2%) and (GlcNAc)3 (76.8%). The ability to degrade p-nitrophenyl N-acetylchitooligosaccharides (pNp-(GlcNAc)n, n=2 -4) fell in the following order: pNp-(GlcNAc)3≫ pNp-(GlcNAc)2 pNp-(GlcNAc)4. Based on these results, we concluded that PaChiB is an endo-type chitinolytic enzyme, and that it preferentially hydrolyzes the third glycosidic bond from the non-reducing end of (GlcNAc)n. Activity toward crystalline α- and β-chitin was activated at 124%-185% in the presence of 0.5 M NaCl. PaChiB exhibited markedly high substrate specificity toward crab-shell α-chitin.

  14. Two-way traffic of glycoside hydrolase family 18 processive chitinases on crystalline chitin

    NASA Astrophysics Data System (ADS)

    Igarashi, Kiyohiko; Uchihashi, Takayuki; Uchiyama, Taku; Sugimoto, Hayuki; Wada, Masahisa; Suzuki, Kazushi; Sakuda, Shohei; Ando, Toshio; Watanabe, Takeshi; Samejima, Masahiro

    2014-06-01

    Processivity refers to the ability of synthesizing, modifying and degrading enzymes to catalyse multiple successive cycles of reaction with polymeric substrates without disengaging from the substrates. Since biomass polysaccharides, such as chitin and cellulose, often form recalcitrant crystalline regions, their degradation is highly dependent on the processivity of degrading enzymes. Here we employ high-speed atomic force microscopy to directly visualize the movement of two processive glycoside hydrolase family 18 chitinases (ChiA and ChiB) from the chitinolytic bacterium Serratia marcescens on crystalline β-chitin. The half-life of processive movement and the velocity of ChiA are larger than those of ChiB, suggesting that asymmetric subsite architecture determines both the direction and the magnitude of processive degradation of crystalline polysaccharides. The directions of processive movements of ChiA and ChiB are observed to be opposite. The molecular mechanism of the two-way traffic is discussed, including a comparison with the processive cellobiohydrolases of the cellulolytic system.

  15. Production of hexaoligochitin from colloidal chitin using a chitinase from Aeromonas schubertii.

    PubMed

    Liu, Chao-Lin; Lan, Chih-Yu; Fu, Chuan-Chieh; Juang, Ruey-Shin

    2014-08-01

    Chitin derivatives, such as those with modified main saccharide chains and deacetylated side chains, exhibit versatile biological functions. The biomedical properties of chitin oligosaccharides depend on their degree of oligomerization. Of the chitin oligosaccharides, chitin hexamers are generally the most potent. In our recent study, N-acetylchitohexaose was obtained by digesting chitin with ASCHI61, a chitinase from Aeromonas schubertii. In this work, the factors involved in the production of chitin hexasaccharide were evaluated experimentally. Using steep map analysis and cross-analysis, the substrate concentration and reaction pH were identified as the key factors in this reaction, and the interactions between these parameters were observed. Using a response surface experimental design, we predicted that a colloidal chitin concentration of 3.4mgmL(-1) and a pH of 6.54 were the optimal conditions for producing hexaoligochitin. These conditions were verified in separate experiments, in which 38.73mmolL(-1) of N-acetylchitohexaose was obtained. The maximum amount of hexamer produced was 42.175mgL(-1), an increase of only 0.27% from the predicted value.

  16. Chitinases and Imaginal disc growth factors organize the extracellular matrix formation at barrier tissues in insects

    PubMed Central

    Pesch, Yanina-Yasmin; Riedel, Dietmar; Patil, Kapil R; Loch, Gerrit; Behr, Matthias

    2016-01-01

    The cuticle forms an apical extracellular-matrix (ECM) that covers exposed organs, such as epidermis, trachea and gut, for organizing morphogenesis and protection of insects. Recently, we reported that cuticle proteins and chitin are involved in ECM formation. However, molecular mechanisms that control assembly, maturation and replacement of the ECM and its components are not well known. Here we investigated the poorly described glyco-18-domain hydrolase family in Drosophila and identified the Chitinases (Chts) and imaginal-disc-growth-factors (Idgfs) that are essential for larval and adult molting. We demonstrate that Cht and idgf depletion results in deformed cuticles, larval and adult molting defects, and insufficient protection against wounding and bacterial infection, which altogether leads to early lethality. We show that Cht2/Cht5/Cht7/Cht9/Cht12 and idgf1/idgf3/idgf4/idgf5/idgf6 are needed for organizing proteins and chitin-matrix at the apical cell surface. Our data indicate that normal ECM formation requires Chts, which potentially hydrolyze chitin-polymers. We further suggest that the non-enzymatic idgfs act as structural proteins to maintain the ECM scaffold against chitinolytic degradation. Conservation of Chts and Idgfs proposes analogous roles in ECM dynamics across the insect taxa, indicating that Chts/Idgfs are new targets for species specific pest control. PMID:26838602

  17. Chitinases and Imaginal disc growth factors organize the extracellular matrix formation at barrier tissues in insects.

    PubMed

    Pesch, Yanina-Yasmin; Riedel, Dietmar; Patil, Kapil R; Loch, Gerrit; Behr, Matthias

    2016-02-03

    The cuticle forms an apical extracellular-matrix (ECM) that covers exposed organs, such as epidermis, trachea and gut, for organizing morphogenesis and protection of insects. Recently, we reported that cuticle proteins and chitin are involved in ECM formation. However, molecular mechanisms that control assembly, maturation and replacement of the ECM and its components are not well known. Here we investigated the poorly described glyco-18-domain hydrolase family in Drosophila and identified the Chitinases (Chts) and imaginal-disc-growth-factors (Idgfs) that are essential for larval and adult molting. We demonstrate that Cht and idgf depletion results in deformed cuticles, larval and adult molting defects, and insufficient protection against wounding and bacterial infection, which altogether leads to early lethality. We show that Cht2/Cht5/Cht7/Cht9/Cht12 and idgf1/idgf3/idgf4/idgf5/idgf6 are needed for organizing proteins and chitin-matrix at the apical cell surface. Our data indicate that normal ECM formation requires Chts, which potentially hydrolyze chitin-polymers. We further suggest that the non-enzymatic idgfs act as structural proteins to maintain the ECM scaffold against chitinolytic degradation. Conservation of Chts and Idgfs proposes analogous roles in ECM dynamics across the insect taxa, indicating that Chts/Idgfs are new targets for species specific pest control.

  18. Amylase and chitinase genes in Streptomyces lividans are regulated by reg1, a pleiotropic regulatory gene.

    PubMed Central

    Nguyen, J; Francou, F; Virolle, M J; Guérineau, M

    1997-01-01

    A regulatory gene, reg1, was identified in Streptomyces lividans. It encodes a 345-amino-acid protein (Reg1) which contains a helix-turn-helix DNA-binding motif in the N-terminal region. Reg1 exhibits similarity with the LacI/GalR family members over the entire sequence. It displays 95% identity with MalR (the repressor of malE in S. coelicolor), 65% identity with ORF-Sl (a putative regulatory gene of alpha-amylase of S. limosus), and 31% identity with CcpA (the carbon catabolite repressor in Bacillus subtilis). In S. lividans, the chromosomal disruption of reg1 affected the expression of several genes. The production of alpha-amylases of S. lividans and that of the alpha-amylase of S. limosus in S. lividans were enhanced in the reg1 mutant strains and relieved of carbon catabolite repression. As a result, the transcription level of the alpha-amylase of S. limosus was noticeably increased in the reg1 mutant strain. Moreover, the induction of chitinase production in S. lividans was relieved of carbon catabolite repression by glucose in the reg1 mutant strain, while the induction by chitin was lost. Therefore, reg1 can be regarded as a pleiotropic regulatory gene in S. lividans. PMID:9335287

  19. Drosophila Chitinase 2 is expressed in chitin producing organs for cuticle formation.

    PubMed

    Pesch, Yanina-Yasmin; Riedel, Dietmar; Behr, Matthias

    2017-01-01

    The architecture of the outer body wall cuticle is fundamental to protect arthropods against invading pathogens and numerous other harmful stresses. Such robust cuticles are formed by parallel running chitin microfibrils. Molting and also local wounding leads to dynamic assembly and disassembly of the chitin-matrix throughout development. However, the underlying molecular mechanisms that organize proper chitin-matrix formation are poorly known. Recently we identified a key region for cuticle thickening at the apical cell surface, the cuticle assembly zone, where Obstructor-A (Obst-A) coordinates the formation of the chitin-matrix. Obst-A binds chitin and the deacetylase Serpentine (Serp) in a core complex, which is required for chitin-matrix maturation and preservation. Here we present evidence that Chitinase 2 (Cht2) could be essential for this molecular machinery. We show that Cht2 is expressed in the chitin-matrix of epidermis, trachea, and the digestive system. There, Cht2 is enriched at the apical cell surface and the dense chitin-matrix. We further show that in Cht2 knockdown larvae the assembly zone is rudimentary, preventing normal cuticle formation and pore canal organization. As sequence similarities of Cht2 and the core complex proteins indicate evolutionarily conserved molecular mechanisms, our findings suggest that Cht2 is involved in chitin formation also in other insects.

  20. Improving the chitinolytic activity of Bacillus pumilus SG2 by random mutagenesis.

    PubMed

    Vahed, Majid; Motalebi, Ebrahim; Rigi, Garshasb; Akbari Noghabi, Kambiz; Soudi, Mohammad Reza; Sadeghi, Mehdi; Ahmadian, Gholamreza

    2013-11-28

    Bacillus pumilus SG2, a halotolerant strain, expresses two major chitinases designated ChiS and ChiL that were induced by chitin and secreted into the supernatant. The present work aimed to obtain a mutant with higher chitinolytic activity through mutagenesis of Bacillus pumilus SG2 using a combination of UV irradiation and nitrous acid treatment. Following mutagenesis and screening on chitin agar and subsequent formation of halos, the mutated strains were examined for degradation of chitin under different conditions. A mutant designated AV2-9 was selected owing to its higher chitinase activity. To search for possible mutations in the whole operon including ChiS and ChiL, the entire chitinase operon, including the intergenic region, promoter, and two areas corresponding to the ChiS and ChiL ORF, was suquenced. Nucleotide sequence analysis of the complete chitinase operon from the SG2 and AV2-9 strains showed the presence of a mutation in the catalytic domain (GH18) of chitinase (ChiL). The results demonstrated that a single base change had occurred in the ChiL sequence in AV2- 9. The wild-type chitinase, ChiL, and the mutant (designated ChiLm) were cloned, expressed, and purified in E. coli. Both enzymes showed similar profiles of activity at different ranges of pH, NaCl concentration, and temperature, but the mutant enzyme showed approximately 30% higher catalytic activity under all the conditions tested. The results obtained in this study showed that the thermal stability of chitinase increased in the mutant strain. Bioinformatics analysis was performed to predict changes in the stability of proteins caused by mutation.

  1. Ectopic expression of the mycorrhiza-specific chitinase gene Mtchit 3-3 in Medicago truncatula root-organ cultures stimulates spore germination of glomalean fungi.

    PubMed

    Elfstrand, Malin; Feddermann, Nadja; Ineichen, Kurt; Nagaraj, Vinay Jantakahalli; Wiemken, Andres; Boller, Thomas; Salzer, Peter

    2005-08-01

    Expression of Mtchit 3-3, a class III chitinase gene, is specifically induced by arbuscular mycorrhizal (AM) fungi in roots of the model legume Medicago truncatula and its transcripts accumulate in cells containing arbuscules. Agrobacterium rhizogenes-transformed roots and root-organ cultures of M. truncatula were used to study effects of Mtchit 3-3 on AM fungi. * This work provides evidence for enzymatic activity of the Mtchit 3-3 gene product and shows with promoter:gus fusions that a 2 kb fragment located 5' upstream from the translational start codon of Mtchit 3-3 is sufficient to confer arbuscule-dependent gene expression. By fusing the Mtchit 3-3 coding region to the CaMV 35S promoter the expression pattern was disrupted. Surprisingly, disruption stimulated spore germination of Glomus intraradices and Glomus constrictum, and in the case of G. intraradices resulted in a higher probability of root colonization and spore formation. However, no effect on the abundance of arbuscules within colonized roots became apparent. These observations demonstrate that disruption of the tight arbuscule-dependent expression pattern of Mtchit 3-3 has effects on the early interaction between roots and AM fungi.

  2. Characterization of chitinase-like proteins (Cg-Clp1 and Cg-Clp2) involved in immune defence of the mollusc Crassostrea gigas.

    PubMed

    Badariotti, Fabien; Lelong, Christophe; Dubos, Marie-Pierre; Favrel, Pascal

    2007-07-01

    Chitinase-like proteins have been identified in insects and mammals as nonenzymatic members of the glycoside hydrolase family 18. Recently, the first molluscan chitinase-like protein, named Crassostrea gigas (Cg)-Clp1, was shown to control the proliferation and synthesis of extracellular matrix components of mammalian chondrocytes. However, the precise physiological roles of Cg-Clp1 in oysters remain unknown. Here, we report the cloning and the characterization of a new chitinase-like protein (Cg-Clp2) from the oyster Crassostrea gigas. Gene expression profiles monitored by quantitative RT-PCR in adult tissues and through development support its involvement in tissue growth and remodelling. Both Cg-Clp1- and Cg-Clp2-encoding genes were transcriptionally stimulated in haemocytes in response to bacterial lipopolysaccharide challenge, strongly suggesting that these two close paralogous genes play a role in oyster immunity.

  3. Biodegradation of shrimp processing bio-waste and concomitant production of chitinase enzyme and N-acetyl-D-glucosamine by marine bacteria: production and process optimization.

    PubMed

    Suresh, P V

    2012-10-01

    A total of 250 chitinolytic bacteria from 68 different marine samples were screened employing enrichment method that utilized native chitin as the sole carbon source. After thorough screening, five bacteria were selected as potential cultures and identified as; Stenotrophomonas sp. (CFR221 M), Vibrio sp. (CFR173 M), Phyllobacteriaceae sp. (CFR16 M), Bacillus badius (CFR198 M) and Bacillus sp. (CFR188 M). All five strains produced extracellular chitinase and GlcNAc in SSF using shrimp bio-waste. Scanning electron microscopy confirmed the ability of these marine bacteria to adsorb onto solid shrimp bio-waste and to degrade chitin microfibers. HPLC analysis of the SSF extract also confirmed presence of 36-65 % GlcNAc as a product of the degradation. The concomitant production of chitinase and GlcNAc by all five strains under SSF using shrimp bio-waste as the solid substrate was optimized by 'one factor at a time' approach. Among the strains, Vibrio sp. CFR173 M produced significantly higher yields of chitinase (4.8 U/g initial dry substrate) and GlcNAc (4.7 μmol/g initial dry substrate) as compared to other cultures tested. A statistically designed experiment was applied to evaluate the interaction of variables in the biodegradation of shrimp bio-waste and concomitant production of chitinase and GlcNAc by Vibrio sp. CFR173 M. Statistical optimization resulted in a twofold increase of chitinase, and a 9.1 fold increase of GlcNAc production. These results indicated the potential of chitinolytic marine bacteria for the reclamation of shrimp bio-waste, as well as the potential for economic production of chitinase and GlcNAc employing SSF using shrimp bio-waste as an ideal substrate.

  4. Transcriptional Regulation of a Chitinase Gene by 20-Hydroxyecdysone and Starvation in the Oriental Fruit Fly, Bactrocera dorsalis

    PubMed Central

    Yang, Wen-Jia; Xu, Kang-Kang; Zhang, Rui-Ying; Dou, Wei; Wang, Jin-Jun

    2013-01-01

    Insect chitinases are hydrolytic enzymes that are required for the degradation of glycosidic bonds of chitin. In this study, we identified and characterized a full-length cDNA of the chitinase gene (BdCht2) in the oriental fruit fly, Bactrocera dorsalis. The cDNA contains an open reading frame (ORF) of 1449 bp that encodes 483 amino acid residues and 126- and 296-bp non-coding regions at the 5′- and 3′-ends, respectively. The BdCht2 genome has four exons and three introns. The predicted molecular mass of the deduced BdCht2 is approximately 54.3 kDa, with an isoelectric point of 5.97. The 977 bp 5′ flanking region was identified and the transcription factor binding sites were predicted. Bioinformatic analyses showed that the deduced amino acid sequence of BdCht2 had 34%–66% identity to that of chitinases identified in other insect species. Quantitative real-time PCR (qPCR) analyses indicated that BdCht2 was mainly expressed during the larval-pupal and pupal-adult transitions. The tissue-specific expression showed that the highest expression was in the integument, followed by the fat body and other tissues. Moreover, the expression of BdCht2 was upregulated significantly upon 20-hydroxyecdysone (20E) at different dose injections after 8 h compared to that of the control. Starvation also increased the expression of BdCht2 in the third-instar larvae and was suppressed again by re-feeding the insects. These results suggest that BdCht2 plays an important role in the molting process of B. dorsalis larvae and can be regulated by 20E. PMID:24113584

  5. Dual silencing of long and short Amblyomma americanum acidic chitinase forms weakens the tick cement cone stability

    PubMed Central

    Kim, Tae K.; Curran, Janet; Mulenga, Albert

    2014-01-01

    This study demonstrates that Amblyomma americanum (Aam) constitutively and ubiquitously expresses the long (L) and short (S) putative acidic chitinases (Ach) that are distinguished by a 210 base pair (bp) deletion in AamAch-S. Full-length AamAch-L and AamAch-S cDNA are 1959 and 1718 bp long, containing 1332 and 1104 bp open reading frames that code for 443 and 367 amino acid residues proteins with the former predicted to be extracellular and the latter intracellular. Both AamAch-L and AamAch-S mRNA are expressed in multiple organs as revealed by qualitative RT-PCR analysis. Furthermore, quantitative reverse transcription polymerase chain reaction analysis revealed that AamAch-L mRNA was downregulated in the mid-gut, but was unchanged in the salivary gland and in other organs in response to feeding. Of significant interest, AamAch-L and/or AamAch-S functions are probably associated with formation and/or maintenance of stability of A. americanum tick cement cone. Dual RNA interference silencing of AamAch-L and/or AamAch-S mRNA caused ticks to loosely attach onto host skin as suggested by bleeding around tick mouthparts and ticks detaching off host skin with a light touch. AamAch-L may apparently encode an inactive chitinase as indicated by Pichia pastoris-expressed recombinant AamAch-L failing to hydrolyse chitinase substrates. Unpublished related work in our laboratory, and published work by others that found AamAch-L in tick saliva, suggest that native AamAch-L is a non-specific immunoglobulin binding tick saliva protein in that rAamAch-L non-specifically bound rabbit, bovine and chicken non-immune sera. We discuss findings in this study with reference to advancing knowledge on tick feeding physiology. PMID:25189365

  6. Correlation of Chitinase 3-Like 1 Single Nucleotide Polymorphisms with Hepatocellular Carcinoma in Taiwan

    PubMed Central

    Huang, Wayne Shih-Wei; Lin, Hung-Yu; Yeh, Chao-Bin; Chen, Li-You; Chou, Ying-Erh; Yang, Shun-Fa; Liu, Yu-Fan

    2017-01-01

    Hepatocellular carcinoma (HCC) is the second leading cause of cancer death in Taiwan. Multiple risk factors, such as chronic hepatitis B or C virus infection, carcinogen exposure, cirrhosis, and various single-nucleotide polymorphisms (SNPs), are considered to contribute to hepatocarcinogenesis. Chitinase-3-like protein 1 (CHI3L1), a biomarker implicated in inflammation and tissue remodeling, plays a promoting role in angiogenesis, antiapoptosis, and cell proliferation. This study investigated the role of CHI3L1 SNPs in HCC susceptibility and clinicopathology. Real-time polymerase chain reaction was used to analyze four SNPs of CHI3L1 in 343 patients with HCC and 686 cancer-free controls. We found associations with HCC susceptibility in CHI3L1 rs880633 polymorphism carriers with genotypes (TC+CC). We observed that HCC patients had lower frequencies of CHI3L1 rs6691378 polymorphisms with the variant genotype GA+AA than the wild-type carriers with distant metastasis and positive HBsAg did. In 200 HBsAg negative HCC patients, we observed that the CHI3L1 rs4950928 polymorphisms carriers with the variant genotype CG+GG had higher frequencies of vascular invasion. Finally, carriers of CHI3L1 rs6691378 and 10399805 polymorphisms with the variant genotypes GA+AA showed lower levels of alpha-fetoprotein in HCC laboratory status. In conclusion, our results indicate that patients with CHI3L1 rs880633 variant genotypes TC+CC are at a higher risk of HCC. CHI3L1 polymorphisms rs880633 or rs4950928 may be potential candidates for predicting poor HCC prognosis and clinical status. PMID:28260989

  7. Discovery and identification of candidate genes from the chitinase gene family for Verticillium dahliae resistance in cotton

    PubMed Central

    Xu, Jun; Xu, Xiaoyang; Tian, Liangliang; Wang, Guilin; Zhang, Xueying; Wang, Xinyu; Guo, Wangzhen

    2016-01-01

    Verticillium dahliae, a destructive and soil-borne fungal pathogen, causes massive losses in cotton yields. However, the resistance mechanism to V. dahilae in cotton is still poorly understood. Accumulating evidence indicates that chitinases are crucial hydrolytic enzymes, which attack fungal pathogens by catalyzing the fungal cell wall degradation. As a large gene family, to date, the chitinase genes (Chis) have not been systematically analyzed and effectively utilized in cotton. Here, we identified 47, 49, 92, and 116 Chis from four sequenced cotton species, diploid Gossypium raimondii (D5), G. arboreum (A2), tetraploid G. hirsutum acc. TM-1 (AD1), and G. barbadense acc. 3–79 (AD2), respectively. The orthologous genes were not one-to-one correspondence in the diploid and tetraploid cotton species, implying changes in the number of Chis in different cotton species during the evolution of Gossypium. Phylogenetic classification indicated that these Chis could be classified into six groups, with distinguishable structural characteristics. The expression patterns of Chis indicated their various expressions in different organs and tissues, and in the V. dahliae response. Silencing of Chi23, Chi32, or Chi47 in cotton significantly impaired the resistance to V. dahliae, suggesting these genes might act as positive regulators in disease resistance to V. dahliae. PMID:27354165

  8. Ultrasonication and steam-explosion as chitin pretreatments for chitin oligosaccharide production by chitinases of Lecanicillium lecanii.

    PubMed

    Villa-Lerma, Guadalupe; González-Márquez, Humberto; Gimeno, Miquel; López-Luna, Alberto; Bárzana, Eduardo; Shirai, Keiko

    2013-10-01

    In this study, chitin oligosaccharides have been successfully produced using chitinases from submerged fermentation of Lecanicillium lecanii. The highest Hex, Chit and Prot production was 0.14, 0.26 and 2.05 U/mg of protein, respectively, which were attained varying pH from 5 to 8 after 96 h. Culture conditions conducted at constant pH of 6 resulted in significantly lower enzyme production. The crude enzyme was partially purified by salting out with (NH4)2SO4 followed by size exclusion chromatography to isolate the chitinase mixture for further chitin hydrolysis assays. In this regard, chitin substrates were pretreated with sonication and steam explosion prior to enzymatic reaction. Structural changes were observed with steam explosion with 11.28% reduction of the crystallinity index attained with the lowest chitin/water ratio (0.1g/mL). Pretreated chitins reached the highest production of reducing sugars (0.37 mg/mL) and GlcNAc (0.59 mg/mL) in 23.6% yield.

  9. Targeting chitinase gene of Helicoverpa armigera by host-induced RNA interference confers insect resistance in tobacco and tomato.

    PubMed

    Mamta; Reddy, K R K; Rajam, M V

    2016-02-01

    Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a devastating agricultural insect pest with broad spectrum of host range, causing million dollars crop loss annually. Limitations in the present conventional and transgenic approaches have made it crucial to develop sustainable and environmental friendly methods for crop improvement. In the present study, host-induced RNA interference (HI-RNAi) approach was used to develop H. armigera resistant tobacco and tomato plants. Chitinase (HaCHI) gene, critically required for insect molting and metamorphosis was selected as a potential target. Hair-pin RNAi construct was prepared from the conserved off-target free partial HaCHI gene sequence and was used to generate several HaCHI-RNAi tobacco and tomato plants. Northern hybridization confirmed the production of HaCHI gene-specific siRNAs in HaCHI-RNAi tobacco and tomato lines. Continuous feeding on leaves of RNAi lines drastically reduced the target gene transcripts and consequently, affected the overall growth and survival of H. armigera. Various developmental deformities were also manifested in H. armigera larvae after feeding on the leaves of RNAi lines. These results demonstrated the role of chitinase in insect development and potential of HI-RNAi for effective management of H. armigera.

  10. SnTox1, a Parastagonospora nodorum necrotrophic effector, is a dual-function protein that facilitates infection while protecting from wheat-produced chitinases.

    PubMed

    Liu, Zhaohui; Gao, Yuanyuan; Kim, Yong Min; Faris, Justin D; Shelver, Weilin L; de Wit, Pierre J G M; Xu, Steven S; Friesen, Timothy L

    2016-08-01

    SnTox1 induces programmed cell death and the up-regulation of pathogenesis-related genes including chitinases. Additionally, SnTox1 has structural homology to several plant chitin-binding proteins. Therefore, we evaluated SnTox1 for chitin binding and localization. We transformed an avirulent strain of Parastagonospora nodorum as well as three nonpathogens of wheat (Triticum aestivum), including a necrotrophic pathogen of barley, a hemibiotrophic pathogen of sugar beet and a saprotroph, to evaluate the role of SnTox1 in infection and in protection from wheat chitinases. SnTox1 bound chitin and an SnTox1-green fluorescent fusion protein localized to the mycelial cell wall. Purified SnTox1 induced necrosis in the absence of the pathogen when sprayed on the leaf surface and appeared to remain on the leaf surface while inducing both epidermal and mesophyll cell death. SnTox1 protected the different fungi from chitinase degradation. SnTox1 was sufficient to change the host range of a necrotrophic pathogen but not a hemibiotroph or saprotroph. Collectively, this work shows that SnTox1 probably interacts with a receptor on the outside of the cell to induce cell death to acquire nutrients, but SnTox1 accomplishes a second role in that it protects against one aspect of the defense response, namely the effects of wheat chitinases.

  11. Chitinase 3-like 1 induces survival and proliferation of intestinal epithelial cells during chronic inflammation and colitis-associated cancer by regulating S100A9

    PubMed Central

    Low, Daren; Subramaniam, Renuka; Lin, Li; Aomatsu, Tomoki; Mizoguchi, Atsushi; Ng, Aylwin; DeGruttola, Arianna K.; Lee, Chun Geun; Elias, Jack A.; Andoh, Akira; Mino-Kenudson, Mari; Mizoguchi, Emiko

    2015-01-01

    Many host-factors are inducibly expressed during the development of inflammatory bowel disease (IBD), each having their unique properties, such as immune activation, bacterial clearance, and tissue repair/remodeling. Dysregulation/imbalance of these factors may have pathogenic effects that can contribute to colitis-associated cancer (CAC). Previous reports showed that IBD patients inducibly express colonic chitinase 3-like 1 (CHI3L1) that is further upregulated during CAC development. However, little is known about the direct pathogenic involvement of CHI3L1 in vivo. Here we demonstrate that CHI3L1 (aka Brp39) knockout (KO) mice treated with azoxymethane (AOM)/dextran sulphate sodium (DSS) developed severe colitis but lesser incidence of CAC as compared to that in wild-type (WT) mice. Highest CHI3L1 expression was found during the chronic phase of colitis, rather than the acute phase, and is essential to promote intestinal epithelial cell (IEC) proliferation in vivo. This CHI3L1-mediated cell proliferation/survival involves partial downregulation of the pro-apoptotic S100A9 protein that is highly expressed during the acute phase of colitis, by binding to the S100A9 receptor, RAGE (Receptor for Advanced Glycation End products). This interaction disrupts the S100A9-associated expression positive feedback loop during early immune activation, creating a CHI3L1hi S100A9low colonic environment, especially in the later phase of colitis, which promotes cell proliferation/survival of both normal IECs and tumor cells. PMID:26431492

  12. Glucanases and Chitinases as Causal Agents in the Protection of Acacia Extrafloral Nectar from Infestation by Phytopathogens1[W][OA

    PubMed Central

    González-Teuber, Marcia; Pozo, María J.; Muck, Alexander; Svatos, Ales; Adame-Álvarez, Rosa M.; Heil, Martin

    2010-01-01

    Nectars are rich in primary metabolites and attract mutualistic animals, which serve as pollinators or as an indirect defense against herbivores. Their chemical composition makes nectars prone to microbial infestation. As protective strategy, floral nectar of ornamental tobacco (Nicotiana langsdorffii × Nicotiana sanderae) contains “nectarins,” proteins producing reactive oxygen species such as hydrogen peroxide. By contrast, pathogenesis-related (PR) proteins were detected in Acacia extrafloral nectar (EFN), which is secreted in the context of defensive ant-plant mutualisms. We investigated whether these PR proteins protect EFN from phytopathogens. Five sympatric species (Acacia cornigera, A. hindsii, A. collinsii, A. farnesiana, and Prosopis juliflora) were compared that differ in their ant-plant mutualism. EFN of myrmecophytes, which are obligate ant-plants that secrete EFN constitutively to nourish specialized ant inhabitants, significantly inhibited the growth of four out of six tested phytopathogenic microorganisms. By contrast, EFN of nonmyrmecophytes, which is secreted only transiently in response to herbivory, did not exhibit a detectable inhibitory activity. Combining two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with nanoflow liquid chromatography-tandem mass spectrometry analysis confirmed that PR proteins represented over 90% of all proteins in myrmecophyte EFN. The inhibition of microbial growth was exerted by the protein fraction, but not the small metabolites of this EFN, and disappeared when nectar was heated. In-gel assays demonstrated the activity of acidic and basic chitinases in all EFNs, whereas glucanases were detected only in EFN of myrmecophytes. Our results demonstrate that PR proteins causally underlie the protection of Acacia EFN from microorganisms and that acidic and basic glucanases likely represent the most important prerequisite in this defensive function. PMID:20023149

  13. Alternative splicing of basic chitinase gene PR3b in the low-nicotine mutants of Nicotiana tabacum L. cv. Burley 21

    PubMed Central

    Ma, Haoran; Wang, Feng; Wang, Wenjing; Yin, Guoying; Zhang, Dingyu; Ding, Yongqiang; Timko, Michael P.; Zhang, Hongbo

    2016-01-01

    Two unlinked semi-dominant loci, A (NIC1) and B (NIC2), control nicotine and related alkaloid biosynthesis in Burley tobaccos. Mutations in either or both loci (nic1 and nic2) lead to low nicotine phenotypes with altered environmental stress responses. Here we show that the transcripts derived from the pathogenesis-related (PR) protein gene PR3b are alternatively spliced to a greater extent in the nic1 and nic2 mutants of Burley 21 tobacco and the nic1nic2 double mutant. The alternative splicing results in a deletion of 65 nucleotides and introduces a premature stop codon into the coding region of PR3b that leads to a significant reduction of PR3b specific chitinase activity. Assays of PR3b splicing in F2 individuals derived from crosses between nic1 and nic2 mutants and wild-type plants showed that the splicing phenotype is controlled by the NIC1 and NIC2 loci, even though NIC1 and NIC2 are unlinked loci. Moreover, the transcriptional analyses showed that the splicing patterns of PR3b in the low-nicotine mutants were differentially regulated by jasmonate (JA) and ethylene (ET). These data suggest that the NIC1 and NIC2 loci display differential roles in regulating the alternative splicing of PR3b in Burley 21. The findings in this study have provided valuable information for extending our understanding of the broader effects of the low-nicotine mutants of Burley 21 and the mechanism by which JA and ET signalling pathways post-transcriptionally regulate the activity of PR3b protein. PMID:27664270

  14. A computational analysis of the binding mode of closantel as inhibitor of the Onchocerca volvulus chitinase: insights on macrofilaricidal drug design

    NASA Astrophysics Data System (ADS)

    Segura-Cabrera, Aldo; Bocanegra-García, Virgilio; Lizarazo-Ortega, Cristian; Guo, Xianwu; Correa-Basurto, José; Rodríguez-Pérez, Mario A.

    2011-12-01

    Onchocerciasis is a leading cause of blindness with at least 37 million people infected and more than 120 million people at risk of contracting the disease; most (99%) of this population, threatened by infection, live in Africa. The drug of choice for mass treatment is the microfilaricidal Mectizan® (ivermectin); it does not kill the adult stages of the parasite at the standard dose which is a single annual dose aimed at disease control. However, multiple treatments a year with ivermectin have effects on adult worms. The discovery of new therapeutic targets and drugs directed towards the killing of the adult parasites are thus urgently needed. The chitinase of filarial nematodes is a new drug target due to its essential function in the metabolism and molting of the parasite. Closantel is a potent and specific inhibitor of chitinase of Onchocerca volvulus (OvCHT1) and other filarial chitinases. However, the binding mode and specificity of closantel towards OvCHT1 remain unknown. In the absence of a crystallographic structure of OvCHT1, we developed a homology model of OvCHT1 using the currently available X-ray structures of human chitinases as templates. Energy minimization and molecular dynamics (MD) simulation of the model led to a high quality of 3D structure of OvCHIT1. A flexible docking study using closantel as the ligand on the binding site of OvCHIT1 and human chitinases was performed and demonstrated the differences in the closantel binding mode between OvCHIT1 and human chitinase. Furthermore, molecular dynamics simulations and free-energy calculation were employed to determine and compare the detailed binding mode of closantel with OvCHT1 and the structure of human chitinase. This comparative study allowed identification of structural features and properties responsible for differences in the computationally predicted closantel binding modes. The homology model and the closantel binding mode reported herein might help guide the rational development of

  15. Density and composition of an insect population in a field trial of chitinase transgenic and wild-type silver birch (Betula pendula) clones.

