Developing cellulolytic Yarrowia lipolytica as a platform for the production of valuable products in consolidated bioprocessing of cellulose.

PubMed

Guo, Zhong-Peng; Robin, Julien; Duquesne, Sophie; O'Donohue, Michael Joseph; Marty, Alain; Bordes, Florence

2018-01-01

Both industrial biotechnology and the use of cellulosic biomass as feedstock for the manufacture of various commercial goods are prominent features of the bioeconomy. In previous work, with the aim of developing a consolidated bioprocess for cellulose bioconversion, we conferred cellulolytic activity of Yarrowia lipolytica , one of the most widely studied "nonconventional" oleaginous yeast species. However, further engineering this strain often leads to the loss of previously introduced heterologous genes due to the presence of multiple LoxP sites when using Cre -recombinase to remove previously employed selection markers. In the present study, we first optimized the strategy of expression of multiple cellulases and rescued selection makers to obtain an auxotrophic cellulolytic Y. lipolytica strain. Then we pursued the quest, exemplifying how this cellulolytic Y. lipolytica strain can be used as a CBP platform for the production of target products. Our results reveal that overexpression of SCD1 gene, encoding stearoyl-CoA desaturase, and DGA1 , encoding acyl-CoA:diacylglycerol acyltransferase, confers the obese phenotype to the cellulolytic Y. lipolytica . When grown in batch conditions and minimal medium, the resulting strain consumed 12 g/L cellulose and accumulated 14% (dry cell weight) lipids. Further enhancement of lipid production was achieved either by the addition of glucose or by enhancing cellulose consumption using a commercial cellulase cocktail. Regarding the latter option, although the addition of external cellulases is contrary to the concept of CBP, the amount of commercial cocktail used remained 50% lower than that used in a conventional process (i.e., without internalized production of cellulases). The introduction of the LIP2 gene into cellulolytic Y. lipolytica led to the production of a strain capable of producing lipase 2 while growing on cellulose. Remarkably, when the strain was grown on glucose, the expression of six cellulases did not

Yarrowia lipolytica vesicle-mediated protein transport pathways

PubMed Central

Swennen, Dominique; Beckerich, Jean-Marie

2007-01-01

Background Protein secretion is a universal cellular process involving vesicles which bud and fuse between organelles to bring proteins to their final destination. Vesicle budding is mediated by protein coats; vesicle targeting and fusion depend on Rab GTPase, tethering factors and SNARE complexes. The Génolevures II sequencing project made available entire genome sequences of four hemiascomycetous yeasts, Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Candida glabrata. Y. lipolytica is a dimorphic yeast and has good capacities to secrete proteins. The translocation of nascent protein through the endoplasmic reticulum membrane was well studied in Y. lipolytica and is largely co-translational as in the mammalian protein secretion pathway. Results We identified S. cerevisiae proteins involved in vesicular secretion and these protein sequences were used for the BLAST searches against Génolevures protein database (Y. lipolytica, C. glabrata, K. lactis and D. hansenii). These proteins are well conserved between these yeasts and Saccharomyces cerevisiae. We note several specificities of Y. lipolytica which may be related to its good protein secretion capacities and to its dimorphic aspect. An expansion of the Y. lipolytica Rab protein family was observed with autoBLAST and the Rab2- and Rab4-related members were identified with BLAST against NCBI protein database. An expansion of this family is also found in filamentous fungi and may reflect the greater complexity of the Y. lipolytica secretion pathway. The Rab4p-related protein may play a role in membrane recycling as rab4 deleted strain shows a modification of colony morphology, dimorphic transition and permeability. Similarly, we find three copies of the gene (SSO) encoding the plasma membrane SNARE protein. Quantification of the percentages of proteins with the greatest homology between S. cerevisiae, Y. lipolytica and animal homologues involved in vesicular transport shows that 40% of Y

Production of Lycopene in the Non-Carotenoid-Producing Yeast Yarrowia lipolytica

PubMed Central

Ketelhot, Markus; Gatter, Michael; Barth, Gerold

2014-01-01

The codon-optimized genes crtB and crtI of Pantoea ananatis were expressed in Yarrowia lipolytica under the control of the TEF1 promoter of Y. lipolytica. Additionally, the rate-limiting genes for isoprenoid biosynthesis in Y. lipolytica, GGS1 and HMG1, were overexpressed to increase the production of lycopene. All of the genes were also expressed in a Y. lipolytica strain with POX1 to POX6 and GUT2 deleted, which led to an increase in the size of lipid bodies and a further increase in lycopene production. Lycopene is located mainly within lipid bodies, and increased lipid body formation leads to an increase in the lycopene storage capacity of Y. lipolytica. Growth-limiting conditions increase the specific lycopene content. Finally, a yield of 16 mg g−1 (dry cell weight) was reached in fed-batch cultures, which is the highest value reported so far for a eukaryotic host. PMID:24375130

Engineering towards a complete heterologous cellulase secretome in Yarrowia lipolytica reveals its potential for consolidated bioprocessing

DOE PAGES

Wei, Hui; Wang, Wei; Alahuhta, Markus; ...

2014-10-16

Background: Yarrowia lipolytica is an oleaginous yeast capable of metabolizing glucose to lipids, which then accumulate intracellularly. However, it lacks the suite of cellulolytic enzymes required to break down biomass cellulose and cannot therefore utilize biomass directly as a carbon source. Toward the development of a direct microbial conversion platform for the production of hydrocarbon fuels from cellulosic biomass, the potential for Y. lipolytica to function as a consolidated bioprocessing strain was investigated by first conducting a genomic search and functional testing of its endogenous glycoside hydrolases. Once the range of endogenous enzymes was determined, the critical cellulases from Trichodermamore » reesei were cloned into Yarrowia. Results: Initially, work to express T. reesei endoglucanase II (EGII) and cellobiohydrolase (CBH) II in Y. lipolytica resulted in the successful secretion of active enzymes. However, a critical cellulase, T. reesei CBHI, while successfully expressed in and secreted from Yarrowia, showed less than expected enzymatic activity, suggesting an incompatibility (probably at the post-translational level) for its expression in Yarrowia. This result prompted us to evaluate alternative or modified CBHI enzymes. Our subsequent expression of a T. reesei-Talaromyces emersonii (Tr-Te) chimeric CBHI, Chaetomium thermophilum CBHI, and Humicola grisea CBHI demonstrated remarkably improved enzymatic activities. Specifically, the purified chimeric Tr-Te CBHI showed a specific activity on Avicel that is comparable to that of the native T. reesei CBHI. Furthermore, the chimeric Tr-Te CBHI also showed significant synergism with EGII and CBHII in degrading cellulosic substrates, using either mixed supernatants or co-cultures of the corresponding Y. lipolytica transformants. The consortia system approach also allows rational volume mixing of the transformant cultures in accordance with the optimal ratio of cellulases required for efficient

Characterization of the two intracellular lipases of Y. lipolytica encoded by TGL3 and TGL4 genes: new insights into the role of intracellular lipases and lipid body organisation.

PubMed

Dulermo, Thierry; Tréton, Brigitte; Beopoulos, Athanasios; Kabran Gnankon, Affoué Philomène; Haddouche, Ramdane; Nicaud, Jean-Marc

2013-09-01

Eukaryotes store lipids in a specialised organelle, the lipid body (LB), mainly as triglycerides (TAGs). Both the rates of synthesis and degradation contribute to the control of the accumulation of TAGs. The synthesis of TAGs in yeasts has been well documented, especially in the model yeast Saccharomyces cerevisiae and in the oleaginous yeast Yarrowia lipolytica. However, descriptions of the processes involved in TAG degradation are more scarce and mostly for S. cerevisiae. Here, we report the characterisation of two Y. lipolytica genes, YlTGL3 and YlTGL4, encoding intracellular lipases involved in TAG degradation. The two proteins are localised in lipid bodies, and YlTgl4 was mainly found at the interface between LBs. Surprisingly, the spatial organisation of YlTgl3 and YlTgl4 depends on the culture medium and on the physiological phase of the cell. Inactivation of one or both genes doubles the lipid accumulation capacity of Y. lipolytica, increasing the cell's capacity to accumulate TAGs. The amino acid sequence of YlTgl4 contains the consensus sequence motif (G/A)XSXG, typical of serine hydrolases, whereas YlTgl3 does not. Single and double mutants are unable to degrade TAGs, and higher expression of YlTgl4 correlates with TAG degradation. Therefore, we propose that YlTgl4 is the main lipase responsible for TAG degradation and that YlTgl3 may act as a positive regulator of YlTgl4 rather than a functional lipase. Thus, contrary to S. cerevisiae, Y. lipolytica possesses two intracellular lipases with distinct roles and with distinct localisations in the LB. © 2013. Published by Elsevier B.V. All rights reserved.

Comparative physiology of forty-five Yarrowia lipolytica strains grown on pretreated switchgrass hydrolysate

USDA-ARS?s Scientific Manuscript database

Yarrowia lipolytica is a well-characterized yeast of the phylum Ascomycota with established use in the biotechnology industry for production of organic acids and enzymes. In addition, the yeast is a model oleaginous organism that accumulates lipids during growth on a variety of carbon sources. The a...

Multi-omics analysis reveals regulators of the response to nitrogen limitation in Yarrowia lipolytica

DOE PAGES

Pomraning, Kyle R.; Kim, Young -Mo; Nicora, Carrie D.; ...

2016-02-25

Yarrowia lipolytica is an oleaginous ascomycete yeast that stores lipids in response to limitation of nitrogen. Furthermore, while the enzymatic pathways responsible for neutral lipid accumulation in Y. lipolytica are well characterized, regulation of these pathways has received little attention. We therefore sought to characterize the response to nitrogen limitation at system-wide levels, including the proteome, phosphoproteome and metabolome, to better understand how this organism regulates and controls lipid metabolism and to identify targets that may be manipulated to improve lipid yield.

Advances in synthetic biology of oleaginous yeast Yarrowia lipolytica for producing non-native chemicals.

PubMed

Darvishi, Farshad; Ariana, Mehdi; Marella, Eko Roy; Borodina, Irina

2018-07-01

Oleaginous yeast Yarrowia lipolytica is an important industrial host for the production of enzymes, oils, fragrances, surfactants, cosmetics, and pharmaceuticals. More recently, improved synthetic biology tools have allowed more extensive engineering of this yeast species, which lead to the production of non-native metabolites. In this review, we summarize the recent advances of genome editing tools for Y. lipolytica, including the application of CRISPR/Cas9 system and discuss case studies, where Y. lipolytica was engineered to produce various non-native chemicals: short-chain fatty alcohols and alkanes as biofuels, polyunsaturated fatty acids for nutritional and pharmaceutical applications, polyhydroxyalkanoates and dicarboxylic acids as precursors for biodegradable plastics, carotenoid-type pigments for food and feed, and campesterol as a precursor for steroid drugs.

Expression and Characterization of Glucose Oxidase from Aspergillus niger in Yarrowia lipolytica.

PubMed

Khadivi Derakshan, Fatemeh; Darvishi, Farshad; Dezfulian, Mehrouz; Madzak, Catherine

2017-08-01

Glucose oxidase (GOX) is currently used in clinical, pharmaceutical, food and chemical industries. The aim of this study was expression and characterization of Aspergillus niger glucose oxidase gene in the yeast Yarrowia lipolytica. For the first time, the GOX gene of A. niger was successfully expressed in Y. lipolytica using a mono-integrative vector containing strong hybrid promoter and secretion signal. The highest total glucose oxidase activity was 370 U/L after 7 days of cultivation. An innovative method was used to cell wall disruption in current study, and it could be recommended to use for efficiently cell wall disruption of Y. lipolytica. Optimum pH and temperature for recombinant GOX activity were 5.5 and 37 °C, respectively. A single band with a molecular weight of 80 kDa similar to the native and pure form of A. niger GOX was observed for the recombinant GOX in SDS-PAGE analysis. Y. lipolytica is a suitable and efficient eukaryotic expression system to production of recombinant GOX in compered with other yeast expression systems and could be used to production of pure form of GOX for industrial applications.

Engineering Promoter Architecture in Oleaginous Yeast Yarrowia lipolytica.