    PubMed

    Vihervuori, Liisa; Pasonen, Hanna-Leena; Lyytikäinen-Saarenmaa, Päivi

    2008-12-01

    Fifteen silver birch (Betula pendula Roth) lines carrying a sugar beet chitinase IV gene and eight wild-type birch clones were grown in a field trial. The composition and density of the insect population and the leaf damage caused by insects were monitored and compared between transgenic and wild-type trees. The most abundant insect group in all trees was aphids, and the variation in total insect densities was mainly explained by the variation in aphid densities. Insect densities were generally higher in the transgenic than in the control trees, indicating that the expression of the sugar beet chitinase IV gene had an influence on the suitability of birch leaves to aphids. The level of leaf damage was higher among transgenic than among control trees. Chewing damage was the most common type of leaf damage in all trees. The number of different damage types was higher among the wild-type clones than among the transgenic lines or their controls. The results indicate that the chitinase transgenic trees are more susceptible to aphids and suffer higher levels of leaf damage than the control trees. In the composition of the damage types, the control trees were more similar to the transgenic than to other wild-type trees, indicating that the composition was mostly linked to the genotype of the tree and not to the expression of the transgene. This study provides important information on the ecological interactions of chitinase transgenic trees in the field trial. No clear harmful effects of transgenic chitinase on the biodiversity of insect population were detected.

  16. Combining chitinase C and N-acetylhexosaminidase from Streptomyces coelicolor A3(2) provides an efficient way to synthesize N-acetylglucosamine from crystalline chitin.

    PubMed

    Nguyen-Thi, Nhung; Doucet, Nicolas

    2016-02-20

    The enzymatic bioconversion of chitin is of considerable interest for the natural production of bioactive compounds such as chitooligosaccharides and N-acetyl-d-glucosamine (GlcNAc). Key enzymes are involved in the natural processing of chitin, hydrolyzing this abundant biopolymer to yield chitooligosaccharides with substantial value to the medicinal and biotechnological fields. In this study, chitinase C (ScChiC) from the soil bacterium and chitin decomposer Streptomyces coelicolor A3(2) was expressed, purified and characterized. We also optimized a Streptomyces lividans system generating ScChiC expression yields nearly 500-fold higher than the previously reported heterologous expression in Escherichia coli. The purified enzyme was found to be stable below 55°C for a broad range of pH values (pH 3.5-9) and exhibited high activity against chitin and chitooligosaccharides to form chitobiose (C2) as main product. Crab shell chitin hydrolysis profiles also revealed that ScChiC catalyzes the bioconversion of chitopolysaccharides through an endo-nonprocessive mode of action. When combining ScChiC with an N-acetylhexosaminidase from S. coelicolor A3(2) (ScHEX) in an assay using crude extracts and crystalline chitin as substrate, GlcNAc was generated as final product with a yield over 90% after 8h incubation. This chitin hydrolysis yield represents one of the most efficient enzyme bioconversion of chitopolysaccharides to GlcNAc characterized to date, making the S. coelicolor ScChiC-ScHEX pair a potentially suitable contender for the viable industrial production of this important bioactive compound.

  17. Slow Off-rates and Strong Product Binding Are Required for Processivity and Efficient Degradation of Recalcitrant Chitin by Family 18 Chitinases.

    PubMed

    Kurašin, Mihhail; Kuusk, Silja; Kuusk, Piret; Sørlie, Morten; Väljamäe, Priit

    2015-11-27

    Processive glycoside hydrolases are the key components of enzymatic machineries that decompose recalcitrant polysaccharides, such as chitin and cellulose. The intrinsic processivity (P(Intr)) of cellulases has been shown to be governed by the rate constant of dissociation from polymer chain (koff). However, the reported koff values of cellulases are strongly dependent on the method used for their measurement. Here, we developed a new method for determining koff, based on measuring the exchange rate of the enzyme between a non-labeled and a (14)C-labeled polymeric substrate. The method was applied to the study of the processive chitinase ChiA from Serratia marcescens. In parallel, ChiA variants with weaker binding of the N-acetylglucosamine unit either in substrate-binding site -3 (ChiA-W167A) or the product-binding site +1 (ChiA-W275A) were studied. Both ChiA variants showed increased off-rates and lower apparent processivity on α-chitin. The rate of the production of insoluble reducing groups on the reduced α-chitin was an order of magnitude higher than koff, suggesting that the enzyme can initiate several processive runs without leaving the substrate. On crystalline chitin, the general activity of the wild type enzyme was higher, and the difference was magnifying with hydrolysis time. On amorphous chitin, the variants clearly outperformed the wild type. A model is proposed whereby strong interactions with polymer in the substrate-binding sites (low off-rates) and strong binding of the product in the product-binding sites (high pushing potential) are required for the removal of obstacles, like disintegration of chitin microfibrils.

  18. Characterization of the starvation-induced chitinase CfcA and α-1,3-glucanase AgnB of Aspergillus niger.

    PubMed

    van Munster, Jolanda M; Dobruchowska, Justyna M; Veloo, Ruud; Dijkhuizen, Lubbert; van der Maarel, Marc J E C

    2015-03-01

    The common saprophyte Aspergillus niger may experience carbon starvation in nature as well as during industrial fermentations. Starvation survival strategies, such as conidiation or the formation of exploratory hyphae, require energy and building blocks, which may be supplied by autolysis. Glycoside hydrolases are key effectors of autolytic degradation of fungal cell walls, but knowledge on their identity and functionality is still limited. We recently identified agnB and cfcA as two genes encoding carbohydrate-active enzymes that had notably increased transcription during carbon starvation in A. niger. Here, we report the biochemical and functional characterization of these enzymes. AgnB is an α-1,3-glucanase that releases glucose from α-1,3-glucan substrates with a minimum degree of polymerization of 4. CfcA is a chitinase that releases dimers from the nonreducing end of chitin. These enzymes thus attack polymers that are found in the fungal cell wall and may have a role in autolytic fungal cell wall degradation in A. niger. Indeed, cell wall degradation during carbon starvation was reduced in the double deletion mutant ΔcfcA ΔagnB compared to the wild-type strain. Furthermore, the cell walls of the carbon-starved mycelium of the mutant contained a higher fraction of chitin or chitosan. The function of at least one of these enzymes, CfcA, therefore appears to be in the recycling of cell wall carbohydrates under carbon limiting conditions. CfcA thus may be a candidate effector for on demand cell lysis, which could be employed in industrial processes for recovery of intracellular products.

  19. Sequence/structural analysis of xylem proteome emphasizes pathogenesis-related proteins, chitinases and β-1, 3-glucanases as key players in grapevine defense against Xylella fastidiosa

    PubMed Central

    Chakraborty, Sandeep; Nascimento, Rafael; Zaini, Paulo A.; Gouran, Hossein; Rao, Basuthkar J.; Goulart, Luiz R.

    2016-01-01

    Background. Xylella fastidiosa, the causative agent of various plant diseases including Pierce’s disease in the US, and Citrus Variegated Chlorosis in Brazil, remains a continual source of concern and economic losses, especially since almost all commercial varieties are sensitive to this Gammaproteobacteria. Differential expression of proteins in infected tissue is an established methodology to identify key elements involved in plant defense pathways. Methods. In the current work, we developed a methodology named CHURNER that emphasizes relevant protein functions from proteomic data, based on identification of proteins with similar structures that do not necessarily have sequence homology. Such clustering emphasizes protein functions which have multiple copies that are up/down-regulated, and highlights similar proteins which are differentially regulated. As a working example we present proteomic data enumerating differentially expressed proteins in xylem sap from grapevines that were infected with X. fastidiosa. Results. Analysis of this data by CHURNER highlighted pathogenesis related PR-1 proteins, reinforcing this as the foremost protein function in xylem sap involved in the grapevine defense response to X. fastidiosa. β-1, 3-glucanase, which has both anti-microbial and anti-fungal activities, is also up-regulated. Simultaneously, chitinases are found to be both up and down-regulated by CHURNER, and thus the net gain of this protein function loses its significance in the defense response. Discussion. We demonstrate how structural data can be incorporated in the pipeline of proteomic data analysis prior to making inferences on the importance of individual proteins to plant defense mechanisms. We expect CHURNER to be applicable to any proteomic data set. PMID:27257535

  20. The Vibrio cholerae extracellular chitinase ChiA2 is important for survival and pathogenesis in the host intestine.

    PubMed

    Mondal, Moumita; Nag, Dhrubajyoti; Koley, Hemanta; Saha, Dhira Rani; Chatterjee, Nabendu Sekhar

    2014-01-01

    In aquatic environments, Vibrio cholerae colonizes mainly on the chitinous surface of copepods and utilizes chitin as the sole carbon and nitrogen source. Of the two extracellular chitinases essential for chitin utilization, the expression of chiA2 is maximally up-regulated in host intestine. Recent studies indicate that several bacterial chitinases may be involved in host pathogenesis. However, the role of V. cholerae chitinases in host infection is not yet known. In this study, we provide evidence to show that ChiA2 is important for V. cholerae survival in intestine as well as in pathogenesis. We demonstrate that ChiA2 de-glycosylates mucin and releases reducing sugars like GlcNAc and its oligomers. Deglycosylation of mucin corroborated with reduced uptake of alcian blue stain by ChiA2 treated mucin. Next, we show that V. cholerae could utilize mucin as a nutrient source. In comparison to the wild type strain, ΔchiA2 mutant was 60-fold less efficient in growth in mucin supplemented minimal media and was also ∼6-fold less competent to survive when grown in the presence of mucin-secreting human intestinal HT29 epithelial cells. Similar results were also obtained when the strains were infected in mice intestine. Infection with the ΔchiA2 mutant caused ∼50-fold less fluid accumulation in infant mice as well as in rabbit ileal loop compared to the wild type strain. To see if the difference in survival of the ΔchiA2 mutant and wild type V. cholerae was due to reduced adhesion of the mutant, we monitored binding of the strains on HT29 cells. The initial binding of the wild type and mutant strain was similar. Collectively these data suggest that ChiA2 secreted by V. cholerae in the intestine hydrolyzed intestinal mucin to release GlcNAc, and the released sugar is successfully utilized by V. cholerae for growth and survival in the host intestine.

  1. Allozyme-specific modification of a maize seed chitinase by a protein secreted by the fungal pathogen Stenocarpella maydis.

    PubMed

    Naumann, Todd A; Wicklow, Donald T

    2010-07-01

    Stenocarpella maydis causes both dry-ear rot and stalk rot of maize. Maize inbred lines have varying levels of resistance to ear rot caused by S. maydis. The genetic basis of resistance appears to rely on multiple genetic factors, none of which are known. The commonly used stiff-stalk inbred line B73 has been shown to be strongly susceptible to ear rot caused by S. maydis. Here, we report that the ChitA protein alloform from B73, ChitA-F, encoded by a known allele of the chiA gene, is susceptible to modification by a protein (Stm-cmp) secreted by S. maydis. We also identify a new allele of chiA (from inbred line LH82) which encodes ChitA-S, an alloform of ChitA that is resistant to Stm-cmp modification. Chitinase zymogram analysis of seed from a commercial field showed the presence of both ChitA alloforms in healthy ears, and showed that ChitA-F but not ChitA-S was modified in ears rotted by S. maydis. The ChitA-F protein was purified from inbred line B73 and ChitA-S from LH82. ChitA-F was modified more efficiently than ChitA-S by S. maydis protein extracts in vitro. The chiA gene from LH82 was cloned and sequenced. It is a novel allele that encodes six polymorphisms relative to the known allele from B73. This is the first demonstration that the susceptibility to modification of a fungal targeted plant chitinase differs among inbred lines. These findings suggest that the LH82 chiA allele may be a specific genetic determinant that contributes to resistance to ear rot caused by S. maydis whereas the B73 allele may contribute to susceptibility.

  2. Feces Derived Allergens of Tyrophagus putrescentiae Reared on Dried Dog Food and Evidence of the Strong Nutritional Interaction between the Mite and Bacillus cereus Producing Protease Bacillolysins and Exo-chitinases

    PubMed Central

    Erban, Tomas; Rybanska, Dagmar; Harant, Karel; Hortova, Bronislava; Hubert, Jan

    2016-01-01

    Tyrophagus putrescentiae (Schrank, 1781) is an emerging source of allergens in stored products and homes. Feces proteases are the major allergens of astigmatid mites (Acari: Acaridida). In addition, the mites are carriers of microorganisms and microbial adjuvant compounds that stimulate innate signaling pathways. We sought to analyze the mite feces proteome, proteolytic activities, and mite-bacterial interaction in dry dog food (DDF). Proteomic methods comprising enzymatic and zymographic analysis of proteases and 2D-E-MS/MS were performed. The highest protease activity was assigned to trypsin-like proteases; lower activity was assigned to chymotrypsin-like proteases, and the cysteine protease cathepsin B-like had very low activity. The 2D-E-MS/MS proteomic analysis identified mite trypsin allergen Tyr p3, fatty acid-binding protein Tyr p13 and putative mite allergens ferritin (Grp 30) and (poly)ubiquitins. Tyr p3 was detected at different positions of the 2D-E. It indicates presence of zymogen at basic pI, and mature-enzyme form and enzyme fragment at acidic pI. Bacillolysins (neutral and alkaline proteases) of Bacillus cereus symbiont can contribute to the protease activity of the mite extract. The bacterial exo-chitinases likely contribute to degradation of mite exuviae, mite bodies or food boluses consisting of chitin, including the peritrophic membrane. Thus, the chitinases disrupt the feces and facilitate release of the allergens. B. cereus was isolated and identified based on amplification and sequencing of 16S rRNA and motB genes. B. cereus was added into high-fat, high-protein (DDF) and low-fat, low-protein (flour) diets to 1 and 5% (w/w), and the diets palatability was evaluated in 21-day population growth test. The supplementation of diet with B. cereus significantly suppressed population growth and the suppressive effect was higher in the high-fat, high-protein diet than in the low-fat, low-protein food. Thus, B. cereus has to coexist with the mite in

  3. Feces Derived Allergens of Tyrophagus putrescentiae Reared on Dried Dog Food and Evidence of the Strong Nutritional Interaction between the Mite and Bacillus cereus Producing Protease Bacillolysins and Exo-chitinases.

    PubMed

    Erban, Tomas; Rybanska, Dagmar; Harant, Karel; Hortova, Bronislava; Hubert, Jan

    2016-01-01

    Tyrophagus putrescentiae (Schrank, 1781) is an emerging source of allergens in stored products and homes. Feces proteases are the major allergens of astigmatid mites (Acari: Acaridida). In addition, the mites are carriers of microorganisms and microbial adjuvant compounds that stimulate innate signaling pathways. We sought to analyze the mite feces proteome, proteolytic activities, and mite-bacterial interaction in dry dog food (DDF). Proteomic methods comprising enzymatic and zymographic analysis of proteases and 2D-E-MS/MS were performed. The highest protease activity was assigned to trypsin-like proteases; lower activity was assigned to chymotrypsin-like proteases, and the cysteine protease cathepsin B-like had very low activity. The 2D-E-MS/MS proteomic analysis identified mite trypsin allergen Tyr p3, fatty acid-binding protein Tyr p13 and putative mite allergens ferritin (Grp 30) and (poly)ubiquitins. Tyr p3 was detected at different positions of the 2D-E. It indicates presence of zymogen at basic pI, and mature-enzyme form and enzyme fragment at acidic pI. Bacillolysins (neutral and alkaline proteases) of Bacillus cereus symbiont can contribute to the protease activity of the mite extract. The bacterial exo-chitinases likely contribute to degradation of mite exuviae, mite bodies or food boluses consisting of chitin, including the peritrophic membrane. Thus, the chitinases disrupt the feces and facilitate release of the allergens. B. cereus was isolated and identified based on amplification and sequencing of 16S rRNA and motB genes. B. cereus was added into high-fat, high-protein (DDF) and low-fat, low-protein (flour) diets to 1 and 5% (w/w), and the diets palatability was evaluated in 21-day population growth test. The supplementation of diet with B. cereus significantly suppressed population growth and the suppressive effect was higher in the high-fat, high-protein diet than in the low-fat, low-protein food. Thus, B. cereus has to coexist with the mite in

  4. Chitin and Its Effects on Inflammatory and Immune Responses.

    PubMed

    Elieh Ali Komi, Daniel; Sharma, Lokesh; Dela Cruz, Charles S

    2017-03-01

    Chitin, a potential allergy-promoting pathogen-associated molecular pattern (PAMP), is a linear polymer composed of N-acetylglucosamine residues which are linked by β-(1,4)-glycosidic bonds. Mammalians are potential hosts for chitin-containing protozoa, fungi, arthropods, and nematodes; however, mammalians themselves do not synthetize chitin and thus it is considered as a potential target for recognition by mammalian immune system. Chitin is sensed primarily in the lungs or gut where it activates a variety of innate (eosinophils, macrophages) and adaptive immune cells (IL-4/IL-13 expressing T helper type-2 lymphocytes). Chitin induces cytokine production, leukocyte recruitment, and alternative macrophage activation. Intranasal or intraperitoneal administration of chitin (varying in size, degree of acetylation and purity) to mice has been applied as a routine approach to investigate chitin's priming effects on innate and adaptive immunity. Structural chitin present in microorganisms is actively degraded by host true chitinases, including acidic mammalian chitinases and chitotriosidase into smaller fragments that can be sensed by mammalian receptors such as FIBCD1, NKR-P1, and RegIIIc. Immune recognition of chitin also involves pattern recognition receptors, mainly via TLR-2 and Dectin-1, to activate immune cells to induce cytokine production and creation of an immune network that results in inflammatory and allergic responses. In this review, we will focus on various immunological aspects of the interaction between chitin and host immune system such as sensing, interactions with immune cells, chitinases as chitin degrading enzymes, and immunologic applications of chitin.

  5. The Role of CHI3L1 (Chitinase-3-Like-1) in the Pathogenesis of Infections in Burns in a Mouse Model.

    PubMed

    Bohr, Stefan; Patel, Suraj J; Vasko, Radovan; Shen, Keyue; Golberg, Alexander; Berthiaume, Francois; Yarmush, Martin L

    2015-01-01

    In severe burn injury the unique setting of a depleted, dysfunctional immune system along with a loss of barrier function commonly results in opportunistic infections that eventually proof fatal. Unfortunately, the dynamic sequence of bacterial contamination, colonization and eventually septic invasion with bacteria such as Pseudomonas species is still poorly understood although a limiting factor in clinical decision making. Increasing evidence supports the notion that inhibition of bacterial translocation into the wound site may be an effective alternative to prevent infection. In this context we investigated the role of the mammalian Chitinase-3-Like-1 (CHI3L1) non-enyzmatic protein predominately expressed on epithelial as well as innate immune cells as a potential bacterial-translocation-mediating factor. We show a strong trend that a modulation of chitinase expression is likely to be effective in reducing mortality rates in a mouse model of burn injury with superinfection with the opportunistic PA14 Pseudomonas strain, thus demonstrating possible clinical leverage.

  6. Silencing of Target Chitinase Genes via Oral Delivery of dsRNA Caused Lethal Phenotypic Effects in Mythimna separata (Lepidoptera: Noctuidae).

    PubMed

    Cao, Budao; Bao, Wenhua; Wuriyanghan, Hada

    2017-02-01

    Mythimna separata walker (Lepidoptera: Noctuidae) is a polyphagous, migratory corn pest. Outbreak of M. separata has led to severe damage to corn production recently in China. RNAi (RNA interference) is a gene silencing technology applied both in model and non-model organisms, and it is especially useful for the latter in which the reverse genetic research tools are not available. RNAi approach was broadly investigated in many plant pathogens and was used for the generation of anti-pest transgenic plants. We are proposing to use this technology to silence M. separata endogenous genes, thereby, providing a biocontrol method for this insect. Feeding of dsRNAs for target Chitinase genes resulted in substantial decreases of their transcript levels in M. separata. Furthermore, silencing of target Chitinase genes led to phenotypic effects such as reduced body weight and increased mortality. Our study provided both reverse genetic research tool and potential control strategy for this insect species.

  7. Turnabout Is Fair Play: Herbivory-Induced Plant Chitinases Excreted in Fall Armyworm Frass Suppress Herbivore Defenses in Maize1[OPEN

    PubMed Central

    Alves, Patrick C.M.S.; Gaffoor, Iffa; Acevedo, Flor E.; Peiffer, Michelle; Jin, Shan; Han, Yang; Shakeel, Samina; Felton, Gary W.

    2016-01-01

    The perception of herbivory by plants is known to be triggered by the deposition of insect-derived factors such as saliva and oral secretions, oviposition materials, and even feces. Such insect-derived materials harbor chemical cues that may elicit herbivore and/or pathogen-induced defenses in plants. Several insect-derived molecules that trigger herbivore-induced defenses in plants are known; however, insect-derived molecules suppressing them are largely unknown. In this study, we identified two plant chitinases from fall armyworm (Spodoptera frugiperda) larval frass that suppress herbivore defenses while simultaneously inducing pathogen defenses in maize (Zea mays). Fall armyworm larvae feed in enclosed whorls of maize plants, where frass accumulates over extended periods of time in close proximity to damaged leaf tissue. Our study shows that maize chitinases, Pr4 and Endochitinase A, are induced during herbivory and subsequently deposited on the host with the feces. These plant chitinases mediate the suppression of herbivore-induced defenses, thereby increasing the performance of the insect on the host. Pr4 and Endochitinase A also trigger the antagonistic pathogen defense pathway in maize and suppress fungal pathogen growth on maize leaves. Frass-induced suppression of herbivore defenses by deposition of the plant-derived chitinases Pr4 and Endochitinase A is a unique way an insect can co-opt the plant’s defense proteins for its own benefit. It is also a phenomenon unlike the induction of herbivore defenses by insect oral secretions in most host-herbivore systems. PMID:26979328

  8. A DFT study of the unusual substrate-assisted mechanism of Serratia marcescens chitinase B reveals the role of solvent and mutational effect on catalysis.

    PubMed

    Jitonnom, Jitrayut; Sattayanon, Chanchai; Kungwan, Nawee; Hannongbua, Supa

    2015-03-01

    Serratia marcescens chitinase B (SmChiB) catalyzes the hydrolysis of β-1,4-glycosidic bond, via an unusual substrate-assisted mechanism, in which the substrate itself acts as an intramolecular nucleophile. In this paper, the catalytic mechanism of SmChiB has been investigated by using density functional theory. The details of two consecutive steps (glycosylation and deglycosylation), the structures and energetics along the whole catalytic reaction, and the roles of solvent molecules as well as some conserved SmChiB residues (Asp142, Tyr214, Asp215, and Arg294) during catalysis are highlighted. Our calculations show that the formation of the oxazolinium cation intermediate in the glycosylation step was found to be a rate-determining step (with a barrier of 23 kcal/mol), in line with our previous computational studies (Jitonnom et al., 2011, 2014). The solvent water molecules have a significant effect on a catalytic efficiency in the degycosylation step: the catalytic water is essentially placed in a perfect position for nucleophic attack by hydrogen bond network, lowering the barrier height of this step from 11.3 kcal/mol to 2.9 kcal/mol when more water molecules were introduced. Upon the in silico mutations of the four conserved residues, their mutational effects on the relative stability of the reaction intermediates and the computed energetics can be obtained by comparing with the wild-type results. Mutations of Tyr214 to Phe or Ala have shown a profound effect on the relative stability of the oxazolinium intermediate, emphasizing a direct role of this residue in destabilizing the intermediate. In line with the experiment that the D142A mutation leads to almost complete loss of SmChiB activity, this mutation greatly decreases the stability of the intermediate, resulting in a very large increase in the activation barrier up to 50 kcal/mol. The salt-bridges residues (Asp215 and Arg294) were also found to play a role in stabilizing the oxazolinium intermediate.

  9. A recessive mutation in the RUB1-conjugating enzyme, RCE1, reveals a requirement for RUB modification for control of ethylene biosynthesis and proper induction of basic chitinase and PDF1.2 in Arabidopsis.

    PubMed

    Larsen, Paul B; Cancel, Jesse D

    2004-05-01

    By screening etiolated Arabidopsis seedlings for mutants with aberrant ethylene-related phenotypes, we identified a mutant that displays features of the ethylene-mediated triple response even in the absence of ethylene. Further characterization showed that the phenotype observed for the dark-grown seedlings of this mutant is reversible by prevention of ethylene perception and is dependent on a modest increase in ethylene production correlated with an increase in 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) activity in the hypocotyl. Molecular characterization of leaves of the mutant revealed severely impaired induction of basic chitinase (chiB) and plant defensin (PDF)1.2 following treatment with jasmonic acid and/or ethylene. Positional cloning of the mutation resulted in identification of a 49-bp deletion in RCE1 (related to ubiquitin 1 (RUB1)-conjugating enzyme), which has been demonstrated to be responsible for covalent attachment of RUB1 to the SCF (Skpl Cdc 53 F-box) ubiquitin ligase complex to modify its activity. Our analyses with rce1-2 demonstrate a previously unknown requirement for RUB1 modification for regulation of ethylene biosynthesis and proper induction of defense-related genes in Arabidopsis.

  10. Exacerbation of Experimental Autoimmune Encephalomyelitis in the Absence of Breast Regression Protein-39/Chitinase 3-like-1

    PubMed Central

    Bonneh-Barkay, Dafna; Wang, Guoji; LaFramboise, William A.; Wiley, Clayton A.; Bissel, Stephanie J.

    2012-01-01

    We previously reported that YKL-40, the human analog of mouse breast regression protein-39 (BRP-39; chitinase 3-like 1), is elevated in the cerebrospinal fluid of patients with a variety of neuroinflammatory conditions, such as multiple sclerosis and traumatic brain injury. YKL-40 expression in the CNS was predominantly associated with reactive astrocytes in the vicinity of inflammatory lesions. Because previous studies have shown that reactive astrocytes play a critical role in limiting immune infiltration in the mouse model of experimental autoimmune encephalomyelitis (EAE), we explored the role of BRP-39 in regulating neuroinflammation in EAE. Using BRP-39-deficient mice (BRP-39−/−), we demonstrate the importance of BRP-39 in modulating the severity of clinical EAE and CNS neuroinflammation. At disease onset, absence of BRP-39 had little effect on clinical disease or lymphocytic infiltrate, but by 14 days post-immunization (dpi), differences in clinical scores were evident. By 28 dpi, BRP-39−/− mice showed more severe and persistent clinical disease than BRP-39+/+ controls. Histopathological evaluation showed that BRP-39−/− mice had more marked lymphocytic and macrophage infiltrates and gliosis vs. BRP-39+/+ mice. These findings support the role of BRP-39 expression in limiting immune cell infiltration into the CNS and offer a new target to modulate neuroinflammation. PMID:23041842

  11. Observation of the controlled assembly of preclick components in the in situ click chemistry generation of a chitinase inhibitor.

    PubMed

    Hirose, Tomoyasu; Maita, Nobuo; Gouda, Hiroaki; Koseki, Jun; Yamamoto, Tsuyoshi; Sugawara, Akihiro; Nakano, Hirofumi; Hirono, Shuichi; Shiomi, Kazuro; Watanabe, Takeshi; Taniguchi, Hisaaki; Sharpless, K Barry; Omura, Satoshi; Sunazuka, Toshiaki

    2013-10-01

    The Huisgen cycloaddition of azides and alkynes, accelerated by target biomolecules, termed "in situ click chemistry," has been successfully exploited to discover highly potent enzyme inhibitors. We have previously reported a specific Serratia marcescens chitinase B (SmChiB)-templated syn-triazole inhibitor generated in situ from an azide-bearing inhibitor and an alkyne fragment. Several in situ click chemistry studies have been reported. Although some mechanistic evidence has been obtained, such as X-ray analysis of [protein]-["click ligand"] complexes, indicating that proteins act as both mold and template between unique pairs of azide and alkyne fragments, to date, observations have been based solely on "postclick" structural information. Here, we describe crystal structures of SmChiB complexed with an azide ligand and an O-allyl oxime fragment as a mimic of a click partner, revealing a mechanism for accelerating syn-triazole formation, which allows generation of its own distinct inhibitor. We have also performed density functional theory calculations based on the X-ray structure to explore the acceleration of the Huisgen cycloaddition by SmChiB. The density functional theory calculations reasonably support that SmChiB plays a role by the cage effect during the pretranslation and posttranslation states of selective syn-triazole click formation.

  12. Study of the intercellular fluid of healthy Lupinus albus organs. Presence of a chitinase and a thaumatin-like protein.

    PubMed Central

    Regalado, A P; Ricardo, C P

    1996-01-01

    Proteins in the intercellular fluid (IF) of healthy Lupinus albus leaves were characterized. Silver staining of the proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed more than 30 polypeptides, with the major ones having a molecular mass lower than 36 kD. After amino-terminal amino acid sequence analysis, one of the major polypeptides, IF4, was shown to have no identity with any of the proteins present in the data bases. Two others, IF1 and IF3, showed identity with previously reported pathogenesis-related proteins, IF1 with an antifungal protein from Hordeum vulgare that belongs to the thaumatin family (PR-5 family), and IF3 with class III chitinase-lysozymes. IF3 was also present in the IF of stem and root and it represents the major polypeptide in the medium of L. albus cell-suspension cultures. The ubiquitous presence of this enzyme in healthy, nonstressed tissues of L. albus cannot be explained. PMID:8587984

  13. Escherichia coli can produce recombinant chitinase in the soil to control the pathogenesis by Fusarium oxysporum without colonization.

    PubMed

    Chung, Soohee; Kim, Sang-Dal

    2007-03-01

    Fusarium wilt of cucumbers was effectively controlled by Escherichia coli expressing an endochitinase gene (chiA), and the rate was as effective (60.0%) as the wildtype strain S. proteamaculans 3095 (55.0%) where the gene was cloned. However, live cells of soil inoculated E. coli host harboring the chiA gene did not proliferate but declined 100-fold from 108 CFU during the first week and showed less than 10 cells after day 14, suggesting that E. coli was able to express and produce the chitinase enzyme to the soil even as the population was gradually decreasing. Because the majority of the strains was alive for only a short period of time and the Fusarium-affected seedlings showed symptoms of wilting within 7-10 days, it seems that the pathogen control was decided early after the introduction of the biocontrol agent, eliminating the survival of the antagonist. These results indicated that soil inoculated E. coli could sufficiently express and produce the recombinant protein to control the pathogen, and root or soil colonization of the antagonist might not be a significant factor in determining the efficacy of biological control.

  14. Purification and characterization of Vibrio parahaemolyticus extracellular chitinase and chitin oligosaccharide deacetylase involved in the production of heterodisaccharide from chitin.

    PubMed

    Kadokura, K; Rokutani, A; Yamamoto, M; Ikegami, T; Sugita, H; Itoi, S; Hakamata, W; Oku, T; Nishio, T

    2007-05-01

    A chitin-degrading bacterial strain, KN1699, isolated from Yatsu dry beach (Narashino, Chiba Prefecture, Japan), was identified as Vibrio parahaemolyticus. Treatment of powdered chitin with crude enzyme solution prepared from the supernatant of KN1699 cultures yielded a disaccharide, beta-D-N-acetylglucosaminyl-(1,4)-D-glucosamine (GlcNAc-GlcN), as the primary chitin degradation product. The extracellular enzymes involved in the production of this heterodisaccharide, chitinase (Pa-Chi; molecular mass, 92 kDa) and chitin oligosaccharide deacetylase (Pa-COD; molecular mass, 46 kDa), were isolated from the crude enzyme solution, and their hydrolysis specificities were elucidated. These studies confirmed that (1) Pa-Chi hydrolyzes chitin to produce (GlcNAc)(2) and (2) Pa-COD hydrolyzes the acetamide group of reducing end GlcNAc residue of (GlcNAc)(2). These findings indicate that GlcNAc-GlcN is produced from chitin by the cooperative hydrolytic reactions of both Pa-Chi and Pa-COD.

  15. The phylogenetically conserved molluscan chitinase-like protein 1 (Cg-Clp1), homologue of human HC-gp39, stimulates proliferation and regulates synthesis of extracellular matrix components of mammalian chondrocytes.

    PubMed

    Badariotti, Fabien; Kypriotou, Magdalini; Lelong, Christophe; Dubos, Marie-Pierre; Renard, Emmanuelle; Galera, Philippe; Favrel, Pascal

    2006-10-06

    Members of chitinase-like proteins (CLPs) have attracted much attention because of their ability to promote cell proliferation in insects (imaginal disc growth factors) and mammals (YKL-40). To gain insights into the molecular processes underlying the physiological control of growth and development in Lophotrochozoa, we report here the cloning and biochemical characterization of the first Lophotrochozoan CLP from the oyster Crassostrea gigas (Cg-Clp1). Gene expression profiles monitored by real time quantitative reverse transcription-PCR in different adult tissues and during development support the involvement of this protein in the control of growth and development in C. gigas. Recombinant Cg-Clp1 demonstrates a strong affinity for chitin but no chitinolytic activity, as was described for the HC-gp39 mammalian homolog. Furthermore, transient expression of Cg-Clp1 in primary cultures of rabbit articular chondrocytes as well as the use of both purified recombinant protein and conditioned medium from Cg-Clp1-expressing rabbit articular chondrocytes established that Cg-Clp1 stimulates cell proliferation and regulates extracellular matrix component synthesis, showing for the first time a possible involvement of a CLP on type II collagen synthesis regulation. These observations together with the fact that Cg-Clp1 gene organization strongly resembles that of its mammalian homologues argue for an early evolutionary origin and a high conservation of this class of proteins at both the structural and functional levels.

  16. Characterization and expression analysis of a chitinase gene (PmChi-4) from black tiger shrimp (Penaeus monodon) under pathogen infection and ambient ammonia nitrogen stress.

    PubMed

    Zhou, Kaimin; Zhou, Falin; Huang, Jianhua; Yang, Qibin; Jiang, Song; Qiu, Lihua; Yang, Lishi; Zhu, Caiyan; Jiang, Shigui

    2017-03-01

    Chitinase is a multi-gene family, which play important physiological roles in crustaceans, involved in several biological processes, including digestion, molting and defense against viruses. In the present study, a chitinase-4 gene (PmChi-4) was cloned from Penaeus monodon by rapid amplification of cDNA ends (RACE). The full length of PmChi-4 cDNA was 2178 bp, including an 1815 bp open reading frame (ORF) which encoded 604 amino acid residues. The predicted PmChi-4 protein was 67.7 kDa and shared 61%-88% identity with the type of Chi-4s from other crustaceans. Quantitative real-time (qRT-PCR) analysis indicated that PmChi-4 was expressed ubiquitously with the high expression level in hepatopancreas. PmChi-4 was expressed throughout the whole larvae stages, and the highest level of PmChi-4 transcripts was detected at Mysis3 stage, which indicated that PmChi-4 may be involved in larval metamorphosis. In order to know whether PmChi-4 was related to the immune response of shrimp, Streptococcus agalactiae and Vibrio harveyi were chosen to challenge the shrimp, PmChi-4 transcripts were significantly increased and reached to the maximum at 6 h in hepatopancreas and at 12 h in gill, respectively. The results suggested that PmChi-4 participated in the immune defenses to pathogen infection. Besides, the ammonia nitrogen stress treatment was also carried out, PmChi-4 transcripts were significantly decreased in hepatopancreas and gill and the result showed that PmChi-4 may be involved in ammonia nitrogen stress in P. monodon. Overall, our present study lay a foundation for further research into the biological function and regulation of chitinase in P. monodon.