PubMed

Shabbir Hussain, Murtaza; Gambill, Lauren; Smith, Spencer; Blenner, Mark A

2016-03-18

Eukaryotic promoters have a complex architecture to control both the strength and timing of gene transcription spanning up to thousands of bases from the initiation site. This complexity makes rational fine-tuning of promoters in fungi difficult to predict; however, this very same complexity enables multiple possible strategies for engineering promoter strength. Here, we studied promoter architecture in the oleaginous yeast, Yarrowia lipolytica. While recent studies have focused on upstream activating sequences, we systematically examined various components common in fungal promoters. Here, we examine several promoter components including upstream activating sequences, proximal promoter sequences, core promoters, and the TATA box in autonomously replicating expression plasmids and integrated into the genome. Our findings show that promoter strength can be fine-tuned through the engineering of the TATA box sequence, core promoter, and upstream activating sequences. Additionally, we identified a previously unreported oleic acid responsive transcription enhancement in the XPR2 upstream activating sequences, which illustrates the complexity of fungal promoters. The promoters engineered here provide new genetic tools for metabolic engineering in Y. lipolytica and provide promoter engineering strategies that may be useful in engineering other non-model fungal systems.

A novel multigene expression construct for modification of glycerol metabolism in Yarrowia lipolytica

PubMed Central

2013-01-01

Background High supply of raw, residual glycerol from biodiesel production plants promote the search for novel biotechnological methods of its utilization. In this study we attempted modification of glycerol catabolism in a nonconventional yeast species Yarrowia lipolytica through genetic engineering approach. Results To address this, we developed a novel genetic construct which allows transferring three heterologous genes, encoding glycerol dehydratase, its reactivator and a wide-spectrum alcohol oxidoreductase under the control of glycerol-induced promoter. The three genes, tandemly arrayed in an expression cassette with a marker gene ura3, regulatory and targeting sequences (G3P dh promoter and XPR-like terminator, 28S rDNA as a target locus), were transferred into Yarrowia lipolytica cells. The obtained recombinant strain NCYC3825 was characterized at the molecular level and with respect to its biotechnological potential. Our experiments indicated that the novel recombinant strain stably borne one copy of the expression cassette and efficiently expressed heterologous alcohol oxidoreductase, while glycerol dehydratase and its reactivator were expressed at lower level. Comparative shake flask cultivations in glucose- and glycerol-based media demonstrated higher biomass production by the recombinant strain when glycerol was the main carbon source. During bioreactor (5 L) fed-batch cultivation in glycerol-based medium, the recombinant strain was characterized by relatively high biomass and lipids accumulation (up to 42 gDCW L-1, and a peak value of 38%LIPIDS of DCW, respectively), and production of high titers of citric acid (59 g L-1) and 2-phenylethanol (up to 1 g L-1 in shake flask cultivation), which are industrially attractive bioproducts. Conclusions Due to heterogeneous nature of the observed alterations, we postulate that the main driving force of the modified phenotype was faster growth in glycerol-based media, triggered by modifications in the red

Fatty alcohol production in Lipomyces starkeyi and Yarrowia lipolytica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Wei; Wei, Hui; Knoshaug, Eric

Current biological pathways to produce biofuel intermediates amenable to separations and catalytic upgrading to hydrocarbon fuels are not cost effective. Previously, oleaginous yeasts have been investigated primarily for lipid production. However, yeasts store neutral lipids intracellularly making recovery difficult and expensive. In addition, once recovered from the cells, lipids are difficult to blend directly with the existing fuels without upgrading. We have, therefore, begun to investigate secreted fatty acid-derived products which can be easily recovered and upgraded to fuels. In this study, we successfully demonstrate the production of fatty alcohols by the oleaginous yeasts, Yarrowia lipolytica and Lipomyces starkeyi, throughmore » expression of the fatty acyl-CoA reductase gene from Marinobactor aquaeolei VT8. This strategy resulted in the production of 167 and 770 mg/L of fatty alcohols in shake flask from Y. lipolytica and L starkeyi, respectively. When using a dodecane overlay during fermentation, 92 and 99% of total fatty alcohols produced by Y. lipolytica and L. starkeyi, respectively, were extracted into the dodecane phase, which compares favorably to the 3 and 50% recovered, respectively, without the dodecane layer. In both oleaginous yeasts, long chain length, saturated fatty alcohols, i.e., hexadecanol (C16:0) and octadecanol (C18:0), were predominant and accounted for more than 85% of the total fatty alcohols produced. To the best of our knowledge, this is the first report of fatty alcohol production in L. starkeyi. Furthermore, this work demonstrates that the oleaginous yeasts, Y. lipolytica and L. starkeyi, can serve as platform organisms for the production of fatty acid-derived biofuels and bioproducts.« less

Fatty alcohol production in Lipomyces starkeyi and Yarrowia lipolytica

DOE PAGES

Wang, Wei; Wei, Hui; Knoshaug, Eric; ...

2016-10-24

Current biological pathways to produce biofuel intermediates amenable to separations and catalytic upgrading to hydrocarbon fuels are not cost effective. Previously, oleaginous yeasts have been investigated primarily for lipid production. However, yeasts store neutral lipids intracellularly making recovery difficult and expensive. In addition, once recovered from the cells, lipids are difficult to blend directly with the existing fuels without upgrading. We have, therefore, begun to investigate secreted fatty acid-derived products which can be easily recovered and upgraded to fuels. In this study, we successfully demonstrate the production of fatty alcohols by the oleaginous yeasts, Yarrowia lipolytica and Lipomyces starkeyi, throughmore » expression of the fatty acyl-CoA reductase gene from Marinobactor aquaeolei VT8. This strategy resulted in the production of 167 and 770 mg/L of fatty alcohols in shake flask from Y. lipolytica and L starkeyi, respectively. When using a dodecane overlay during fermentation, 92 and 99% of total fatty alcohols produced by Y. lipolytica and L. starkeyi, respectively, were extracted into the dodecane phase, which compares favorably to the 3 and 50% recovered, respectively, without the dodecane layer. In both oleaginous yeasts, long chain length, saturated fatty alcohols, i.e., hexadecanol (C16:0) and octadecanol (C18:0), were predominant and accounted for more than 85% of the total fatty alcohols produced. To the best of our knowledge, this is the first report of fatty alcohol production in L. starkeyi. Furthermore, this work demonstrates that the oleaginous yeasts, Y. lipolytica and L. starkeyi, can serve as platform organisms for the production of fatty acid-derived biofuels and bioproducts.« less

Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system.

PubMed

Gao, Shuliang; Tong, Yangyang; Wen, Zhiqiang; Zhu, Li; Ge, Mei; Chen, Daijie; Jiang, Yu; Yang, Sheng

2016-08-01

Yarrowia lipolytica is categorized as a generally recognized as safe (GRAS) organism and is a heavily documented, unconventional yeast that has been widely incorporated into multiple industrial fields to produce valuable biochemicals. This study describes the construction of a CRISPR-Cas9 system for genome editing in Y. lipolytica using a single plasmid (pCAS1yl or pCAS2yl) to transport Cas9 and relevant guide RNA expression cassettes, with or without donor DNA, to target genes. Two Cas9 target genes, TRP1 and PEX10, were repaired by non-homologous end-joining (NHEJ) or homologous recombination, with maximal efficiencies in Y. lipolytica of 85.6 % for the wild-type strain and 94.1 % for the ku70/ku80 double-deficient strain, within 4 days. Simultaneous double and triple multigene editing was achieved with pCAS1yl by NHEJ, with efficiencies of 36.7 or 19.3 %, respectively, and the pCASyl system was successfully expanded to different Y. lipolytica breeding strains. This timesaving method will enable and improve synthetic biology, metabolic engineering and functional genomic studies of Y. lipolytica.

Metabolic peculiarities of the citric acid overproduction from glucose in yeasts Yarrowia lipolytica.

PubMed

Kamzolova, Svetlana V; Morgunov, Igor G

2017-11-01

Comparative study of 43 natural yeast strains belonging to 20 species for their capability for overproduction of citric acid (CA) from glucose under nitrogen limitation of cell growth was carried out. As a result, natural strain Yarrowia lipolytica VKM Y-2373 was selected. The effect of growth limitation by biogenic macroelements (nitrogen, phosphorus, or sulfur) on the CA production by the selected strain was studied. It was shown that yeasts Y. lipolytica grown under deficiency of nitrogen, phosphorus, or sulfur were able to excrete CA in industrially sufficient amounts (80-85g/L with the product yield (Y CA ) of 0.70-0.75g/g and the process selectivity of 92.5-95.3%). Based on the obtained data on activities of enzymes involved in the initial stages of glucose oxidation, the cycle of tricarboxylic acids, and the glyoxylate cycle, the conception of the mechanism responsible for the CA overproduction from glucose in Y. lipolytica was formulated. Copyright © 2017 Elsevier Ltd. All rights reserved.

Impacts of environmental conditions on product formation and morphology of Yarrowia lipolytica.

PubMed

Timoumi, Asma; Guillouet, Stéphane E; Molina-Jouve, Carole; Fillaudeau, Luc; Gorret, Nathalie

2018-05-01

The yeast Yarrowia lipolytica is an industrially important microorganism with distinctive physiological and metabolic characteristics. A variety of external factors (e.g., pH, temperature, and nutrient availability) influences the behavior of the yeast and may act as stress conditions which the cells must withstand and adapt. In this mini review, the impacts of environmental factors on the morphology and metabolite production by Y. lipolytica are summarized. In this regard, detailed insights into the effectors involved in the dimorphic transition of Y. lipolytica, the cultivation conditions employed, as well as the methods applied for the morphological characterization are highlighted. Concerning the metabolism products, a special focus is addressed on lipid and citric acid metabolites which have attracted significant attention in recent years. The dependence of lipid and citric acid productivity on key process parameters, such as media composition and physico-chemical variables, is thoroughly discussed. This review attempts to provide a recent update on the topic and will serve as a meaningful resource for researchers working in the field.

Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica.

PubMed

Beneyton, Thomas; Thomas, Stéphane; Griffiths, Andrew D; Nicaud, Jean-Marc; Drevelle, Antoine; Rossignol, Tristan

2017-01-31

Droplet-based microfluidics is becoming an increasingly attractive alternative to microtiter plate techniques for enzymatic high-throughput screening (HTS), especially for exploring large diversities with lower time and cost footprint. In this case, the assayed enzyme has to be accessible to the substrate within the water-in-oil droplet by being ideally extracellular or displayed at the cell surface. However, most of the enzymes screened to date are expressed within the cytoplasm of Escherichia coli cells, which means that a lysis step must take place inside the droplets for enzyme activity to be assayed. Here, we take advantage of the excellent secretion abilities of the yeast Yarrowia lipolytica to describe a highly efficient expression system particularly suitable for the droplet-based microfluidic HTS. Five hydrolytic genes from Aspergillus niger genome were chosen and the corresponding five Yarrowia lipolytica producing strains were constructed. Each enzyme (endo-β-1,4-xylanase B and C; 1,4-β-cellobiohydrolase A; endoglucanase A; aspartic protease) was successfully overexpressed and secreted in an active form in the crude supernatant. A droplet-based microfluidic HTS system was developed to (a) encapsulate single yeast cells; (b) grow yeast in droplets; (c) inject the relevant enzymatic substrate; (d) incubate droplets on chip; (e) detect enzymatic activity; and (f) sort droplets based on enzymatic activity. Combining this integrated microfluidic platform with gene expression in Y. lipolytica results in remarkably low variability in the enzymatic activity at the single cell level within a given monoclonal population (<5%). Xylanase, cellobiohydrolase and protease activities were successfully assayed using this system. We then used the system to screen for thermostable variants of endo-β-1,4-xylanase C in error-prone PCR libraries. Variants displaying higher thermostable xylanase activities compared to the wild-type were isolated (up to 4.7-fold improvement

Metabolic engineering of oleaginous yeast Yarrowia lipolytica for limonene overproduction.

PubMed

Cao, Xuan; Lv, Yu-Bei; Chen, Jun; Imanaka, Tadayuki; Wei, Liu-Jing; Hua, Qiang

2016-01-01

Limonene, a monocyclic monoterpene, is known for its using as an important precursor of many flavoring, pharmaceutical, and biodiesel products. Currently, d-limonene has been produced via fractionation from essential oils or as a byproduct of orange juice production, however, considering the increasing need for limonene and a certain amount of pesticides may exist in the limonene obtained from the citrus industry, some other methods should be explored to produce limonene. To construct the limonene synthetic pathway in Yarrowia lipolytica , two genes encoding neryl diphosphate synthase 1 (NDPS1) and limonene synthase (LS) were codon-optimized and heterologously expressed in Y. lipolytica . Furthermore, to maximize limonene production, several genes involved in the MVA pathway were overexpressed, either in different copies of the same gene or in combination. Finally with the optimized pyruvic acid and dodecane concentration in flask culture, a maximum limonene titer and content of 23.56 mg/L and 1.36 mg/g DCW were achieved in the final engineered strain Po1f-LN-051, showing approximately 226-fold increase compared with the initial yield 0.006 mg/g DCW. This is the first report on limonene biosynthesis in oleaginous yeast Y. lipolytica by heterologous expression of codon-optimized tLS and tNDPS1 genes. To our knowledge, the limonene production 23.56 mg/L, is the highest limonene production level reported in yeast. In short, we demonstrate that Y. lipolytica provides a compelling platform for the overproduction of limonene derivatives, and even other monoterpenes.

Optimization of odd chain fatty acid production by Yarrowia lipolytica.

PubMed

Park, Young-Kyoung; Dulermo, Thierry; Ledesma-Amaro, Rodrigo; Nicaud, Jean-Marc

2018-01-01

Odd chain fatty acids (odd FAs) have a wide range of applications in therapeutic and nutritional industries, as well as in chemical industries including biofuel. Yarrowia lipolytica is an oleaginous yeast considered a preferred microorganism for the production of lipid-derived biofuels and chemicals. However, it naturally produces negligible amounts of odd chain fatty acids. The possibility of producing odd FAs using Y. lipolytica was investigated. Y. lipolytica wild-type strain was shown able to grow on weak acids; acetate, lactate, and propionate. Maximal growth rate on propionate reached 0.24 ± 0.01 h -1 at 2 g/L, and growth inhibition occurred at concentration above 10 g/L. Wild-type strain accumulated lipids ranging from 7.39 to 8.14% (w/w DCW) depending on the carbon source composition, and odd FAs represented only 0.01-0.12 g/L. We here proved that the deletion of the PHD1 gene improved odd FAs production, which reached a ratio of 46.82% to total lipids. When this modification was transferred to an obese strain, engineered for improving lipid accumulation, further increase odd FAs production reaching a total of 0.57 g/L was shown. Finally, a fed-batch co-feeding strategy was optimized for further increase odd FAs production, which generated 0.75 g/L, the best production described so far in Y. lipolytica . A Y. lipolytica strain able to accumulate high level of odd chain fatty acids, mainly heptadecenoic acid, has been successfully developed. In addition, a fed-batch co-feeding strategy was optimized to further improve lipid accumulation and odd chain fatty acid content. These lipids enriched in odd chain fatty acid can (1) improve the properties of the biodiesel generated from Y. lipolytica lipids and (2) be used as renewable source of odd chain fatty acid for industrial applications. This work paves the way for further improvements in odd chain fatty acids and fatty acid-derived compound production.

YALI0E32769g (DGA1) and YALI0E16797g (LRO1) encode major triacylglycerol synthases of the oleaginous yeast Yarrowia lipolytica.

PubMed

Athenstaedt, Karin

2011-10-01

The oleaginous yeast Yarrowia lipolytica has an outstanding capacity to produce and store triacylglycerols resembling adipocytes of higher eukaryotes. Here, the identification of two genes YALI0E32769g (DGA1) and YALI0E16797g (LRO1) encoding major triacylglycerol synthases of Yarrowia lipolytica is reported. Heterologous expression of either DGA1 or LRO1 in a mutant of the budding yeast Saccharomyces cerevisiae defective in triacylglycerol synthesis restores the formation of this neutral lipid. Whereas Dga1p requires acyl-CoA as a substrate for acylation of diacylglycerol, Lro1p is an acyl-CoA independent triacylglycerol synthase using phospholipids as acyl-donor. Growth of Yarrowia lipolytica strains deleted of DGA1 and/or LRO1 on glucose containing medium significantly decreases triacylglycerol accumulation. Most interestingly, when oleic acid serves as the carbon source the ratio of triacylglycerol accumulation in mutants to wild-type is significantly increased in strains defective in DGA1 but not in lro1Δ. In vitro experiments revealed that under these conditions an additional acyl-CoA dependent triacylglycerol synthase contributes to triacylglycerol synthesis in the respective mutants. Taken together, evidence is provided that Yarrowia lipolytica contains at least four triacylglycerol synthases, namely Lro1p, Dga1p and two additional triacylglycerol synthases whereof one is acyl-CoA dependent and specifically induced upon growth on oleic acid. Copyright © 2011 Elsevier B.V. All rights reserved.

Yarrowia lipolytica morphological mutant enables lasting in situ immobilization in bioreactor.

PubMed

Vandermies, Marie; Kar, Tambi; Carly, Frédéric; Nicaud, Jean-Marc; Delvigne, Frank; Fickers, Patrick

2018-04-26

In the present study, we have isolated and characterized a Yarrowia lipolytica morphological mutant growing exclusively in the pseudohyphal morphology. The gene responsible for this phenotype, YALI0E06519g, was identified as homologous to the mitosis regulation gene HSL1 from Saccharomyces cerevisiae. Taking advantage of its morphology, we achieved the immobilization of the Δhsl1 mutant on the metallic structured packing of immobilized-cell bioreactors. We obtained significant cell retention and growth on the support during shake flask and bioreactor experiments without an attachment step prior to the culture. The system of medium aspersion on the packing ensured oxygen availability in the absence of agitation and minimized the potential release of cells in the culture medium. Additionally, the metallic packing proved its facility of cleaning and sterilization after fermentation. This combined use of morphological mutation and bioreactor design is a promising strategy to develop continuous processes for the production of recombinant protein and metabolites using Y. lipolytica. Graphical Abstract.

Engineering ..beta..-Oxidation in Yarrowia lipolytica for Methyl Ketone Production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sanchez i Nogue, Violeta; Ramirez, Kelsey J; Singer, Christine

Medium- and long-chain methyl ketones are fatty acid-derived compounds that can be used as biofuel blending agents, flavors and fragrances. However, their large-scale production from sustainable feedstocks is currently limited due to the lack of robust microbial biocatalysts. The oleaginous yeast Yarrowia lipolytica is a promising biorefinery platform strain for the production of methyl ketones from renewable lignocellulosic biomass due to its natively high flux towards fatty acid biosynthesis. In this study, we report the metabolic engineering of Y. lipolytica to produce long- and very long-chain methyl ketones. Truncation of peroxisomal ..beta..-oxidation by chromosomal deletion of pot1 resulted in themore » biosynthesis of saturated, mono-, and diunsaturated methyl ketones in the C13-C23 range. Additional overexpression and peroxisomal targeting of a heterologous bacterial methyl ketone biosynthesis pathway yielded an initial titer of 151.5 mg/L of saturated methyl ketones. Dissolved oxygen concentrations in the cultures were found to substantially impact cell morphology and methyl ketone biosynthesis. Bioreactor cultivation under optimized conditions resulted in a titer of 314.8 mg/L of total methyl ketones, representing more than a 6000-fold increase over the parental strain. This work highlights the potential of Y. lipolytica to serve as chassis organism for the biosynthesis of acyl-thioester derived long- and very long-chain methyl ketones.« less

Production of Medium Chain Fatty Acids by Yarrowia lipolytica: Combining Molecular Design and TALEN to Engineer the Fatty Acid Synthase.

PubMed

Rigouin, Coraline; Gueroult, Marc; Croux, Christian; Dubois, Gwendoline; Borsenberger, Vinciane; Barbe, Sophie; Marty, Alain; Daboussi, Fayza; André, Isabelle; Bordes, Florence

2017-10-20

Yarrowia lipolytica is a promising organism for the production of lipids of biotechnological interest and particularly for biofuel. In this study, we engineered the key enzyme involved in lipid biosynthesis, the giant multifunctional fatty acid synthase (FAS), to shorten chain length of the synthesized fatty acids. Taking as starting point that the ketoacyl synthase (KS) domain of Yarrowia lipolytica FAS is directly involved in chain length specificity, we used molecular modeling to investigate molecular recognition of palmitic acid (C16 fatty acid) by the KS. This enabled to point out the key role of an isoleucine residue, I1220, from the fatty acid binding site, which could be targeted by mutagenesis. To address this challenge, TALEN (transcription activator-like effector nucleases)-based genome editing technology was applied for the first time to Yarrowia lipolytica and proved to be very efficient for inducing targeted genome modifications. Among the generated FAS mutants, those having a bulky aromatic amino acid residue in place of the native isoleucine at position 1220 led to a significant increase of myristic acid (C14) production compared to parental wild-type KS. Particularly, the best performing mutant, I1220W, accumulates C14 at a level of 11.6% total fatty acids. Overall, this work illustrates how a combination of molecular modeling and genome-editing technology can offer novel opportunities to rationally engineer complex systems for synthetic biology.

Impact of culture conditions on β-carotene encapsulation using Yarrowia lipolytica cells

NASA Astrophysics Data System (ADS)

Dang, Tran Hai; Minh, Ho Thi Thu; Van Nhi, Tran Nguyen; Ngoc, Ta Thi Minh

2017-09-01

Yeast cell was reported as an effective natural preformed material for use in encapsulation of hydrophobic compounds. The encapsulation process was normally considered as passive transfer through cellular wall and cellular membrane. Beside solubility of hydrophobic compound in phospholipid membrane or plasmolysis, membrane characteristics of yeast cell which are differed between strains and influenced by culture conditions are main factors involving the accumulation of hydrophobic compound into yeast cell. In this study, the oleaginous yeast Yarrowia lipolytica was used as micro-container shell to encapsulate a high hydrophobic compound - β-carotene. Yeast cell was cultured under different conditions and wet yeast biomass was incubated with β-carotene which was dissolved in soybean oil overnight. β-carotene accumulation was then extracted and evaluated by UV-VIS spectrometry. Optimization of culture condition was investigated using the Box-Behnken model. β-carotene encapsulation efficiency in Y. lipolytica was showed to be affected by both pH of medium and agitation conditions. The highest β-carotene encapsulation efficiency was optimized at 42.8 μg/g with Y. lipolytica cultured at pH 4.5, medium volume equal to 115 ml and agitation speed at 211 rpm.

Draft Genome Sequence of the Dimorphic Yeast Yarrowia lipolytica Strain W29

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pomraning, Kyle R.; Baker, Scott E.

Here, we present the draft genome sequence of the dimorphic ascomycete yeastYarrowia lipolyticastrain W29 (ATCC 20460).Y. lipolyticais a commonly employed model for the industrial production of lipases, small molecules, and more recently for its ability to accumulate lipids.

Prospect for Developing a Consolidated Bioprocessing (CBP) Strain Using Xylan as the Substrate: the Case Study of Yarrowia lipolytica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Wei; Wei, Hui; Alahuhta, Markus

2016-07-08

To achieve the goal of developing a direct microbial sugar conversion platform for the production of lipids and drop-in fuels from cellulosic biomass substrate, Yarrowia lipolytica was used to investigate its potential for being developed as CBP strain by expressing cellulase and xylanase enzymes. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing glucose and xylose to produce lipids; however, due to the lack of the biomass degrading enzymes, it cannot directly utilize lignocellulosic substrates as carbon sources. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in thismore » study, we present successful expression of several xylanases in Y. lipolytica. To the best of our knowledge, this is the first study introducing heterologous hemicellulose genes into the genome of Y. lipolytica. SDS-PAGE and western blotting analysis showed that the endo-xylanase gene XynII and exo-xylosidase gene XlnD were successfully expressed and secreted, and the expressed xylanases were likely either not or sparsely glycosylated, which is advantageous for expression of heterologous proteins from any species. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action on converting xylan to xylose was observed when XlnD worked in concert with XynII. XlnD was able to work on the xylo-oligomers generated by XynII, enhancing the xylan conversion to monomeric xylose. The successful expression of these xylanases in Yarrowia further advances us towards our goal to develop a direct microbial conversion process using this organism. and xylose to produce lipids; however, due to the lack of the biomass degrading enzymes, it cannot directly utilize lignocellulosic substrates as carbon sources. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful

Engineering β-oxidation in Yarrowia lipolytica for methyl ketone production.

PubMed

Hanko, Erik K R; Denby, Charles M; Sànchez I Nogué, Violeta; Lin, Weiyin; Ramirez, Kelsey J; Singer, Christine A; Beckham, Gregg T; Keasling, Jay D

2018-05-28

Medium- and long-chain methyl ketones are fatty acid-derived compounds that can be used as biofuel blending agents, flavors and fragrances. However, their large-scale production from sustainable feedstocks is currently limited due to the lack of robust microbial biocatalysts. The oleaginous yeast Yarrowia lipolytica is a promising biorefinery platform strain for the production of methyl ketones from renewable lignocellulosic biomass due to its natively high flux towards fatty acid biosynthesis. In this study, we report the metabolic engineering of Y. lipolytica to produce long- and very long-chain methyl ketones. Truncation of peroxisomal β-oxidation by chromosomal deletion of pot1 resulted in the biosynthesis of saturated, mono-, and diunsaturated methyl ketones in the C 13 -C 23 range. Additional overexpression and peroxisomal targeting of a heterologous bacterial methyl ketone biosynthesis pathway yielded an initial titer of 151.5 mg/L of saturated methyl ketones. Dissolved oxygen concentrations in the cultures were found to substantially impact cell morphology and methyl ketone biosynthesis. Bioreactor cultivation under optimized conditions resulted in a titer of 314.8 mg/L of total methyl ketones, representing more than a 6000-fold increase over the parental strain. This work highlights the potential of Y. lipolytica to serve as chassis organism for the biosynthesis of acyl-thioester derived long- and very long-chain methyl ketones. Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Yarrowia lipolytica: a model yeast for citric acid production.