  17. The impact of acute aerobic exercise on chitinase 3-like protein 1 and intelectin-1 expression in obesity.

    PubMed

    Huang, Chun-Jung; Slusher, Aaron L; Whitehurst, Michael; Wells, Marie; Maharaj, Arun; Shibata, Yoshimi

    2016-01-01

    Chitinase 3-like 1 (CHI3L1) and intelectin 1 (ITLN-1) recognize microbial N-acetylglucosamine polymer and galactofuranosyl carbohydrates, respectively. Both lectins are highly abundant in plasma and seem to play pro- and anti-inflammatory roles, respectively, in obesity and inflammatory-related illnesses. The aim of this study was to examine whether plasma levels of these lectins in obese subjects are useful for monitoring inflammatory conditions immediately influenced by acute aerobic exercise. Plasma interleukin-6, a pro-inflammatory cytokine, was also examined. Twenty-two (11 obese and 11 normal-weight) healthy subjects, ages 18-30 years, were recruited to perform a 30 min bout of acute aerobic exercise at 75% VO2max. We confirmed higher baseline levels of plasma CHI3L1, but lower ITLN-1, in obese subjects than in normal-weight subjects. The baseline levels of CHI3L1 were negatively correlated with cardiorespiratory fitness (relative VO2max). However, when controlled for BMI, the relationship between baseline level of CHI3L1 and relative VO2max was no longer observed. While acute aerobic exercise elicited an elevation in these parameters, we found a lower ITLN-1 response in obese subjects compared to normal-weight subjects. Our study clearly indicates that acute aerobic exercise elicits a pro-inflammatory response (e.g. CHI3L1) with a lower anti-inflammatory effect (e.g. ITLN-1) in obese individuals. Furthermore, these lectins could be predictors of outcome of exercise interventions in obesity-associated inflammation.

  18. Overexpression of a Chitinase Gene from Trichoderma asperellum Increases Disease Resistance in Transgenic Soybean.

    PubMed

    Zhang, Fuli; Ruan, Xianle; Wang, Xian; Liu, Zhihua; Hu, Lizong; Li, Chengwei

    2016-12-01

    In the present study, a chi gene from Trichoderma asperellum, designated Tachi, was cloned and functionally characterized in soybean. Firstly, the effects of sodium thiosulfate on soybean Agrobacterium-mediated genetic transformation with embryonic tip regeneration system were investigated. The transformation frequency was improved by adding sodium thiosulfate in co-culture medium for three soybean genotypes. Transgenic soybean plants with constitutive expression of Tachi showed increased resistance to Sclerotinia sclerotiorum compared to WT plants. Meanwhile, overexpression of Tachi in soybean exhibited increased reactive oxygen species (ROS) level as well as peroxidase (POD) and catalase (SOD) activities, decreased malondialdehyde (MDA) content, along with diminished electrolytic leakage rate after S. sclerotiorum inoculation. These results suggest that Tachi can improve disease resistance in plants by enhancing ROS accumulation and activities of ROS scavenging enzymes and then diminishing cell death. Therefore, Tachi represents a candidate gene with potential application for increasing disease resistance in plants.

  19. Identification of fungus-responsive cis-acting element in the promoter of Brassica juncea chitinase gene, BjCHI1.

    PubMed

    Gao, Ying; Zan, Xin-Li; Wu, Xue-Feng; Yao, Lei; Chen, Yu-Ling; Jia, Shuang-Wei; Zhao, Kai-Jun

    2014-02-01

    Chitinases are a group of pathogenesis-related proteins. The Brassica juncea chitinase gene BjCHI1 is highly inducible by pathogenic fungal infection, suggesting that the promoter of BjCHI1 might contain specific cis-acting element responsive to fungal attack. To identify the fungus-responsive element in BjCHI1 promoter (BjC-P), a series of binary plant transformation vectors were constructed by fusing the BjC-P or its deletion-derivatives to β-glucuronidase (GUS) reporter gene. Expression of the GUS reporter gene was systematically assayed by a transient gene expression system in Nicotiana benthamiana leaves treated with fungal elicitor Hexa-N-Acetyl-Chitohexaose, as well as in transgenic Arabidopsis plants inoculated with fungus Botrytis cinerea. The histochemical and quantitative GUS assays showed that the W-box-like element (GTAGTGACTCAT) in the region (-668 to -657) was necessary for the fungus-response, although there were another five W-box-like elements in BjC-P. In addition, gain-of-function analysis demonstrated that the fragment (-409 to -337) coupled to the W-box-like element was needed for full magnitude of the fungal induction. These results revealed the existence of a novel regulation mechanism of W-box-like element involved in plant pathogenic resistance, and will benefit the potential application of BjC-P in engineering crops.

  20. The Role of CHI3L1 (Chitinase-3-Like-1) in the Pathogenesis of Infections in Burns in a Mouse Model

    PubMed Central

    Bohr, Stefan; Patel, Suraj J.; Vasko, Radovan; Shen, Keyue; Golberg, Alexander; Berthiaume, Francois; Yarmush, Martin L.

    2015-01-01

    In severe burn injury the unique setting of a depleted, dysfunctional immune system along with a loss of barrier function commonly results in opportunistic infections that eventually proof fatal. Unfortunately, the dynamic sequence of bacterial contamination, colonization and eventually septic invasion with bacteria such as Pseudomonas species is still poorly understood although a limiting factor in clinical decision making. Increasing evidence supports the notion that inhibition of bacterial translocation into the wound site may be an effective alternative to prevent infection. In this context we investigated the role of the mammalian Chitinase-3-Like-1 (CHI3L1) non-enyzmatic protein predominately expressed on epithelial as well as innate immune cells as a potential bacterial-translocation-mediating factor. We show a strong trend that a modulation of chitinase expression is likely to be effective in reducing mortality rates in a mouse model of burn injury with superinfection with the opportunistic PA14 Pseudomonas strain, thus demonstrating possible clinical leverage. PMID:26528713

  1. Preliminary Study on Gene Expression of Chitinase-Like Cytokines in Human Airway Epithelial Cell Under Chitin and Chitosan Microparticles Treatment.

    PubMed

    Alimohammadi, Masumeh; Yeganeh, Farshid; Haji Molla Hoseini, Mostafa

    2016-06-01

    Small-sized chitin and chitosan microparticles (MPs) reduce allergic inflammation. We examined the capacity of these glycans to stimulate A549 human airway epithelial cells to determine the feasibility of using of these glycans as allergic therapeutic modality. A549 cells were treated with MPs and then expressions levels of chitinase domain-containing 1 (CHID1) and chitinase 3-like 1 (CHI3L1) genes were determined by quantitative real-time PCR. IL-6 production was measured by ELISA. Chitin MPs resulted in upregulation of CHI3L1 expression by 35.7-fold while mRNA expression did not change with chitosan MPs. Compared to the untreated group, production of IL-6 was significantly decreased in the chitosan MPs-treated group, but chitin MPs treatment cause elevation of IL-6 level. This study demonstrates that chitin potently induces CHI3L1 expression, but chitosan is relatively inert. This effect and inhibition of pro-inflammatory cytokine (IL-6) suggest that chitosan MPs may possess more potential for therapeutic uses in human airway allergic inflammation.

  2. Interaction of di-N-acetylchitobiosyl moranoline with a family GH19 chitinase from moss, Bryum coronatum.

    PubMed

    Shinya, Shoko; Urasaki, Atsushi; Ohnuma, Takayuki; Taira, Toki; Suzuki, Akari; Ogata, Makoto; Usui, Taichi; Lampela, Outi; Juffer, André H; Fukamizo, Tamo

    2014-10-01

    Tri-N-acetylchitotriosyl moranoline, (GlcNAc)3-M, was previously shown to strongly inhibit lysozyme (Ogata M, Umemoto N, Ohnuma T, Numata T, Suzuki A, Usui T, Fukamizo T. 2013. A novel transition-state analogue for lysozyme, 4-O-β-tri-Nacetylchitotriosyl moranoline, provided evidence supporting the covalent glycosyl-enzyme intermediate. J Biol Chem. 288:6072-6082). The findings prompted us to examine the interaction of di-N-acetylchitobiosyl moranoline, (GlcNAc)2-M, with a family GH19 chitinase from moss, Bryum coronatum (BcChi19A). Thermal unfolding experiments using BcChi19A and the catalytic acid-deficient mutant (BcChi19A-E61A) revealed that the transition temperature (Tm) was elevated by 4.3 and 5.8°C, respectively, upon the addition of (GlcNAc)2-M, while the chitin dimer, (GlcNAc)2, elevated Tm only by 1.0 and 1.4°C, respectively. By means of isothermal titration calorimetry, binding free energy changes for the interactions of (GlcNAc)3 and (GlcNAc)2-M with BcChi19A-E61A were determined to be -5.2 and -6.6 kcal/mol, respectively, while (GlcNAc)2 was found to interact with BcChi19A-E61A with markedly lower affinity. nuclear magnetic resonance titration experiments using (15)N-labeled BcChi19A and BcChi19A-E61A revealed that both (GlcNAc)2 and (GlcNAc)2-M interact with the region surrounding the catalytic center of the enzyme and that the interaction of (GlcNAc)2-M is markedly stronger than that of (GlcNAc)2 for both enzymes. However, (GlcNAc)2-M was found to moderately inhibit the hydrolytic reaction of chitin oligosaccharides catalyzed by BcChi19A (IC50 = 130-620 μM). A molecular dynamics simulation of BcChi19A in complex with (GlcNAc)2-M revealed that the complex is quite stable and the binding mode does not significantly change during the simulation. The moranoline moiety of (GlcNAc)2-M did not fit into the catalytic cleft (subsite -1) but was rather in contact with subsite +1. This situation may result in the moderate inhibition toward the BcChi19A

  3. Chitin stimulates expression of acidic mammalian chitinase and eotaxin-3 by human sinonasal epithelial cells in vitro

    PubMed Central

    Lalaker, Ashley; Nkrumah, Louis; Lee, Won-Kyung; Ramanathan, Murugappan; Lane, Andrew P.

    2010-01-01

    Background Sinonasal epithelial cells participate in host defense by initiating innate immune mechanisms against potential pathogens. Antimicrobial innate mechanisms have been shown to involve Th1-like inflammatory responses. Although epithelial cells can also be induced by Th2 cytokines to express proeosinophilic mediators, no environmental agents have been identified that promote this effect. Methods Human sinonasal epithelial cells from patients with chronic rhinosinusitis with nasal polyps (CRSwNPs) and controls were harvested and grown in primary culture. Cell cultures were exposed to a range of concentrations of chitin for 24 hours, and mRNA for acidic mammalian chitinase (AMCase), eotaxin-3, and thymic stromal-derived lymphopoietin (TSLP) were assessed. Other cultures were exposed to interleukin 4 (IL- 4) alone and in combination with dust-mite antigen (DMA) for 36 hours. Extracted mRNA and cell culture supernatant were analyzed for expression of AMCase and eotaxin-3. Results Chitin induced a dose-dependent expression of AMCase and eotaxin-3 mRNA but not TSLP. Patients with recalcitrant CRSwNPs showed lower baseline expression of AMCase when compared with treatment-responsive CRSwNP and less induction of AMCase expression by chitin. DMA did not directly induce expression of AMCase or eotaxin-3. Expression of eotaxin-3 was stimulated by IL-4 and further enhanced with the addition of DMA. Levels of AMCase were not significantly affected by either IL-4 or DMA exposure. In some cases, the combination of IL-4 and DMA was able to induce AMCase expression in cell cultures not producing AMCase at baseline. Conclusion The abundant biopolymer chitin appears to be recognized by a yet uncharacterized receptor on sinonasal epithelial cells. Chitin stimulates production of AMCase and eotaxin-3, two pro-Th2 effector proteins. This finding suggests the existence of a novel innate immune pathway for local defense against chitin-containing organisms in the sinonasal tract

  4. Genetic ontogeny of pancreatic enzymes in Labrus bergylta larvae and the effect of feed type on enzyme activities and gene expression.

    PubMed

    Hansen, Truls Wergeland; Folkvord, Arild; Grøtan, Espen; Sæle, Øystein

    2013-03-01

    A newly cultivated wrasse species, Labrus bergylta, have shown great potential for use in Atlantic salmon (Salmo salar) farms in the battle against sea lice (Lepeoptheirus salmonis) infections. Hatchery reared L. bergylta were studied from 2 to 55 DPH to examine the molecular basis of digestive ontogeny related to the pancreas. An isolated feeding trial was performed on 27-34 DPH larvae to compare the effect of diet on enzyme activity and the possible exogenous contribution by live feed. The following genes coding for key pancreatic enzymes were analyzed by qPCR: trypsin, Cyp7 A1, BAL, sPLA(2) 1B, amylase and pancreatic chitinase. Enzyme activity was measured on trypsin, neutral lipase, sPLA(2), amylase and chitinase in fed and unfed larvae. We did not observe any effects of the formulated diet v.s. rotifers on enzyme activities of neutral lipase, chitinase and sPLA(2). However, a probable feed-dependency was observed at a transcriptional level, where rotifers seem to stimulate upregulation. The regulation of BAL was the only exception, where an upregulation was observed after weaning both in the ontogeny series and the experimental part. Our data on pancreatic chitinase and amylase mRNA levels suggest the importance of carbohydrates in the diet of early larval and juvenile L. bergylta.

  5. Construction of a Streptomyces lydicus A01 transformant with a chit42 gene from Trichoderma harzianum P1 and evaluation of its biocontrol activity against Botrytis cinerea.

    PubMed

    Wu, Qiong; Bai, Linquan; Liu, Weicheng; Li, Yingying; Lu, Caige; Li, Yaqian; Fu, Kehe; Yu, Chuanjin; Chen, Jie

    2013-04-01

    Streptomyces lydicus A01 and Trichoderma harzianum P1 are potential biocontrol agents of fungal diseases in plants. S. lydicus A01 produces natamycin to bind the ergosterol of the fungal cell membrane and inhibits the growth of Botrytis cinerea. T. harzianum P1, on the other hand, features high chitinase activity and decomposes the chitin in the cell wall of B. cinerea. To obtain the synergistic biocontrol effects of chitinase and natamycin on Botrytis cinerea, this study transformed the chit42 gene from T. harzianum P1 to S. lydicus A01. The conjugal transformant (CT) of S. lydicus A01 with the chit42 gene was detected using polymerase chain reaction (PCR). Associated chitinase activity and natamycin production were examined using the 3, 5-dinitrosalicylic acid (DNS) method and ultraviolet spectrophotometry, respectively. The S. lydicus A01-chit42 CT showed substantially higher chitinase activity and natamycin production than its wild type strain (WT). Consequently, the biocontrol effects of S. lydicus A01-chit42 CT on B. cinerea, including inhibition to spore germination and mycelial growth, were highly improved compared with those of the WT. Our research indicates that the biocontrol effect of Streptomyces can be highly improved by transforming the exogenous resistance gene, i.e. chit42 from Trichoderma, which not only enhances the production of antibiotics, but also provides a supplementary function by degrading the cell walls of the pathogens.

  6. The presence and role of bacterial quorum sensing in activated sludge

    PubMed Central

    Chong, Grace; Kimyon, Onder; Rice, Scott A.; Kjelleberg, Staffan; Manefield, Mike

    2012-01-01

    Summary Activated sludge used for wastewater treatment globally is composed of a high‐density microbial community of great biotechnological significance. In this study the presence and purpose of quorum sensing via N‐acylated‐l‐homoserine lactones (AHLs) in activated sludge was explored. The presence of N‐heptanoyl‐l‐homoserine lactone in organic extracts of sludge was demonstrated along with activation of a LuxR‐based AHL monitor strain deployed in sludge, indicating AHL‐mediated gene expression is active in sludge flocculates but not in the bulk aqueous phase. Bacterial isolates from activated sludge were screened for AHL production and expression of phenotypes commonly but not exclusively regulated by AHL‐mediated gene transcription. N‐acylated‐l‐homoserine lactone and exoenzyme production were frequently observed among the isolates. N‐acylated‐l‐homoserine lactone addition to sludge upregulated chitinase activity and an AHL‐ and chitinase‐producing isolate closely related to Aeromonas hydrophila was shown to respond to AHL addition with upregulation of chitinase activity. N‐acylated‐l‐homoserine lactones produced by this strain were identified and genes ahyI/R and chiA, encoding AHL production and response and chitinase activity respectively, were sequenced. These experiments provide insight into the relationship between AHL‐mediated gene expression and exoenzyme activity in activated sludge and may ultimately create opportunities to improve sludge performance. PMID:22583685

  7. Extracellular enzyme activity at the air-water interface of an estuarine lake

    NASA Astrophysics Data System (ADS)

    Mudryk, Z. J.; Skórczewski, P.

    2004-01-01

    Variations in hydrolytic activity of eight extracellular enzymes in surface and subsurface waters in estuarine Lake Gardno were measured. The ranking of potential activity rates of the assayed enzymes was the same in both surface and subsurface water, i.e. esterase > lipase > aminopeptidase > phosphatase > β-glucosidase > α-glucosidase > chitinase > β-lactosidase. The vertical activity profiles show that esterase, aminopeptidase, α-glucosidase, β-glucosidase and β-lactosidase reached the highest values in surface layer, whereas lipase, phosphatase and chitinase showed maximum activity in subsurface water. Significant differences in enzyme activity between different parts of the studied lake were demonstrated, with higher values in the seawater zone, and lower values in the freshwater zone.

  8. Anthelmintic activity of Leucaena leucocephala protein extracts on Haemonchus contortus.

    PubMed

    Soares, Alexandra Martins dos Santos; de Araújo, Sandra Alves; Lopes, Suzana Gomes; Costa Junior, Livio Martins

    2015-01-01

    The objective of this study was to evaluate the effects of protein extracts obtained from the plant Leucaena leucocephala on the nematode parasite Haemonchus contortus. The seeds, shell and cotyledon of L. leucocephala were separated and their proteins extracted using a sodium phosphate buffer, and named as TE (total seed extract), SE (shell extract) and CE (cotyledon extract). Soluble protein content, protease, protease inhibitory and chitinase activity assays were performed. Exsheathment inhibition of H. contortus larvae were performed at concentrations of 0.6 mg mL-1, and egg hatch assays were conducted at protein concentrations of 0.8, 0.4, 0.2, 0.1 and 0.05 mg mL-1. The effective concentration for 50% hatching inhibition (EC50) was estimated by probit. Different proportions of soluble proteins, protease and chitinase were found in TE and CE. Protease inhibitory activity was detected in all extracts. The EC50 of the CE and TE extracts were 0.48 and 0.33 mg mL-1, respectively. No ovicidal effects on H. contortus were detected in SE extracts, and none of the protein extracts demonstrated larvicidal effects on H. contortus. We therefore conclude that protein extracts of L. leucocephala had a detrimental effect on nematode eggs, which can be correlated with the high protease and chitinase activity of these extracts.

  9. miR-71 and miR-263 Jointly Regulate Target Genes Chitin synthase and Chitinase to Control Locust Molting

    PubMed Central

    Jiang, Feng; Song, Tianqi; Wang, Huimin; Liu, Qing; Zhang, Jie; Zhang, Jianzhen; Kang, Le

    2016-01-01

    Chitin synthase and chitinase play crucial roles in chitin biosynthesis and degradation during insect molting. Silencing of Dicer-1 results in reduced levels of mature miRNAs and severely blocks molting in the migratory locust. However, the regulatory mechanism of miRNAs in the molting process of locusts has remained elusive. In this study, we found that in chitin metabolism, two crucial enzymes, chitin synthase (CHS) and chitinase (CHT) were regulated by miR-71 and miR-263 during nymph molting. The coding sequence of CHS1 and the 3’-untranslated region of CHT10 contain functional binding sites for miR-71 and miR-263, respectively. miR-71/miR-263 displayed cellular co-localization with their target genes in epidermal cells and directly interacted with CHS1 and CHT10 in the locust integument, respectively. Injections of miR-71 and miR-263 agomirs suppressed the expression of CHS1 and CHT10, which consequently altered chitin production of new and old cuticles and resulted in a molting-defective phenotype in locusts. Unexpectedly, reduced expression of miR-71 and miR-263 increased CHS1 and CHT10 mRNA expression and led to molting defects similar to those induced by miRNA delivery. This study reveals a novel function and balancing modulation pattern of two miRNAs in chitin biosynthesis and degradation, and it provides insight into the underlying molecular mechanisms of the molting process in locusts. PMID:27532544

  10. In-vitro selection of Vitis vinifera 'Chardonnay' with Elsinoe ampelina culture filtrate is accompanied by fungal resistance and enhanced secretion of chitinase.

    PubMed

    Jayasankar, S; Li, Z; Gray, D J

    2000-07-01

    Proembryogenic masses of grapevine (Vitis vinifera L.) 'Chardonnay' (clone 02Ch) were exposed to the culture filtrate of Elsinoe ampelina (deBary) Shear, the causal agent of anthracnose disease. After four or five cycles of recurrent in-vitro selection with medium containing 40% fungal culture filtrate, putative resistant lines RC1 and RC2 respectively, were established. The selected lines inhibited the growth of E. ampelina and Fusarium oxysporium (Schlecht.) (isolated from watermelon) in a dual-culture assay and reduced the growth of mycelium on a conditioned-medium test, thus suggesting the involvement of extracellular compounds in resistance. Sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis of extracellular proteins from spent suspension-culture medium showed enhanced secretion of new proteins by selected lines. A 36-kDa protein was immunodetected by a chitinase antiserum. This chitinase continued to express constitutively in differentiated somatic embryos and also in the intercellular fluids of plants regenerated from the selected lines. Somatic embryos from selected lines grew uninhibitedly in a medium containing 40% fungal culture filtrate, whereas non-selected (control) somatic embryos became necrotic and died within a few days. Plants regenerated from selected lines exhibited resistance to infection by E. ampelina in both greenhouse tests and detached leaf bioassays. Results suggest that embryogenic cells can be selected for resistance following in-vitro selection, resulting in resistant plants. Whether or not resistant cells pre-existed in the original embryogenic culture or were induced by the selection pressure could not be determined.

  11. Latex-allergic patients sensitized to the major allergen hevein and hevein-like domains of class I chitinases show no increased frequency of latex-associated plant food allergy

    PubMed Central

    Radauer, Christian; Adhami, Farzaneh; Fürtler, Irene; Wagner, Stefan; Allwardt, Dorothee; Scala, Enrico; Ebner, Christof; Hafner, Christine; Hemmer, Wolfgang; Mari, Adriano; Breiteneder, Heimo

    2011-01-01

    Allergies to certain fruits such as banana, avocado, chestnut and kiwi are described in 30–70% of latex-allergic patients. This association is attributed to the cross-reactivity between the major latex allergen hevein and hevein-like domains (HLDs) from fruit class I chitinases. We aimed to assess the extent of cross-reactivity between hevein and HLDs using sera from latex-allergic patients with and without plant food allergy. Hevein and HLDs of latex, banana, and avocado chitinases were expressed in Escherichia coli as fusion proteins with the maltose-binding protein and purified by affinity chromatography. IgE binding to these proteins was studied in sera from 59 latex-allergic patients and 20 banana-allergic patients without latex allergy by ELISA and ELISA inhibition. Additionally, 16,408 allergic patients’ sera were tested for IgE binding to hevein, latex chitinase, and wheat germ agglutinin using an allergen microarray. Hevein-specific IgE was detected in 34/59 (58%) latex-allergic patients’ sera. HLDs of latex, banana, and avocado chitinases were recognized by 21 (36%), 20 (34%), and 9 (15%) sera, respectively. In contrast, only one of 20 banana-allergic patients without latex allergy was sensitized to chitinase HLDs. In most tested latex-allergic patients’ sera, IgE binding to hevein was only partially reduced by preincubation with HLDs. Among hevein-sensitized, latex-allergic patients, the percentage of plant food allergy (15/34 = 44%) was equal to latex-allergic patients without hevein sensitization (11/25 = 44%). In the general allergic population, 230 of 16,408 sera (1.4%) reacted to hevein and/or a hevein-like allergen. Of these, 128 sera showed an isolated sensitization to hevein, whereas only 17 bound to latex chitinase or wheat germ agglutinin without hevein sensitization. In conclusion, the IgE response to HLDs is elicited by hevein as sensitizing allergen in most cases. Despite considerable cross-reactivity between these allergens, no

  12. Influence of soil reaction on diversity and antifungal activity of fluorescent pseudomonads in crop rhizospheres.

    PubMed

    Verma, Rajni; Naosekpam, Ajit Singh; Kumar, Sanjay; Prasad, Ramdeen; Shanmugam, V

    2007-05-01

    The diversity and antifungal activity of fluorescent pseudomonads isolated from rhizospheres of tea, gladiolus, carnation and black gram grown in acidic soils with similar texture and climatic conditions were studied. Biochemical characterisation including antibiotic resistance assay, RAPD and PCR-RFLP studies revealed a largely homogenous population. At soil pH (5.2), the isolates exhibited growth with varying levels of siderophore production, irrespective of crop rhizospheres. Two isolates with maximum chitinase production showed antagonism. The bacterial populations in general lacked the ability to produce deleterious traits such as cellulase, pectinase and hydrogen cyanide. However, increased pH levels beyond 5.2 caused reduction in metabolite production with reduced antifungal activity. The homogeneity of the bacterial population irrespective of crop rhizospheres together with decreased secondary metabolite production at higher pH levels reinstated the importance of soil over host plant in influencing rhizosphere populations. The studies also yielded acid tolerant chitinase producing antagonistic fluorescent pseudomonads.

  13. Visualization of enzyme activities inside earthworm pores

    NASA Astrophysics Data System (ADS)

    Hoang, Duyen; Razavi, Bahar S.

    2015-04-01

    In extremely dynamic microhabitats as bio-pores made by earthworm, the in situ enzyme activities are assumed as a footprint of complex biotic interactions. Our study focused on the effect of earthworm on the enzyme activities inside bio-pores and visualizing the differences between bio-pores and earthworm-free soil by zymography technique (Spohn and Kuzyakov, 2013). For the first time, we aimed at quantitative imaging of enzyme activities in bio-pores. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). After two weeks when bio-pore systems were formed by earthworms, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine-aminopeptidase, and phosphatase. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. However, the differences in activity of cellobiohydrolase and leucine aminopeptidase between bio-pore and bulk soil were less pronounced. This demonstrated an applicability of zymography approach to monitor and to distinguish the in situ activity of hydrolytic enzymes in soil biopores.

  14. Evaluation of Mosquito Repellent Activity of Isolated Oleic Acid, Eicosyl Ester from Thalictrum javanicum

    PubMed Central

    Gurunathan, Abinaya; Senguttuvan, Jamuna; Paulsamy, S.

    2016-01-01

    To evaluate the traditional use, the mosquito repellent property of Thalictrum javanicum and to confirm the predicted larvicidal activity of the isolated compound, oleic acid, eicosyl ester from its aerial parts by PASS software, the present study was carried out using 4th instar stage larvae of the mosquitoes, Aedes aegypti (dengue vector) and Culex quinquefasciatus (filarial vector). Insecticidal susceptibility tests were conducted and the mortality rate was observed after 24 h exposure. The chitinase activity of isolated compound was assessed by using purified β-N-acetyl glucosaminidase (chitinase). Ecdysone 20-monooxygenase assay (radioimmuno assay) was made using the same larval stage of A. aegyptiand C. quinquefasciatus. The results were compared with the crude methanol extract of the whole plant. The isolated compound, oleic acid, eicosyl ester was found to be the most effective larvicide against A. aegypti (LC50/24 h -8.51 ppm) and C. quinquefasciatus (LC50/24 h - 12.5 ppm) than the crude methanol extract (LC50/24 h - 257.03 ppm and LC50/24 h - 281.83 ppm, respectively). The impact of oleic acid, eicosyl ester on reducing the activity of chitinase and ecdysone 20-monooxygenase was most prominent in both the target species, A. aegyptiand C. quinquefasciatus than the control. The results therefore suggest that the compound, oleic acid, eicosyl ester from Thalictrum javanicum may be considered as a potent source of mosquito larvicidal property. PMID:27168688

  15. Agrobacterium tumefaciens-mediated transformation of taro (Colocasia esculenta (L.) Schott) with a rice chitinase gene for improved tolerance to a fungal pathogen Sclerotium rolfsii.

    PubMed

    He, Xiaoling; Miyasaka, Susan C; Fitch, Maureen M M; Moore, Paul H; Zhu, Yun J

    2008-05-01

    Taro (Colocasia esculenta) is one of the most important crops in the Pacific Islands, however, taro yields have been declining in Hawaii over the past 30 years partly due to diseases caused by oomycete and fungal pathogens. In this study, an efficient Agrobacterium tumefaciens-mediated transformation method for taro is first reported. In total, approximately 200 pieces (8 g) of embryogenic calluses were infected with the super-virulent A. tumefaciens strain EHA105 harboring the plant transformation plasmid pBI121/ricchi11 that contains the rice chitinase gene ricchi11. The presence and expression of the transgene ricchi11 in six independent transgenic lines was confirmed using polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR). Southern blot analysis of the six independent lines indicated that three out of six (50%) had integrated a single copy of the transgene, and the other three lines had two or three copies of the transgene. Compared to the particle bombardment transformation of taro method, which was used in the previous studies, the Agrobacterium-mediated transformation method obtained 43-fold higher transformation efficiency. In addition, these six transgenic lines via Agrobacterium may be more effective for transgene expression as a result of single-copy or low-copy insertion of the transgene than the single line with multiple copies of the transgene via particle bombardment. In a laboratory bioassay, all six transgenic lines exhibited increased tolerance to the fungal pathogen Sclerotium rolfsii, ranging from 42 to 63% reduction in lesion expansion.

  16. CHITINASE-LIKE1/POM-POM1 and Its Homolog CTL2 Are Glucan-Interacting Proteins Important for Cellulose Biosynthesis in Arabidopsis[W][OA

    PubMed Central

    Sánchez-Rodríguez, Clara; Bauer, Stefan; Hématy, Kian; Saxe, Friederike; Ibáñez, Ana Belén; Vodermaier, Vera; Konlechner, Cornelia; Sampathkumar, Arun; Rüggeberg, Markus; Aichinger, Ernst; Neumetzler, Lutz; Burgert, Ingo; Somerville, Chris; Hauser, Marie-Theres; Persson, Staffan

    2012-01-01

    Plant cells are encased by a cellulose-containing wall that is essential for plant morphogenesis. Cellulose consists of β-1,4-linked glucan chains assembled into paracrystalline microfibrils that are synthesized by plasma membrane–located cellulose synthase (CESA) complexes. Associations with hemicelluloses are important for microfibril spacing and for maintaining cell wall tensile strength. Several components associated with cellulose synthesis have been identified; however, the biological functions for many of them remain elusive. We show that the chitinase-like (CTL) proteins, CTL1/POM1 and CTL2, are functionally equivalent, affect cellulose biosynthesis, and are likely to play a key role in establishing interactions between cellulose microfibrils and hemicelluloses. CTL1/POM1 coincided with CESAs in the endomembrane system and was secreted to the apoplast. The movement of CESAs was compromised in ctl1/pom1 mutant seedlings, and the cellulose content and xyloglucan structures were altered. X-ray analysis revealed reduced crystalline cellulose content in ctl1 ctl2 double mutants, suggesting that the CTLs cooperatively affect assembly of the glucan chains, which may affect interactions between hemicelluloses and cellulose. Consistent with this hypothesis, both CTLs bound glucan-based polymers in vitro. We propose that the apoplastic CTLs regulate cellulose assembly and interaction with hemicelluloses via binding to emerging cellulose microfibrils. PMID:22327741

  17. Co-bombardment, integration and expression of rice chitinase and thaumatin-like protein genes in barley (Hordeum vulgare cv. Conlon).

    PubMed

    Tobias, Dennis J; Manoharan, Muthusamy; Pritsch, Clara; Dahleen, Lynn S

    2007-05-01

    Pathogenesis-related (PR) proteins associated with degradation of structural components of pathogenic filamentous fungi were overexpressed in the two-rowed malting barley (Hordeum vulgare L.) cultivar Conlon. Transgenes were introduced by co-bombardment with two plasmids, one carrying a rice (Oryza sativa L.) chitinase gene (chi11) and another carrying a rice thaumatin-like protein gene (tlp). Each gene was under the control of the maize ubiquitin (Ubi1) promoter. Fifty-eight primary transformants from three independent transformation events were regenerated. T(1) plants with high rice chi11 and tlp protein expression levels were advanced to identify T(2) homozygotes by herbicide spray and subjected to further molecular analyses. T(3) progeny from one event (E2) had stable integration and expression of the rice chi11 and tlp while those from the other events (E1 and E3) showed stable integration only of tlp. The successful production of these lines overexpressing the antifungal chi and tlp proteins provides materials to test the effects of these genes on a variety of fungal diseases that attack barley and to serve as potential additional sources of disease resistance.

  18. Expression of CHI3L1 and CHIT1 in Osteoarthritic Rat Cartilage Model. A Morphological Study

    PubMed Central

    Di Rosa, M.; Szychlinska, M.A.; Tibullo, D.; Malaguarnera, L.