PubMed

Cavallo, Ema; Charreau, Hernán; Cerrutti, Patricia; Foresti, María Laura

2017-12-01

Every year more than 2 million tons of citric acid (CA) are produced around the world for industrial uses. Although initially extracted from citrus, the low profitability of the process and the increasing demand soon stimulated the search for more efficient methods to produce CA. Currently, most world CA demand (99%) is satisfied by fermentations with microorganisms, especially filamentous fungi and yeasts. CA production with yeasts has certain advantages over molds (e.g. higher productivity and easier cultivation), which in the last two decades have triggered a clear increase in publications and patents devoted to the use of yeasts in this field. Yarrowia lipolytica has become a model yeast that proved to be successful in different production systems. Considering the current interest evidenced in the literature, the most significant information on CA production using Y. lipolytica is summarized. The relevance on CA yields of key factors such as strains, media formulation, environmental conditions and production regimes is thoroughly discussed, with particular focus on increasing CA productivity. Besides, the possibility of tuning the mentioned variables to reduce concomitant isocitric acid production-the biggest disadvantage of using yeasts-is analyzed. Available methods for CA purification/quantification are also discussed. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Engineering Yarrowia lipolytica for Use in Biotechnological Applications: A Review of Major Achievements and Recent Innovations.

PubMed

Madzak, Catherine

2018-06-25

Yarrowia lipolytica is an oleaginous saccharomycetous yeast with a long history of industrial use. It aroused interest several decades ago as host for heterologous protein production. Thanks to the development of numerous molecular and genetic tools, Y. lipolytica is now a recognized system for expressing heterologous genes and secreting the corresponding proteins of interest. As genomic and transcriptomic tools increased our basic knowledge on this yeast, we can now envision engineering its metabolic pathways for use as whole-cell factory in various bioconversion processes. Y. lipolytica is currently being developed as a workhorse for biotechnology, notably for single-cell oil production and upgrading of industrial wastes into valuable products. As it becomes more and more difficult to keep up with an ever-increasing literature on Y. lipolytica engineering technology, this article aims to provide basic and actualized knowledge on this research area. The most useful reviews on Y. lipolytica biology, use, and safety will be evoked, together with a resume of the engineering tools available in this yeast. This mini-review will then focus on recently developed tools and engineering strategies, with a particular emphasis on promoter tuning, metabolic pathways assembly, and genome editing technologies.

Development of a novel rDNA based plasmid for enhanced cell surface display on Yarrowia lipolytica.

PubMed

Bulani, Siyavuya Ishmael; Moleleki, Lucy; Albertyn, Jacobus; Moleleki, Ntsane

2012-05-20

In this study, a novel rDNA based plasmid was developed for display of heterologous proteins on the cell surface of Yarrowia lipolytica using the C-terminal end of the glycosylphosphatidylinositol (GPI) anchored Y. lipolytica cell wall protein 1 (YlCWP1). mCherry was used as a model protein to assess the efficiency of the constructed plasmid. Y. lipolytica transformants harbouring the expression cassettes showed a purple colour phenotype on selective YNB-casamino plates as compared to control cells indicating that mCherry was displayed on the cells. Expression of mCherry on cells of Y. lipolytica was confirmed by both fluorescent microscopy and flow cytometry. Furthermore, SDS-PAGE analysis and matrix-assisted laser desorption/ionization (MALDI)-time-of (TOF)-mass spectrometry (MS) peptide mass fingerprinting (PMF) confirmed that the protein cleaved from the yeast cells using enterokinase was mCherry. Efficient cleavage of mCherry reported in this work offers an alternative purification method for displayed heterologous proteins on Y. lipolytica cells using the plasmid constructed in this study. The developed displaying system offers great potential for industrial production and purification of heterologous proteins at low cost.

Comprehensive metabolomic, lipidomic and microscopic profiling of Yarrowia lipolytica during lipid accumulation identifies targets for increased lipogenesis

DOE PAGES

Pomraning, Kyle R.; Wei, Siwei; Karagiosis, Sue A.; ...

2015-04-23

Yarrowia lipolytica is an oleaginous ascomycete yeast that accumulates large amounts of lipids and has potential as a biofuel producing organism. Despite a growing scientific literature focused on lipid production by Y. lipolytica, there remain significant knowledge gaps regarding the key biological processes involved. We applied a combination of metabolomic and lipidomic profiling approaches as well as microscopic techniques to identify and characterize the key pathways involved in de novo lipid accumulation from glucose in batch cultured, wild-type Y. lipolytica. We found that lipids accumulated rapidly and peaked at 48 hours during the five day experiment, concurrent with a shiftmore » in amino acid metabolism. We also report that Y. lipolytica secretes disaccharides early in batch culture and reabsorbs them when extracellular glucose is depleted. Exhaustion of extracellular sugars coincided with thickening of the cell wall, suggesting that genes involved in cell wall biogenesis may be a useful target for improving the efficiency of lipid producing yeast strains.« less

Extracellular expression of YlLip11 with a native signal peptide from Yarrowia lipolytica MSR80 in three different yeast hosts.

PubMed

Kumari, Arti; Baronian, Keith; Kunze, Gotthard; Gupta, Rani

2015-06-01

Lipase YlLip11 from Yarrowia lipolytica was expressed with a signal peptide encoding sequence in Arxula adeninivorans, Saccharomyces cerevisiae and Hansenula polymorpha using the Xplor®2 transformation/expression platform and an expression module with the constitutive Arxula-derived TEF1 promoter. The YlLip11 signal peptide was functional in all of the yeast hosts with 97% of the recombinant enzyme being secreted into the culture medium. However, recombinant YlLip11 with His Tag fused at C-terminal was not active. The best recombinant YlLip11 producing A. adeninivorans G1212/YRC102-YlLip11 transformant cultivated in shake flasks produced 2654 U/L lipase, followed by S. cerevisiae SEY6210/YRC103-YlLip11 (1632U/L) and H. polymorpha RB11/YRC103-YlLip11 (1144U/L). Although the biochemical parameters of YlLip11 synthesized in different hosts were similar, their glycosylation level and thermo stability differed. The protein synthesized by the H. polymorpha transformant had the highest degree of glycosylation and with a t1/2 of 60min at 70°C, exhibited the highest thermostability. Copyright © 2015 Elsevier Inc. All rights reserved.

Urea and urine are a viable and cost-effective nitrogen source for Yarrowia lipolytica biomass and lipid accumulation.

PubMed

Brabender, Matthew; Hussain, Murtaza Shabbir; Rodriguez, Gabriel; Blenner, Mark A

2018-03-01

Yarrowia lipolytica is an industrial yeast that has been used in the sustainable production of fatty acid-derived and lipid compounds due to its high growth capacity, genetic tractability, and oleaginous properties. This investigation examines the possibility of utilizing urea or urine as an alternative to ammonium sulfate as a nitrogen source to culture Y. lipolytica. The use of a stoichiometrically equivalent concentration of urea in lieu of ammonium sulfate significantly increased cell growth when glucose was used as the carbon source. Furthermore, Y. lipolytica growth was equally improved when grown with synthetic urine and real human urine. Equivalent or better lipid production was achieved when cells are grown on urea or urine. The successful use of urea and urine as nitrogen sources for Y. lipolytica growth highlights the potential of using cheaper media components as well as exploiting and recycling non-treated human waste streams for biotechnology processes.

Genome-scale model-driven strain design for dicarboxylic acid production in Yarrowia lipolytica.

PubMed

Mishra, Pranjul; Lee, Na-Rae; Lakshmanan, Meiyappan; Kim, Minsuk; Kim, Byung-Gee; Lee, Dong-Yup

2018-03-19

Recently, there have been several attempts to produce long-chain dicarboxylic acids (DCAs) in various microbial hosts. Of these, Yarrowia lipolytica has great potential due to its oleaginous characteristics and unique ability to utilize hydrophobic substrates. However, Y. lipolytica should be further engineered to make it more competitive: the current approaches are mostly intuitive and cumbersome, thus limiting its industrial application. In this study, we proposed model-guided metabolic engineering strategies for enhanced production of DCAs in Y. lipolytica. At the outset, we reconstructed genome-scale metabolic model (GSMM) of Y. lipolytica (iYLI647) by substantially expanding the previous models. Subsequently, the model was validated using three sets of published culture experiment data. It was finally exploited to identify genetic engineering targets for overexpression, knockout, and cofactor modification by applying several in silico strain design methods, which potentially give rise to high yield production of the industrially relevant long-chain DCAs, e.g., dodecanedioic acid (DDDA). The resultant targets include (1) malate dehydrogenase and malic enzyme genes and (2) glutamate dehydrogenase gene, in silico overexpression of which generated additional NADPH required for fatty acid synthesis, leading to the increased DDDA fluxes by 48% and 22% higher, respectively, compared to wild-type. We further investigated the effect of supplying branched-chain amino acids on the acetyl-CoA turn-over rate which is key metabolite for fatty acid synthesis, suggesting their significance for production of DDDA in Y. lipolytica. In silico model-based strain design strategies allowed us to identify several metabolic engineering targets for overproducing DCAs in lipid accumulating yeast, Y. lipolytica. Thus, the current study can provide a methodological framework that is applicable to other oleaginous yeasts for value-added biochemical production.

Synthetic Biology Expands the Industrial Potential of Yarrowia lipolytica.

PubMed

Markham, Kelly A; Alper, Hal S

2018-06-04

The oleaginous yeast Yarrowia lipolytica is quickly emerging as the most popular non-conventional (i.e., non-model organism) yeast in the bioproduction field. With a high propensity for flux through tricarboxylic acid (TCA) cycle intermediates and biological precursors such as acetyl-CoA and malonyl-CoA, this host is especially well suited to meet our industrial chemical production needs. Recent progress in synthetic biology tool development has greatly enhanced our ability to rewire this organism, with advances in genetic component design, CRISPR technologies, and modular cloning strategies. In this review we investigate recent developments in metabolic engineering and describe how the new tools being developed help to realize the full industrial potential of this host. Finally, we conclude with our vision of the developments that will be necessary to enhance future engineering efforts. Copyright © 2018 Elsevier Ltd. All rights reserved.

Engineering Yarrowia lipolytica to express secretory invertase with strong FBA1IN promoter.

PubMed

Hong, Seung-Pyo; Seip, John; Walters-Pollak, Dana; Rupert, Ross; Jackson, Raymond; Xue, Zhixiong; Zhu, Quinn

2012-02-01

Oleaginous yeast Yarrowia lipolytica is an important host for the production of lipid-derived compounds or heterologous proteins. Selection of strong promoters and effective expression systems is critical for heterologous protein secretion. To search for a strong promoter in Y. lipolytica, activities of FBA1, TDH1 and GPM1 promoters were compared to that of TEF1 promoter by constructing GUS reporter fusions. The FBA1 promoter activity was 2.2 and 5.5 times stronger than the TDH1 and GPM1 promoters, respectively. The FBA1IN promoter (FBA1 sequence of -826 to +169) containing an intron (+64 to +165) showed five-fold higher expression than the FBA1 promoter (-831 to -1). The transcriptional enhancement by the 5'-region within the FBA1 gene was confirmed by GPM1::FBA1 chimeric promoter construction. Using the strong FBA1IN promoter, four different S. cerevisiae SUC2 expression cassettes were tested for the SUC+ phenotype in Y. lipolytica. Functional invertase secretion was facilitated by the Xpr2 prepro-region with an additional 13 amino acids of mature Xpr2, or by the native Suc2 signal sequence. However, these two secretory signals in tandem, or the mature Suc2 with no secretory signal, did not direct secretion of functional invertase. Unlike previously reported Y. lipolytica SUC+ strains, our engineered stains secreted most of invertase into the medium. Copyright © 2011 John Wiley & Sons, Ltd.

Selection of Yarrowia lipolytica strains with high protein content from yeasts isolated from different marine environments

NASA Astrophysics Data System (ADS)

Chi, Zhenming; Wang, Fang; Wang, Lin; Li, Jing; Wang, Xianghong

2007-10-01

A total of 78 Yarrowia lipolytica yeast strains from seawater, sediments, mud of salterns, the guts of marine fish, and marine algae were obtained. After the crude protein of the yeasts was estimated by the method of Kjehldahl, we found that seven strains of the marine yeasts grown in soy bean cake hydrolysate with 20 g L-1 of glucose for 48 h at 28°C contained more than 41.0 g protein per 100 g of cell dry weight and the cell dry weight was more than 4.4 g per L of the culture. Among them, strain SWJ-1b contained the highest crude protein. The results of Biolog identification and molecular methods further confirmed that they indeed belonged to Y. lipolytica.

[Genetic system for maintaining the mitochondrial human genome in yeast Yarrowia lipolytica].

PubMed

Isakova, E P; Deryabina, Yu I; Velyakova, A V; Biryukova, J K; Teplova, V V; Shevelev, A B

2016-01-01

For the first time, the possibility of maintaining an intact human mitochondrial genome in a heterologous system in the mitochondria of yeast Yarrowia lipolytica is shown. A method for introducing directional changes into the structure of the mitochondrial human genome replicating in Y. lipolytica by an artificially induced ability of yeast mitochondria for homologous recombination is proposed. A method of introducing and using phenotypic selection markers for the presence or absence of defects in genes tRNA-Lys and tRNA-Leu of the mitochondrial genome is developed. The proposed system can be used to correct harmful mutations of the human mitochondrial genome associated with mitochondrial diseases and for preparative amplification of intact mitochondrial DNA with an adjusted sequence in yeast cells. The applicability of the new system for the correction of mutations in the genes of Lys- and Leu-specific tRNAs of the human mitochondrial genome associated with serious and widespread human mitochondrial diseases such as myoclonic epilepsy with lactic acidosis (MELAS) and myoclonic epilepsy with ragged-red fibers (MERRF) is shown.

CRISPR-Cas9-Mediated Genome Editing and Transcriptional Control in Yarrowia lipolytica.

PubMed

Schwartz, Cory; Wheeldon, Ian

2018-01-01

The discovery and adaptation of RNA-guided nucleases has resulted in the rapid development of efficient, scalable, and easily accessible synthetic biology tools for targeted genome editing and transcriptional control. In these systems, for example CRISPR-Cas9 from Streptococcus pyogenes, a protein with nuclease activity is targeted to a specific nucleotide sequence by a short RNA molecule, whereupon binding it cleaves the targeted nucleotide strand. To extend this genome-editing ability to the industrially important oleaginous yeast Yarrowia lipolytica, we developed a set of easily usable and effective CRISPR-Cas9 episomal vectors. In this protocols chapter, we first present a method by which arbitrary protein-coding genes can be disrupted via indel formation after CRISPR-Cas9 targeting. A second method demonstrates how the same CRISPR-Cas9 system can be used to induce markerless gene cassette integration into the genome by inducing homologous recombination after DNA cleavage by Cas9. Finally, we describe how a catalytically inactive form of Cas9 fused to a transcriptional repressor can be used to control transcription of native genes in Y. lipolytica. The CRISPR-Cas9 tools and strategies described here greatly increase the types of genome editing and transcriptional control that can be achieved in Y. lipolytica, and promise to facilitate more advanced engineering of this important oleaginous host.

Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates

USDA-ARS?s Scientific Manuscript database

Increased interest in sustainable production of renewable diesel and other valuable bioproducts is redoubling efforts to improve economic feasibility of microbial-based oil production. The yeast Yarrowia lipolytica is capable of employing a wide variety of substrates to produce oil and valuable co-p...

An ortholog of farA of Aspergillus nidulans is implicated in the transcriptional activation of genes involved in fatty acid utilization in the yeast Yarrowia lipolytica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poopanitpan, Napapol; Kobayashi, Satoshi; Fukuda, Ryouichi

2010-11-26

Research highlights: {yields} POR1 is a Yarrowia lipolytica ortholog of farA involved in fatty acid response in A. nidulans. {yields} Deletion of POR1 caused growth defects on fatty acids. {yields} {Delta}por1 strain exhibited defects in the induction of genes involved in fatty acid utilization. -- Abstract: The yeast Yarrowia lipolytica effectively utilizes hydrophobic substrates such as fatty acids and n-alkanes. To identify a gene(s) regulating fatty acid utilization in Y. lipolytica, we first studied homologous genes to OAF1 and PIP2 of Saccharomyces cerevisiae, but their disruption did not change growth on oleic acid at all. We next characterized a Y.more » lipolytica gene, POR1 (primary oleate regulator 1), an ortholog of farA encoding a transcriptional activator that regulates fatty acid utilization in Aspergillus nidulans. The deletion mutant of POR1 was defective in the growth on various fatty acids, but not on glucose, glycerol, or n-hexadecane. It exhibited slight defect on n-decane. The transcriptional induction of genes involved in {beta}-oxidation and peroxisome proliferation by oleate was distinctly diminished in the {Delta}por1 strains. These data suggest that POR1 encodes a transcriptional activator widely regulating fatty acid metabolism in Y. lipolytica.« less

[The Engineering of a Yarrowia lipolytica Yeast Strain Capable of Homologous Recombination of the Mitochondrial Genome].

PubMed

Isakova, E P; Epova, E Yu; Sekova, V Yu; Trubnikova, E V; Kudykina, Yu K; Zylkova, M V; Guseva, M A; Deryabina, Yu I

2015-01-01

None of the studied eukaryotic species has a natural system for homologous recombination of the mitochondrial genome. We propose an integrated genetic construct pQ-SRUS, which allows introduction of the recA gene from Bacillus subtilis into the nuclear genome of an extremophilic yeast, Yarrowia lipolytica. The targeting of recombinant RecA to the yeast mitochondria is provided by leader sequences (5'-UTR and 3'-UTR) derived from the SOD2 gene mRNA, which exhibits affinity to the outer mitochondrial membrane and thus provides cotranslational transport of RecA to the inner space of the mitochondria. The Y. lipolytica strain bearing the pQ-SRUS construct has the unique ability to integrate DNA constructs into the mitochondrial genome. This fact was confirmed using a tester construct, pQ-NIHN, intended for the introduction of the EYFP gene into the translation initiation region of the Y. lipolytica ND1 mitochondrial gene. The Y. lipolytica strain bearing pQ-SRUS makes it possible to engineer recombinant producers based on Y. lipolytica bearing transgenes in the mitochondrial genome. They are promising for the construction of a genetic system for in vivo replication and modification of the human mitochondrial genome. These strains may be used as a tool for the treatment of human mitochondrial diseases (including genetically inherited ones).

Robust signal peptides for protein secretion in Yarrowia lipolytica: identification and characterization of novel secretory tags.

PubMed

Celińska, Ewelina; Borkowska, Monika; Białas, Wojciech; Korpys, Paulina; Nicaud, Jean-Marc

2018-06-01

Upon expression of a given protein in an expression host, its secretion into the culture medium or cell-surface display is frequently advantageous in both research and industrial contexts. Hence, engineering strategies targeting folding, trafficking, and secretion of the proteins gain considerable interest. Yarrowia lipolytica has emerged as an efficient protein expression platform, repeatedly proved to be a competitive secretor of proteins. Although the key role of signal peptides (SPs) in secretory overexpression of proteins and their direct effect on the final protein titers are widely known, the number of reports on manipulation with SPs in Y. lipolytica is rather scattered. In this study, we assessed the potential of ten different SPs for secretion of two heterologous proteins in Y. lipolytica. Genomic and transcriptomic data mining allowed us to select five novel, previously undescribed SPs for recombinant protein secretion in Y. lipolytica. Their secretory potential was assessed in comparison with known, widely exploited SPs. We took advantage of Golden Gate approach, for construction of expression cassettes, and micro-volume enzymatic assays, for functional screening of large libraries of recombinant strains. Based on the adopted strategy, we identified novel secretory tags, characterized their secretory capacity, indicated the most potent SPs, and suggested a consensus sequence of a potentially robust synthetic SP to expand the molecular toolbox for engineering Y. lipolytica.

Bioconversion of R-(+)-limonene to perillic acid by the yeast Yarrowia lipolytica

PubMed Central

Ferrara, Maria Antonieta; Almeida, Débora S.; Siani, Antonio C.; Lucchetti, Leonardo; Lacerda, Paulo S.B.; Freitas, André; Tappin, Marcelo R.R.; Bon, Elba P.S.

2013-01-01

Perillyl derivatives are increasingly important due to their flavouring and antimicrobial properties as well as their potential as anticancer agents. These terpenoid species, which are present in limited amounts in plants, may be obtained via bioconversion of selected monoterpene hydrocarbons. In this study, seventeen yeast strains were screened for their ability to oxidize the exocyclic methyl group in the p-menthene moiety of limonene into perillic acid. Of the yeast tested, the highest efficiency was observed for Yarrowia lipolytica ATCC 18942. The conversion of R (+)-limonene by Y. lipolytica was evaluated by varying the pH (3 to 8) and the temperature (25 to 30 °C) in a reaction medium containing 0.5% v/v limonene and 10 g/L of stationary phase cells (dry weight). The best results, corresponding to 564 mg/L of perillic acid, were obtained in buffered medium at pH 7.1 that was incubated at 25 °C for 48 h. The stepwise addition of limonene increased the perillic acid concentration by over 50%, reaching 855 mg/L, whereas the addition of glucose or surfactant to the reaction medium did not improve the bioconversion process. The use of Y. lipolytica showed promise for ease of further downstream processing, as perillic acid was the sole oxidised product of the bioconversion reaction. Moreover, bioprocesses using safe and easy to cultivate yeast cells have been favoured in industry. PMID:24688495

Engineering Yarrowia lipolytica as a platform for synthesis of drop-in transportation fuels and oleochemicals

PubMed Central

Xu, Peng; Qiao, Kangjian; Ahn, Woo Suk; Stephanopoulos, Gregory

2016-01-01

Harnessing lipogenic pathways and rewiring acyl-CoA and acyl-ACP (acyl carrier protein) metabolism in Yarrowia lipolytica hold great potential for cost-efficient production of diesel, gasoline-like fuels, and oleochemicals. Here we assessed various pathway engineering strategies in Y. lipolytica toward developing a yeast biorefinery platform for sustainable production of fuel-like molecules and oleochemicals. Specifically, acyl-CoA/acyl-ACP processing enzymes were targeted to the cytoplasm, peroxisome, or endoplasmic reticulum to generate fatty acid ethyl esters and fatty alkanes with tailored chain length. Activation of endogenous free fatty acids and the subsequent reduction of fatty acyl-CoAs enabled the efficient synthesis of fatty alcohols. Engineering a hybrid fatty acid synthase shifted the free fatty acids to a medium chain-length scale. Manipulation of alternative cytosolic acetyl-CoA pathways partially decoupled lipogenesis from nitrogen starvation and unleashed the lipogenic potential of Y. lipolytica. Taken together, the strategies reported here represent promising steps to develop a yeast biorefinery platform that potentially upgrades low-value carbons to high-value fuels and oleochemicals in a sustainable and environmentally friendly manner. PMID:27621436

Yarrowia lipolytica possesses two plasma membrane alkali metal cation/H+ antiporters with different functions in cell physiology.

PubMed

Papouskova, Klara; Sychrova, Hana

2006-04-03

The family of Nha antiporters mediating the efflux of alkali metal cations in exchange for protons across the plasma membrane is conserved in all yeast species. Yarrowia lipolytica is a dimorphic yeast, phylogenetically very distant from the model yeast Saccharomyces cerevisiae. A search in its sequenced genome revealed two genes (designated as YlNHA1 and YlNHA2) with homology to the S. cerevisiae NHA1 gene, which encodes a plasma membrane alkali metal cation/H+ antiporter. Upon heterologous expression of both YlNHA genes in S. cerevisiae, we showed that Y. lipolytica antiporters differ not only in length and sequence, but also in their affinity for individual substrates. While the YlNha1 protein mainly increased cell tolerance to potassium, YlNha2p displayed a remarkable transport capacity for sodium. Thus, Y. lipolytica is the first example of a yeast species with two plasma membrane alkali metal cation/H+ antiporters differing in their putative functions in cell physiology; cell detoxification vs. the maintenance of stable intracellular pH, potassium content and cell volume.