    2014-01-01

    Osteoarthritis is a degenerative joint disease, which affects millions of people around the world. It occurs when the protective cartilage at the end of bones wears over time, leading to loss of flexibility of the joint, pain and stiffness. The cause of osteoarthritis is unknown, but its development is associated with different factors, such as metabolic, genetic, mechanical and inflammatory ones. In recent years the biological role of chitinases has been studied in relation to different inflammatory diseases and more in particular the elevated levels of human cartilage glycoprotein 39 (CHI3L1) and chitotriosidase (CHIT1) have been reported in a variety of diseases including chronic inflammation and degenerative disorders. The aim of this study was to investigate, by immunohistochemistry, the distribution of CHI3L1 and CHIT1 in osteoarthritic and normal rat articular cartilage, to discover their potential role in the development of this disease. The hypothesis was that the expression of chitinases could increase in OA disease. Immunohistochemical analysis showed that CHI3L1 and CHIT1 staining was very strong in osteoarthritic cartilage, especially in the superficial areas of the cartilage most exposed to mechanical load, while it was weak or absent in normal cartilage. These findings suggest that these two chitinases could be functionally associated with the development of osteoarthritis and could be used as markers, so in the future they could have a role in the daily clinical practice to stage the severity of the disease. However, the longer-term in vivoand in vitro studies are needed to understand the exact mechanism of these molecules, their receptors and activities on cartilage tissue. PMID:25308850

  19. Enzyme activities along a latitudinal transect in Western Siberia

    NASA Astrophysics Data System (ADS)

    Schnecker, Jörg; Wild, Birgit; Eloy Alves, Ricardo J.; Gentsch, Norman; Gittel, Antje; Knoltsch, Anna; Lashchinskiy, Nikolay; Mikutta, Robert; Takriti, Mounir; Richter, Andreas

    2014-05-01

    Decomposition of soil organic matter (SOM) and thus carbon and nutrient cycling in soils is mediated by the activity of extracellular enzymes. The specific activities of these enzymes and their ratios to each other represent the link between the composition of soil organic matter and the nutrient demand of the microbial community. Depending on the difference between microbial nutrient demand and substrate availability, extracellular enzymes can enhance or slow down different nutrient cycles in the soil. We investigated activities of six extracellular enzymes (cellobiohydrolase, leucine-amino-peptidase, N-acetylglucosaminidase, chitotriosidase, phosphatase and phenoloxidase) in the topsoil organic horizon, topsoil mineral horizon and subsoil horizon in seven ecosystems along a 1,500 km-long North-South transect in Western Siberia. The transect included sites in the southern tundra, northern taiga, middle taiga, southern taiga, forest-steppe (in forested patches as well as in adjacent meadows) and Steppe. We found that enzyme patterns varied stronger with soil depth than between ecosystems. Differences between horizons were mainly based on the increasing ratio of oxidative enzymes to hydrolytic enzymes. Differences between sites were more pronounced in topsoil than in subsoil mineral horizons, but did not reflect the north-south transect and the related gradients in temperature and precipitation. The observed differences between sites in topsoil horizons might therefore result from differences in vegetation rather than climatic factors. The decreasing variability in the enzyme pattern with depth might also indicate that the composition of soil organic matter becomes more similar with soil depth, most likely by an increasing proportion of microbial remains compared to plant derived constituents of SOM. This also indicates, that SOM becomes less divers the more it is processed by soil microorganisms. Our findings highlight the importance of soil depth on enzyme

  20. Investigations on hydrolytic activities from Stachybotrys microspora and their use as an alternative in yeast DNA extraction.

    PubMed

    Abdeljalil, Salma; Ben Hmad, Ines; Saibi, Walid; Amouri, Bahia; Maalej, Wiem; Kaaniche, Marwa; Koubaa, Aida; Gargouri, Ali

    2014-02-01

    Stachybotrys microspora is a filamentous fungus characterized by the secretion of multiple hydrolytic activities (cellulolytic and non-cellulolytic enzymes). The production of these biocatalysts was studied under submerged culture using glucose, cellulose, and wheat bran as carbon sources. Endoglucanases, pectinases, xylanases, β-glucanases, chitinases, and proteases were induced on cellulose-based medium and repressed on glucose in both strains with higher amounts produced by the mutant. β-glucosidases were roughly equally produced by both strains under glucose and cellulose conditions. The yield of chitinases, β-glucanases, and proteases produced by Stachybotrys strains was as much higher than the commercialized lysing enzyme called "zymolyase," currently used in yeast DNA extraction. In this context, we showed that S. microspora hydrolases can be successfully applied in the extraction of yeast DNA.

  1. The abundant class III chitinase homolog in young developing banana fruits behaves as a transient vegetative storage protein and most probably serves as an important supply of amino acids for the synthesis of ripening-associated proteins.

    PubMed

    Peumans, Willy J; Proost, Paul; Swennen, Rony L; Van Damme, Els J M

    2002-10-01

    Analyses of the protein content and composition revealed dramatic changes in gene expression during in situ banana (Musa spp.) fruit formation/ripening. The total banana protein content rapidly increases during the first 60 to 70 d, but remains constant for the rest of fruit formation/ripening. During the phase of rapid protein accumulation, an inactive homolog of class III chitinases accounts for up to 40% (w/v) of the total protein. Concomitant with the arrest of net protein accumulation, the chitinase-related protein (CRP) progressively decreases and several novel proteins appear in the electropherograms. Hence, CRP behaves as a fruit-specific vegetative storage protein that accumulates during early fruit formation and serves as a source of amino acids for the synthesis of ripening-associated proteins. Analyses of individual proteins revealed that a thaumatin-like protein, a beta-1,3-glucanase, a class I chitinase, and a mannose-binding lectin are the most abundant ripening-associated proteins. Because during the ripening of prematurely harvested bananas, similar changes take place as in the in situ ripening bananas, CRP present in immature fruits is a sufficient source of amino acids for a quasi-normal synthesis of ripening-associated proteins. However, it is evident that the conversion of CRP in ripening-associated proteins takes place at an accelerated rate, especially when climacteric ripening is induced by ethylene. The present report also includes a discussion of the accumulation of the major banana allergens and the identification of suitable promoters for the production of vaccines in transgenic bananas.

  2. The Abundant Class III Chitinase Homolog in Young Developing Banana Fruits Behaves as a Transient Vegetative Storage Protein and Most Probably Serves as an Important Supply of Amino Acids for the Synthesis of Ripening-Associated Proteins1

    PubMed Central

    Peumans, Willy J.; Proost, Paul; Swennen, Rony L.; Van Damme, Els J.M.

    2002-01-01

    Analyses of the protein content and composition revealed dramatic changes in gene expression during in situ banana (Musa spp.) fruit formation/ripening. The total banana protein content rapidly increases during the first 60 to 70 d, but remains constant for the rest of fruit formation/ripening. During the phase of rapid protein accumulation, an inactive homolog of class III chitinases accounts for up to 40% (w/v) of the total protein. Concomitant with the arrest of net protein accumulation, the chitinase-related protein (CRP) progressively decreases and several novel proteins appear in the electropherograms. Hence, CRP behaves as a fruit-specific vegetative storage protein that accumulates during early fruit formation and serves as a source of amino acids for the synthesis of ripening-associated proteins. Analyses of individual proteins revealed that a thaumatin-like protein, a β-1,3-glucanase, a class I chitinase, and a mannose-binding lectin are the most abundant ripening-associated proteins. Because during the ripening of prematurely harvested bananas, similar changes take place as in the in situ ripening bananas, CRP present in immature fruits is a sufficient source of amino acids for a quasi-normal synthesis of ripening-associated proteins. However, it is evident that the conversion of CRP in ripening-associated proteins takes place at an accelerated rate, especially when climacteric ripening is induced by ethylene. The present report also includes a discussion of the accumulation of the major banana allergens and the identification of suitable promoters for the production of vaccines in transgenic bananas. PMID:12376669

  3. Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74∆sp chitinase inclusions, Cry crystals and spores for applied use

    PubMed Central

    2014-01-01

    Background The endochitinase ChiA74 is a soluble secreted enzyme produced by Bacillus thuringiensis that synergizes the entomotoxigenecity of Cry proteins that accumulate as intracellular crystalline inclusion during sporulation. The purpose of this study was to produce alkaline-soluble ChiA74∆sp inclusions in B. thuringiensis, and to determine its effect on Cry crystal production, sporulation and toxicity to an important agronomical insect, Manduca sexta. To this end we deleted the secretion signal peptide-coding sequence of chiA74 (i.e. chiA74∆sp) and expressed it under its native promoter (pEHchiA74∆sp) or strong chimeric sporulation-dependent cytA-p/STAB-SD promoter (pEBchiA74∆sp) in Escherichia coli, acrystalliferous B. thuringiensis (4Q7) and B. thuringiensis HD1. Results Based on mRNA analyses, up to ~9-fold increase in expression of chiA74∆sp was observed using the cytA-p/STAB-SD promoter. ChiA74∆sp (~70 kDa) formed intracellular inclusions that frequently accumulated at the poles of cells. ChiA74∆sp inclusions were dissolved in alkali and reducing conditions, similar to Cry crystals, and retained its activity in a wide range of pH (5 to 9), but showed a drastic reduction (~70%) at pH 10. Chitinase activity of E. coli-pEHchiA74∆sp was ~150 mU/mL, and in E. coli-pEBchiA74∆sp, 250 mU/mL. 4Q7-pEBchiA74∆sp and 4Q7-pEHchiA74∆sp had activities of ~127 mU/mL and ~41 mU/mL, respectively. The endochitinase activity in HD1-pEBchiA74∆sp increased 42x when compared to parental HD1 strain. HD1-pEBchiA74∆sp and HD1 harbored typical bipyramidal Cry inclusions, but crystals in the recombinant were ~30% smaller. Additionally, a 3x increase in the number of viable spores was observed in cultures of the recombinant strain when compared to HD1. Bioassays against first instar larvae of M. sexta with spore-crystals of HD1 or spore-crystal-ChiA74∆sp inclusions of HD1-pEBchiA74∆sp showed LC50s of 67.30 ng/cm2 and 41.45 ng/cm2, respectively

  4. Increased Obesity-Associated Circulating Levels of the Extracellular Matrix Proteins Osteopontin, Chitinase-3 Like-1 and Tenascin C Are Associated with Colon Cancer

    PubMed Central

    Catalán, Victoria; Gómez-Ambrosi, Javier; Rodríguez, Amaia; Ramírez, Beatriz; Izaguirre, Maitane; Hernández-Lizoain, José Luis; Baixauli, Jorge; Martí, Pablo; Valentí, Víctor; Moncada, Rafael; Silva, Camilo; Salvador, Javier; Frühbeck, Gema

    2016-01-01

    Background Excess adipose tissue represents a major risk factor for the development of colon cancer with inflammation and extracellular matrix (ECM) remodeling being proposed as plausible mechanisms. The aim of this study was to investigate whether obesity can influence circulating levels of inflammation-related extracellular matrix proteins in patients with colon cancer (CC), promoting a microenvironment favorable for tumor growth. Methods Serum samples obtained from 79 subjects [26 lean (LN) and 53 obese (OB)] were used in the study. Enrolled subjects were further subclassified according to the established diagnostic protocol for CC (44 without CC and 35 with CC). Anthropometric measurements as well as circulating metabolites and hormones were determined. Circulating concentrations of the ECM proteins osteopontin (OPN), chitinase-3-like protein 1 (YKL-40), tenascin C (TNC) and lipocalin-2 (LCN-2) were determined by ELISA. Results Significant differences in circulating OPN, YKL-40 and TNC concentrations between the experimental groups were observed, being significantly increased due to obesity (P<0.01) and colon cancer (P<0.05). LCN-2 levels were affected by obesity (P<0.05), but no differences were detected regarding the presence or not of CC. A positive association (P<0.05) with different inflammatory markers was also detected. Conclusions To our knowledge, we herein show for the first time that obese patients with CC exhibit increased circulating levels of OPN, YKL-40 and TNC providing further evidence for the influence of obesity on CC development via ECM proteins, representing promising diagnostic biomarkers or target molecules for therapeutics. PMID:27612200

  5. Rhizoxin analogs, orfamide A and chitinase production contribute to the toxicity of Pseudomonas protegens strain Pf-5 to Drosophila melanogaster

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pseudomonas protegens strain Pf-5 is a soil bacterium that was first described for its activity in biological control of plant diseases and has since been shown to be lethal to certain insects. Among these is the fruit fly Drosophila melanogaster, a well-established model organism for studies evalu...

  6. Microbial dynamics and enzyme activities in tropical Andosols depending on land use and nutrient inputs

    NASA Astrophysics Data System (ADS)

    Mganga, Kevin; Razavi, Bahar; Kuzyakov, Yakov

    2015-04-01

    Microbial decomposition of soil organic matter is mediated by enzymes and is a key source of terrestrial CO2 emissions. Microbial and enzyme activities are necessary to understand soil biochemical functioning and identify changes in soil quality. However, little is known about land use and nutrients availability effects on enzyme activities and microbial processes, especially in tropical soils of Africa. This study was conducted to examine how microbial and enzyme activities differ between different land uses and nutrient availability. As Andosols of Mt. Kilimanjaro are limited by nutrient concentrations, we hypothesize that N and P additions will stimulate enzyme activity. N and P were added to soil samples (0-20 cm) representing common land use types in East Africa: (1) savannah, (2) maize fields, (3) lower montane forest, (4) coffee plantation, (5) grasslands and (6) traditional Chagga homegardens. Total CO2 efflux from soil, microbial biomass and activities of β-glucosidase, cellobiohydrolase, chitinase and phosphatase involved in C, N and P cycling, respectively was monitored for 60 days. Total CO2 production, microbial biomass and enzyme activities varied in the order forest soils > grassland soils > arable soils. Increased β-glucosidase and cellobiohydrolase activities after N addition of grassland soils suggest that microorganisms increased N uptake and utilization to produce C-acquiring enzymes. Low N concentration in all soils inhibited chitinase activity. Depending on land use, N and P addition had an inhibitory or neutral effect on phosphatase activity. We attribute this to the high P retention of Andosols and low impact of N and P on the labile P fractions. Enhanced CO2 production after P addition suggests that increased P availability could stimulate soil organic matter biodegradation in Andosols. In conclusion, land use and nutrients influenced soil enzyme activities and microbial dynamics and demonstrated the decline in soil quality after landuse

  7. QM/MM free-energy simulations of reaction in Serratia marcescens Chitinase B reveal the protonation state of Asp142 and the critical role of Tyr214.

    PubMed

    Jitonnom, Jitrayut; Limb, Michael A L; Mulholland, Adrian J

    2014-05-08

    Serratia marcescens Chitinase B (ChiB), belonging to the glycosidase family 18 (GH18), catalyzes the hydrolysis of β-1,4-glycosidic bond, with retention of configuration, via an unusual substrate-assisted mechanism, in which the substrate itself acts as an intramolecular nucleophile. Here, both elementary steps (glycosylation and deglycosylation) of the ChiB-catalyzed reaction are investigated by means of combined quantum mechanics/molecular mechanics (QM/MM) umbrella sampling molecular dynamics (MD) simulations at the SCC-DFTB/CHARMM22 level of theory. We examine the influence of the Asp142 protonation state on the reaction and the role that this residue performs in the reaction. Our simulations show that reaction with a neutral Asp142 is preferred and demonstrate that this residue provides electrostatic stabilization of the oxazolinium ion intermediate formed in the reaction. Insight into the conformational itinerary ((1,4)B↔(4)H5↔(4)C1) adopted by the substrate (bound in subsite -1) along the preferred reaction pathway is also provided by the simulations. The relative energies of the stationary points found along the reaction pathway calculated with SCC-DFTB and B3LYP were compared. The results suggest that SCC-DFTB is an accurate method for estimating the relative barriers for both steps of the reaction; however, it was found to overestimate the relative energy of an intermediate formed in the reaction when compared with the higher level of theory. Glycosylation is suggested to be a rate-determining step in the reaction with calculated overall reaction free-energy barrier of 20.5 kcal/mol, in a reasonable agreement with the 16.1 kcal/mol barrier derived from the experiment. The role of Tyr214 in catalysis was also investigated with the results, indicating that the residue plays a critical role in the deglycosylation step of the reaction. Simulations of the enzyme-product complex were also performed with an unbinding event suggested to have been observed

  8. Direct repeat sequences in the Streptomyces chitinase-63 promoter direct both glucose repression and chitin induction

    PubMed Central

    Ni, Xiangyang; Westpheling, Janet

    1997-01-01

    The chi63 promoter directs glucose-sensitive, chitin-dependent transcription of a gene involved in the utilization of chitin as carbon source. Analysis of 5′ and 3′ deletions of the promoter region revealed that a 350-bp segment is sufficient for wild-type levels of expression and regulation. The analysis of single base changes throughout the promoter region, introduced by random and site-directed mutagenesis, identified several sequences to be important for activity and regulation. Single base changes at −10, −12, −32, −33, −35, and −37 upstream of the transcription start site resulted in loss of activity from the promoter, suggesting that bases in these positions are important for RNA polymerase interaction. The sequences centered around −10 (TATTCT) and −35 (TTGACC) in this promoter are, in fact, prototypical of eubacterial promoters. Overlapping the RNA polymerase binding site is a perfect 12-bp direct repeat sequence. Some base changes within this direct repeat resulted in constitutive expression, suggesting that this sequence is an operator for negative regulation. Other base changes resulted in loss of glucose repression while retaining the requirement for chitin induction, suggesting that this sequence is also involved in glucose repression. The fact that cis-acting mutations resulted in glucose resistance but not inducer independence rules out the possibility that glucose repression acts exclusively by inducer exclusion. The fact that mutations that affect glucose repression and chitin induction fall within the same direct repeat sequence module suggests that the direct repeat sequence facilitates both chitin induction and glucose repression. PMID:9371809

  9. One-pot synthesis and antifungal activity against plant pathogens of quinazolinone derivatives containing an amide moiety.

    PubMed

    Zhang, Jin; Liu, Jia; Ma, Yangmin; Ren, Decheng; Cheng, Pei; Zhao, Jiawen; Zhang, Fan; Yao, Yuan

    2016-05-01

    An efficient one-pot, three-component synthesis of quinazolinone derivatives containing 3-acrylamino motif was carried out using CeO2 nanoparticles as catalyst. Thirty-nine synthesized compounds were obtained with satisfied yield and elucidated by spectroscopic analysis. Four phytopathogenic fungi were chosen to test the antifungal activities by minimum inhibitory concentration (MIC) method. Compounds 4ag, 4bb, 4bc showed broad antifungal activities against at least three fungi, and dramatic effects of substituents on the activities were observed. Docking studies were established to explore the potential antifungal mechanism of quinazolinone derivatives as the chitinase inhibitors, and also verified the importance of the amide moiety.

  10. Synthesis of Long-Chain Chitooligosaccharides by a Hypertransglycosylating Processive Endochitinase of Serratia proteamaculans 568

    PubMed Central

    Purushotham, Pallinti

    2012-01-01

    We describe the heterologous expression and characterization of a 407-residue single-domain glycosyl hydrolase family 18 chitinase (SpChiD) from Gram-negative Serratia proteamaculans 568 that has unprecedented catalytic properties. SpChiD was optimally active at pH 6.0 and 40°C, where it showed a Km of 83 mg ml−1, a kcat of 3.9 × 102 h−1, and a kcat/Km of 4.7 h mg−1 ml−1 on colloidal chitin. On chitobiose, the Km, kcat, and kcat/Km were 203 μM, 1.3 × 102 h−1, and 0.62 h−1 μM−1, respectively. Hydrolytic activity on chitooligosaccharides (CHOS) and colloidal chitin indicated that SpChiD was an endo-acting processive enzyme, with the unique ability to convert released chitobiose to N-acetylglucosamine, the major end product. SpChiD showed hyper transglycosylation (TG) with trimer-hexamer CHOS substrates, generating considerable amounts of long-chain CHOS. The TG activity of SpChiD was dependent on both the length and concentration of the oligomeric substrate and also on the enzyme concentration. The length and amount of accumulated TG products increased with increases in the length of the substrate and its concentration and decreased with increases in the enzyme concentration. The SpChiD bound to insoluble and soluble chitin substrates despite the absence of accessory domains. Sequence alignments and structural modeling indicated that SpChiD would have a deep substrate-binding groove lined with aromatic residues, which is characteristic of processive enzymes. SpChiD shows a combination of properties that seems rare among family 18 chitinases and that may resemble the properties of human chitotriosidase. PMID:22685288

  11. Stochastic and nonstochastic post-transcriptional silencing of chitinase and beta-1,3-glucanase genes involves increased RNA turnover-possible role for ribosome-independent RNA degradation.

    PubMed Central

    Holtorf, H; Schöb, H; Kunz, C; Waldvogel, R; Meins, F

    1999-01-01

    Stochastic and nonstochastic post-transcriptional gene silencing (PTGS) in Nicotiana sylvestris plants carrying tobacco class I chitinase (CHN) and beta-1,3-glucanase transgenes differs in incidence, stability, and pattern of expression. Measurements with inhibitors of RNA synthesis (cordycepin, actinomycin D, and alpha-amanitin) showed that both forms of PTGS are associated with increased sequence-specific degradation of transcripts, suggesting that increased RNA turnover may be a general feature of PTGS. The protein synthesis inhibitors cycloheximide and verrucarin A did not inhibit degradation of CHN RNA targeted for PTGS, confirming that PTGS-related RNA degradation does not depend on ongoing protein synthesis. Because verrucarin A, unlike cycloheximide, dissociates mRNA from ribosomes, our results also suggest that ribosome-associated RNA degradation pathways may not be involved in CHN PTGS. PMID:10072405

  12. Soil zymography - A novel technique for mapping enzyme activity in the rhizosphere

    NASA Astrophysics Data System (ADS)

    Spohn, Marie

    2014-05-01

    The effect plant roots on microbial activity in soil at the millimeter scale is poorly understood. One reason for this is that spatially explicit methods for the study of microbial activity in soil are limited. Here we present a quantitative in situ technique for mapping the distribution of exoenzymes in soil along with some results about the effects of roots on exoenzyme activity in soil. In the first study we showed that both acid and alkaline phosphatase activity were up to 5.4-times larger in the rhizosphere of Lupinus albus than in the bulk soil. While acid phosphatase activity (produced by roots and microorganisms) was closely associated with roots, alkaline phosphatase activity (produced only by microorganisms) was more widely distributed, leading to a 2.5-times larger area of activity of alkaline than of acid phosphatase. These results indicate a spatial differentiation of different ecophysiological groups of organic phosphorus mineralizing organisms in the rhizosphere which might alleviate a potential competition for phosphorus between them. In a second study cellulase, chitinase and phosphatase activities were analyzed in the presence of living Lupinus polyphyllus roots and dead/dying roots (in the same soils 10, 20 and 30 days after cutting the L. polyphyllus shoots). The activity of all three enzymes was 9.0 to 13.9-times higher at the living roots compared to the bulk soil. Microhotspots of cellulase, chitinase and phosphatase activity in the soil were found up to 60 mm away from the living roots. 10 days after shoot cutting, the areas of high activities of cellulase and phosphatase activity were extend up to 55 mm away from the next root, while the extension of the area of chitinase activity did not change significantly. At the root, cellulase and chitinase activity increased first at the root tips after shoot cutting and showed maximal activity 20 days after shoot cutting. The number and activity of microhotspots of chitinase activity was maximal 10

  13. Influence of protoplast fusion between two Trichoderma spp. on extracellular enzymes production and antagonistic activity

    PubMed Central

    Hassan, Mohamed M.

    2014-01-01

    Biological control plays a crucial role in grapevine pathogens disease management. The cell-wall degrading enzymes chitinase, cellulase and β-glucanase have been suggested to be essential for the mycoparasitism activity of Trichoderma species against grapevine fungal pathogens. In order to develop a useful strain as a single source of these vital enzymes, it was intended to incorporate the characteristics of two parental fungicides tolerant mutants of Trichoderma belonging to the high chitinase producing species T. harzianum and the high cellulase producing species T. viride, by fusing their protoplasts. The phylogeny of the parental strains was carried out using a sequence of the 5.8S-ITS region. The BLAST of the obtained sequence identified these isolates as T. harzianum and T. viride. Protoplasts were isolated using lysing enzymes and were fused using polyethylene glycol. The fused protoplasts have been regenerated on protoplast regeneration minimal medium supplemented with two selective fungicides. Among the 40 fast growing fusants, 17 fusants were selected based on their enhanced growth on selective media for further studies. The fusant strains were growing 60%–70% faster than the parents up to third generation. All the 17 selected fusants exhibited morphological variations. Some fusant strains displayed threefold increased chitinase enzyme activity and twofold increase in β-glucanase enzyme activity compared to the parent strains. Most fusants showed powerful antagonistic activity against Macrophomin aphaseolina, Pythium ultimum and Sclerotium rolfsii pathogens. Fusant number 15 showed the highest inhibition percentage (92.8%) against M. phaseolina and P. ultimum, while fusant number 9 showed the highest inhibition percentage (98.2%) against the growth of S. rolfsii. A hyphal intertwining and degradation phenomenon was observed by scanning electron microscope. The Trichoderma antagonistic effect against pathogenic fungal mycelia was due to the

  14. Soil Microbial Activity Provides Insight to Carbon Cycling in Shrub Ecotones of Sub-Arctic Sweden

    NASA Astrophysics Data System (ADS)

    Marek, E.; Kashi, N. N.; Chen, J.; Hobbie, E. A.; Schwan, M. R.; Varner, R. K.

    2015-12-01

    Shrubs are expanding in Arctic and sub-Arctic regions due to rising atmospheric temperatures. Microbial activity increases as growing temperatures cause permafrost warming and subsequent thaw, leading to a greater resource of soil nutrients enabling shrub growth. Increased carbon inputs from shrubs is predicted to result in faster carbon turnover by microbial decomposition. Further understanding of microbial activity underneath shrubs could uncover how microbes and soil processes interact to promote shrub expansion and carbon cycling. To address how higher soil carbon input from shrubs influences decomposition, soil samples were taken across a heath, shrub, and forest ecotone gradient at two sites near Abikso, Sweden. Samples were analyzed for soluble carbon and nitrogen, microbial abundance, and microbial activity of chitinase, glucosidase, and phosphatase to reflect organic matter decomposition and availability of nitrogen, carbon, and phosphate respectively. Chitinase activity positively correlated with shrub cover, suggesting microbial demands for nitrogen increase with higher shrub cover. Glucosidase activity negatively correlated with shrub cover and soluble carbon, suggesting decreased microbial demand for carbon as shrub cover and carbon stores increase. Lower glucosidase activity in areas with high carbon input from shrubs implies that microbes are decomposing carbon less readily than carbon is being put into the soil. Increasing soil carbon stores in shrub covered areas can lead to shrubs becoming a net carbon sink and a negative feedback to changing climate.

  15. Activities of Aureobasidium pullulans cell filtrates against Monilinia laxa of peaches.

    PubMed

    Di Francesco, Alessandra; Roberti, Roberta; Martini, Camilla; Baraldi, Elena; Mari, Marta

    2015-12-01

    The Aureobasidium pullulans L1 and L8 strains are known as efficient biocontrol agents against several postharvest fungal pathogens. In order to better understand the mechanism of action underneath the antifungal activity of L1 and L8 strains, yeast cell filtrates grown at different times were evaluated in vivo against Monilinia laxa on peach. Lesion diameters on peach fruit were reduced by L1 and L8 culture filtrates of 42.5% and 67% respectively. The ability of these filtrates to inhibit M. laxa conidia germination and germ tube elongation was studied by in vitro assays. The results showed a 70% reduction of conidia germination for both strains while for germ tube elongation, it was 52% and 41% for L1 and L8 culture filtrates respectively. Finally, the activity of cell wall hydrolytic enzymes such as chitinase and glucanase in cell filtrates was analysed and the expression of genes encoding these activities was quantified during yeast growth. From 24h onward, both culture filtrates contained β,1-3,glucanase and. chitinase activities, the most pronounced of which was N-β-acetylglucosaminidase. Gene expression level encoding for these enzymes in L1 and L8 varied according to the strain. These results indicate that L1 and L8 strains culture filtrates retain the yeast antagonistic activity and suggest that the production of hydrolytic enzymes plays an important role in this activity.

  16. The influence of repeated administration of poloxamer 407 on serum lipoproteins and protease activity in mouse liver and heart.

    PubMed

    Korolenko, Tatyana A; Tuzikov, Fedor V; Johnston, Thomas P; Tuzikova, Natalia A; Kisarova, Yana A; Zhanaeva, Svetlana Ya; Alexeenko, Tatyana V; Zhukova, Natalia A; Brak, Ivan V; Spiridonov, Victor K; Filjushina, Elena E; Cherkanova, Marina S; Monoszon, Anna A

    2012-11-01

    The effects of repeated administration of poloxamer 407 (P-407) on lipoprotein-cholesterol (LP-C) and lipoprotein-triglyceride (LP-TG) fractions and subfractions, as well as the effect on liver and heart proteases, were studied. Repeated administration of P-407 to male CBA mice resulted in a model of atherosclerosis with increased diastolic blood pressure; there was a drastic increase in total serum cholesterol and especially TG. A novel small-angle X-ray scattering method for the determination of the fractional and subfractional composition of LP-C and LP-TG was used. In chronically P-407-treated mice, P-407 significantly increased atherogenic low-density lipoprotein C (LDL-C) fractions, as well as intermediate-density lipoprotein C (IDL-C), and LDL₁₋₃-C subfractions, and very-low-density lipoprotein-C (VLDL-C) fractions, as well as VLDL₁₋₂-C and VLDL₃₋₅-C subfractions), to a lesser extent, the total anti-atherogenic high-density lipoprotein C (HDL-C) fraction, as well as HDL₂-C and HDL₃-C subfractions. Additionally, we demonstrated an increase in the serum chitotriosidase activity, without significant changes in serum matrix metalloprotease (MMP) activity. Morphological changes observed in P-407-treated mice included atherosclerosis in the heart and storage syndrome in the liver macrophages. P-407 significantly increased the activity of cysteine, aspartate proteases, and MMPs in the heart, and only the activity of cathepsin B and MMPs in the liver of mice. Thus, repeated administration of P-407 to mice induced atherosclerosis secondary to sustained dyslipidemia and formation of foamy macrophages in liver, and also modulated the activity of heart and liver proteases.

  17. Chitins and chitosans as immunoadjuvants and non-allergenic drug carriers.

    PubMed

    Muzzarelli, Riccardo A A

    2010-02-21

    amplified during many infections and diseases, the common feature of chitinase-like proteins and chitinase activity in all organisms appears to be the biochemical defense of the host. Unfortunately, conceptual and methodological errors are present in certain recent articles dealing with chitin and allergy, i.e., (1) omitted consideration of mammalian chitinase and/or chitotriosidase secretion, accompanied by inactive chitinase-like proteins, as an ancestral defensive means against invasion, capable to prevent the insurgence of allergy; (2) omitted consideration of the fact that the mammalian organism recognizes more promptly the secreted water soluble chitinase produced by a pathogen, rather than the insoluble and well protected chitin within the pathogen itself; (3) superficial and incomplete reports and investigations on chitin as an allergen, without mentioning the potent allergen from crustacean flesh, tropomyosine; (4) limited perception of the importance of the chemical/biochemical characteristics of the isolated chitin or chitosan for the replication of experiments and optimization of results; and (5) lack of interdisciplinarity. There is quite a large body of knowledge today on the use of chitosans as biomaterials, and more specifically as drug carriers for a variety of applications: the delivery routes being the same as those adopted for the immunological studies. Said articles, that devote attention to the safety and biocompatibility aspects, never reported intolerance or allergy in individuals and animals, even when the quantities of chitosan used in single experiments were quite large. Therefore, it is concluded that crab, shrimp, prawn and lobster chitins, as well as chitosans of all grades, once purified, should not be considered as "crustacean derivatives", because the isolation procedures have removed proteins, fats and other contaminants to such an extent as to allow them to be classified as chemicals regardless of their origin.

  18. Active invasion of bacteria into living fungal cells.

    PubMed

    Moebius, Nadine; Üzüm, Zerrin; Dijksterhuis, Jan; Lackner, Gerald; Hertweck, Christian

    2014-09-02

    The rice seedling blight fungus Rhizopus microsporus and its endosymbiont Burkholderia rhizoxinica form an unusual, highly specific alliance to produce the highly potent antimitotic phytotoxin rhizoxin. Yet, it has remained a riddle how bacteria invade the fungal cells. Genome mining for potential symbiosis factors and functional analyses revealed that a type 2 secretion system (T2SS) of the bacterial endosymbiont is required for the formation of the endosymbiosis. Comparative proteome analyses show that the T2SS releases chitinolytic enzymes (chitinase, chitosanase) and chitin-binding proteins. The genes responsible for chitinolytic proteins and T2SS components are highly expressed during infection. Through targeted gene knock-outs, sporulation assays and microscopic investigations we found that chitinase is essential for bacteria to enter hyphae. Unprecedented snapshots of the traceless bacterial intrusion were obtained using cryo-electron microscopy. Beyond unveiling the pivotal role of chitinolytic enzymes in the active invasion of a fungus by bacteria, these findings grant unprecedented insight into the fungal cell wall penetration and symbiosis formation.