Steroid biotransformations in biphasic systems with Yarrowia lipolytica expressing human liver cytochrome P450 genes

PubMed Central

2012-01-01

Background Yarrowia lipolytica efficiently metabolizes and assimilates hydrophobic compounds such as n-alkanes and fatty acids. Efficient substrate uptake is enabled by naturally secreted emulsifiers and a modified cell surface hydrophobicity and protrusions formed by this yeast. We were examining the potential of recombinant Y. lipolytica as a biocatalyst for the oxidation of hardly soluble hydrophobic steroids. Furthermore, two-liquid biphasic culture systems were evaluated to increase substrate availability. While cells, together with water soluble nutrients, are maintained in the aqueous phase, substrates and most of the products are contained in a second water-immiscible organic solvent phase. Results For the first time we have co-expressed the human cytochromes P450 2D6 and 3A4 genes in Y. lipolytica together with human cytochrome P450 reductase (hCPR) or Y. lipolytica cytochrome P450 reductase (YlCPR). These whole-cell biocatalysts were used for the conversion of poorly soluble steroids in biphasic systems. Employing a biphasic system with the organic solvent and Y. lipolytica carbon source ethyl oleate for the whole-cell bioconversion of progesterone, the initial specific hydroxylation rate in a 1.5 L stirred tank bioreactor was further increased 2-fold. Furthermore, the product formation was significantly prolonged as compared to the aqueous system. Co-expression of the human CPR gene led to a 4-10-fold higher specific activity, compared to the co-overexpression of the native Y. lipolytica CPR gene. Multicopy transformants showed a 50-70-fold increase of activity as compared to single copy strains. Conclusions Alkane-assimilating yeast Y. lipolytica, coupled with the described expression strategies, demonstrated its high potential for biotransformations of hydrophobic substrates in two-liquid biphasic systems. Especially organic solvents which can be efficiently taken up and/or metabolized by the cell might enable more efficient bioconversion as compared

Enhanced α-ketoglutaric acid production and recovery in Yarrowia lipolytica yeast by effective pH controlling.

PubMed

Morgunov, Igor G; Kamzolova, Svetlana V; Samoilenko, Vladimir A

2013-10-01

The replacement of chemical synthesis by environmentally friendly energy-efficient technologies for production of valuable metabolites is a principal strategy of developing biotechnological industry all over the world. In the present study, we develop a method for α-ketoglutaric acid (KGA) production from rapeseed oil with the use of Yarrowia lipolytica yeast. Sixty strains of Y. lipolytica yeasts were tested for their ability to produce KGA, and the strain Y. lipolytica 212 (Y. lipolytica VKM Y-2412) was selected as a promising KGA producer. Using a three-stage pH controlling, in which pH was 4.5 in the growth phase, then since 72 to 144 h, pH was maintained at 3.5 and in the later phase of acid production, the titration by KOH was switch off, selected strain produced 106.5 g l(-1) of KGA with mass yield of 0.95 g g(-1). KGA in the form of monopotassium salt was isolated from the culture broth and purified. The isolation procedure involved separation of biomass, extraction of residual triglycerides, filtrate bleaching, and acidification with mineral acid (to pH 2.8-3.4), concentration, precipitation of mineral salts, and crystallization of the product. The purity of KGA isolated from the culture filtrate reached 99.1 %.

Development of recombinant Yarrowia lipolytica producing virus-like particles of a fish nervous necrosis virus.

PubMed

Luu, Van-Trinh; Moon, Hye Yun; Hwang, Jee Youn; Kang, Bo-Kyu; Kang, Hyun Ah

2017-08-01

Nervous necrosis virus (NNV) causes viral encephalopathy and retinopathy, a devastating disease of many species of cultured marine fish worldwide. In this study, we used the dimorphic non-pathogenic yeast Yarrowia lipolytica as a host to express the capsid protein of red-spotted grouper nervous necrosis virus (RGNNV-CP) and evaluated its potential as a platform for vaccine production. An initial attempt was made to express the codon-optimized synthetic genes encoding intact and N-terminal truncated forms of RGNNV-CP under the strong constitutive TEF1 promoter using autonomously replicating sequence (ARS)-based vectors. The full-length recombinant capsid proteins expressed in Y. lipolytica were detected not only as monomers and but also as trimers, which is a basic unit for formation of NNV virus-like particles (VLPs). Oral immunization of mice with whole recombinant Y. lipolytica harboring the ARS-based plasmids was shown to efficiently induce the formation of IgG against RGNNV-CP. To increase the number of integrated copies of the RGNNV-CP expression cassette, a set of 26S ribosomal DNA-based multiple integrative vectors was constructed in combination with a series of defective Ylura3 with truncated promoters as selection markers, resulting in integrants harboring up to eight copies of the RGNNV-CP cassette. Sucrose gradient centrifugation and transmission electron microscopy of this high-copy integrant were carried out to confirm the expression of RGNNV-CPs as VLPs. This is the first report on efficient expression of viral capsid proteins as VLPs in Y. lipolytica, demonstrating high potential for the Y. lipolytica expression system as a platform for recombinant vaccine production based on VLPs.

The ‘LipoYeasts’ project: using the oleaginous yeast Yarrowia lipolytica in combination with specific bacterial genes for the bioconversion of lipids, fats and oils into high‐value products

PubMed Central

Sabirova, Julia S.; Haddouche, R.; Van Bogaert, I. N.; Mulaa, F.; Verstraete, W.; Timmis, K. N.; Schmidt‐Dannert, C.; Nicaud, J. M.; Soetaert, W.

2011-01-01

Summary The oleochemical industry is currently still dominated by conventional chemistry, with biotechnology only starting to play a more prominent role, primarily with respect to the biosurfactants or lipases, e.g. as detergents, or for biofuel production. A major bottleneck for all further biotechnological applications is the problem of the initial mobilization of cheap and vastly available lipid and oil substrates, which are then to be transformed into high‐value biotechnological, nutritional or pharmacological products. Under the EU‐sponsored LipoYeasts project we are developing the oleaginous yeast Yarrowia lipolytica into a versatile and high‐throughput microbial factory that, by use of specific enzymatic pathways from hydrocarbonoclastic bacteria, efficiently mobilizes lipids by directing its versatile lipid metabolism towards the production of industrially valuable lipid‐derived compounds like wax esters (WE), isoprenoid‐derived compounds (carotenoids, polyenic carotenoid ester), polyhydroxyalkanoates (PHAs) and free hydroxylated fatty acids (HFAs). Different lipid stocks (petroleum, alkane, vegetable oil, fatty acid) and combinations thereof are being assessed as substrates in combination with different mutant and recombinant strains of Y. lipolytica, in order to modulate the composition and yields of the produced added‐value products. PMID:21255371

Investigating Proteome and Transcriptome Defense Response of Apples Induced by Yarrowia lipolytica.

PubMed

Zhang, Hongyin; Chen, Liangliang; Sun, Yiwen; Zhao, Lina; Zheng, Xiangfeng; Yang, Qiya; Zhang, Xiaoyun

2017-04-01

A better understanding of the mode of action of postharvest biocontrol agents on fruit surfaces is critical for the advancement of successful implementation of postharvest biocontrol products. This is due to the increasing importance of biological control of postharvest diseases over chemical and other control methods. However, most of the mechanisms involved in biological control remain unknown and need to be explored. Yarrowia lipolytica significantly inhibited blue mold decay of apples caused by Penicillium expansum. The findings also demonstrated that Y. lipolytica stimulated the activities of polyphenoloxidase, peroxidase, chitinase, l-phenylalanine ammonia lyase involved in enhancing defense responses in apple fruit tissue. Proteomic and transcriptomic analysis revealed a total of 35 proteins identified as up- and down-regulated in response to the Y. lipolytica inducement. These proteins were related to defense, biotic stimulus, and stress responses, such as pathogenesis-related proteins and dehydrin. The analysis of the transcriptome results proved that the induced resistance was mediated by a crosstalk between salicylic acid (SA) and ethylene/jasmonate (ET/JA) pathways. Y. lipolytica treatment activated the expression of isochorismate synthase gene in the SA pathway, which up-regulates the expression of PR4 in apple. The expression of 1-aminocyclopropane-1-carboxylate oxidase gene and ET-responsive transcription factors 2 and 4, which are involved in the ET pathway, were also activated. In addition, cytochrome oxidase I, which plays an important role in JA signaling for resistance acquisition, was also activated. However, not all of the genes had a positive effect on the SA and ET/JA signal pathways. As transcriptional repressors in JA signaling, TIFY3B and TIFY11B were triggered by the yeast, but the gene expression levels were relatively low. Taken together, Y. lipolytica induced the SA and ET/JA signal mediating the defense pathways by stimulating

Optimized invertase expression and secretion cassette for improving Yarrowia lipolytica growth on sucrose for industrial applications.

PubMed

Lazar, Zbigniew; Rossignol, Tristan; Verbeke, Jonathan; Crutz-Le Coq, Anne-Marie; Nicaud, Jean-Marc; Robak, Małgorzata

2013-11-01

Yarrowia lipolytica requires the expression of a heterologous invertase to grow on a sucrose-based substrate. This work reports the construction of an optimized invertase expression cassette composed of Saccharomyces cerevisiae Suc2p secretion signal sequence followed by the SUC2 sequence and under the control of the strong Y. lipolytica pTEF promoter. This new construction allows a fast and optimal cleavage of sucrose into glucose and fructose and allows cells to reach the maximum growth rate. Contrary to pre-existing constructions, the expression of SUC2 is not sensitive to medium composition in this context. The strain JMY2593, expressing this new cassette with an optimized secretion signal sequence and a strong promoter, produces 4,519 U/l of extracellular invertase in bioreactor experiments compared to 597 U/l in a strain expressing the former invertase construction. The expression of this cassette strongly improved production of invertase and is suitable for simultaneously high production level of citric acid from sucrose-based media.

A Rac Homolog Is Required for Induction of Hyphal Growth in the Dimorphic Yeast Yarrowia lipolytica

PubMed Central

Hurtado, Cleofe A. R.; Beckerich, Jean-Marie; Gaillardin, Claude; Rachubinski, Richard A.

2000-01-01

Dimorphism in fungi is believed to constitute a mechanism of response to adverse conditions and represents an important attribute for the development of virulence by a number of pathogenic fungal species. We have isolated YlRAC1, a gene encoding a 192-amino-acid protein that is essential for hyphal growth in the dimorphic yeast Yarrowia lipolytica and which represents the first Rac homolog described for fungi. YlRAC1 is not an essential gene, and its deletion does not affect the ability to mate or impair actin polarization in Y. lipolytica. However, strains lacking functional YlRAC1 show alterations in cell morphology, suggesting that the function of YlRAC1 may be related to some aspect of the polarization of cell growth. Northern blot analysis showed that transcription of YlRAC1 increases steadily during the yeast-to-hypha transition, while Southern blot analysis of genomic DNA suggested the presence of several RAC family members in Y. lipolytica. Interestingly, strains lacking functional YlRAC1 are still able to grow as the pseudohyphal form and to invade agar, thus pointing to a function for YlRAC1 downstream of MHY1, a previously isolated gene encoding a C2H2-type zinc finger protein with the ability to bind putative stress response elements and whose activity is essential for both hyphal and pseudohyphal growth in Y. lipolytica. PMID:10762235

Unraveling fatty acid transport and activation mechanisms in Yarrowia lipolytica.

PubMed

Dulermo, Rémi; Gamboa-Meléndez, Heber; Ledesma-Amaro, Rodrigo; Thévenieau, France; Nicaud, Jean-Marc

2015-09-01

Fatty acid (FA) transport and activation have been extensively studied in the model yeast species Saccharomyces cerevisiae but have rarely been examined in oleaginous yeasts, such as Yarrowia lipolytica. Because the latter begins to be used in biodiesel production, understanding its FA transport and activation mechanisms is essential. We found that Y. lipolytica has FA transport and activation proteins similar to those of S. cerevisiae (Faa1p, Pxa1p, Pxa2p, Ant1p) but mechanism of FA peroxisomal transport and activation differs greatly with that of S. cerevisiae. While the ScPxa1p/ScPxa2p heterodimer is essential for growth on long-chain FAs, ΔYlpxa1 ΔYlpxa2 is not impaired for growth on FAs. Meanwhile, ScAnt1p and YlAnt1p are both essential for yeast growth on medium-chain FAs, suggesting they function similarly. Interestingly, we found that the ΔYlpxa1 ΔYlpxa2 ΔYlant1 mutant was unable to grow on short-, medium-, or long-chain FAs, suggesting that YlPxa1p, YlPxa2p, and YlAnt1p belong to two different FA degradation pathways. We also found that YlFaa1p is involved in FA storage in lipid bodies and that FA remobilization largely depended on YlFat1p, YlPxa1p and YlPxa2p. This study is the first to comprehensively examine FA intracellular transport and activation in oleaginous yeast. Copyright © 2015. Published by Elsevier B.V.

Biomass production by novel strains of Yarrowia lipolytica using raw glycerol, derived from biodiesel production.

PubMed

Juszczyk, Piotr; Tomaszewska, Ludwika; Kita, Agnieszka; Rymowicz, Waldemar

2013-06-01

This study demonstrated the potential applicability of the isolated strains of Yarrowia lipolytica for the valorization of glycerol waste generated during biodiesel production, throughout biomass production. Twenty-one strains were isolated from different environments and identified as Y. lipolytica. Biomass production from pure glycerol (25 g L(-1)) was performed in the shake-flasks experiment. Eight strains with the best biomass production ability were chosen for studies in bioreactor (pH 3.5). The analysis of technological process parameters and biomass chemical composition demonstrated that S6 strain was the most suitable for biomass production. Its application allowed obtaining 11.7 and 12.3 g L(-1) of the biomass with 1.30 and 1.37 g L(-1) h(-1) productivity, respectively when pure and raw glycerol (25 g L(-1)) was used. In the yeast protein amino acid profile the contents of lysine, threonine and phenylalanine/tyrosine were higher than required by FAO/WHO. According to the EAAI, the nutritional value of the biomass reached up to 72.3%. Copyright © 2013 Elsevier Ltd. All rights reserved.

Sugar versus fat: elimination of glycogen storage improves lipid accumulation in Yarrowia lipolytica.

PubMed

Bhutada, Govindprasad; Kavšcek, Martin; Ledesma-Amaro, Rodrigo; Thomas, Stéphane; Rechberger, Gerald N; Nicaud, Jean-Marc; Natter, Klaus

2017-05-01

Triacylglycerol (TAG) and glycogen are the two major metabolites for carbon storage in most eukaryotic organisms. We investigated the glycogen metabolism of the oleaginous Yarrowia lipolytica and found that this yeast accumulates up to 16% glycogen in its biomass. Assuming that elimination of glycogen synthesis would result in an improvement of lipid accumulation, we characterized and deleted the single gene coding for glycogen synthase, YlGSY1. The mutant was grown under lipogenic conditions with glucose and glycerol as substrates and we obtained up to 60% improvement in TAG accumulation compared to the wild-type strain. Additionally, YlGSY1 was deleted in a background that was already engineered for high lipid accumulation. In this obese background, TAG accumulation was also further increased. The highest lipid content of 52% was found after 3 days of cultivation in nitrogen-limited glycerol medium. Furthermore, we constructed mutants of Y. lipolytica and Saccharomyces cerevisiae that are deleted for both glycogen and TAG synthesis, demonstrating that the ability to store carbon is not essential. Overall, this work showed that glycogen synthesis is a competing pathway for TAG accumulation in oleaginous yeasts and that deletion of the glycogen synthase has beneficial effects on neutral lipid storage. © FEMS 2017.

Functional analysis of recombinant human and Yarrowia lipolytica O-GlcNAc transferases expressed in Saccharomyces cerevisiae.

PubMed

Oh, Hye Ji; Moon, Hye Yun; Cheon, Seon Ah; Hahn, Yoonsoo; Kang, Hyun Ah

2016-10-01

O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is an important post-translational modification in many cellular processes. It is mediated by O-GlcNAc transferases (OGTs), which catalyze the addition of O-GlcNAc to serine or threonine residues of the target proteins. In this study, we expressed a putative Yarrowia lipolytica OGT (YlOGT), the only homolog identified in the subphylum Saccharomycotina through bioinformatics analysis, and the human OGT (hOGT) as recombinant proteins in Saccharomyces cerevisiae, and performed their functional characterization. Immunoblotting assays using antibody against O-GlcNAc revealed that recombinant hOGT (rhOGT), but not the recombinant YlOGT (rYlOGT), undergoes auto-O-GlcNAcylation in the heterologous host S. cerevisiae. Moreover, the rhOGT expressed in S. cerevisiae showed a catalytic activity during in vitro assays using casein kinase II substrates, whereas no such activity was obtained in rYlOGT. However, the chimeric human-Y. lipolytica OGT, carrying the human tetratricopeptide repeat (TPR) domain along with the Y. lipolytica catalytic domain (CTD), mediated the transfer of O-GlcNAc moiety during the in vitro assays. Although the overexpression of full-length OGTs inhibited the growth of S. cerevisiae, no such inhibition was obtained upon overexpression of only the CTD fragment, indicating the role of TPR domain in growth inhibition. This is the first report on the functional analysis of the fungal OGT, indicating that the Y. lipolytica OGT retains its catalytic activity, although the physiological role and substrates of YlOGT remain to be elucidated.

Multi-omics analysis reveals regulators of the response to nitrogen limitation in Yarrowia lipolytica.

PubMed

Pomraning, Kyle R; Kim, Young-Mo; Nicora, Carrie D; Chu, Rosalie K; Bredeweg, Erin L; Purvine, Samuel O; Hu, Dehong; Metz, Thomas O; Baker, Scott E

2016-02-25

Yarrowia lipolytica is an oleaginous ascomycete yeast that stores lipids in response to limitation of nitrogen. While the enzymatic pathways responsible for neutral lipid accumulation in Y. lipolytica are well characterized, regulation of these pathways has received little attention. We therefore sought to characterize the response to nitrogen limitation at system-wide levels, including the proteome, phosphoproteome and metabolome, to better understand how this organism regulates and controls lipid metabolism and to identify targets that may be manipulated to improve lipid yield. We found that ribosome structural genes are down-regulated under nitrogen limitation, during which nitrogen containing compounds (alanine, putrescine, spermidine and urea) are depleted and sugar alcohols and TCA cycle intermediates accumulate (citrate, fumarate and malate). We identified 1219 novel phosphorylation sites in Y. lipolytica, 133 of which change in their abundance during nitrogen limitation. Regulatory proteins, including kinases and DNA binding proteins, are particularly enriched for phosphorylation. Within lipid synthesis pathways, we found that ATP-citrate lyase, acetyl-CoA carboxylase and lecithin cholesterol acyl transferase are phosphorylated during nitrogen limitation while many of the proteins involved in β-oxidation are down-regulated, suggesting that storage lipid accumulation may be regulated by phosphorylation of key enzymes. Further, we identified short DNA elements that associate specific transcription factor families with up- and down-regulated genes. Integration of metabolome, proteome and phosphoproteome data identifies lipid accumulation in response to nitrogen limitation as a two-fold result of increased production of acetyl-CoA from excess citrate and decreased capacity for β-oxidation.

Study of trans-trans farnesol effect on hyphae formation by Yarrowia lipolytica.

PubMed

Nunes, Patrícia Martins Botelho; da Rocha, Silvia Maria; Amaral, Priscilla Filomena Fonseca; da Rocha-Leão, Maria Helena Miguez

2013-12-01

Dimorphism is an ability of certain fungi related to its adaptation to the environment and provides a selective advantage under stress conditions and is associated to the development of human diseases. Hyphae inducing- and inhibitory-effect of farnesol on hyphae formation by the dimorphic yeast Yarrowia lipolytica was evaluated through digital image analysis. The agitation speed of the culture was the most effective hyphae inducer in comparison to bovine calf serum and N-acetylglucosamine. In low agitation system, bovine calf serum was more effective for hyphae formation inducing 57 % of hyphae transition. Farnesol inhibited hyphae formation even in low concentration (300 μM) and this effect increased with increasing concentrations. In the presence of N-acetylglucosamine, this effect was more evident in comparison to the presence of bovine calf serum, which might have protected the cells from farnesol. Digital image analysis was an important tool to evaluate this phenomenon.

New Insights into Sulfur Metabolism in Yeasts as Revealed by Studies of Yarrowia lipolytica

PubMed Central

Hébert, Agnès; Forquin-Gomez, Marie-Pierre; Roux, Aurélie; Aubert, Julie; Junot, Christophe; Heilier, Jean-François; Landaud, Sophie; Bonnarme, Pascal

2013-01-01

Yarrowia lipolytica, located at the frontier of hemiascomycetous yeasts and fungi, is an excellent candidate for studies of metabolism evolution. This yeast, widely recognized for its technological applications, in particular produces volatile sulfur compounds (VSCs) that fully contribute to the flavor of smear cheese. We report here a relevant global vision of sulfur metabolism in Y. lipolytica based on a comparison between high- and low-sulfur source supplies (sulfate, methionine, or cystine) by combined approaches (transcriptomics, metabolite profiling, and VSC analysis). The strongest repression of the sulfate assimilation pathway was observed in the case of high methionine supply, together with a large accumulation of sulfur intermediates. A high sulfate supply seems to provoke considerable cellular stress via sulfite production, resulting in a decrease of the availability of the glutathione pathway's sulfur intermediates. The most limited effect was observed for the cystine supply, suggesting that the intracellular cysteine level is more controlled than that of methionine and sulfate. Using a combination of metabolomic profiling and genetic experiments, we revealed taurine and hypotaurine metabolism in yeast for the first time. On the basis of a phylogenetic study, we then demonstrated that this pathway was lost by some of the hemiascomycetous yeasts during evolution. PMID:23220962

The expression of the Cuphea palustris thioesterase CpFatB2 in Yarrowia lipolytica triggers oleic acid accumulation.

PubMed

Stefan, Alessandra; Hochkoeppler, Alejandro; Ugolini, Luisa; Lazzeri, Luca; Conte, Emanuele

2016-01-01

The conversion of industrial by-products into high-value added compounds is a challenging issue. Crude glycerol, a by-product of the biodiesel production chain, could represent an alternative carbon source for the cultivation of oleaginous yeasts. Here, we developed five minimal synthetic glycerol-based media, with different C/N ratios, and we analyzed the production of biomass and fatty acids by Yarrowia lipolytica Po1g strain. We identified two media at the expense of which Y. lipolytica was able to accumulate ∼5 g L(-1) of biomass and 0.8 g L(-1) of fatty acids (0.16 g of fatty acids per g of dry weight). These optimized media contained 0.5 g L(-1) of urea or ammonium sulfate and 20 g L(-1) of glycerol, and were devoid of yeast extract. Moreover, Y. lipolytica was engineered by inserting the FatB2 gene, coding for the CpFatB2 thioesterase from Cuphea palustris, in order to modify the fatty acid composition towards the accumulation of medium-chain fatty acids. Contrary to the expected, the expression of the heterologous gene increased the production of oleic acid, and concomitantly decreased the level of saturated fatty acids. © 2015 American Institute of Chemical Engineers.

Substrates and oxygen dependent citric acid production by Yarrowia lipolytica: insights through transcriptome and fluxome analyses.

PubMed

Sabra, Wael; Bommareddy, Rajesh Reddy; Maheshwari, Garima; Papanikolaou, Seraphim; Zeng, An-Ping

2017-05-08

Unlike the well-studied backer yeast where catabolite repression represents a burden for mixed substrate fermentation, Yarrowia lipolytica, an oleaginous yeast, is recognized for its potential to produce single cell oils and citric acid from different feedstocks. These versatilities of Y. lipolytica with regards to substrate utilization make it an attractive host for biorefinery application. However, to develop a commercial process for the production of citric acid by Y. lipolytica, it is necessary to better understand the primary metabolism and its regulation, especially for growth on mixed substrate. Controlling the dissolved oxygen concentration (pO 2 ) in Y. lipolytica cultures enhanced citric acid production significantly in cultures grown on glucose in mono- or dual substrate fermentations, whereas with glycerol as mono-substrate no significant effect of pO 2 was found on citrate production. Growth on mixed substrate with glucose and glycerol revealed a relative preference of glycerol utilization by Y. lipolytica. Under optimized conditions with pO 2 control, the citric acid titer on glucose in mono- or in dual substrate cultures was 55 and 50 g/L (with productivity of 0.6 g/L*h in both cultures), respectively, compared to a maximum of 18 g/L (0.2 g/L*h) with glycerol in monosubstrate culture. Additionally, in dual substrate fermentation, glycerol limitation was found to trigger citrate consumption despite the presence of enough glucose in pO 2 -limited culture. The metabolic behavior of this yeast on different substrates was investigated at transcriptomic and 13 C-based fluxomics levels. Upregulation of most of the genes of the pentose phosphate pathway was found in cultures with highest citrate production with glucose in mono- or in dual substrate fermentation with pO 2 control. The activation of the glyoxylate cycle in the oxygen limited cultures and the imbalance caused by glycerol limitation might be the reason for the re-consumption of citrate in

Citric acid production from hydrolysate of pretreated straw cellulose by Yarrowia lipolytica SWJ-1b using batch and fed-batch cultivation.

PubMed

Liu, Xiaoyan; Lv, Jinshun; Zhang, Tong; Deng, Yuanfang

2015-01-01

In this study, crude cellulase produced by Trichoderma reesei Rut-30 was used to hydrolyze pretreated straw. After the compositions of the hydrolysate of pretreated straw were optimized, the study showed that natural components of pretreated straw without addition of any other components such as (NH4)2SO4, KH2PO4, or Mg(2+) were suitable for citric acid production by Yarrowia lipolytica SWJ-1b, and the optimal ventilatory capacity was 10.0 L/min/L medium. Batch and fed-batch production of citric acid from the hydrolysate of pretreated straw by Yarrowia lipolytica SWJ-1b has been investigated. In the batch cultivation, 25.4 g/L and 26.7 g/L citric acid were yields from glucose and hydrolysate of straw cellulose, respectively, while the cultivation time was 120 hr. In the three-cycle fed-batch cultivation, citric acid (CA) production was increased to 42.4 g/L and the cultivation time was extended to 240 hr. However, iso-citric acid (ICA) yield in fed-batch cultivation (4.0 g/L) was similar to that during the batch cultivation (3.9 g/L), and only 1.6 g/L of reducing sugar was left in the medium at the end of fed-batch cultivation, suggesting that most of the added carbon was used in the cultivation.