  19. Active invasion of bacteria into living fungal cells

    PubMed Central

    Moebius, Nadine; Üzüm, Zerrin; Dijksterhuis, Jan; Lackner, Gerald; Hertweck, Christian

    2014-01-01

    The rice seedling blight fungus Rhizopus microsporus and its endosymbiont Burkholderia rhizoxinica form an unusual, highly specific alliance to produce the highly potent antimitotic phytotoxin rhizoxin. Yet, it has remained a riddle how bacteria invade the fungal cells. Genome mining for potential symbiosis factors and functional analyses revealed that a type 2 secretion system (T2SS) of the bacterial endosymbiont is required for the formation of the endosymbiosis. Comparative proteome analyses show that the T2SS releases chitinolytic enzymes (chitinase, chitosanase) and chitin-binding proteins. The genes responsible for chitinolytic proteins and T2SS components are highly expressed during infection. Through targeted gene knock-outs, sporulation assays and microscopic investigations we found that chitinase is essential for bacteria to enter hyphae. Unprecedented snapshots of the traceless bacterial intrusion were obtained using cryo-electron microscopy. Beyond unveiling the pivotal role of chitinolytic enzymes in the active invasion of a fungus by bacteria, these findings grant unprecedented insight into the fungal cell wall penetration and symbiosis formation. DOI: http://dx.doi.org/10.7554/eLife.03007.001 PMID:25182414

  20. Activity.

    ERIC Educational Resources Information Center

    Clearing: Nature and Learning in the Pacific Northwest, 1984

    1984-01-01

    Presents three activities: (1) investigating succession in a schoolground; (2) investigating oak galls; and (3) making sun prints (photographs made without camera or darkroom). Each activity includes a list of materials needed and procedures used. (JN)

  1. Potential association between TLR4 and chitinase 3-like 1 (CHI3L1/YKL-40) signaling on colonic epithelial cells in inflammatory bowel disease and colitis-associated cancer

    PubMed Central

    Kamba, Alan; Lee, In-Ah; Mizoguchi, Emiko

    2013-01-01

    Inflammatory bowel disease (IBD) is a group of inflammatory disorders in the small and large intestines. Several studies have proved that persistent and disregulated host/microbial interactions are required for the development of IBD. It is well known that chronic IBD is strongly associated with an increased risk of developing colorectal cancer by 0.5–1% annually, 8–10 years after the initial diagnosis. To detect the tiny dysplasia or early stage of cancer in chronic IBD patients, a tremendous amount of effort is currently directed for improving colonoscopic technology and noninvasive serological marker development. However, there is only a limited amount of data available to understand the exact mechanism of how long term chronic colitis is connected to the development of colorectal tumors. Recently, our group has identified that significantly increased expression of chitinase 3-like 1 (CHI3L1) molecule in non-dysplastic mucosa from patients with IBD and remote dysplasia/cancer, compared to patients with IBD without dysplasia or healthy controls. CHI3L1 seems to contribute to the proliferation, migration, and neoplastic progression of colonic epithelial cells (CECs) under inflammatory conditions. Furthermore, the CHI3L1-mediated intracellular signaling cascade is likely to interact with TLR4 signaling in CECs. In this review article, we have concisely summarized the cellular and molecular mechanisms underlining the development of IBD and colitis-associated cancer, with particular focus on the CHI3L1-and TLR4-signaling pathways in CECs. PMID:23170831

  2. Activities.

    ERIC Educational Resources Information Center

    Bippert, Judy

    1993-01-01

    Presents activities designed to give students an opportunity to solve concrete problems involving spatial relationships and logical thinking utilizing hands-on manipulatives. Provides teacher instructions and four reproducible worksheets. (MDH)

  3. The herbicide flumioxazin stimulates pathogenesis-related gene expression and enzyme activities in Vitis vinifera.

    PubMed

    Castro, Antonio Jesús; Saladin, Gäelle; Bézier, Annie; Mazeyrat-Gourbeyre, Florence; Baillieul, Fabienne; Clément, Christophe

    2008-11-01

    In this work, the capacity of the soil-applied herbicide flumioxazin (fmx) to trigger defence mechanisms was assessed using 6-week-old in vitro grown Vitis vinifera L. plantlets. Time-course studies demonstrated that the herbicide induced the expression of basic beta-1,3-glucanase (Vvglu), basic chitinase (Vvchit1b) and PR10 (VvPR10.3) genes encoding three pathogenesis-related (PR) proteins involved in grapevine defence against pathogens. Thus, all transcripts accumulated in grapevine tissues to reach maximum values after 24-72 h of herbicide exposure, except for VvPR10.3 gene expression, which was induced in roots and stems but not in leaves. Induction of PR genes was observed to a greater extent in roots and leaves, and its intensity diminished in the stems although still remained noteworthy. The activities of beta-1,3-glucanase and chitinase enzymes significantly increased in the whole plant after herbicide exposure and were still stimulated 21 days after the beginning of treatments. Similarly, the most remarkable effect occurred in roots. However, all enzyme activities tested were stimulated in the upper aerial tissues as well, indicating that fmx or a derived product acts systemically, likely via root uptake.

  4. Activities.

    ERIC Educational Resources Information Center

    Kincaid, Charlene; And Others

    1993-01-01

    Presents an activity in which students collect and organize data from a real-world simulation of the scientific concept of half life. Students collect data using a marble sifter, analyze the data using a graphing calculator, and determine an appropriate mathematical model. Includes reproducible worksheets. (MDH)

  5. Visualization of enzyme activities inside earthworm biopores by in situ soil zymography

    NASA Astrophysics Data System (ADS)

    Thu Duyen Hoang, Thi; Razavi, Bahar. S.; Blagodatskaya, Evgenia; Kuzyakov, Yakov

    2015-04-01

    Earthworms can strongly activate microorganisms, increase microbial and enzyme activities and consequently the turnover of native soil organic matter. In extremely dynamic microhabitats and hotspots as biopores made by earthworms, the in situ enzyme activities are a footprint of complex biotic interactions. The effect of earthworms on the alteration of enzyme activities inside biopores and the difference between bio-pores and earthworm-free soil was visualized by in situ soil zymography (Spohn and Kuzyakov, 2014). For the first time, we prepared quantitative imaging of enzyme activities in biopores. Furthermore, we developed the zymography technique by direct application of a substrate saturated membrane to the soil to obtain better spatial resolution. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). Simultaneously, maize seed was sown in the soil. Control soil box with maize and without earthworm was prepared in the same way. After two weeks when bio-pore systems were formed by earthworm, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine aminopeptidase) and phosphatase. Followed by non-destructive zymography, biopore samples and control soil were destructively collected to assay enzyme kinetics by fluorogenically labeled substrates method. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. These differences were further confirmed by fluorimetric microplate enzyme assay detected significant difference of Vmax in four above mentioned enzymes. Vmax of β-glucosidase, chitinase, xylanase and phosphatase in biopores is 68%, 108%, 50% and 49% higher than that of control soil. However, no difference in cellobiohydrolase and leucine aminopeptidase kinetics between biopores and control soil were detected. This indicated little effect of earthworms on protein and cellulose transformation in soil

  6. The Capsicum annuum class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses.

    PubMed

    Kim, Dae Sung; Kim, Nak Hyun; Hwang, Byung Kook

    2015-04-01

    The pepper receptor-like cytoplasmic protein kinase, CaPIK1, which mediates signalling of plant cell death and defence responses was previously identified. Here, the identification of a class IV chitinase, CaChitIV, from pepper plants (Capsicum annuum), which interacts with CaPIK1 and promotes CaPIK1-triggered cell death and defence responses, is reported. CaChitIV contains a signal peptide, chitin-binding domain, and glycol hydrolase domain. CaChitIV expression was up-regulated by Xanthomonas campestris pv. vesicatoria (Xcv) infection. Notably, avirulent Xcv infection rapidly induced CaChitIV expression in pepper leaves. Bimolecular fluorescence complementation and co-immunoprecipitation revealed that CaPIK1 interacts with CaChitIV in planta, and that the CaPIK1-CaChitIV complex is localized mainly in the cytoplasm and plasma membrane. CaChitIV is also localized in the endoplasmic reticulum. Transient co-expression of CaChitIV with CaPIK1 enhanced CaPIK1-triggered cell death response and reactive oxygen species (ROS) and nitric oxide (NO) bursts. Co-silencing of both CaChitIV and CaPIK1 in pepper plants conferred enhanced susceptibility to Xcv infection, which was accompanied by a reduced induction of cell death response, ROS and NO bursts, and defence response genes. Ectopic expression of CaPIK1 in Arabidopsis enhanced basal resistance to Hyaloperonospora arabidopsidis infection. Together, the results suggest that CaChitIV positively regulates CaPIK1-triggered cell death and defence responses through its interaction with CaPIK1.

  7. Isolation of a New Mexican Strain of Bacillus subtilis with Antifungal and Antibacterial Activities

    PubMed Central

    Basurto-Cadena, M. G. L.; Vázquez-Arista, M.; García-Jiménez, J.; Salcedo-Hernández, R.; Bideshi, D. K.; Barboza-Corona, J. E.

    2012-01-01

    Although several strains of B. subtilis with antifungal activity have been isolated worldwide, to date there are no published reports regarding the isolation of a native B. subtilis strain from strawberry plants in Mexico. A native bacterium (Bacillus subtilis 21) demonstrated in vitro antagonistic activity against different plant pathogenic fungi. Under greenhouse conditions, it was shown that plants infected with Rhizoctonia solani and Fusarium verticillioides and treated with B. subtilis 21 produced augment in the number of leaves per plant and an increment in the length of healthy leaves in comparison with untreated plants. In addition, B. subtilis 21 showed activity against pathogenic bacteria. Secreted proteins by B. subtilis 21 were studied, detecting the presence of proteases and bacteriocin-like inhibitor substances that could be implicated in its antagonistic activity. Chitinases and zwittermicin production could not be detected. Then, B. subtilis 21 could potentially be used to control phytopathogenic fungi that infect strawberry plants. PMID:22593682

  8. Isolation of a new Mexican strain of Bacillus subtilis with antifungal and antibacterial activities.

    PubMed

    Basurto-Cadena, M G L; Vázquez-Arista, M; García-Jiménez, J; Salcedo-Hernández, R; Bideshi, D K; Barboza-Corona, J E

    2012-01-01

    Although several strains of B. subtilis with antifungal activity have been isolated worldwide, to date there are no published reports regarding the isolation of a native B. subtilis strain from strawberry plants in Mexico. A native bacterium (Bacillus subtilis 21) demonstrated in vitro antagonistic activity against different plant pathogenic fungi. Under greenhouse conditions, it was shown that plants infected with Rhizoctonia solani and Fusarium verticillioides and treated with B. subtilis 21 produced augment in the number of leaves per plant and an increment in the length of healthy leaves in comparison with untreated plants. In addition, B. subtilis 21 showed activity against pathogenic bacteria. Secreted proteins by B. subtilis 21 were studied, detecting the presence of proteases and bacteriocin-like inhibitor substances that could be implicated in its antagonistic activity. Chitinases and zwittermicin production could not be detected. Then, B. subtilis 21 could potentially be used to control phytopathogenic fungi that infect strawberry plants.

  9. Activation of Pathogenesis-related Genes by the Rhizobacterium, Bacillus sp. JS, Which Induces Systemic Resistance in Tobacco Plants.

    PubMed

    Kim, Ji-Seong; Lee, Jeongeun; Lee, Chan-Hui; Woo, Su Young; Kang, Hoduck; Seo, Sang-Gyu; Kim, Sun-Hyung

    2015-06-01

    Plant growth promoting rhizobacteria (PGPR) are known to confer disease resistance to plants. Bacillus sp. JS demonstrated antifungal activities against five fungal pathogens in in vitro assays. To verify whether the volatiles of Bacillus sp. JS confer disease resistance, tobacco leaves pre-treated with the volatiles were damaged by the fungal pathogen, Rhizoctonia solani and oomycete Phytophthora nicotianae. Pre-treated tobacco leaves had smaller lesion than the control plant leaves. In pathogenesis-related (PR) gene expression analysis, volatiles of Bacillus sp. JS caused the up-regulation of PR-2 encoding β-1,3-glucanase and acidic PR-3 encoding chitinase. Expression of acidic PR-4 encoding chitinase and acidic PR-9 encoding peroxidase increased gradually after exposure of the volatiles to Bacillus sp. JS. Basic PR-14 encoding lipid transfer protein was also increased. However, PR-1 genes, as markers of salicylic acid (SA) induced resistance, were not expressed. These results suggested that the volatiles of Bacillus sp. JS confer disease resistance against fungal and oomycete pathogens through PR genes expression.

  10. The platelet-activating factor acetylhydrolase gene derived from Trichoderma harzianum induces maize resistance to Curvularia lunata through the jasmonic acid signaling pathway.

    PubMed

    Yu, Chuanjin; Fan, Lili; Gao, Jinxin; Wang, Meng; Wu, Qiong; Tang, Jun; Li, Yaqian; Chen, Jie

    2015-01-01

    Platelet-activating factor acetylhydrolase (PAF-AH) derived from Trichoderma harzianum was upregulated by the interaction of T. harzianum with maize roots or the foliar pathogen Curvularia lunata. PAF-AH was associated with chitinase and cellulase expressions, but especially with chitinase, because its activity in the KO40 transformant (PAF-AH disruption transformant) was lower, compared with the wild-type strain T28. The result demonstrated that the colonization of maize roots by T. harzianum induced systemic protection of leaves inoculated with C. lunata. Such protection was associated with the expression of inducible jasmonic acid pathway-related genes. Moreover, the data from liquid chromatography-mass spectrometry confirmed that the concentration of jasmonic acid in maize leaves was associated with the expression level of defense-related genes, suggesting that PAF-AH induced resistance to the foliar pathogen. Our findings showed that PAF-AH had an important function in inducing systemic resistance to maize leaf spot pathogen.

  11. The endochitinase VDECH from Verticillium dahliae inhibits spore germination and activates plant defense responses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chitinases function in the digestion of chitin molecules, which are present principally in insects and fungi. In plants, chitinase genes play important roles in defense, and their expression can be triggered in response to both biotic and abiotic stresses. In this study, we cloned and characterized ...

  12. Antifungal and antiviral products of marine organisms

    PubMed Central

    Cheung, Randy Chi Fai; Pan, Wen Liang; Chan, Yau Sang; Yin, Cui Ming; Dan, Xiu Li; Wang, He Xiang; Fang, Evandro Fei; Lam, Sze Kwan; Ngai, Patrick Hung Kui; Xia, Li Xin; Liu, Fang; Ye, Xiu Yun; Zhang, Guo Qing; Liu, Qing Hong; Sha, Ou; Lin, Peng; Ki, Chan; Bekhit, Adnan A; Bekhit, Alaa El-Din; Wan, David Chi Cheong

    2017-01-01

    Marine organisms including bacteria, fungi, algae, sponges, echinoderms, mollusks, and cephalochordates produce a variety of products with antifungal activity including bacterial chitinases, lipopeptides, and lactones; fungal (−)-sclerotiorin and peptaibols, purpurides B and C, berkedrimane B and purpuride; algal gambieric acids A and B, phlorotannins; 3,5-dibromo-2-(3,5-dibromo-2-methoxyphenoxy)phenol, spongistatin 1, eurysterols A and B, nortetillapyrone, bromotyrosine alkaloids, bis-indole alkaloid, ageloxime B and (−)-ageloxime D, haliscosamine, hamigeran G, hippolachnin A from sponges; echinoderm triterpene glycosides and alkene sulfates; molluscan kahalalide F and a 1485-Da peptide with a sequence SRSELIVHQR; and cepalochordate chitotriosidase and a 5026.9-Da antifungal peptide. The antiviral compounds from marine organisms include bacterial polysaccharide and furan-2-yl acetate; fungal macrolide, purpurester A, purpurquinone B, isoindolone derivatives, alterporriol Q, tetrahydroaltersolanol C and asperterrestide A, algal diterpenes, xylogalactofucan, alginic acid, glycolipid sulfoquinovosyldiacylglycerol, sulfated polysaccharide p-KG03, meroditerpenoids, methyl ester derivative of vatomaric acid, lectins, polysaccharides, tannins, cnidarian zoanthoxanthin alkaloids, norditerpenoid and capilloquinol; crustacean antilipopolysaccharide factors, molluscan hemocyanin; echinoderm triterpenoid glycosides; tunicate didemnin B, tamandarins A and B and; tilapia hepcidin 1–5 (TH 1–5), seabream SauMx1, SauMx2, and SauMx3, and orange-spotted grouper β-defensin. Although the mechanisms of antifungal and antiviral activities of only some of the afore-mentioned compounds have been elucidated, the possibility to use those known to have distinctly different mechanisms, good bioavailability, and minimal toxicity in combination therapy remains to be investigated. It is also worthwhile to test the marine antimicrobials for possible synergism with existing drugs. The

  13. In Vitro and In Vivo Plant Growth Promoting Activities and DNA Fingerprinting of Antagonistic Endophytic Actinomycetes Associates with Medicinal Plants.

    PubMed

    Passari, Ajit Kumar; Mishra, Vineet Kumar; Gupta, Vijai Kumar; Yadav, Mukesh Kumar; Saikia, Ratul; Singh, Bhim Pratap

    2015-01-01

    Endophytic actinomycetes have shown unique plant growth promoting as well as antagonistic activity against fungal phytopathogens. In the present study forty-two endophytic actinomycetes recovered from medicinal plants were evaluated for their antagonistic potential and plant growth-promoting abilities. Twenty-two isolates which showed the inhibitory activity against at least one pathogen were subsequently tested for their plant-growth promoting activities and were compared genotypically using DNA based fingerprinting, including enterobacterial repetitive intergenic consensus (ERIC) and BOX repetitive elements. Genetic relatedness based on both ERIC and BOX-PCR generates specific patterns corresponding to particular genotypes. Exponentially grown antagonistic isolates were used to evaluate phosphate solubilization, siderophores, HCN, ammonia, chitinase, indole-3-acetic acid production, as well as antifungal activities. Out of 22 isolates, the amount of indole-3-acetic acid (IAA) ranging between 10-32 μg/ml was produced by 20 isolates and all isolates were positive for ammonia production ranging between 5.2 to 54 mg/ml. Among 22 isolates tested, the amount of hydroxamate-type siderophores were produced by 16 isolates ranging between 5.2 to 36.4 μg/ml, while catechols-type siderophores produced by 5 isolates ranging from 3.2 to 5.4 μg/ml. Fourteen isolates showed the solubilisation of inorganic phosphorous ranging from 3.2 to 32.6 mg/100ml. Chitinase and HCN production was shown by 19 and 15 different isolates, respectively. In addition, genes of indole acetic acid (iaaM) and chitinase (chiC) were successively amplified from 20 and 19 isolates respectively. The two potential strains Streptomyces sp. (BPSAC34) and Leifsonia xyli (BPSAC24) were tested in vivo and improved a range of growth parameters in chilli (Capsicum annuum L.) under greenhouse conditions. This study is the first published report that actinomycetes can be isolated as endophytes from within these

  14. In Vitro and In Vivo Plant Growth Promoting Activities and DNA Fingerprinting of Antagonistic Endophytic Actinomycetes Associates with Medicinal Plants

    PubMed Central

    Passari, Ajit Kumar; Mishra, Vineet Kumar; Gupta, Vijai Kumar; Yadav, Mukesh Kumar; Saikia, Ratul; Singh, Bhim Pratap

    2015-01-01

    Endophytic actinomycetes have shown unique plant growth promoting as well as antagonistic activity against fungal phytopathogens. In the present study forty-two endophytic actinomycetes recovered from medicinal plants were evaluated for their antagonistic potential and plant growth-promoting abilities. Twenty-two isolates which showed the inhibitory activity against at least one pathogen were subsequently tested for their plant-growth promoting activities and were compared genotypically using DNA based fingerprinting, including enterobacterial repetitive intergenic consensus (ERIC) and BOX repetitive elements. Genetic relatedness based on both ERIC and BOX-PCR generates specific patterns corresponding to particular genotypes. Exponentially grown antagonistic isolates were used to evaluate phosphate solubilization, siderophores, HCN, ammonia, chitinase, indole-3-acetic acid production, as well as antifungal activities. Out of 22 isolates, the amount of indole-3-acetic acid (IAA) ranging between 10–32 μg/ml was produced by 20 isolates and all isolates were positive for ammonia production ranging between 5.2 to 54 mg/ml. Among 22 isolates tested, the amount of hydroxamate-type siderophores were produced by 16 isolates ranging between 5.2 to 36.4 μg/ml, while catechols-type siderophores produced by 5 isolates ranging from 3.2 to 5.4 μg/ml. Fourteen isolates showed the solubilisation of inorganic phosphorous ranging from 3.2 to 32.6 mg/100ml. Chitinase and HCN production was shown by 19 and 15 different isolates, respectively. In addition, genes of indole acetic acid (iaaM) and chitinase (chiC) were successively amplified from 20 and 19 isolates respectively. The two potential strains Streptomyces sp. (BPSAC34) and Leifsonia xyli (BPSAC24) were tested in vivo and improved a range of growth parameters in chilli (Capsicum annuum L.) under greenhouse conditions. This study is the first published report that actinomycetes can be isolated as endophytes from within these

  15. Burdock fructooligosaccharide induces fungal resistance in postharvest Kyoho grapes by activating the salicylic acid-dependent pathway and inhibiting browning.

    PubMed

    Sun, Fei; Zhang, Pengying; Guo, Moran; Yu, Wenqian; Chen, Kaoshan

    2013-05-01

    Burdock fructooligosaccharide (BFO) is a natural elicitor from Arcitum lappa. The effects of BFO in controlling postharvest disease in grape, apple, banana, kiwi, citrus, strawberry, and pear were investigated. The disease index, decay percentage, and area under the disease progress curve indicated that BFO has general control effects on postharvest disease of fruits. Kyoho grapes were studied to elucidate the mechanism of BFO in boosting the resistance of grapes to Botrytis cinerea infection. BFO treatment induced upregulation of the npr1, pr1, pal, and sts genes, and inhibited the total phenol content decrease, which activated chitinase and β-1,3-glucanase. These results indicated that the salicylic acid-dependent signalling pathway was induced. The delayed colour change and peroxidase and polyphenoloxidase activity suggested that BFO delayed grape browning. The reduced respiration rate, weight loss, and titratable acidity prolonged the shelf life of postharvest grapes. BFO is a promising elicitor in postharvest disease control.

  16. Expression profiles for macrophage alternative activation genes in AD and in mouse models of AD

    PubMed Central

    Colton, Carol A; Mott, Ryan T; Sharpe, Hayley; Xu, Qing; Van Nostrand, William E; Vitek, Michael P

    2006-01-01

    Background Microglia are associated with neuritic plaques in Alzheimer disease (AD) and serve as a primary component of the innate immune response in the brain. Neuritic plaques are fibrous deposits composed of the amyloid beta-peptide fragments (Abeta) of the amyloid precursor protein (APP). Numerous studies have shown that the immune cells in the vicinity of amyloid deposits in AD express mRNA and proteins for pro-inflammatory cytokines, leading to the hypothesis that microglia demonstrate classical (Th-1) immune activation in AD. Nonetheless, the complex role of microglial activation has yet to be fully explored since recent studies show that peripheral macrophages enter an "alternative" activation state. Methods To study alternative activation of microglia, we used quantitative RT-PCR to identify genes associated with alternative activation in microglia, including arginase I (AGI), mannose receptor (MRC1), found in inflammatory zone 1 (FIZZ1), and chitinase 3-like 3 (YM1). Results Our findings confirmed that treatment of microglia with anti-inflammatory cytokines such as IL-4 and IL-13 induces a gene profile typical of alternative activation similar to that previously observed in peripheral macrophages. We then used this gene expression profile to examine two mouse models of AD, the APPsw (Tg-2576) and Tg-SwDI, models for amyloid deposition and for cerebral amyloid angiopathy (CAA) respectively. AGI, MRC1 and YM1 mRNA levels were significantly increased in the Tg-2576 mouse brains compared to age-matched controls while TNFα and NOS2 mRNA levels, genes commonly associated with classical activation, increased or did not change, respectively. Only TNFα mRNA increased in the Tg-SwDI mouse brain. Alternative activation genes were also identified in brain samples from individuals with AD and were compared to age-matched control individuals. In AD brain, mRNAs for TNFα, AGI, MRC1 and the chitinase-3 like 1 and 2 genes (CHI3L1; CHI3L2) were significantly increased

  17. Biological activity of phenylpropionic acid isolated from a terrestrial Streptomycetes.

    PubMed

    Narayana, Kolla J P; Prabhakar, Peddikotla; Vijayalakshmi, Muvva; Venkateswarlu, Yenamandra; Krishna, Palakodety S J

    2007-01-01

    The strain ANU 6277 was isolated from laterite soil and identified as Streptomyces sp. closely related to Streptomyces albidoflavus cluster by 16S rRNA analysis. The cultural, morphological and physiological characters of the strain were recorded. The strain exhibited resistance to chloramphenicol, penicillin and streptomycin. It had the ability to produce enzymes such as amylase and chitinase. A bioactive compound was isolated from the strain at stationary phase of culture and identified as 3-phenylpropionic acid (3-PPA) by FT-IR, EI-MS, 1H NMR and 13C NMR spectral studies. It exhibited antimicrobial activity against different bacteria like Bacillus cereus, B. subtilis, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, P. flourescens, Staphylococcus aureus and some fungi including Aspergillus flavus, A. niger, Candida albicans, Fusarium oxysporum, F. udum and Penicillium citrinum. The antifungal activity of 3-PPA of the strain was evaluated in in vivo and in vitro conditions against Fusarium udum causing wilt disease in pigeon pea. The compound 3-PPA is an effective antifungal agent when compared to tricyclozole (fungicide) to control wilt caused by F. udum, but it exhibited less antifungal activity than carbendazim.

  18. The Use of Fluorogenic Substrates To Measure Fungal Presence and Activity in Soil

    PubMed Central

    Miller, Morten; Palojärvi, Ansa; Rangger, Andrea; Reeslev, Morten; Kjøller, Annelise

    1998-01-01

    Our objective was to determine if 4-methylumbelliferyl-labelled enzyme substrates could be used to detect and quantify specific components of chitinase and cellulase activities as specific indicators of the presence and activity of fungal biomass. The fluorogenic substrates 4-methylumbelliferyl (MUF) N-acetyl-β-d-glucosaminide and MUF β-d-lactoside were used for the detection and quantification of β-N-acetylglucosaminidase (EC 3.2.1.30) (NAGase) and endo 1,4-β-glucanase (EC 3.2.1.4)/cellobiohydrolase (EC 3.2.1.91) (CELase), respectively. Culture screenings on solid media showed a widespread ability to produce NAGase among a taxonomically diverse selection of fungi on media with and without added chitin. NAGase activity was expressed only in a limited number of bacteria and on media supplemented with chitin. The CELase activity was observed only in a limited number of fungi and bacteria. Bacterial CELase activity was expressed on agar media containing a cellulose-derived substrate. In soil samples, NAGase activity was significantly correlated with estimates of fungal biomass, based on the content of two fungus-specific indicator molecules, 18:2ω6 phospholipid fatty acid (PLFA) and ergosterol. CELase activity was significantly correlated with the PLFA-based estimate of fungal biomass in the soil, but no correlation was found with ergosterol-based estimates of fungal biomass. PMID:9464399

  19. Ascalin, a new anti-fungal peptide with human immunodeficiency virus type 1 reverse transcriptase-inhibiting activity from shallot bulbs.

    PubMed

    Wang, H X; Ng, T B

    2002-06-01

    An isolation procedure comprising ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Sepharose and gel filtration on Superdex 75 was used to isolate an anti-fungal peptide from the bulbs of the shallot Allium ascalonicum. The peptide demonstrated a molecular weight of 9.5kDa, and possessed an N-terminal sequence YQCGQGG somewhat similar to chitinases from other Allium species which are however much larger in molecular weight. The peptide designated ascalin manifested a unique specific anti-fungal activity. It inhibited mycelial growth in the fungus Botrytis cinerea but not in the fungi Mycosphaerella arachidicola and Fusarium oxysporum. Ascalin inhibited HIV-1 reverse transcriptase with an IC(50) of 10 microM, much more potently than Allium tuberosum anti-fungal protein and other anti-fungal proteins.

  20. Antifungal activity of gallic acid purified from Terminalia nigrovenulosa bark against Fusarium solani.

    PubMed

    Nguyen, Dang-Minh-Chanh; Seo, Dong-Jun; Lee, Hyang-Burm; Kim, In-Seon; Kim, Kil-Yong; Park, Ro-Dong; Jung, Woo-Jin

    2013-03-01

    The antifungal activities of methanolic extracts from Terminalia nigrovenulosa bark (TNB) was investigated for effects on the initial growth of mycelia against Fusarium solani. The ethyl acetate fraction separated from TNB demonstrated the highest antifungal activity against F. solani. The antifungal compound was isolated from TNB using silica gel column and Sephadex LH-20 chromatography combined with thin-layer chromatography and high performance liquid chromatography. Structural identification of the antifungal compound was conducted using (1)H NMR, (13)C NMR, and liquid chromatography-tandem mass spectrometry. The purified antifungal compound was gallic acid (GA) or 3,4,5-trihydroxy benzoic acid. Purified-GA possesses the high antifungal activity against F. solani, and that antifungal activity was dosage-dependent. The hyphae became collapsed and shrunken after 24 h incubation with GA (500 ppm). In pot experiments, the application of TNB crude extract was found to be effective in controlling the cucumber Fusarium root rot disease by enhancing activities of chitinase, peroxidase thereby promoting the growth of plants. The applied TNB extract significantly suppressed root rot disease compared to control. It resulted in 33, 75 and 81% disease suppression with 100, 500 and 1000 ppm of TNB crude extract, respectively. The study effectively demonstrated biological activities of the TNB extract, therefore suggesting the application of TNB for the control of soil-borne diseases of cucumber plants.

  1. Proteins and enzymatic activities in Erbaluce grape berries with different response to the withering process.

    PubMed

    Vincenzi, Simone; Tolin, Serena; Cocolin, Luca; Rantsiou, Kalliopi; Curioni, Andrea; Rolle, Luca

    2012-06-30

    During the off-vine natural withering process of Erbaluce (white) grapes to obtain "Erbaluce Caluso" Passito wine, some berries change in color from green-yellow to blue. This phenomenon appears at different extents in different years and might be related to several parameters, such as temperature and humidity during withering, grape composition and Botrytis cinerea loading. To better understand the mechanism involved in color variation, the metabolic changes corresponding to this event were studied. At the end of the withering process berries with different colors were separated using a reflectance spectrophotometer, obtaining three color classes identified as "green" (L*=40.3, a*=-0.56, b*=15.20), "gold" (L*=37.7, a*=5.01, b*=14.12) and "blue" (L*=28.6, a*=0.89, b*=-0.67). The three groups of berries had different water contents, the blue berries containing about 30% less water than the green ones. Samples were crushed and the juices were analyzed. The juice yield for blue berries was less than 50% of that of the other two classes, confirming their higher dehydration level. Protein extraction from de-seeded berries was carried out using two different protocols, the first involving a treatment with phenol (to remove polyphenolic substances) and the second based on an extraction with a mild detergent (to recover the proteins to be used for enzymatic analyses). No trace of laccase activity was found in any of the samples, although DNA analysis, by quantitative PCR, suggested the presence of B. cinerea infection in the blue grapes. Chitinase activity of the blue berries was only 30% of that of the other two samples, as confirmed also by zymographic analysis on electrophoretic gels. The same was found also for esterase activity, which was lower (of about 85%) in the blue berries, which, in contrast, showed the highest beta-glucosidase activity. The electrophoretic analysis of the protein extracts revealed strong differences among the samples. Compared to the green and

  2. Determination of lytic enzyme activities of indigenous Trichoderma isolates from Pakistan.

    PubMed

    Asad, Saeed Ahmad; Tabassum, Ayesha; Hameed, Abdul; Hassan, Fayyaz Ul; Afzal, Aftab; Khan, Sabaz Ali; Ahmed, Rafiq; Shahzad, Muhammad

    2015-01-01

    This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, β-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. β-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract.

  3. Digestive organ sizes and enzyme activities of refueling western sandpipers (Calidris mauri): contrasting effects of season and age.

    PubMed

    Stein, R Will; Place, Allen R; Lacourse, Terri; Guglielmo, Christopher G; Williams, Tony D

    2005-01-01

    We examined seasonal and age-related variation in digestive organ sizes and enzyme activities in female western sandpipers (Calidris mauri) refueling at a coastal stopover site in southern British Columbia. Adult sandpipers exhibited seasonal variation in pancreatic and intestinal enzyme activities but not in digestive system or organ sizes. Spring migrants had 22% higher total and 67% higher standardized pancreatic lipase activities but 37% lower total pancreatic amylase activity than fall migrants, which suggests that the spring diet was enriched with lipids but low in glycogen. Spring migrants also had 47% higher total intestinal maltase activity as well as 56% higher standardized maltase and 13% higher standardized aminopeptidase-N activities. Spring migrants had higher total enzymic capacity than fall migrants, due primarily to higher total lipase and maltase activities. During fall migration, the juvenile's digestive system was 10% larger than the adult's, and it was composed differently: juveniles had a 16% larger small intestine but a 27% smaller proventriculus. The juvenile's larger digestive system was associated with lower total enzymic capacity than the adult's due to 20% lower total chitinase and 23% lower total lipase activities. These results suggest that juvenile western sandpipers may process food differently from adults and/or have a lower-quality diet.

  4. The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa

    PubMed Central

    2014-01-01

    Background The production and accumulation of pathogenesis-related proteins (PR proteins) in plants in response to biotic or abiotic stresses is well known and is considered as a crucial mechanism for plant defense. A pathogenesis-related protein 4 cDNA was identified from a cacao-Moniliophthora perniciosa interaction cDNA library and named TcPR-4b. Results TcPR-4b presents a Barwin domain with six conserved cysteine residues, but lacks the chitin-binding site. Molecular modeling of TcPR-4b confirmed the importance of the cysteine residues to maintain the protein structure, and of several conserved amino acids for the catalytic activity. In the cacao genome, TcPR-4b belonged to a small multigene family organized mainly on chromosome 5. TcPR-4b RT-qPCR analysis in resistant and susceptible cacao plants infected by M. perniciosa showed an increase of expression at 48 hours after infection (hai) in both cacao genotypes. After the initial stage (24-72 hai), the TcPR-4b expression was observed at all times in the resistant genotypes, while in the susceptible one the expression was concentrated at the final stages of infection (45-90 days after infection). The recombinant TcPR-4b protein showed RNase, and bivalent ions dependent-DNase activity, but no chitinase activity. Moreover, TcPR-4b presented antifungal action against M. perniciosa, and the reduction of M. perniciosa survival was related to ROS production in fungal hyphae. Conclusion To our knowledge, this is the first report of a PR-4 showing simultaneously RNase, DNase and antifungal properties, but no chitinase activity. Moreover, we showed that the antifungal activity of TcPR-4b is directly related to RNase function. In cacao, TcPR-4b nuclease activities may be related to the establishment and maintenance of resistance, and to the PCD mechanism, in resistant and susceptible cacao genotypes, respectively. PMID:24920373

  5. Different Effects of Metarhizium anisopliae Strains IMI330189 and IBC200614 on Enzymes Activities and Hemocytes of Locusta migratoria L.

    PubMed Central

    Cao, Guangchun; Jia, Miao; Zhao, Xia; Wang, Lei; Tu, Xiongbing; Wang, Guangjun; Nong, Xiangqun; Zhang, Zehua

    2016-01-01

    Background Metarhizium is an important class of entomopathogenic fungi in the biocontrol of insects, but its virulence is affected by insect immunity. To clarify the mechanism in virulence of Metarhizium, we compared the immunological differences in Locusta migratoria L. when exposed to two strains of Metarhizium anisopliae (Ma). Results The virulence of Ma IMI330189 was significantly higher than that of Ma IBC200614 to locust, and IMI330189 overcame the hemocytes and began destroying the hemocytes of locust at 72 h after spray, while locust is immune to IBC200614. IMI330189 could overcome the humoral immunity of locust by inhibiting the activities of phenol oxidase (PO), esterases, multi-function oxidases (MFOs) and acetylcholinesterases in locust while increasing the activities of glutathione-S-transferases (GSTs), catalase and aryl-acylamidase (AA). However IBC200614 inhibit the activities of GSTs and AA in locust and increase the activities of MFOs, PO, superoxide dismutase, peroxidase and chitinase in locust. The changes of enzymes activities in period of infection showed that the time period between the 2nd and the 5th day after spray is critical in the pathogenic process. Conclusion These results found the phenomenon that Ma initiatively broke host hemocytes, revealed the correlation between the virulence of Ma and the changes of enzymes activities in host induced by Ma, and clarified the critical period in the infection of Ma. So, these results should provide guidance for the construction of efficient biocontrol Ma strains. PMID:27227835

  6. Cold active hydrolytic enzymes production by psychrotrophic Bacilli isolated from three sub-glacial lakes of NW Indian Himalayas.

    PubMed

    Yadav, Ajar Nath; Sachan, Shashwati Ghosh; Verma, Priyanka; Kaushik, Rajeev; Saxena, Anil Kumar

    2016-03-01

    The diversity of culturable, cold-active enzymes producing Bacilli was investigated from three sub-glacial lakes of north western Indian Himalayas. Amplified ribosomal DNA restriction analysis (ARDRA) using three restriction enzymes Alu I, Msp I, and Hae III led to the clustering of 136 Bacilli into 26, 23, and 22 clusters at 75% similarity index from Chandratal Lake, Dashair Lake, and Pangong Lake, respectively. Phylogenetic analysis based on 16S rRNA gene sequencing led to the identification of 35 Bacilli that could be grouped in seven families viz.: Bacillaceae (48%), Staphylococcaceae (14%), Bacillales incertae sedis (13%), Planococcaceae (12%), Paenibacillaceae (9%), Sporolactobacillaceae (3%), and Carnobacteriaceae (1%), which included twelve different genera Bacillus, Desemzia, Exiguobacterium, Jeotgalicoccus, Lysinibacillus, Paenibacillus, Planococcus, Pontibacillus, Sinobaca, Sporosarcina, Staphylococcus, and Virgibacillus. Based on their optimal temperature for growth, 35 Bacilli were grouped as psychrophilic (11 strains), psychrotrophic (17 strains), or psychrotolerant (7 strains), respectively. The representative isolates from each cluster were screened for cold-active enzyme activities. Amylase, β-glucosidase, pectinase, and protease activities at 4 °C were detected in more than 80% of the strains while approximately 40, 31, 23, 14, 11, and 9% of strains possessed cellulase, xylanase, β-galactosidase, laccase, chitinase, and lipase activity, respectively. Among 35 Bacilli, Bacillus amyloliquefaciens, Bacillus marisflavi, Exiguobacterium indicum, Paenibacillus terrae, Pontibacillus sp., Sporosarcina globispora, and Sporosarcina psychrophila were efficient producers of different cold-active enzymes. These cold-adapted Bacilli could play an important role in industrial and agricultural processes.

  7. 3D structure of the Yersinia entomophaga toxin complex and implications for insecticidal activity

    PubMed Central

    Landsberg, Michael J.; Jones, Sandra A.; Rothnagel, Rosalba; Busby, Jason N.; Marshall, Sean D. G.; Simpson, Robert M.; Lott, J. Shaun; Hankamer, Ben; Hurst, Mark R. H.

    2011-01-01

    Toxin complex (Tc) proteins are a class of bacterial protein toxins that form large, multisubunit complexes. Comprising TcA, B, and C components, they are of great interest because many exhibit potent insecticidal activity. Here we report the structure of a novel Tc, Yen-Tc, isolated from the bacterium Yersinia entomophaga MH96, which differs from the majority of bacterially derived Tcs in that it exhibits oral activity toward a broad range of insect pests, including the diamondback moth (Plutella xylostella). We have determined the structure of the Yen-Tc using single particle electron microscopy and studied its mechanism of toxicity by comparative analyses of two variants of the complex exhibiting different toxicity profiles. We show that the A subunits form the basis of a fivefold symmetric assembly that differs substantially in structure and subunit arrangement from its most well characterized homologue, the Xenorhabdus nematophila toxin XptA1. Histopathological and quantitative dose response analyses identify the B and C subunits, which map to a single, surface-accessible region of the structure, as the sole determinants of toxicity. Finally, we show that the assembled Yen-Tc has endochitinase activity and attribute this to putative chitinase subunits that decorate the surface of the TcA scaffold, an observation that may explain the oral toxicity associated with the complex. PMID:22158901

  8. Strategies to improve the insecticidal activity of Cry toxins from Bacillus thuringiensis.

    PubMed

    Pardo-López, L; Muñoz-Garay, C; Porta, H; Rodríguez-Almazán, C; Soberón, M; Bravo, A

    2009-03-01

    Bacillus thuringiensis Cry toxins have been widely used in the control of insect pests either as spray products or expressed in transgenic crops. These proteins are pore-forming toxins with a complex mechanism of action that involves the sequential interaction with several toxin-receptors. Cry toxins are specific against susceptible larvae and although they are often highly effective, some insect pests are not affected by them or show low susceptibility. In addition, the development of resistance threatens their effectiveness, so strategies to cope with all these problems are necessary. In this review we will discuss and compare the different strategies that have been used to improve insecticidal activity of Cry toxins. The activity of Cry toxins can be enhanced by using additional proteins in the bioassay like serine protease inhibitors, chitinases, Cyt toxins, or a fragment of cadherin receptor containing a toxin-binding site. On the other hand, different modifications performed in the toxin gene such as site-directed mutagenesis, introduction of cleavage sites in specific regions of the protein, and deletion of small fragments from the amino-terminal region lead to improved toxicity or overcome resistance, representing interesting alternatives for insect pest control.

  9. Activation of Host Defense Mechanisms by Elevated Production of H2O2 in Transgenic Plants.

    PubMed Central

    Wu, G.; Shortt, B. J.; Lawrence, E. B.; Leon, J.; Fitzsimmons, K. C.; Levine, E. B.; Raskin, I.; Shah, D. M.

    1997-01-01

    Active oxygen species have been postulated to perform multiple functions in plant defense, but their exact role in plant resistance to diseases is not fully understood. We have recently demonstrated H2O2-mediated disease resistance in transgenic potato (Solanum tuberosum) plants expressing a foreign gene encoding glucose oxidase. In this study we provide further evidence that the H2O2-mediated disease resistance in potato is effective against a broad range of plant pathogens. We have investigated mechanisms underlying the H2O2-mediated disease resistance in transgenic potato plants. The constitutively elevated levels of H2O2 induced the accumulation of total salicylic acid severalfold in the leaf tissue of transgenic plants, although no significant change was detected in the level of free salicylic acid. The mRNAs of two defense-related genes encoding the anionic peroxidase and acidic chitinase were also induced. In addition, an increased accumulation of several isoforms of extracellular peroxidase, including a newly induced one, was observed. This was accompanied by a significant increase in the lignin content of stem and root tissues of the transgenic plants. The results suggest that constitutively elevated sublethal levels of H2O2 are sufficient to activate an array of host defense mechanisms, and these defense mechanisms may be a major contributing factor to the H2O2-mediated disease resistance in transgenic plants. PMID:12223817

  10. Depth profiles of bacterioplankton assemblages and their activities in the Ross Sea

    NASA Astrophysics Data System (ADS)

    Celussi, Mauro; Cataletto, Bruno; Fonda Umani, Serena; Del Negro, Paola

    2009-12-01

    The identification of bacterial community structure has led, since the beginning of the 1990s, to the idea that bacterioplankton populations are stratified in the water column and that diverse lineages with mostly unknown phenotypes dominate marine microbial communities. The diversity of depth-related assemblages is also reflected in their patterns of activities, as bacteria affiliated to different groups can express different activities in a given ecosystem. We analysed bacterial assemblages (DGGE fingerprinting) and their activities (prokaryotic carbon production, protease, phosphatase, chitinase, beta-glucosidase and lipase activities) in two areas in the Ross Sea, differing mainly in their productivity regime: two stations are located in the Terra Nova Bay polynya area (highly productive during summer) and two close to Cape Adare (low phytoplankton biomass and activity). At every station a pronounced stratification of bacterial assemblages was identified, highlighting epipelagic communities differing substantially from the mesopelagic and the bathypelagic communities. Multivariate analysis suggested that pressure and indirectly light-affected variables (i.e. oxygen and fluorescence) had a great effect on the bacterial communities outcompeting the possible influences of temperature and dissolved organic carbon concentration. Generally activities decreased with depth even though a signal of the Circumpolar Deep Water (CDW) at one of the northern stations corresponded to an increase in some of the degradative activities, generating some 'hot spots' in the profile. We also found that similar assemblages express similar metabolic requirements reflected in analogous patterns of activity (similar degradative potential and leucine uptake rate). Furthermore, the presence of eukaryotic chloroplasts' 16S rDNA in deep samples highlighted how in some cases the dense surface-water formation (in this case High Salinity Shelf Water—HSSW) and downwelling can affect, at least

  11. The cell factory approach toward biotechnological production of high-value chitosan oligomers and their derivatives: an update.

    PubMed

    Naqvi, Shoa; Moerschbacher, Bruno M

    2017-02-01

    Chitin is one of the most abundant renewable resources, and chitosans, the partially deacetylated derivatives of chitin, are among the most promising functional biopolymers, with superior material properties and versatile biological functionalities. Elucidating molecular structure-function relationships and cellular modes of action of chitosans, however, it is challenging due to the micro-heterogeneity and structural complexity of polysaccharides. Lately, it has become apparent that many of the biological activities of chitosan polymers, such as in agricultural plant disease protection or in mediating scar-free wound healing, may be attributed to oligomeric break-down products generated by the action of chitosanolytic hydrolases present in the target tissues, such as human chitotriosidase. Consequently, the focus of current research is shifting toward chitosan oligomers so that the availability of well-defined chitosan oligosaccharides (COS) becomes a bottleneck. Well-known ways of producing COS use physical and/or chemical means for the partial depolymerization of chitosan polymers, typically leading to broad mixtures of COS varying in their degrees of polymerization (DP) and acetylation (DA), and with more or less random patterns of acetylation (PAs). Even after chromatographic separation according to DP and DA, such mixtures are of limited value to elucidate structure-function relationships and modes of action. More recently, enzymatic means using chitinases and/or chitosanases, and sometimes chitin deacetylases, have been proposed as these can be more tightly controlled and yield slightly better defined mixtures of COS. An alternative would be chemical synthesis of COS which in principle would allow for full structural control, but protocols for it are lengthy, costly, and not yet well developed, and yields are low. Synthetic biology now allows to develop today's in vitro bio-refinery approaches into in vivo cell factory approaches for the biotechnological

  12. The Glycolytic Enzymes Activity in the Midgut of Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) adult and their Seasonal Changes

    PubMed Central

    Guzik, Joanna; Nakonieczny, Mirosław; Tarnawska, Monika; Bereś, Paweł K.; Drzewiecki, Sławomir; Migula, Paweł

    2015-01-01

    The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) is an important pest of maize. The diet of the D. virgifera imago is rich in starch and other polysaccharides present in cereals such as maize. Therefore, knowledge about enzymes involved in digestion of such specific food of this pest seems to be important. The paper shows, for the first time, the activities of main glycolytic enzymes in the midgut of D. virgifera imago: endoglycosidases (α-amylase, cellulase, chitinase, licheninase, laminarinase); exoglycosidases (α- and β-glucosidases, α- and β-galactosidases) and disaccharidases (maltase, isomaltase, sucrase, trehalase, lactase, and cellobiase). Activities of α-amylase, α-glucosidase, and maltase were the highest among assayed endoglycosidases, exoglycosidases, and disaccharidases, respectively. This indicates that in the midgut of D. virgifera imago α-amylase, α-glucosidase and maltase are important enzymes in starch hydrolysis and products of its digestion. These results lead to conclusion that inhibition of most active glycolytic enzymes of D. virgifera imago may be another promising method for chemical control of this pest of maize.

  13. Response of microbial extracellular enzyme activities and r- vs. K- selected microorganisms to elevated atmospheric CO2 depends on soil aggregate size

    NASA Astrophysics Data System (ADS)

    Dorodnikov, Maxim; Blagodatskaya, Evgenia; Blagodatskiy, Sergey; Kuzyakov, Yakov

    2014-05-01

    Increased belowground carbon (C) transfer by plant roots under elevated atmospheric CO2 and the contrasting environment in soil macro- and microaggregates could affect properties of the microbial community in the rhizosphere. We evaluated the effect of 5 years of elevated CO2 (550 ppm) on four extracellular enzymes: ß-glucosidase, chitinase, phosphatase, and sulfatase along with the contribution of fast- (r-strategists) and slow-growing microorganisms (K-strategists) in soil aggregates. We fractionated the bulk soil from the ambient and elevated CO2 treatments of FACE-Hohenheim (Stuttgart) into large macro- (>2 mm), small macro- (0.25-2.00 mm), and microaggregates (<0.25 mm) using a modified dry sieving. Microbial biomass (C-mic by SIR), the maximal specific growth rate (µ), growing microbial biomass (GMB) and lag-period (t-lag) were estimated by the kinetics of CO2 emission from bulk soil and aggregates amended with glucose and nutrients. In the bulk soil and isolated aggregates before and after activation with glucose, the actual and the potential enzyme activities were measured. Although C-org and C-mic as well as the activities of ß-glucosidase, phosphatase, and sulfatase were unaffected in bulk soil and in aggregate-size classes by elevated CO2, significant changes were observed in potential enzyme production after substrate amendment. After adding glucose, enzyme activities under elevated CO2 were 1.2-1.9-fold higher than under ambient CO2. In addition, µ values were significantly higher under elevated than ambient CO2 for bulk soil, small macroaggregates, and microaggregates. Based on changes in µ, GMB, and lag-period, we conclude that elevated atmospheric CO2 stimulated the r-selected microorganisms, especially in soil microaggregates. In contrast, significantly higher chitinase activity in bulk soil and in large macroaggregates under elevated CO2 revealed an increased contribution of fungi to turnover processes. We conclude that quantitative and

  14. An insecticidal GroEL protein with chitin binding activity from Xenorhabdus nematophila.

    PubMed

    Joshi, Mohan Chandra; Sharma, Animesh; Kant, Sashi; Birah, Ajanta; Gupta, Gorakh Prasad; Khan, Sharik R; Bhatnagar, Rakesh; Banerjee, Nirupama

    2008-10-17

    Xenorhabdus nematophila secretes insecticidal proteins to kill its larval prey. We have isolated an approximately 58-kDa GroEL homolog, secreted in the culture medium through outer membrane vesicles. The protein was orally insecticidal to the major crop pest Helicoverpa armigera with an LC50 of approximately 3.6 microg/g diet. For optimal insecticidal activity all three domains of the protein, apical, intermediate, and equatorial, were necessary. The apical domain alone was able to bind to the larval gut membranes and manifest low level insecticidal activity. At equimolar concentrations, the apical domain contained approximately one-third and the apical-intermediate domain approximately one-half bioactivity of that of the full-length protein. Interaction of the protein with the larval gut membrane was specifically inhibited by N-acetylglucosamine and chito-oligosaccharides. Treatment of the larval gut membranes with chitinase abolished protein binding. Based on the three-dimensional structural model, mutational analysis demonstrated that surface-exposed residues Thr-347 and Ser-356 in the apical domain were crucial for both binding to the gut epithelium and insecticidal activity. Double mutant T347A,S356A was 80% less toxic (p < 0.001) than the wild type protein. The GroEL homolog showed alpha-chitin binding activity with Kd approximately 0.64 microm and Bmax approximately 4.68 micromol/g chitin. The variation in chitin binding activity of the mutant proteins was in good agreement with membrane binding characteristics and insecticidal activity. The less toxic double mutant XnGroEL showed an approximately 8-fold increase of Kd in chitin binding assay. Our results demonstrate that X. nematophila secretes an insecticidal GroEL protein with chitin binding activity.

  15. An Insecticidal GroEL Protein with Chitin Binding Activity from Xenorhabdus nematophila*

    PubMed Central

    Joshi, Mohan Chandra; Sharma, Animesh; Kant, Sashi; Birah, Ajanta; Gupta, Gorakh Prasad; Khan, Sharik R.; Bhatnagar, Rakesh; Banerjee, Nirupama

    2008-01-01

    Xenorhabdus nematophila secretes insecticidal proteins to kill its larval prey. We have isolated an ∼58-kDa GroEL homolog, secreted in the culture medium through outer membrane vesicles. The protein was orally insecticidal to the major crop pest Helicoverpa armigera with an LC50 of ∼3.6 μg/g diet. For optimal insecticidal activity all three domains of the protein, apical, intermediate, and equatorial, were necessary. The apical domain alone was able to bind to the larval gut membranes and manifest low level insecticidal activity. At equimolar concentrations, the apical domain contained approximately one-third and the apical-intermediate domain approximately one-half bioactivity of that of the full-length protein. Interaction of the protein with the larval gut membrane was specifically inhibited by N-acetylglucosamine and chito-oligosaccharides. Treatment of the larval gut membranes with chitinase abolished protein binding. Based on the three-dimensional structural model, mutational analysis demonstrated that surface-exposed residues Thr-347 and Ser-356 in the apical domain were crucial for both binding to the gut epithelium and insecticidal activity. Double mutant T347A,S356A was 80% less toxic (p < 0.001) than the wild type protein. The GroEL homolog showed α-chitin binding activity with Kd ∼ 0.64 μm and Bmax ∼ 4.68 μmol/g chitin. The variation in chitin binding activity of the mutant proteins was in good agreement with membrane binding characteristics and insecticidal activity. The less toxic double mutant XnGroEL showed an ∼8-fold increase of Kd in chitin binding assay. Our results demonstrate that X. nematophila secretes an insecticidal GroEL protein with chitin binding activity. PMID:18667427

  16. Expression, refolding, and purification of active diacetylchitobiose deacetylase from Pyrococcus horikoshii.

    PubMed

    Mine, Shouhei; Ikegami, Takahisa; Kawasaki, Kazunori; Nakamura, Tsutomu; Uegaki, Koichi

    2012-08-01

    A chitinase from the hyperthermophilic archaeon Pyrococcus furiosus degrades chitin to produce diacetylchitobiose [(GlcNAc)(2)] as the end product. To further investigate the degradation mechanism of (GlcNAc)(2) in Pyrococcus spp., we cloned the gene of PH0499 from Pyrococcus horikoshii, which encodes a protein homologous to the diacetylchitobiose deacetylase of Thermococcus kodakaraensis. The deacetylase (Ph-Dac) was overexpressed as inclusion bodies in Escherichia coli Rosetta (DE3) pLys. The insoluble inclusion body was solubilized and reactivated through a refolding procedure. After several purification steps, 40 mg of soluble, thermostable (up to 80°C) Ph-Dac was obtained from 1L of culture. The apparent molecular mass of the refolded Ph-Dac was 180 kDa, indicating Ph-Dac to be a homohexamer. The refolded Ph-Dac also exhibited deacetylase activity toward (GlcNAc)(2), and the deacetylation site was revealed to be specific to the nonreducing end residue of (GlcNAc)(2). These expression and purification systems are useful for further characterization of Ph-Dac.

  17. Comparison of the White-Nose Syndrome Agent Pseudogymnoascus destructans to Cave-Dwelling Relatives Suggests Reduced Saprotrophic Enzyme Activity

    PubMed Central

    Reynolds, Hannah T.; Barton, Hazel A.

    2014-01-01

    White-nose Syndrome (WNS) is an emerging infectious mycosis that has impacted multiple species of North American bats since its initial discovery in 2006, yet the physiology of the causal agent, the psychrophilic fungus Pseudogymnoascus destructans ( = Geomyces destructans), is not well understood. We investigated the ability of P. destructans to secrete enzymes that could permit environmental growth or affect pathogenesis and compared enzyme activity across several Pseudogymnoascus species isolated from both hibernating bats and cave sediments. We found that P. destructans produced enzymes that could be beneficial in either a pathogenic or saprotrophic context, such as lipases, hemolysins, and urease, as well as chitinase and cellulases, which could aid in saprotrophic growth. The WNS pathogen showed significantly lower activity for urease and endoglucanase compared to con-generic species (Pseudogymnoascus), which may indicate a shift in selective pressure to the detriment of P. destructans’ saprotrophic ability. Based on the positive function of multiple saprotrophic enzymes, the causal agent of White-nose Syndrome shows potential for environmental growth on a variety of substrates found in caves, albeit at a reduced level compared to environmental strains. Our data suggest that if P. destructans emerged as an opportunistic infection from an environmental source, co-evolution with its host may have led to a reduced capacity for saprotrophic growth. PMID:24466096

  18. Characterization of maize chitinase-A, a tough allergenic molecule

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Food allergy is recognized as a major health concern with a steady increasing trend in Western countries. Food allergens are proteins belonging to a small group of about 30 families, with restricted biochemical functions. This leads to the assumption that allergens must meet specific, but not yet co...

  19. A Recombinant Anticarsia gemmatalis MNPV Harboring chiA and v-cath Genes from Choristoneura fumiferana Defective NPV Induce Host Liquefaction and Increased Insecticidal Activity

    PubMed Central

    Lima, Anabele Azevedo; Aragão, Clara Wandenkolck Silva; de Castro, Maria Elita Batista; Oliveira, Juliana Velasco de Castro; Sosa Gómez, Daniel Ricardo; Ribeiro, Bergmann Morais

    2013-01-01

    One of the interesting features of Anticarsia gemmatalis multiple nucleopolyhedrovirus isolate 2D (AgMNPV-2D) genome is the absence of chitinase (chiA) and cathepsin (v-cath) genes. This characteristic may be responsible for the lack of liquefaction and melanization in A. gemmatalis larvae killed by AgMNPV-2D infection. This study aimed to test the hypothesis that CHIA and V-CATH proteins from Choristonera fumiferana DEF multiple nucleopolyhedrovirus (CfDEFNPV) are able to liquefy and melanize the cuticle of A. gemmatalis larvae infected by a recombinant AgMNPV containing chiA and v-cath genes inserted in its genome. A fragment from the CfDefNPV genome containing chiA and v-cath genes was inserted into the genome of AgMNPV-2D. The recombinant virus (vAgp2100Cf.chiA/v-cath) was purified and used to infect insect cells and larvae. Transcripts of v-cath and chiA genes were detected along the infection of insect cells by qRT-PCR, from early to late phases of infection. The analysis of A. gemmatalis larvae killed by vAgp2100Cf.chiA/v-cath infection confirmed the hypothesis proposed. The vAgp2100Cf.chiA/v-cath showed higher insecticidal activity against third instar A. gemmatalis larvae when compared to AgMNPV-2D. The mean time to death was also lower for the vAgp2100Cf.chiA/v-cath when compared to AgMNPV-2D at 10 days post infection. Occlusion body production was higher in A. gemmatalis larvae infected with vAgp2100Cf.chiA/v-cath when compared to AgMNPV-2D. Enzyme assays showed higher chitinase and cysteine protease activities in insect cells and insects infected with vAgp2100Cf.chiA/v-cath when compared to AgMNPV-2D. The introduction of chiA and v-cath genes into the genome of AgMNPV improves its insecticidal activity against A. gemmatalis larvae and this recombinant virus could be used as an alternative to the wild type virus to control this important insect pest. PMID:24086357

  20. A recombinant Anticarsia gemmatalis MNPV harboring chiA and v-cath genes from Choristoneura fumiferana defective NPV induce host liquefaction and increased insecticidal activity.

    PubMed

    Lima, Anabele Azevedo; Aragão, Clara Wandenkolck Silva; de Castro, Maria Elita Batista; Oliveira, Juliana Velasco de Castro; Sosa Gómez, Daniel Ricardo; Ribeiro, Bergmann Morais

    2013-01-01

    One of the interesting features of Anticarsia gemmatalis multiple nucleopolyhedrovirus isolate 2D (AgMNPV-2D) genome is the absence of chitinase (chiA) and cathepsin (v-cath) genes. This characteristic may be responsible for the lack of liquefaction and melanization in A. gemmatalis larvae killed by AgMNPV-2D infection. This study aimed to test the hypothesis that CHIA and V-CATH proteins from Choristonera fumiferana DEF multiple nucleopolyhedrovirus (CfDEFNPV) are able to liquefy and melanize the cuticle of A. gemmatalis larvae infected by a recombinant AgMNPV containing chiA and v-cath genes inserted in its genome. A fragment from the CfDefNPV genome containing chiA and v-cath genes was inserted into the genome of AgMNPV-2D. The recombinant virus (vAgp2100Cf.chiA/v-cath) was purified and used to infect insect cells and larvae. Transcripts of v-cath and chiA genes were detected along the infection of insect cells by qRT-PCR, from early to late phases of infection. The analysis of A. gemmatalis larvae killed by vAgp2100Cf.chiA/v-cath infection confirmed the hypothesis proposed. The vAgp2100Cf.chiA/v-cath showed higher insecticidal activity against third instar A. gemmatalis larvae when compared to AgMNPV-2D. The mean time to death was also lower for the vAgp2100Cf.chiA/v-cath when compared to AgMNPV-2D at 10 days post infection. Occlusion body production was higher in A. gemmatalis larvae infected with vAgp2100Cf.chiA/v-cath when compared to AgMNPV-2D. Enzyme assays showed higher chitinase and cysteine protease activities in insect cells and insects infected with vAgp2100Cf.chiA/v-cath when compared to AgMNPV-2D. The introduction of chiA and v-cath genes into the genome of AgMNPV improves its insecticidal activity against A. gemmatalis larvae and this recombinant virus could be used as an alternative to the wild type virus to control this important insect pest.

  1. Effects of stoichiometry and temperature perturbations on beech litter decomposition, enzyme activities and protein expression

    NASA Astrophysics Data System (ADS)

    Keiblinger, K. M.; Schneider, T.; Roschitzki, B.; Schmid, E.; Eberl, L.; Hämmerle, I.; Leitner, S.; Richter, A.; Wanek, W.; Riedel, K.; Zechmeister-Boltenstern, S.

    2011-12-01

    Microbes are major players in leaf litter decomposition and therefore advances in the understanding of their control on element cycling are of paramount importance. Our aim was to investigate the influence of leaf litter stoichiometry in terms of carbon (C) : nitrogen (N) : phosphorus (P) on the decomposition process, and to follow changes in microbial community structure and function in response to temperature-stress treatments. To elucidate how the stoichiometry of beech litter (Fagus sylvatica L.) and stress treatments interactively affect the decomposition processes, a terrestrial microcosm experiment was conducted. Beech litter from different Austrian sites covering C:N ratios from 39 to 61 and C:P ratios from 666 to 1729 were incubated at 15 °C and 60% moisture for six months. Part of the microcosms were then subjected to severe changes in temperature (+30 °C and -15 °C) to monitor the influence of temperature stress. Extracellular enzyme activities were assayed and respiratory activities measured. A semi-quantitative metaproteomics approach (1D-SDS PAGE combined with liquid chromatography and tandem mass-spectrometry; unique spectral counting) was employed to investigate the impact of the applied stress treatments in dependency of litter stoichiometry on structure and function of the decomposing community. In litter with narrow C:nutrient ratios microbial decomposers were most abundant. Cellulase, chitinase, phosphatase and protease activity decreased after heat and frost treatments. Decomposer communities and specific functions varied with site i.e. stoichiometry. The applied stress evoked strong changes of enzyme activities, dissolved organic nitrogen and litter pH. Freeze treatments resulted in a decline in residual plant litter material, and increased fungal abundance indicating slightly accelerated decomposition. Overall, we could detect a strong effect of litter stoichiometry on microbial community structure as well as function. Temperature

  2. Candida albicans Chitin Increases Arginase-1 Activity in Human Macrophages, with an Impact on Macrophage Antimicrobial Functions

    PubMed Central

    MacCallum, Donna M.; Brown, Gordon D.

    2017-01-01

    ABSTRACT   The opportunistic human fungal pathogen Candida albicans can cause a variety of diseases, ranging from superficial mucosal infections to life-threatening systemic infections. Phagocytic cells of the innate immune response, such as neutrophils and macrophages, are important first-line responders to an infection and generate reactive oxygen and nitrogen species as part of their protective antimicrobial response. During an infection, host cells generate nitric oxide through the enzyme inducible nitric oxide synthase (iNOS) to kill the invading pathogen. Inside the phagocyte, iNOS competes with the enzyme arginase-1 for a common substrate, the amino acid l-arginine. Several pathogenic species, including bacteria and parasitic protozoans, actively modulate the production of nitric oxide by inducing their own arginases or the host’s arginase activity to prevent the conversion of l-arginine to nitric oxide. We report here that C. albicans blocks nitric oxide production in human-monocyte-derived macrophages by induction of host arginase activity. We further determined that purified chitin (a fungal cell wall polysaccharide) and increased chitin exposure at the fungal cell wall surface induces this host arginase activity. Blocking the C. albicans-induced arginase activity with the arginase-specific substrate inhibitor Nω-hydroxy-nor-arginine (nor-NOHA) or the chitinase inhibitor bisdionin F restored nitric oxide production and increased the efficiency of fungal killing. Moreover, we determined that C. albicans influences macrophage polarization from a classically activated phenotype toward an alternatively activated phenotype, thereby reducing antimicrobial functions and mediating fungal survival. Therefore, C. albicans modulates l-arginine metabolism in macrophages during an infection, potentiating its own survival. PMID:28119468

  3. Hydrolytic ectoenzyme activity associated with suspended and sinking organic particles within the anoxic Cariaco Basin

    NASA Astrophysics Data System (ADS)

    Taylor, Gordon T.; Thunell, Robert; Varela, Ramon; Benitez-Nelson, Claudia; Scranton, Mary I.

    2009-08-01

    Ectohydrolase activities of suspended microbiota were compared to those associated with sinking particles (sed-POM) retrieved from sediment traps deployed in the permanently anoxic Cariaco Basin. In shore-based assays, activities of aminopeptidase, β-glucosidase, chitinase and alkaline phosphatase were measured in samples obtained from oxic and anoxic depths using MUF- and MCA-labeled fluorogenic substrate analogs. Hydrolysis potentials for these enzymes in the seston varied widely over the nine cruises sampled (8 Nov 1996-3 May 2000) and among depths (15-1265 m); from <10 to over 1600 nM d -1 hydrolysate released, generally co-varying with one another and with suspended particulate organic carbon (POC) and particulate nitrogen (PN). Hydrolytic potentials, prokaryotic abundances and POC/PN concentrations in sinking debris were 400-1.3×10 7 times higher than in comparable volumes of seawater. However when normalized to PN, hydrolytic potentials in sediment trap samples were not demonstrably higher than in Niskin bottle samples. We estimate that PN pools in sediment trap samples were turned over 2-1400 times (medians=7-26 x) slower by hydrolysis than were suspended PN pools. Median prokaryotic growth rates (divisions d -1) in sinking debris were also ˜150 times slower than for bacterioplankton. Hydrolytic potentials in surface oxic waters were generally faster than in underlying anoxic waters on a volumetric basis (nM hydrolysate d -1), but were not significantly ( p>0.05) different when normalized to PN or prokaryote abundances. Alkaline phosphatase was consistently the most active ectohydrolase in both sample types, suggesting that Cariaco Basin assemblages were adapted to decomposing phosphate esters in organic polymers. However, phosphorus limitation was not evident from nutrient inventories in the water column. Results support the hypothesis that efficiencies of polymer hydrolysis in anoxic waters are not inherently lower than in oxic waters.

  4. Biocontrol activity and primed systemic resistance by compost water extracts against anthracnoses of pepper and cucumber.

    PubMed

    Sang, Mee Kyung; Kim, Ki Deok

    2011-06-01

    We investigated direct and indirect effects of compost water extracts (CWEs) from Iljuk-3, Iljuk-7, Shinong-8, and Shinong-9 for the control of anthracnoses caused by Colletotrichum coccodes on pepper and C. orbiculare on cucumber. All tested CWEs significantly (P < 0.05) inhibited in vitro conidial germination and appressorium formation of the fungal pathogens; however, DL-β-amino-n-butyric acid (BABA) failed to inhibit the conidial development of the pathogens. Direct treatments of the CWEs and BABA on pepper and cucumber leaves at 1 and 3 days before or after inoculation significantly (P < 0.05) reduced anthracnose severities; Iljuk-3, Shinong-9, and BABA for pepper and Iljuk-7 for cucumber had more protective activities than curative activities. In addition, root treatment of CWEs suppressed anthracnoses on the plants by the pathogens; however, CWE treatment on lower leaves failed to reduce the diseases on the upper leaves of the plants. The CWE root treatments enhanced not only the expression of the pathogenesis-related (PR) genes CABPR1, CABGLU, CAChi2, CaPR-4, CAPO1, and CaPR-10 in pepper and PR1-1a, PR-2, PR-3, and APOX in cucumber but also the activity of β-1,3-glucanase, chitinase, and peroxidase and the generation of hydrogen peroxide in pepper and cucumber under pathogen-inoculated conditions. However, the CWE treatments failed to induce the plant responses under pathogen-free conditions. These results indicated that the CWEs had direct effects, reducing anthracnoses by C. coccodes on pepper leaves and C. orbiculare on cucumber leaves through protective and curative effects. In addition, CWE root treatments could induce systemic resistance in the primed state against pathogens on plant leaves that enhanced PR gene expression, defense-related enzyme production, and hydrogen peroxide generation rapidly and effectively immediately after pathogen infection. Thus, the CWEs might suppress anthracnoses on leaves of both pepper and cucumber through primed

  5. Bacterial diversity and bioprospecting for cold-active hydrolytic enzymes from culturable bacteria associated with sediment from Nella Fjord, Eastern Antarctica.

    PubMed

    Yu, Yong; Li, Hui-Rong; Zeng, Yin-Xin; Chen, Bo

    2011-01-31

    The diversity and cold-active hydrolytic enzymes of culturable bacteria associated with sandy sediment from Nella Fjord, Eastern Antarctica (69°22'6″ S, 76°21'45″ E) was investigated. A total of 33 aerobic heterotrophic bacterial strains were isolated at 4 °C. These bacterial isolates could be sorted into 18 phylotypes based on the 16S rRNA gene sequence belonging to four phyla, namely Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes and Actinobacteria. Only seven isolates were psychrophilic, 15 isolates were moderately psychrophilic, and 11 isolates were psychrotolerant. More than 72% of the isolates required sodium chloride to grow. Esterase, β-glucosidase and proteases activities at 4 °C were detected in more than 45% of the strains while approximately 21%, 15% and 12% of the strains possessed lipase, amylase and chitinase, respectively. These results indicate that a relatively high culturable bacterial diversity is present within marine sediment of Nella Fjord and it could serve as an ideal candidate region for bioprospecting.

  6. Diversity and extracellular enzymatic activities of yeasts isolated from King George Island, the sub-Antarctic region

    PubMed Central

    2012-01-01

    Background Antarctica has been successfully colonized by microorganisms despite presenting adverse conditions for life such as low temperatures, high solar radiation, low nutrient availability and dryness. Although these “cold-loving” microorganisms are recognized as primarily responsible for nutrient and organic matter recycling/mineralization, the yeasts, in particular, remain poorly characterized and understood. The aim of this work was to study the yeast microbiota in soil and water samples collected on King George Island. Results A high number of yeast isolates was obtained from 34 soil and 14 water samples. Molecular analyses based on rDNA sequences revealed 22 yeast species belonging to 12 genera, with Mrakia and Cryptococcus genera containing the highest species diversity. The species Sporidiobolus salmonicolor was by far the most ubiquitous, being identified in 24 isolates from 13 different samples. Most of the yeasts were psychrotolerant and ranged widely in their ability to assimilate carbon sources (consuming from 1 to 27 of the 29 carbon sources tested). All species displayed at least 1 of the 8 extracellular enzyme activities tested. Lipase, amylase and esterase activity dominated, while chitinase and xylanase were less common. Two yeasts identified as Leuconeurospora sp. and Dioszegia fristingensis displayed 6 enzyme activities. Conclusions A high diversity of yeasts was isolated in this work including undescribed species and species not previously isolated from the Antarctic region, including Wickerhamomyces anomalus, which has not been isolated from cold regions in general. The diversity of extracellular enzyme activities, and hence the variety of compounds that the yeasts may degrade or transform, suggests an important nutrient recycling role of microorganisms in this region. These yeasts are of potential use in industrial applications requiring high enzyme activities at low temperatures. PMID:23131126

  7. Active-R filter

    DOEpatents

    Soderstrand, Michael A.

    1976-01-01

    An operational amplifier-type active filter in which the only capacitor in the circuit is the compensating capacitance of the operational amplifiers, the various feedback and coupling elements being essentially solely resistive.

  8. Effects of polyacrylamide, biopolymer, and biochar on decomposition of soil organic matter and 14C-labeled plant residues as determined by enzyme activities

    NASA Astrophysics Data System (ADS)

    Mahmoud Awad, Yasser; Ok, Young Sik; Kuzyakov, Yakov

    2014-05-01

    Application of polymers for the improvement of aggregate structure and reduction of soil erosion may alter the availability and decomposition of plant residues. In this study, we assessed the effects of anionic polyacrylamide (PAM), synthesized biopolymer (BP), and biochar (BC) on the decomposition of 14C-labeled maize residue in sandy and sandy loam soils. Specifically, PAM and BP with or without 14C-labeled plant residue were applied at 400 kg ha-1, whereas BC was applied at 5000 kg ha-1, after which the soils were incubated for 80 days at 22 oC. Initially, plant residue decomposition was much higher in untreated sandy loam soil than in sandy soil. Nevertheless, the stimulating effects of BP and BC on the decomposition of plant residue were more pronounced in sandy soil, where it accounted for 13.4% and 23.4% of 14C input, respectively, whereas in sandy loam soil, the acceleration of plant residue decomposition by BP and BC did not exceed 2.6% and 14.1%, respectively, compared to untreated soil with plant residue. The stimulating effects of BP and BC on the decomposition of plant residue were confirmed based on activities of β-cellobiohydrolase, β-glucosidase, and chitinase in both soils. In contrast to BC and BP, PAM did not increase the decomposition of native or added C in both soils.

  9. Get Active

    MedlinePlus

    ... Basics: Health Benefits What are the benefits of physical activity? Physical activity increases your chances of living longer. ... pain Help you feel better about yourself Is physical activity for everyone? Yes! Physical activity is good for ...

  10. Biocontrol activity and induction of systemic resistance in pepper by compost water extracts against Phytophthora capsici.

    PubMed

    Sang, Mee Kyung; Kim, Jeong-Gyu; Kim, Ki Deok

    2010-08-01

    We investigated the effects of water extracts of composts (CWE) from commercial compost facilities for controlling root and foliar infection of pepper plants by Phytophthora capsici. Among 47 CWE tested, CWE from composts Iljuk-3, Iljuk-7, Shinong-8, and Shinong-9 significantly (P < 0.05) inhibited zoospore germination, germ tube elongation, mycelial growth, and population of P. capsici. All selected CWE significantly (P < 0.05) reduced the disease incidence and severity in the seedling and plant assays compared with the controls. However, there were no significant differences in zoospore germination, disease incidence, and disease severity between treatments of untreated, autoclaved, and filtered CWE. In addition, CWE significantly (P < 0.05) suppressed leaf infection of P. capsici through induced systemic resistance (ISR) in plants root-drenched with CWE. The tested CWE enhanced the expression of the pathogenesis-related genes, CABPR1, CABGLU, CAChi2, CaPR-4, CAPO1, or CaPR-10 as well as beta-1,3-glucanase, chitinase, and peroxidase activities, which resulted in enhanced plant defense against P. capsici in pepper plants. Moreover, the CWE enhanced the chemical and structural defenses of the plants, including H(2)O(2) generation in the leaves and lignin accumulation in the stems. The CWE could also suppress other fungal pathogens (Colletotrichum coccodes in pepper leaves and C. orbiculare in cucumber leaves) through ISR; however, it failed to inhibit other bacterial pathogens (Xanthomonas campestris pv. vesicatoria in pepper leaves and Pseudomonas syringae pv. lachrymans in cucumber leaves). These results suggest that a heat-stable chemical(s) in the CWE can suppress root and foliar infection by P. capsici in pepper plants. In addition, these suppressions might result from direct inhibition of development and population of P. capsici for root infection, as well as indirect inhibition of foliar infection through ISR with broad-spectrum protection.

  11. Differences in the activities of eight enzymes from ten soil fungi and their possible influences on the surface structure, functional groups, and element composition of soil colloids.

    PubMed

    Wang, Wenjie; Li, Yanhong; Wang, Huimei; Zu, Yuangang

    2014-01-01

    How soil fungi function in soil carbon and nutrient cycling is not well understood by using fungal enzymatic differences and their interactions with soil colloids. Eight extracellular enzymes, EEAs (chitinase, carboxymethyl cellulase, β-glucosidase, protease, acid phosphatase, polyphenol oxidase, laccase, and guaiacol oxidase) secreted by ten fungi were compared, and then the fungi that showed low and high enzymatic activity were co-cultured with soil colloids for the purpose of finding fungi-soil interactions. Some fungi (Gomphidius rutilus, Russula integra, Pholiota adiposa, and Geastrum mammosum) secreted 3-4 enzymes with weak activities, while others (Cyathus striatus, Suillus granulate, Phallus impudicus, Collybia dryophila, Agaricus sylvicola, and Lactarius deliciosus) could secret over 5 enzymes with high activities. The differences in these fungi contributed to the alterations of functional groups (stretching bands of O-H, N-H, C-H, C = O, COO- decreased by 11-60%, while P = O, C-O stretching, O-H bending and Si-O-Si stretching increased 9-22%), surface appearance (disappearance of adhesive organic materials), and elemental compositions (11-49% decreases in C1s) in soil colloids. Moreover, more evident changes were generally in high enzymatic fungi (C. striatus) compared with low enzymatic fungi (G. rutilus). Our findings indicate that inter-fungi differences in EEA types and activities might be responsible for physical and chemical changes in soil colloids (the most active component of soil matrix), highlighting the important roles of soil fungi in soil nutrient cycling and functional maintenance.

  12. Differences in the Activities of Eight Enzymes from Ten Soil Fungi and Their Possible Influences on the Surface Structure, Functional Groups, and Element Composition of Soil Colloids

    PubMed Central

    Wang, Wenjie; Li, Yanhong; Wang, Huimei; Zu, Yuangang

    2014-01-01

    How soil fungi function in soil carbon and nutrient cycling is not well understood by using fungal enzymatic differences and their interactions with soil colloids. Eight extracellular enzymes, EEAs (chitinase, carboxymethyl cellulase, β-glucosidase, protease, acid phosphatase, polyphenol oxidase, laccase, and guaiacol oxidase) secreted by ten fungi were compared, and then the fungi that showed low and high enzymatic activity were co-cultured with soil colloids for the purpose of finding fungi-soil interactions. Some fungi (Gomphidius rutilus, Russula integra, Pholiota adiposa, and Geastrum mammosum) secreted 3–4 enzymes with weak activities, while others (Cyathus striatus, Suillus granulate, Phallus impudicus, Collybia dryophila, Agaricus sylvicola, and Lactarius deliciosus) could secret over 5 enzymes with high activities. The differences in these fungi contributed to the alterations of functional groups (stretching bands of O-H, N-H, C-H, C = O, COO- decreased by 11–60%, while P = O, C-O stretching, O-H bending and Si-O-Si stretching increased 9–22%), surface appearance (disappearance of adhesive organic materials), and elemental compositions (11–49% decreases in C1s) in soil colloids. Moreover, more evident changes were generally in high enzymatic fungi (C. striatus) compared with low enzymatic fungi (G. rutilus). Our findings indicate that inter-fungi differences in EEA types and activities might be responsible for physical and chemical changes in soil colloids (the most active component of soil matrix), highlighting the important roles of soil fungi in soil nutrient cycling and functional maintenance. PMID:25398013

  13. A constitutively expressed 36 kDa exochitinase from Bacillus thuringiensis HD-1.

    PubMed

    Arora, Naresh; Ahmad, Tarannum; Rajagopal, R; Bhatnagar, Raj K

    2003-08-01

    A 36 kDa chitinase was purified by ion exchange and gel filtration chromatography from the culture supernatant of Bacillus thuringiensis HD-1. The chitinase production was independent of the presence of chitin in the growth medium and was produced even in the presence of glucose. The purified chitinase was active at acidic pH, had an optimal activity at pH 6.5, and showed maximum activity at 65 degrees C. Of the various substrates, the enzyme catalyzed the hydrolysis of the disaccharide 4-MU(GlnAc)(2) most efficiently and was therefore classified as an exochitinase. The sequence of the tryptic peptides showed extensive homology with Bacillus cereus 36 kDa exochitinase. The 1083 bp open reading frame encoding 36 kDa chitinase was amplified with primers based on the gene sequence of B. cereus 36 kDa exochitinase. The deduced amino-acid sequence showed that the protein contained an N-terminal signal peptide and consisted of a single catalytic domain. The two conserved signature sequences characteristic of family 18 chitinases were mapped at positions 105-109 and 138-145 of Chi36. The recombinant chitinase was expressed in a catalytically active form in Escherichia coli in the vector pQE-32. The expressed 36 kDa chitinase potentiated the insecticidal effect of the vegetative insecticidal protein (Vip) when used against neonate larvae of Spodoptera litura.

  14. Isolation and characterization of multifunctional Streptomyces species with antimicrobial, nematicidal and phytohormone activities from marine environments in Egypt.

    PubMed

    Rashad, Ferial M; Fathy, Hayam M; El-Zayat, Ayatollah S; Elghonaimy, Ahlam M

    2015-06-01

    Different strategies have been employed for selective isolation of Streptomycetes from 20 marine samples varied in their biological nature. The recovery of Streptomycetes isolates (112) was influenced preferentially by different strategies; sediment samples were the best source of potential candidate Streptomycetes. All isolates exhibited antimicrobial activities with variable spectrum; the most promising isolates (31) were phenotypically characterized and identified as Streptomyces sp.; these isolates exhibited variable capacity for secretion of numerous hydrolytic enzymes such as catalase, protease, amylase, lipase, lecithinase, asparaginase, chitinase and pectinase. All the strains resisted both penicillin and streptomycin, 29 were sensitive to neomycin; the majority of strains (25) showed multiple antibiotic resistance index greater than 0.2; 23, 22 and 13 degraded the shrimp shell, chicken feather and corn cob, respectively, producing bioactive substance(s) which indicates their diversity and their ecological role in the marine ecosystem. At least 28 strains exhibited nematicidal activity in vitro and in vivo against root-knot nematode and supported plant growth. In vitro, the assessed Streptomyces species exhibited the ability to produce gibberellic acid, indole acetic acid, abscisic acid, kinetin and benzyladenine. Except for indole acetic acid, this is the first report concerning the ability of marine Streptomyces to produce such phytohormones and the use of shrimp shell waste as a mono component medium for production of phytohormones. The study is efficacious in selecting effective biodiverse strains of marine Streptomyces that may work under diverse agro-ecological conditions as a useful element in plant nutrition and as biocontrol agents involved in integrated management programs.

  15. Complete genome sequences of the Serratia plymuthica strains 3Rp8 and 3Re4-18, two rhizosphere bacteria with antagonistic activity towards fungal phytopathogens and plant growth promoting abilities.

    PubMed

    Adam, Eveline; Müller, Henry; Erlacher, Armin; Berg, Gabriele

    2016-01-01

    The Serratia plymuthica strains 3Rp8 and 3Re4-18 are motile, Gram-negative, non-sporulating bacteria. Strain 3Rp8 was isolated from the rhizosphere of Brassica napus L. and strain 3Re4-18 from the endorhiza of Solanum tuberosum L. Studies have shown in vitro activity against the soil-borne fungi Verticillium dahliae Kleb., Rhizoctonia solani Kühn, and Sclerotinia sclerotiorum. Here, we announce and describe the complete genome sequence of S. plymuthica 3Rp8 consisting of a single circular chromosome of 5.5 Mb that encodes 4954 protein-coding and 108 RNA-only encoding genes and of S. plymuthica 3Re4-18 consisting of a single circular chromosome of 5.4 Mb that encodes 4845 protein-coding and 109 RNA-only encoding genes. The whole genome sequences and annotations are available in NCBI under the locus numbers CP012096 and CP012097, respectively. The genome analyses revealed genes putatively responsible for the promising plant growth promoting and biocontrol properties including predicting factors such as secretion systems, iron scavenging siderophores, chitinases, secreted proteases, glucanases and non-ribosomal peptide synthetases, as well as unique genomic islands.

  16. Activation detector

    DOEpatents

    Bell, Zane William [Oak Ridge, TN; Boatner, Lynn Allen [Oak Ridge, TN

    2009-12-08

    A method of detecting an activator, the method including impinging with an activator a receptor material lacking a photoluminescent material and generating a by-product of a radioactive decay due to the activator impinging the reeptor material. The method further including, generating light from the by-product via the Cherenkov effect and identifying a characteristic of the activator based on the light.

  17. Did the temporary shortage in supply of imiglucerase have clinical consequences? Retrospective observational study on 34 italian Gaucher type I patients.

    PubMed

    Deroma, Laura; Sechi, Annalisa; Dardis, Andrea; Macor, Daniela; Liva, Giulia; Ciana, Giovanni; Bembi, Bruno

    2013-01-01

    Background. Enzyme Replacement Therapy (ERT) is the standard of care in Gaucher disease. The effects of withdrawal or reduced doses are debated, thus a retrospective cohort study was conducted to investigate clinical and laboratory differences in 34 Gaucher type 1 patients experiencing an ERT dosage reduction after the forced temporary imiglucerase shortage in 2009. Methods. Haemoglobin concentration, leukocytes and platelets counts, and chitotriosidase activity were assessed at baseline and after 6 and 12 months (t0, t6, t12), while bone pain, energy, work or school performance, concentration, memory and social life every 3 months. Results. The cohort was made up of 18 males and 16 females (medians: age 41.8 years, therapy duration 14.1 years, dosage reduction 35.5%). Haemoglobin, leukocytes and platelets remained substantially stable, while chitotriosidase activity showed an increase, especially after t6. Age, splenectomy or genotype were not associated with laboratory parameters changes, except for a significant median increase of chitotriosidase activity in non-splenectomised patients after 12 months (p = 0.01). At 3, 6, 9 and 12 months, more than 50% patients reported at least one problem in subjective well-being (56%, 65%, 70%, 58%, respectively), while bone pain occurred or worsened in 13/33, 13/32, 7/28 and 5/26 patients, respectively. No bone crises were reported. Conclusions. Drug reduction did not induce substantial modification in the laboratory values but seems to have influenced the well-being perception of some Gaucher patients. Thus, bone pain, general health and quality of life should be carefully monitored during ERT reductions.

  18. Effects of stoichiometry and temperature perturbations on beech leaf litter decomposition, enzyme activities and protein expression

    NASA Astrophysics Data System (ADS)

    Keiblinger, K. M.; Schneider, T.; Roschitzki, B.; Schmid, E.; Eberl, L.; Hämmerle, I.; Leitner, S.; Richter, A.; Wanek, W.; Riedel, K.; Zechmeister-Boltenstern, S.

    2012-11-01

    Microbes are major players in leaf litter decomposition and therefore advances in the understanding of their control on element cycling are of paramount importance. Our aim was to investigate the influence of leaf litter stoichiometry in terms of carbon (C) : nitrogen (N) : phosphorus (P) ratios on the decomposition processes and to track changes in microbial community structures and functions in response to temperature stress treatments. To elucidate how the stoichiometry of beech leaf litter (Fagus sylvatica L.) and stress treatments interactively affect the microbial decomposition processes, a terrestrial microcosm experiment was conducted. Beech litter from different Austrian sites covering C:N ratios from 39 to 61 and C:P ratios from 666 to 1729 were incubated at 15 °C and 60% moisture for six months. Part of the microcosms were then subjected to severe changes in temperature (+30 °C and -15 °C) to monitor the influence of temperature stress. Extracellular enzyme activities were assayed and respiratory activities measured. A semi-quantitative metaproteomics approach (1D-SDS PAGE combined with liquid chromatography and tandem mass spectrometry; unique spectral counting) was employed to investigate the impact of the applied stress treatments in dependency of litter stoichiometry on structure and function of the decomposing community. In litter with narrow C:nutrient (C:N, C:P) ratios, microbial decomposers were most abundant. Cellulase, chitinase, phosphatase and protease activity decreased after heat and freezing treatments. Decomposer communities and specific functions varied with site, i.e. stoichiometry. The applied stress combined with the respective time of sampling evoked changes of enzyme activities and litter pH. Freezing treatments resulted in a decline in residual plant litter material and increased fungal abundance, indicating slightly accelerated decomposition. Overall, a strong effect of litter stoichiometry on microbial community structures and

  19. Antimicrobial activity of engineered shrimp ovarian peritrophin fragments from Fenneropenaeus merguiensis.

    PubMed

    Anuchan, Sujunya; Deachamag, Panchalika; Siammai, Nareerat; Phongpaichit, Souwalak; Chotigeat, Wilaiwan

    2015-01-01

    Shrimp ovarian peritrophin (SOP), a major protein in jelly layer and cortical rods, plays a role in egg protection after spawning. Previous study, sequence of SOP gene from Fenneropenaeus merguiensis (Fm-SOP) was composed of domain A and domain B. The SOP domain A contains amino acid sequences between 1-80 of Fm-SOP. The domain A had six conserved cysteines which have been found in many antimicrobial peptides. The molecular weight of purified rSOP-A protein was about 9 kDa. The SOP domain B contains amino acid sequences 81-329 of Fm-SOP while SOP-B1 was amino acid sequence 182-275 of Fm-SOP. The molecular weight of purified rHis-SOP-B and rHis-SOP-B1 protein were about 38.5 and 18.0 kDa, respectively. Antimicrobial activities of rSOP-A, rHis-SOP-B and rHis-SOP-B1 protein were investigated by liquid growth inhibition assay. Minimal Inhibition Concentration (MIC) of rSOP-A against Staphylococcus aureus, Escherichia coli, Vibrio harveyi, Candida albicans and Fusarium oxysporum were 35, 280, 280, 570 and 15 µg/mL, respectively. The MIC of rHis-SOP-B against S. aureus, V. harveyi and F. oxysporum were 30, 270 and 500 µg/mL, respectively. And the MIC of rHis-SOP-B1 against S. aureus, V. harveyi and F. oxysporum were 20, 470 and 250 µg/mL, respectively. The rHis-SOP-B and rHis-SOP-B1 (1000 µg/mL) did not show antimicrobial activity against E. coli and C. albicans. Three purified proteins were able to agglutinate V. harveyi in vitro, displayed a chitinase activity and proteinase inhibition. In addition the stability of the proteins was tested and found decrease antimicrobial activity after incubation at 50 °C for 5 h.

  20. Genomic Analyses and Transcriptional Profiles of the Glycoside Hydrolase Family 18 Genes of the Entomopathogenic Fungus Metarhizium anisopliae

    PubMed Central

    Junges, Ângela; Boldo, Juliano Tomazzoni; Souza, Bárbara Kunzler; Guedes, Rafael Lucas Muniz; Sbaraini, Nicolau; Kmetzsch, Lívia; Thompson, Claudia Elizabeth; Staats, Charley Christian; de Almeida, Luis Gonzaga Paula; de Vasconcelos, Ana Tereza Ribeiro; Vainstein, Marilene Henning; Schrank, Augusto

    2014-01-01

    Fungal chitin metabolism involves diverse processes such as metabolically active cell wall maintenance, basic nutrition, and different aspects of virulence. Chitinases are enzymes belonging to the glycoside hydrolase family 18 (GH18) and 19 (GH19) and are responsible for the hydrolysis of β-1,4-linkages in chitin. This linear homopolymer of N-acetyl-β-D-glucosamine is an essential constituent of fungal cell walls and arthropod exoskeletons. Several chitinases have been directly implicated in structural, morphogenetic, autolytic and nutritional activities of fungal cells. In the entomopathogen Metarhizium anisopliae, chitinases are also involved in virulence. Filamentous fungi genomes exhibit a higher number of chitinase-coding genes than bacteria or yeasts. The survey performed in the M. anisopliae genome has successfully identified 24 genes belonging to glycoside hydrolase family 18, including three previously experimentally determined chitinase-coding genes named chit1, chi2 and chi3. These putative chitinases were classified based on domain organization and phylogenetic analysis into the previously described A, B and C chitinase subgroups, and into a new subgroup D. Moreover, three GH18 proteins could be classified as putative endo-N-acetyl-β-D-glucosaminidases, enzymes that are associated with deglycosylation and were therefore assigned to a new subgroup E. The transcriptional profile of the GH18 genes was evaluated by qPCR with RNA extracted from eight culture conditions, representing different stages of development or different nutritional states. The transcripts from the GH18 genes were detected in at least one of the different M. anisopliae developmental stages, thus validating the proposed genes. Moreover, not all members from the same chitinase subgroup presented equal patterns of transcript expression under the eight distinct conditions studied. The determination of M. anisopliae chitinases and ENGases and a more detailed study concerning the enzymes

  1. Exposure of Daphnia magna to trichloroethylene (TCE) and vinyl chloride (VC): evaluation of gene transcription, cellular activity, and life-history parameters.

    PubMed

    Houde, Magali; Douville, Mélanie; Gagnon, Pierre; Sproull, Jim; Cloutier, François

    2015-06-01

    Trichloroethylene (TCE) is a ubiquitous contaminant classified as a human carcinogen. Vinyl chloride (VC) is primarily used to manufacture polyvinyl chloride and can also be a degradation product of TCE. Very few data exist on the toxicity of TCE and VC in aquatic organisms particularly at environmentally relevant concentrations. The aim of this study was to evaluate the sub-lethal effects (10 day exposure; 0.1; 1; 10 µg/L) of TCE and VC in Daphnia magna at the gene, cellular, and life-history levels. Results indicated impacts of VC on the regulation of genes related to glutathione-S-transferase (GST), juvenile hormone esterase (JHE), and the vitelline outer layer membrane protein (VMO1). On the cellular level, exposure to 0.1, 1, and 10 µg/L of VC significantly increased the activity of JHE in D. magna and TCE increased the activity of chitinase (at 1 and 10 µg/L). Results for life-history parameters indicated a possible tendency of TCE to affect the number of molts at the individual level in D. magna (p=0.051). Measurement of VG-like proteins using the alkali-labile phosphates (ALP) assay did not show differences between TCE treated organisms and controls. However, semi-quantitative measurement using gradient gel electrophoresis (213-218 kDa) indicated significant decrease in VG-like protein levels following exposure to TCE at all three concentrations. Overall, results indicate effects of TCE and VC on genes and proteins related to metabolism, reproduction, and growth in D. magna.

  2. Catalyst activator

    DOEpatents

    McAdon, Mark H.; Nickias, Peter N.; Marks, Tobin J.; Schwartz, David J.

    2001-01-01

    A catalyst activator particularly adapted for use in the activation of metal complexes of metals of Group 3-10 for polymerization of ethylenically unsaturated polymerizable monomers, especially olefins, comprising two Group 13 metal or metalloid atoms and a ligand structure including at least one bridging group connecting ligands on the two Group 13 metal or metalloid atoms.

  3. Outdoor Activities.

    ERIC Educational Resources Information Center

    Minneapolis Independent School District 275, Minn.

    Twenty-four activities suitable for outdoor use by elementary school children are outlined. Activities designed to make children aware of their environment include soil painting, burr collecting, insect and pond water collecting, studies of insect galls and field mice, succession studies, and a model of natural selection using dyed toothpicks. A…

  4. Physical activity

    MedlinePlus

    ... activity -- which includes an active lifestyle and routine exercise -- plus eating well, is the best way to stay healthy. ... goal. Your goal might be to: Manage a health condition Reduce stress ... other benefits, such as: Better control of your weight and ...

  5. Astronomy Activities.

    ERIC Educational Resources Information Center

    Greenstone, Sid

    This document consists of activities and references for teaching astronomy. The activities (which include objectives, list of materials needed, and procedures) focus on: observing the Big Dipper and locating the North Star; examining the Big Dipper's stars; making and using an astrolabe; examining retograde motion of Mars; measuring the Sun's…

  6. Activated Charcoal

    MedlinePlus

    ... is used to treat poisonings, reduce intestinal gas (flatulence), lower cholesterol levels, prevent hangover, and treat bile ... lower cholesterol levels in the blood. Decreasing gas (flatulence). Some studies show that activated charcoal is effective ...

  7. Activity Theory.

    ERIC Educational Resources Information Center

    Koschmann, Timothy; Roschelle, Jeremy; Nardi, Bonnie A.

    1998-01-01

    Includes three articles that discuss activity theory, based on "Context and Consciousness." Topics include human-computer interaction; computer interfaces; hierarchical structuring; mediation; contradictions and development; failure analysis; and designing educational technology. (LRW)

  8. A Role for the GCC-Box in Jasmonate-Mediated Activation of the PDF1.2 Gene of Arabidopsis1

    PubMed Central

    Brown, Rebecca L.; Kazan, Kemal; McGrath, Ken C.; Maclean, Don J.; Manners, John M.

    2003-01-01

    The PDF1.2 gene of Arabidopsis encoding a plant defensin is commonly used as a marker for characterization of the jasmonate-dependent defense responses. Here, using PDF1.2 promoter-deletion lines linked to the β-glucoronidase-reporter gene, we examined putative promoter elements associated with jasmonate-responsive expression of this gene. Using stably transformed plants, we first characterized the extended promoter region that positively regulates basal expression from the PDF1.2 promoter. Second, using promoter deletion constructs including one from which the GCC-box region was deleted, we observed a substantially lower response to jasmonate than lines carrying this motif. In addition, point mutations introduced into the core GCC-box sequence substantially reduced jasmonate responsiveness, whereas addition of a 20-nucleotide-long promoter element carrying the core GCC-box and flanking nucleotides provided jasmonate responsiveness to a 35S minimal promoter. Taken together, these results indicated that the GCC-box plays a key role in conferring jasmonate responsiveness to the PDF1.2 promoter. However, deletion or specific mutations introduced into the core GCC-box did not completely abolish the jasmonate responsiveness of the promoter, suggesting that the other promoter elements lying downstream from the GCC-box region may also contribute to jasmonate responsiveness. In other experiments, we identified a jasmonate- and pathogen-responsive ethylene response factor transcription factor, AtERF2, which when overexpressed in transgenic Arabidopsis plants activated transcription from the PDF1.2, Thi2.1, and PR4 (basic chitinase) genes, all of which contain a GCC-box sequence in their promoters. Our results suggest that in addition to their roles in regulating ethylene-mediated gene expression, ethylene response factors also appear to play important roles in regulating jasmonate-responsive gene expression, possibly via interaction with the GCC-box. PMID:12805630

  9. CSF markers of Alzheimer’s pathology and microglial activation are associated with altered white matter microstructure in asymptomatic adults at risk for Alzheimer’s disease

    PubMed Central

    Melah, Kelsey E; Lu, Sharon Yuan-Fu; Hoscheidt, Siobhan M; Alexander, Andrew L; Adluru, Nagesh; Destiche, Daniel J; Carlsson, Cynthia M; Zetterberg, Henrik; Blennow, Kaj; Okonkwo, Ozioma C; Gleason, Carey E; Dowling, N Maritza; Bratzke, Lisa C; Rowley, Howard A; Sager, Mark A; Asthana, Sanjay; Johnson, Sterling C; Bendlin, Barbara B

    2015-01-01

    Background The immune response in Alzheimer’s disease (AD) involves activation of microglia which may remove β-amyloid. However, overproduction of inflammatory compounds may exacerbate neural damage in Alzheimer’s disease. AD pathology accumulates years before diagnosis, yet the extent to which neuroinflammation is involved in the earliest disease stages is unknown. Objective To determine whether neuroinflammation exacerbates neural damage in preclinical AD. Methods We utilized cerebrospinal fluid (CSF) and magnetic resonance imaging collected in 192 asymptomatic late-middle-aged adults (mean age=60.98 years). Neuroinflammatory markers chitinase-3-like protein 1 (YKL-40) and monocyte chemoattractant protein-1 (MCP-1) in CSF were utilized as markers of neuroinflammation. Neural cell damage was assessed using CSF neurofilament light chain protein (NFL), CSF total tau (T-Tau), and neural microstructure assessed with diffusion tensor imaging (DTI). With regard to AD pathology, CSF Aβ42 and tau phosphorylated at threonine 181 (P-Tau181) were used as markers of amyloid and tau pathology, respectively. We hypothesized that higher YKL-40 and MCP-1 in the presence of AD pathology would be associated with higher NFL, T-Tau, and altered microstructure on DTI. Results Neuroinflammation was associated with markers of neural damage. Higher CSF YKL-40 was associated with both higher CSF NFL and T-Tau. Inflammation interacted with AD pathology, such that greater MCP-1 and lower Aβ42 was associated with altered microstructure in bilateral frontal and right temporal lobe and that greater MCP-1 and greater P-Tau181 was associated with altered microstructure in precuneus. Conclusion Inflammation may play a role in neural damage in preclinical AD. PMID:26836182

  10. Pathogenic and enzyme activities of the entomopathogenic fungus Tolypocladium cylindrosporum (Ascomycota: Hypocreales) from Tierra del Fuego, Argentina.

    PubMed

    Scorsetti, Ana C; Elíades, Lorena A; Stenglein, Sebastián A; Cabello, Marta N; Pelizza, Sebastián A; Saparrat, Mario C N

    2012-06-01

    Tolypocladium cylindrosporum is an entomopathogenic fungi that has been studied as a biological control agent against insects of several orders. The fungus has been isolated from the soil as well as from insects of the orders Coleoptera, Lepidoptera, Diptera and Hymenoptera. In this study, we analyzed the ability of a strain of T cylindrosporum, isolated from soil samples taken in Tierra del Fuego, Argentina, to produce hydrolytic enzymes, and to study the relationship of those activities to the fungus pathogenicity against pest aphids. We have made the traditional and molecular characterization of this strain of T cylindrosporum. The expression of hydrolase activity in the fungal strain was estimated at three incubation temperatures (4 degreeC, 12 degreeC and 24 degreeC), on different agar media supplemented with the following specific substrates: chitin azure, Tween 20, casein, and urea for chitinase, lipase, protease, and urease activity, respectively. The hydrolytic-enzyme activity was estimated qualitatively according to the presence of a halo of clarification through hydrolase action, besides was expressed semi-quantitatively as the ratio between the hydrolytic-halo and colony diameters. The pathogenicity of the fungus was tested on adults of the aphid Rhopalosiphum padi at three temperatures of incubation (4 degree C, 12 degree C and 24 degree C). The suspension was adjusted to a concentration of 1x10(7) conidia/ml. In pathogenicity assays at seven days post-inoculation, the fungus caused the mortality of adults of Ropalosiphum padi at different temperatures also showed a broad ability to grow on several agar-culture media, supplemented with different carbon sources at the three incubation temperatures tested. Although, the growth was greater with higher incubation temperatures (with maximum levels at 24 degreeC), the fungus reached similar colony diameters after 15 days of incubation on the medium supplemented with Tween 20 at the lower two incubation

  11. Plant metacaspase activation and activity.

    PubMed

    Minina, Elena A; Stael, Simon; Van Breusegem, Frank; Bozhkov, Peter V

    2014-01-01

    Metacaspases are essential for cell death regulation in plants. Further understanding of biochemistry of metacaspases and their molecular function in plant biology requires a set of robust methods for detection of metacaspase activation and quantitative analysis of corresponding proteolytic activity. Here we describe methods for purification of recombinant metacaspases, measurement of enzymatic activity of recombinant and endogenous metacaspases in vitro and in cell lysates, respectively, and finally detection of metacaspase activation in vivo. Additionally, an in vitro metacaspase protein substrate cleavage assay based on the cell-free production of substrate protein followed by proteolysis with recombinant metacaspase is presented. These methods have been originally developed for type II metacaspases from Arabidopsis and Norway spruce (Picea abies), but they can be used as templates for type I metacaspases, as well as for type II metacaspases from other species.

  12. Active colloids

    NASA Astrophysics Data System (ADS)

    Aranson, Igor S.

    2013-01-01

    A colloidal suspension is a heterogeneous fluid containing solid microscopic particles. Colloids play an important role in our everyday life, from food and pharmaceutical industries to medicine and nanotechnology. It is useful to distinguish two major classes of colloidal suspensions: equilibrium and active, i.e., maintained out of thermodynamic equilibrium by external electric or magnetic fields, light, chemical reactions, or hydrodynamic shear flow. While the properties of equilibrium colloidal suspensions are fairly well understood, active colloids pose a formidable challenge, and the research is in its early exploratory stage. One of the most remarkable properties of active colloids is the possibility of dynamic self-assembly, a natural tendency of simple building blocks to organize into complex functional architectures. Examples range from tunable, self-healing colloidal crystals and membranes to self-assembled microswimmers and robots. Active colloidal suspensions may exhibit material properties not present in their equilibrium counterparts, e.g., reduced viscosity and enhanced self-diffusivity, etc. This study surveys the most recent developments in the physics of active colloids, both in synthetic and living systems, with the aim of elucidation of the fundamental physical mechanisms governing self-assembly and collective behavior.

  13. Active microwaves

    NASA Technical Reports Server (NTRS)

    Evans, D.; Vidal-Madjar, D.

    1994-01-01

    Research on the use of active microwaves in remote sensing, presented during plenary and poster sessions, is summarized. The main highlights are: calibration techniques are well understood; innovative modeling approaches have been developed which increase active microwave applications (segmentation prior to model inversion, use of ERS-1 scatterometer, simulations); polarization angle and frequency diversity improves characterization of ice sheets, vegetation, and determination of soil moisture (X band sensor study); SAR (Synthetic Aperture Radar) interferometry potential is emerging; use of multiple sensors/extended spectral signatures is important (increase emphasis).

  14. Activated Sludge.

    ERIC Educational Resources Information Center

    Saunders, F. Michael

    1978-01-01

    Presents the 1978 literature review of wastewater treatment. This review covers: (1) activated sludge process; (2) process control; (3) oxygen uptake and transfer; (4) phosphorus removal; (5) nitrification; (6) industrial wastewater; and (7) aerobic digestion. A list of 136 references is also presented. (HM)

  15. Activities: Spirolaterals.

    ERIC Educational Resources Information Center

    Brannan, Richard; McFadden, Scott

    1981-01-01

    A set of activities designed to help students discover properties about order-3 spirolaterals on square grid paper is presented. The materials are prepared on worksheets designed for easy duplication. The lessons can lead to investigations involving spirolaterals of many other orders and shapes. (MP)

  16. Leaf Activities.

    ERIC Educational Resources Information Center

    Mingie, Walter

    Leaf activities can provide a means of using basic concepts of outdoor education to learn in elementary level subject areas. Equipment needed includes leaves, a clipboard with paper, and a pencil. A bag of leaves may be brought into the classroom if weather conditions or time do not permit going outdoors. Each student should pick a leaf, examine…

  17. Learning Activities.

    ERIC Educational Resources Information Center

    Tipton, Tom, Ed.

    1983-01-01

    Presents a flow chart for naming inorganic compounds. Although it is not necessary for students to memorize rules, preliminary skills needed before using the chart are outlined. Also presents an activity in which the mass of an imaginary atom is determined using lead shot, Petri dishes, and a platform balance. (JN)

  18. Activity report

    SciTech Connect

    Yu, S W

    2008-08-11

    This report is aimed to show the author's activities to support the LDRD. The title is 'Investigation of the Double-C Behavior in the Pu-Ga Time-Temperature-Transformation Diagram' The sections are: (1) Sample Holder Test; (2) Calculation of x-ray diffraction patterns; (3) Literature search and preparing publications; (4) Tasks Required for APS Experiments; and (5) Communications.

  19. Effects of switching from a reduced dose imiglucerase to velaglucerase in type 1 Gaucher disease: clinical and biochemical outcomes

    PubMed Central

    van Dussen, Laura; Cox, Timothy M.; Hendriks, Erik J.; Morris, Elizabeth; Akkerman, Erik M.; Maas, Mario; Groener, Johanna E. M.; Aerts, Johannes M.F.G.; Deegan, Patrick B.; Hollak, Carla E. M.

    2012-01-01

    This paper describes the effects of a switch to velaglucerase alfa in a group of adult patients with type 1 Gaucher disease, all of whom had previously had their dose reduced as a consequence of the worldwide imiglucerase shortage. Thirty-two patients from two large European Gaucher centers switched to treatment with velaglucerase alfa after 1-8.5 months of dose reduction. The course of important Gaucher disease parameters was studied at four time points: one year before the shortage, just before the shortage, before a switch to velaglucerase and after up to one year of treatment with velaglucerase. These parameters included hemoglobin concentration, platelet count, plasma chitotriosidase activity in all patients, and spleen and liver volumes (as well as bone marrow fat fraction images) in 10 patients. Decreases in platelet counts as a result of reduced treatment with imiglucerase were quickly restored on treatment with velaglucerase alfa. Chitotriosidase activity declined overall after switching. Five out of 10 patients had an increase in liver volume of at least 10% after six months of velaglucerase treatment, which was reversible in 3. Most patients received infusions at home and no important side effects were observed. Velaglucerase alfa appears to be a safe and effective alternative for imiglucerase. PMID:22773601

  20. Organic matter recycling in a shallow coastal zone (NW Mediterranean): The influence of local and global climatic forcing and organic matter lability on hydrolytic enzyme activity

    NASA Astrophysics Data System (ADS)

    Misic, Cristina; Harriague, Anabella Covazzi

    2008-12-01

    Seawater and sediment were collected on a monthly basis from a shallow (10.5 m depth) coastal site in the Ligurian Sea (NW Mediterranean) from November 1993 to December 1994 to determine the main environmental forces that influenced the biogeochemical processes and to study the relationships between the availability and lability of the organic matter (OM) and hydrolytic enzymatic activity. The current direction throughout the sampling year was influenced by the climatic conditions, which showed significant correlations with north atlantic oscillation (NAO) index values. The current generally flowed northwards in spring. This could cause significantly lower transparency values than in the summer, when an eastward current probably reduced the allochthonous input of material from the main local watercourse and contributed to turning the conditions from mesotrophic to oligotrophic. Spring and summer were separated by transitional periods more than by the canonical autumn and winter seasons. These transitions were characterised by a reduction in salinity values and by resuspension caused by water column mixing and a current flowing towards the southwest. The significant inverse correlations of the chlorophyll- a and protein concentrations, bacterial abundance and proteolysis of the bottom seawater and transparency showed the direct influence of resuspension on the organic matter dynamics. Moreover, OM trophic quality influenced the bacterial parameters and the enzymatic activities. The glycolytic β glucosidase and chitinase activities and their bacterial cell-specific hydrolytic rates were higher when substrates such as hydrolysable proteins were available, while they decreased when refractory compounds were abundant. The low leucine aminopeptidase: β glucosidase ratio values observed in the water column were presumably related to the potential ease with which microbes obtained protein-derived materials and energy, the protein hydrolysable fraction being estimated at

  1. Antimicrobial Activity.

    PubMed

    2016-01-01

    Natural products of higher plants may possess a new source of antimicrobial agents with possibly novel mechanisms of action. They are effective in the treatment of infectious diseases while simultaneously mitigating many of the side effects that are often associated with conventional antimicrobials. A method using scanning electron microscope (SEM) to study the morphology of the bacterial and fungal microbes and thus determining antimicrobial activity is presented in the chapter.

  2. Mineral vs. Organic Amendments: Microbial Community Structure, Activity and Abundance of Agriculturally Relevant Microbes Are Driven by Long-Term Fertilization Strategies

    PubMed Central

    Francioli, Davide; Schulz, Elke; Lentendu, Guillaume; Wubet, Tesfaye; Buscot, François; Reitz, Thomas

    2016-01-01

    Soil management is fundamental to all agricultural systems and fertilization practices have contributed substantially to the impressive increases in food production. Despite the pivotal role of soil microorganisms in agro-ecosystems, we still have a limited understanding of the complex response of the soil microbiota to organic and mineral fertilization in the very long-term. Here, we report the effects of different fertilization regimes (mineral, organic and combined mineral and organic fertilization), carried out for more than a century, on the structure and activity of the soil microbiome. Organic matter content, nutrient concentrations, and microbial biomass carbon were significantly increased by mineral, and even more strongly by organic fertilization. Pyrosequencing revealed significant differences between the structures of bacterial and fungal soil communities associated to each fertilization regime. Organic fertilization increased bacterial diversity, and stimulated microbial groups (Firmicutes, Proteobacteria, and Zygomycota) that are known to prefer nutrient-rich environments, and that are involved in the degradation of complex organic compounds. In contrast, soils not receiving manure harbored distinct microbial communities enriched in oligotrophic organisms adapted to nutrient-limited environments, as Acidobacteria. The fertilization regime also affected the relative abundances of plant beneficial and detrimental microbial taxa, which may influence productivity and stability of the agroecosystem. As expected, the activity of microbial exoenzymes involved in carbon, nitrogen, and phosphorous mineralization were enhanced by both types of fertilization. However, in contrast to comparable studies, the highest chitinase and phosphatase activities were observed in the solely mineral fertilized soil. Interestingly, these two enzymes showed also a particular high biomass-specific activities and a strong negative relation with soil pH. As many soil parameters

  3. Cultural conditions on the production of extracellular enzymes by Trichoderma isolates from tobacco rhizosphere.

    PubMed

    Mallikharjuna Rao, K L N; Siva Raju, K; Ravisankar, H

    2016-01-01

    Twelve isolates of Trichoderma spp. isolated from tobacco rhizosphere were evaluated for their ability to produce chitinase and β-1,3-glucanase extracellular hydrolytic enzymes. Isolates ThJt1 and TvHt2, out of 12 isolates, produced maximum activities of chitinase and β-1,3-glucanase, respectively. In vitro production of chitinase and β-1,3-glucanase by isolates ThJt1 and TvHt2 was tested under different cultural conditions. The enzyme activities were significantly influenced by acidic pH and the optimum temperature was 30°C. The chitin and cell walls of Sclerotium rolfsii, as carbon sources, supported the maximum and significantly higher chitinase activity by both isolates. The chitinase activity of isolate ThJt1 was suppressed significantly by fructose (80.28%), followed by glucose (77.42%), whereas the β-1,3-glucanase activity of ThJt1 and both enzymes of isolate TvHt2 were significantly suppressed by fructose, followed by sucrose. Ammonium nitrate as nitrogen source supported the maximum activity of chitinase in both isolates, whereas urea was a poor nitrogen source. Production of both enzymes by the isolates was significantly influenced by the cultural conditions. Thus, the isolates ThJt1 and TvHt2 showed higher levels of chitinase and β-1,3-glucanase activities and were capable of hydrolyzing the mycelium of S. rolfsii infecting tobacco. These organisms can be used therefore for assessment of their synergism in biomass production and biocontrol efficacy and for their field biocontrol ability against S. rolfsii and Pythium aphanidermatum infecting tobacco.

  4. Cultural conditions on the production of extracellular enzymes by Trichoderma isolates from tobacco rhizosphere

    PubMed Central

    Mallikharjuna Rao, K.L.N.; Siva Raju, K.; Ravisankar, H.

    2016-01-01

    Twelve isolates of Trichoderma spp. isolated from tobacco rhizosphere were evaluated for their ability to produce chitinase and β-1,3-glucanase extracellular hydrolytic enzymes. Isolates ThJt1 and TvHt2, out of 12 isolates, produced maximum activities of chitinase and β-1,3-glucanase, respectively. In vitro production of chitinase and β-1,3-glucanase by isolates ThJt1 and TvHt2 was tested under different cultural conditions. The enzyme activities were significantly influenced by acidic pH and the optimum temperature was 30 °C. The chitin and cell walls of Sclerotium rolfsii, as carbon sources, supported the maximum and significantly higher chitinase activity by both isolates. The chitinase activity of isolate ThJt1 was suppressed significantly by fructose (80.28%), followed by glucose (77.42%), whereas the β-1,3-glucanase activity of ThJt1 and both enzymes of isolate TvHt2 were significantly suppressed by fructose, followed by sucrose. Ammonium nitrate as nitrogen source supported the maximum activity of chitinase in both isolates, whereas urea was a poor nitrogen source. Production of both enzymes by the isolates was significantly influenced by the cultural conditions. Thus, the isolates ThJt1 and TvHt2 showed higher levels of chitinase and β-1,3-glucanase activities and were capable of hydrolyzing the mycelium of S. rolfsii infecting tobacco. These organisms can be used therefore for assessment of their synergism in biomass production and biocontrol efficacy and for their field biocontrol ability against S. rolfsii and Pythium aphanidermatum infecting tobacco. PMID:26887223

  5. Analgesic Activity.

    PubMed

    2016-01-01

    Analgesics are agents which selectively relieve pain by acting in the CNS and peripheral pain mediators without changing consciousness. Analgesics may be narcotic or non-narcotic. The study of pain in animals raises ethical, philosophical, and technical problems. Both peripheral and central pain models are included to make the test more evident for the analgesic property of the plant. This chapter highlights methods such as hot plate and formalin and acetic acid-induced pain models to check the analgesic activity of medicinal plants.

  6. Active packaging with antifungal activities.

    PubMed

    Nguyen Van Long, N; Joly, Catherine; Dantigny, Philippe

    2016-03-02

    There have been many reviews concerned with antimicrobial food packaging, and with the use of antifungal compounds, but none provided an exhaustive picture of the applications of active packaging to control fungal spoilage. Very recently, many studies have been done in these fields, therefore it is timely to review this topic. This article examines the effects of essential oils, preservatives, natural products, chemical fungicides, nanoparticles coated to different films, and chitosan in vitro on the growth of moulds, but also in vivo on the mould free shelf-life of bread, cheese, and fresh fruits and vegetables. A short section is also dedicated to yeasts. All the applications are described from a microbiological point of view, and these were sorted depending on the name of the species. Methods and results obtained are discussed. Essential oils and preservatives were ranked by increased efficacy on mould growth. For all the tested molecules, Penicillium species were shown more sensitive than Aspergillus species. However, comparison between the results was difficult because it appeared that the efficiency of active packaging depended greatly on the environmental factors of food such as water activity, pH, temperature, NaCl concentration, the nature, the size, and the mode of application of the films, in addition to the fact that the amount of released antifungal compounds was not constant with time.

  7. Active tectonics

    SciTech Connect

    Not Available

    1986-01-01

    This study is part of a series of Studies in Geophysics that have been undertaken for the Geophysics Research Forum by the Geophysics Study Committee. One purpose of each study is to provide assessments from the scientific community to aid policymakers in decisions on societal problems that involve geophysics. An important part of such assessments is an evaluation of the adequacy of current geophysical knowledge and the appropriateness of current research programs as a source of information required for those decisions. The study addresses our current scientific understanding of active tectonics --- particularly the patterns and rates of ongoing tectonic processes. Many of these processes cannot be described reasonably using the limited instrumental or historical records; however, most can be described adequately for practical purposes using the geologic record of the past 500,000 years. A program of fundamental research focusing especially on Quaternary tectonic geology and geomorphology, paleoseismology, neotectonics, and geodesy is recommended to better understand ongoing, active tectonic processes. This volume contains 16 papers. Individual papers are indexed separately on the Energy Database.

  8. Elevated atmospheric CO2 increases microbial growth rates and enzymes activity in soil

    NASA Astrophysics Data System (ADS)

    Blagodatskaya, Evgenia; Blagodatsky, Sergey; Dorodnikov, Maxim; Kuzyakov, Yakov

    2010-05-01

    Increasing the belowground translocation of assimilated carbon by plants grown under elevated CO2 can cause a shift in the structure and activity of the microbial community responsible for the turnover of organic matter in soil. We investigated the long-term effect of elevated CO2 in the atmosphere on microbial biomass and specific growth rates in root-free and rhizosphere soil. The experiments were conducted under two free air carbon dioxide enrichment (FACE) systems: in Hohenheim and Braunschweig, as well as in the intensively managed forest mesocosm of the Biosphere 2 Laboratory (B2L) in Oracle, AZ. Specific microbial growth rates (μ) were determined using the substrate-induced respiration response after glucose and/or yeast extract addition to the soil. We evaluated the effect of elevated CO2 on b-glucosidase, chitinase, phosphatase, and sulfatase to estimate the potential enzyme activity after soil amendment with glucose and nutrients. For B2L and both FACE systems, up to 58% higher μ were observed under elevated vs. ambient CO2, depending on site, plant species and N fertilization. The μ-values increased linearly with atmospheric CO2 concentration at all three sites. The effect of elevated CO2 on rhizosphere microorganisms was plant dependent and increased for: Brassica napus=Triticum aestivumactivities under elevated CO2 were

  9. Production of chitinase from shellfish waste by Pseudomonas aeruginosa K-187.

    PubMed

    Wang, S L; Chiou, S H; Chang, W T

    1997-04-01

    The production of chitinolytic enzyme by Pseudomonas aeruginosa K-187, using shrimp and crab shell powder (SCSP) as the carbon source, was studied. It was observed that chemically treated SCSP induced a significant increase of enzyme production, as compared with untreated SCSP. Spent HCl and NaOH from the chitin production industry was used to process SCSP. Various strategies of SCSP processing are examined and compared in terms of chitinolytic enzyme production. A three-and-one-half-fold increase of enzyme production (0.68 U/ml to 2.4 U/ml) was attained using HCl/NaOH treated SCSP. The microorganism (K-187) was isolated from soil in Taiwan and has been characterized and reported in a previous paper.

  10. Field tolerance to fungal pathogens of Brassica napus constitutively expressing a chimeric chitinase gene

    SciTech Connect

    Grison, R.; Grezes-Besset, B.; Lucante, N.

    1996-05-01

    Constitutive overexpression of a protein involved in plant defense mechanisms to disease is one of the strategies proposed to increase plant tolerance to fungal pathogens. A hybrid endochitinase gene under a constitutive promoter was introduced by Agrobacterium-mediated transformation into a winter-type oilseed rape (Brassica napus var. oleifera) inbred line. Progeny from transformed plants was challenged using three different fungal pathogens (Cylindrosporium concentricum, Phoma lingam, Sclerotinia sclerotiorum) in field trials at two different geographical locations. These plants exhibited an increased tolerance to disease as compared with the nontransgenic parental plants. 31 refs., 1 fig., 2 tabs.

  11. IASS Activity

    NASA Astrophysics Data System (ADS)

    Hojaev, Alisher S.; Ibragimova, Elvira M.

    2015-08-01

    It’s well known, astronomy in Uzbekistan has ancient roots and traditions (e.g., Mirzo Ulugh Beg, Abū al-Rayhān al-Bīrūnī, Abū ‘Abdallāh al-Khwārizmī) and astronomical heritage carefully preserved. Nowadays uzbek astronomers play a key role in scientific research but also in OAD and Decadal Plan activity in the Central Asia region. International Aerospace School (IASS) is an amazing and wonderful event held annually about 30 years. IASS is unique project in the region, and at the beginning we spent the Summer and Winter Schools. At present in the summer camp we gather about 50 teenage and undergraduate students over the country and abroad (France, Malaysia, Turkey, Azerbaijan, Pakistan, Russia, etc.). They are selected on the basis of tests of astronomy and space issues. During two weeks of IASS camp the invited scientists, cosmonauts and astronauts as well as other specialists give lectures and engage in practical exercises with IASS students in astronomy, including daily observations of the Sun and night sky observations with meniscus telescope, space research and exploration, aerospace modelling, preparation and presentation of original projects. This is important that IASS gives not theoretical grounds only but also practically train the students and the hands-on training is the major aims of IASS. Lectures and practice in the field of astronomy carried out with the direct involvement and generous assistance of Uranoscope Association (Paris, France). The current 26-th IASS is planned to held in July 2015.

  12. Physical Activity (Exercise)

    MedlinePlus

    ... Physical activity (exercise) fact sheet ePublications Physical activity (exercise) fact sheet How can physical activity improve my ... recent hip surgery More information on physical activity (exercise) For more information about physical activity (exercise), call ...

  13. Impact of repeated dry-wet cycles on soil greenhouse gas emissions, extracellular enzyme activity and nutrient cycling in a temperate forest

    NASA Astrophysics Data System (ADS)

    Leitner, Sonja; Zimmermann, Michael; Bockholt, Jan; Schartner, Markus; Brugner, Paul; Holtermann, Christian; Zechmeister-Boltenstern, Sophie

    2014-05-01

    , chitinase, phosphatase and protease) and phenoloxidase responded strongly to rewetting events with significantly increased activities. Furthermore, we observed a pulsed release of inorganic nitrogen which resulted in high concentrations of NH4 and NO3 in the first 24h after soil rewetting, especially in summer when soil temperatures were high. Emissions of CO2 were increased in the first 24 to 48h after rewetting, and then slowly decreased again. Overall, our results indicate that repeated dry-wet cycles strongly influence microbial soil processes, even in the first year of experimental rainfall manipulation. The next 2 years will show whether these changes are permanent, or if the system adapts to the new precipitation regime.

  14. Activation Energy

    NASA Technical Reports Server (NTRS)

    Gadeken, Owen

    2002-01-01

    Teaming is so common in today's project management environment that most of us assume it comes naturally. We further assume that when presented with meaningful and challenging work, project teams will naturally engage in productive activity to complete their tasks. This assumption is expressed in the simple (but false) equation: Team + Work = Teamwork. Although this equation appears simple and straightforward, it is far from true for most project organizations whose reality is a complex web of institutional norms based on individual achievement and rewards. This is illustrated by the very first successful team experience from my early Air Force career. As a young lieutenant, I was sent to Squadron Officer School, which was the first in the series of Air Force professional military education courses I was required to complete during my career. We were immediately formed into teams of twelve officers. Much of the course featured competition between these teams. As the most junior member of my team, I quickly observed the tremendous pressure to show individual leadership capability. At one point early in the course, almost everyone in our group was vying to become the team leader. This conflict was so intense that it caused us to fail miserably in our first outdoor team building exercise. We spent so much time fighting over leadership that we were unable to complete any of the events on the outdoor obstacle course. This complete lack of success was so disheartening to me that I gave our team little hope for future success. What followed was a very intense period of bickering, conflict, and even shouting matches as our dysfunctional team tried to cope with our early failures and find some way to succeed. British physician and researcher Wilfred Bion (Experiences in Groups, 1961) discovered that there are powerful psychological forces inherent in all groups that divert from accomplishing their primary tasks. To overcome these restraining forces and use the potential

  15. Staying Active: Physical Activity and Exercise

    MedlinePlus

    ... How much physical activity should I do each week? The Centers for Disease Control and Prevention recommend ... 150 minutes of moderate-intensity aerobic activity a week, along with muscle-strengthening activities on 2 days ...

  16. Enhancement of Exochitinase Production by Bacillus licheniformis AT6 Strain and Improvement of N-Acetylglucosamine Production.

    PubMed

    Aounallah, Mohamed Amine; Slimene-Debez, Imen Ben; Djebali, Kais; Gharbi, Dorra; Hammami, Majdi; Azaiez, Sana; Limam, Ferid; Tabbene, Olfa

    2017-02-01

    A strain producing chitinase, isolated from potato stem tissue, was identified as Bacillus licheniformis by biochemical properties and 16S RNA sequence analysis. Statistical experimental designs were used to optimize nine independent variables for chitinase production by B. licheniformis AT6 strain in submerged fermentation. Using Plackett-Burman design, (NH4)2SO4, MgSO4.7H2O, colloidal chitin, MnCl2 2H2O, and temperature were found to influence chitinase production significantly. According to Box-Behnken response surface methodology, the optimal fermentation conditions allowing maximum chitinase production were (in gram per liter): (NH4)2SO4, 7; K2HPO4, 1; NaCl, 1; MgSO4.7H2O, 0.1; yeast extract, 0.5; colloidal chitin, 7.5; MnCl2.2H2O, 0.2; temperature 35 °C; pH medium 7. The optimization strategy led to a 10-fold increase in chitinase activity (505.26 ± 22.223 mU/mL versus 50.35 ± 19.62 mU/mL for control basal medium). A major protein band with a molecular weight of 61.9 kDa corresponding to chitinase activity was clearly detected under optimized conditions. Chitinase activity produced in optimized medium mainly releases N-acetyl glucosamine (GlcNAc) monomer from colloidal chitin. This enzyme also acts as an exochitinase with β-N-acetylglucosaminidase. These results suggest that B. licheniformis AT6 secreting exochitinase is highly efficient in GlcNAc production which could in turn be envisaged as a therapeutic agent or as a conservator against the alteration of several ailments.

  17. The role of the chi1 gene from the endophytic bacteria Serratia proteamaculans 336x in the biological control of wheat take-all.

    PubMed

    Wang, Miao; Xing, Yuwan; Wang, Junfang; Xu, Yubin; Wang, Gang

    2014-08-01

    Take-all, a disease caused by the fungus Gaeumannomyces graminis var. tritici, is the most important root disease of wheat and causes severe yield losses worldwide. Using microorganisms as biological agents to control the disease is important because no resistant cultivars or effective chemical fungicides are available. In this study, we tested the biological control capability of a chitinase produced by the endophytic bacterium Serratia proteamaculans 336x against wheat take-all. The chitinase gene chi1 of S. proteamaculans 336x was cloned and heterologously expressed in Escherichia coli. The recombinant protein exhibited chitinase activity and in vitro antifungal activity against G. graminis var. tritici. With in-frame deletion of the chi1 gene by homologous recombination, the chi1-deleted mutant was devoid of chitinase activity and the biocontrol efficacy was reduced by 42.5%. The complementation of the Δchi1 mutant strain by the chi1 gene resulted in the partial restoration of the chitinase activity and biocontrol efficacy. These results support a role for the Chi1 protein in the biocontrol process of S. proteamaculans 336x against wheat take-all.

  18. Regulation of defense and cryoprotective proteins by high levels of CO(2) in Annona fruit stored at chilling temperature.

    PubMed

    Goñi, Oscar; Sanchez-Ballesta, María T; Merodio, Carmen; Escribano, María I

    2009-02-15

    This study focuses on how the length of exposure to chilling temperature and atmosphere storage conditions regulate the hydrolytic activity and expression of chitinase (PR-Q) and 1,3-beta-glucanase (PR-2) isoenzymes in cherimoyas (Annona cherimola Mill.). Storage at 6 degrees C modified the expression of constitutive isoenzymes and induced the appearance of novel acidic chitinases, AChi26 and AChi24, at the onset of the storage period, and of a basic chitinase, BChi33, after prolonged storage. The induction of this basic isoenzyme was concomitant with the accumulation of basic constitutive 1,3-beta-glucanases. These low-temperature-induced chitinases modified the growth inhibition in vitro of Botrytis cinerea. Short-term high CO(2) treatment activated a coordinated response of acidic chitinases and 1,3-beta-glucanases after prolonged storage at chilling temperature. Moreover, the high in vitro cryoprotective activity of CO(2)-treated protein extracts was associated with the induction of two low molecular mass isoenzymes, AGlu19 and BChi14. Thus, exposure to high concentrations of CO(2) modified the response of fruit to low temperature, inducing the synthesis of cryoprotectant proteins such as specific pathogenesis-related isoenzymes that could be functionally associated with an increase in chilling tolerance in vivo.

  19. Adolescent sexual activity.

    PubMed

    Braverman, P K; Strasburger, V C

    1993-11-01

    Adolescents are becoming sexually active at younger ages. One half of the adolescents in the United States are sexually active. This article reviews adolescent sexual activity, including rates of sexual activity, sexual practices, gay and lesbian youth, and factors affecting the initiation of sexual activity. In addition, adolescent pregnancy, with possible outcomes and effects on teen parents and their offspring, is discussed.

  20. Guide to Physical Activity

    MedlinePlus

    ... Families ( We Can! ) Health Professional Resources Guide to Physical Activity Physical activity is an important part of your ... to injury. Examples of moderate-intensity amounts of physical activity Common Chores Washing and waxing a car for ...

  1. Physical Activity Assessment

    Cancer.gov

    Current evidence convincingly indicates that physical activity reduces the risk of colon and breast cancer. Physical activity may also reduce risk of prostate cancer. Scientists are also evaluating potential relationships between physical activity and other cancers.

  2. Political activity for physical activity: health advocacy for active transport

    PubMed Central

    2011-01-01

    Effective health advocacy is a priority for efforts to increase population participation in physical activity. Local councils are an important audience for this advocacy. The aim of the current study was to describe features of advocacy for active transport via submissions to city council annual plans in New Zealand, and the impact of an information sheet to encourage the health sector to be involved in this process. Written submissions to city council's annual consultation process were requested for 16 city councils over the period of three years (2007/08, 2008/09, and 2009/10). Submissions were reviewed and categories of responses were created. An advocacy information sheet encouraging health sector participation and summarising some of the evidence-base related to physical activity, active transport and health was released just prior to the 2009/10 submission time. Over the period of the study, city councils received 47,392 submissions, 17% of which were related to active transport. Most submissions came from city residents, with a small proportion (2%) from the health sector. The largest category of submissions was in support of pedestrian and cycling infrastructure, design and maintenance of facilities and additional features to support use of these transport modes. Health arguments featured prominently in justifications for active transport initiatives, including concerns about injury risk, obesity, physical inactivity, personal safety and facilities for people with disabilities. There was evidence that the information sheet was utilised by some health sector submitters (12.5%), providing tentative support for initiatives of this nature. In conclusion, the study provides novel information about the current nature of health advocacy for active transport and informs future advocacy efforts about areas for emphasis, such as health benefits of active transport, and potential alliances with other sectors such as environmental sustainability, transport and urban

  3. Effect of colostrum immunoglobulin concentration on immunity in Majorera goat kids.

    PubMed

    Rodríguez, C; Castro, N; Capote, J; Morales-Delanuez, A; Moreno-Indias, I; Sánchez-Macías, D; Argüello, A

    2009-04-01

    The aim of the research was to evaluate the effects of immunoglobulin G (IgG) colostrum concentration on goat kid immune status when the total amount of IgG fed was constant. Majorera goat kids (n = 56) were randomly assigned to 1 of 4 groups, and kids received 4 g of IgG per kg of body weight of atomized colostrum at 4 different IgG concentrations: 20 (AC-20), 40 (AC-40), 60 (AC-60), and 80 (AC-80) mg/mL. Blood samples were obtained on d 0, 1, 2, 3, 4, and 5 postpartum. Immunoglobulin G, IgA, and IgM plasma concentrations, apparent efficiency of absorption of IgG, plasma chitotriosidase activity, plasma complement activity, and plasma proteinogram were measured. Plasma IgG and IgM concentrations were highest on d 1 in AC-80 animals, and IgA plasma concentration was lower in AC-20 than in AC-80. The apparent efficiency of absorption was higher in AC-80 (24.4%) than in the other treatment groups (by an average of 13.8%). Chitotriosidase plasma activity on d 5 (1,488 nmol/mL per hour) was higher than on d 0 and 1 (average of 1,183 nmol/mL per hour). There were no effects of colostrum IgG concentration on complement activity and plasma protein distribution, but gamma-globulin and alpha-globulin were lower on d 0 than on d 1, 2, 3, 4, and 5. Increasing the immunoglobulin concentration in colostrum using atomized colostrum improves the immunoglobulin absorption at the same amount of immunoglobulin fed.

  4. Home Activities for Fours.

    ERIC Educational Resources Information Center

    Ferguson-Florissant School District, Ferguson, MO.

    These home learning activity guides have been developed for parents to use with their 4-year-old children. Most of the activities require only household items that are often thrown away and can be recycled for learning activities. Some require no materials at all. The guides frequently begin with a discussion of home activities; progress through…

  5. Active commuting to school

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Declines in physical activity levels have coincided with increasing rates of obesity in children. This is problematic because physical activity has been shown to attenuate weight gain in children. Active commuting to school is one way of increasing children's physical activity. However, given the hi...

  6. Civil Law: 12 Activities.

    ERIC Educational Resources Information Center

    Dresbach, Debra

    These learning activities on civil law are intended to supplement the secondary level Scholastic materials "Living Law." Case studies, simulations, and role-play activities are included. Information provided for each activity includes a brief overview, background information, teacher instructions and a description of each activity.…

  7. Increasing Youth Physical Activity with Activity Calendars

    ERIC Educational Resources Information Center

    Eckler, Seth

    2016-01-01

    Physical educators often struggle with ways to get their students to be active beyond the school day. One strategy to accomplish this is the use of physical activity calendars (PACs). The purpose of this article is to support the use of PACs and give practical advice for creating effective PACs.

  8. Cultural Activation of Consumers.

    PubMed

    Siegel, Carole E; Reid-Rose, Lenora; Joseph, Adriana M; Hernandez, Jennifer C; Haugland, Gary

    2016-02-01

    This column discusses "cultural activation," defined as a consumer's recognition of the importance of providing cultural information to providers about cultural affiliations, challenges, views about, and attitudes toward behavioral health and general medical health care, as well as the consumer's confidence in his or her ability to provide this information. An aid to activation, "Cultural Activation Prompts," and a scale that measures a consumer's level of activation, the Cultural Activation Measurement Scale, are described. Suggestions are made about ways to introduce cultural activation as a component of usual care.

  9. Smoking, physical activity, and active life expectancy.

    PubMed

    Ferrucci, L; Izmirlian, G; Leveille, S; Phillips, C L; Corti, M C; Brock, D B; Guralnik, J M

    1999-04-01

    The effect of smoking and physical activity on active and disabled life expectancy was estimated using data from the Established Populations for Epidemiologic Studies of the Elderly (EPESE). Population-based samples of persons aged > or = 65 years from the East Boston, Massachusetts, New Haven, Connecticut, and Iowa sites of the EPESE were assessed at baseline between 1981 and 1983 and followed for mortality and disability over six annual follow-ups. A total of 8,604 persons without disability at baseline were classified as "ever" or "never" smokers and doing "low," "moderate," or "high" level physical activity. Active and disabled life expectancies were estimated using a Markov chain model. Compared with smokers, men and women nonsmokers survived 1.6-3.9 and 1.6-3.6 years longer, respectively, depending on level of physical activity. When smokers were disabled and close to death, most nonsmokers were still nondisabled. Physical activity, from low to moderate to high, was significantly associated with more years of life expectancy in both smokers (9.5, 10.5, 12.9 years in men and 11.1, 12.6, 15.3 years in women at age 65) and nonsmokers (11.0, 14.4, 16.2 years in men and 12.7, 16.2, 18.4 years in women at age 65). Higher physical activity was associated with fewer years of disability prior to death. These findings provide strong and explicit evidence that refraining from smoking and doing regular physical activity predict a long and healthy life.

  10. Population Education. Awareness Activities.

    ERIC Educational Resources Information Center

    Brouse, Deborah E.

    1990-01-01

    Described are awareness activities that deal with human population growth, resources, and the environment. Activities include simulations, mathematical exercises, and discussions of the topic. Specific examples of what individuals can do to help are listed. (KR)

  11. Major operations and activities

    SciTech Connect

    Black, D