Dual CRISPR-Cas9 Cleavage Mediated Gene Excision and Targeted Integration in Yarrowia lipolytica.

PubMed

Gao, Difeng; Smith, Spencer; Spagnuolo, Michael; Rodriguez, Gabriel; Blenner, Mark

2018-05-29

CRISPR-Cas9 technology has been successfully applied in Yarrowia lipolytica for targeted genomic editing including gene disruption and integration; however, disruptions by existing methods typically result from small frameshift mutations caused by indels within the coding region, which usually resulted in unnatural protein. In this study, a dual cleavage strategy directed by paired sgRNAs is developed for gene knockout. This method allows fast and robust gene excision, demonstrated on six genes of interest. The targeted regions for excision vary in length from 0.3 kb up to 3.5 kb and contain both non-coding and coding regions. The majority of the gene excisions are repaired by perfect nonhomologous end-joining without indel. Based on this dual cleavage system, two targeted markerless integration methods are developed by providing repair templates. While both strategies are effective, homology mediated end joining (HMEJ) based method are twice as efficient as homology recombination (HR) based method. In both cases, dual cleavage leads to similar or improved gene integration efficiencies compared to gene excision without integration. This dual cleavage strategy will be useful for not only generating more predictable and robust gene knockout, but also for efficient targeted markerless integration, and simultaneous knockout and integration in Y. lipolytica. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Omega-3 production by fermentation of Yarrowia lipolytica: From fed-batch to continuous.

PubMed

Xie, Dongming; Miller, Edward; Sharpe, Pamela; Jackson, Ethel; Zhu, Quinn

2017-04-01

The omega-3 fatty acid, cis-5,8,11,14,17-eicosapentaenoic acid (C20:5; EPA) has wide-ranging benefits in improving heart health, immune function, and mental health. A sustainable source of EPA production through fermentation of metabolically engineered Yarrowia lipolytica has been developed. In this paper, key fed-batch fermentation conditions were identified to achieve 25% EPA in the yeast biomass, which is so far the highest EPA titer reported in the literature. Dynamic models of the EPA fermentation process were established for analyzing, optimizing, and scaling up the fermentation process. In addition, model simulations were used to develop a two-stage continuous process and compare to single-stage continuous and fed- batch processes. The two stage continuous process, which is equipped with a smaller growth fermentor (Stage 1) and a larger production fermentor (Stage 2), was found to be a superior process to achieve high titer, rate, and yield of EPA. A two-stage continuous fermentation experiment with Y. lipolytica strain Z7334 was designed using the model simulation and then tested in a 2 L and 5 L fermentation system for 1,008 h. Compared with the standard 2 L fed-batch process, the two-stage continuous fermentation process improved the overall EPA productivity by 80% and EPA concentration in the fermenter by 40% while achieving comparable EPA titer in biomass and similar conversion yield from glucose. During the long-term experiment it was also found that the Y. lipolytica strain evolved to reduce byproduct and increase lipid production. This is one of the few continuous fermentation examples that demonstrated improved productivity and concentration of a final product with similar conversion yield compared with a fed-batch process. This paper suggests the two-stage continuous fermentation could be an effective process to achieve improved production of omega-3 and other fermentation products where non-growth or partially growth associated kinetics

Three alcohol dehydrogenase genes and one acetyl-CoA synthetase gene are responsible for ethanol utilization in Yarrowia lipolytica.

PubMed

Gatter, Michael; Ottlik, Stephanie; Kövesi, Zsolt; Bauer, Benjamin; Matthäus, Falk; Barth, Gerold

2016-10-01

The non-conventional yeast Yarrowia lipolytica is able to utilize a wide range of different substrates like glucose, glycerol, ethanol, acetate, proteins and various hydrophobic molecules. Although most metabolic pathways for the utilization of these substrates have been clarified by now, it was not clear whether ethanol is oxidized by alcohol dehydrogenases or by an alternative oxidation system inside the cell. In order to detect the genes that are required for ethanol utilization in Y. lipolytica, eight alcohol dehydrogenase (ADH) genes and one alcohol oxidase gene (FAO1) have been identified and respective deletion strains were tested for their ability to metabolize ethanol. As a result of this, we found that the availability of ADH1, ADH2 or ADH3 is required for ethanol utilization in Y. lipolytica. A strain with deletions in all three genes is lacking the ability to utilize ethanol as sole carbon source. Although Adh2p showed by far the highest enzyme activity in an in vitro assay, the availability of any of the three genes was sufficient to enable a decent growth. In addition to ADH1, ADH2 and ADH3, an acetyl-CoA synthetase encoding gene (ACS1) was found to be essential for ethanol utilization. As Y. lipolytica is a non-fermenting yeast, it is neither able to grow under anaerobic conditions nor to produce ethanol. To investigate whether Y. lipolytica may produce ethanol, the key genes of alcoholic fermentation in S. cerevisiae, ScADH1 and ScPDC1, were overexpressed in an ADH and an ACS1 deletion strain. However, instead of producing ethanol, the respective strains regained the ability to use ethanol as single carbon source and were still not able to grow under anaerobic conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

Characterization of phosphatidic acid phosphatase activity in the oleaginous yeast Yarrowia lipolytica and its role in lipid biosynthesis.

PubMed

Hardman, Derell; McFalls, Daniel; Fakas, Stylianos

2017-02-01

Phosphatidic acid phosphatase (PAP) catalyses the committed step of triacylglycerol (TAG) biosynthesis and thus regulates the amounts of TAG produced by the cell. TAG is the target of biotechnological processes developed for the production of food lipids or biofuels. These processes are using oleaginous microorganisms like the yeast Yarrowia lipolytica as the TAG producers. Thus manipulating key enzymatic activities like PAP in Y. lipolytica could drive lipid biosynthesis towards TAG production and increase TAG yields. In this study, PAP activity in Y. lipolytica was characterized in detail and its role in lipid biosynthesis was addressed. PAP activity increased 2.5-fold with the addition of Mg 2+ (1 mm) in the assay mixture, which means that most of the PAP activity was due to Mg 2+ -dependent PAP enzymes (e.g. Pah1, App1). In contrast, N-ethylmaleimide (NEM) potently inhibited PAP activity, indicating the presence of NEM-sensitive PAP enzymes (e.g. App1, Lpp1). Localization studies revealed that the majority of PAP activity resides in the membrane fraction, while the cytosolic fraction harbours only a small amount of activity. PAP activity was regulated in a growth-dependent manner, being induced at the early exponential phase and declining thereafter. PAP activity did not correlate with TAG synthesis, which increased as cells progressed from the exponential phase to the early stationary phase. In stationary phase, TAG was mobilized with the concomitant synthesis of sterols and sterol esters. These results provide the first insights into the role of PAP in lipid biosynthesis by Y. lipolytica. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Citric acid production in Yarrowia lipolytica SWJ-1b yeast when grown on waste cooking oil.

PubMed

Liu, Xiaoyan; Lv, Jinshun; Xu, Jiaxing; Zhang, Tong; Deng, Yuanfang; He, Jianlong

2015-03-01

In this study, citric acid was produced from waste cooking oil by Yarrowia lipolytica SWJ-1b. To get the maximal yield of citric acid, the compositions of the medium for citric acid production were optimized, and our results showed that extra nitrogen and magnesium rather than vitamin B1 and phosphate were needed for CA accumulation when using waste cooking oil. The results also indicated that the optimal initial concentration of the waste cooking oil in the medium for citric acid production was 80.0 g/l, and the ideal inoculation size was 1 × 10(7) cells/l of medium. We also reported that during 10-l fermentation, 31.7 g/l of citric acid, 6.5 g/l of isocitric acid, 5.9 g/l of biomass, and 42.1 g/100.0 g cell dry weight of lipid were attained from 80.0 g/l of waste cooking oil within 336 h. At the end of the fermentation, 94.6 % of the waste cooking oil was utilized by the cells of Y. lipolytica SWJ-1b, and the yield of citric acid was 0.4 g/g waste cooking oil, which suggested that waste cooking oil was a suitable carbon resource for citric acid production.

The RAD52 ortholog of Yarrowia lipolytica is essential for nuclear integrity and DNA repair.

PubMed

Campos-Góngora, Eduardo; Andaluz, Encarnación; Bellido, Alberto; Ruiz-Herrera, José; Larriba, German

2013-08-01

Yarrowia lipolytica (Yl) is a dimorphic fungus that has become a well-established model for a number of biological processes, including secretion of heterologous and chimerical proteins. However, little is known on the recombination machinery responsible for the integration in the genome of the exogenous DNA encoding for those proteins. We have carried out a phenotypic analysis of rad52 deletants of Y. lipolytica. YlRad52 exhibited 20-30% identity with Rad52 homologues of other eukaryotes, including Saccharomyces cerevisiae and Candida albicans. Ylrad52-Δ strains formed colonies on YPD-agar plates which were spinier and smaller than those from wild type, whereas in YPD liquid cultures they exhibited a decreased grow rate and contained cells with aberrant morphology and fragmented chromatin, supporting a role for homologous recombination (HR) in genome stability under nondamaging conditions. In addition, Ylrad52 mutants showed moderate to high sensitivity to UV light, oxidizing agents and compounds that cause single- (SSB) and double-strand breaks (DSB), indicating an important role for Rad52 in DNA repair. These findings extend to Yl previous observations indicating that RAD52 is a crucial gene for DNA repair in other fungi, including S. cerevisiae, C. albicans and Schizosaccharomyces pombe. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Sustainable source of omega-3 eicosapentaenoic acid from metabolically engineered Yarrowia lipolytica: from fundamental research to commercial production.

PubMed

Xie, Dongming; Jackson, Ethel N; Zhu, Quinn

2015-02-01

The omega-3 fatty acids, cis-5, 8, 11, 14, and 17-eicosapentaenoic acid (C20:5; EPA) and cis-4, 7, 10, 13, 16, and 19-docosahexaenoic acid (C22:6; DHA), have wide-ranging benefits in improving heart health, immune function, mental health, and infant cognitive development. Currently, the major source for EPA and DHA is from fish oil, and a minor source of DHA is from microalgae. With the increased demand for EPA and DHA, DuPont has developed a clean and sustainable source of the omega-3 fatty acid EPA through fermentation using metabolically engineered strains of Yarrowia lipolytica. In this mini-review, we will focus on DuPont's technology for EPA production. Specifically, EPA biosynthetic and supporting pathways have been introduced into the oleaginous yeast to synthesize and accumulate EPA under fermentation conditions. This Yarrowia platform can also produce tailored omega-3 (EPA, DHA) and/or omega-6 (ARA, GLA) fatty acid mixtures in the cellular lipid profiles. Fundamental research such as metabolic engineering for strain construction, high-throughput screening for strain selection, fermentation process development, and process scale-up were all needed to achieve the high levels of EPA titer, rate, and yield required for commercial application. Here, we summarize how we have combined the fundamental bioscience and the industrial engineering skills to achieve large-scale production of Yarrowia biomass containing high amounts of EPA, which led to two commercial products, New Harvest™ EPA oil and Verlasso® salmon.

Influence of oxygen availability on the metabolism and morphology of Yarrowia lipolytica: insights into the impact of glucose levels on dimorphism.

PubMed

Timoumi, Asma; Bideaux, Carine; Guillouet, Stéphane E; Allouche, Yohan; Molina-Jouve, Carole; Fillaudeau, Luc; Gorret, Nathalie

2017-10-01

Dynamic behavior of Yarrowia lipolytica W29 strain under conditions of fluctuating, low, and limited oxygen supply was characterized in batch and glucose-limited chemostat cultures. In batch cultures, transient oscillations between oxygen-rich and -deprived environments induced a slight citric acid accumulation (lower than 29 mg L -1 ). By contrast, no citric acid was detected in continuous fermentations for all stress conditions: full anoxia (zero pO 2 value, 100% N 2 ), limited (zero pO 2 value, 75% of cell needs), and low (pO 2 close to 2%) dissolved oxygen (DO) levels. The macroscopic behavior (kinetic parameters, yields, viability) of Y. lipolytica was not significantly affected by the exposure to DO fluctuations under both modes of culture. Nevertheless, conditions of oxygen limitation resulted in the destabilization of the glucose-limited growth during the continuous cultivations. Morphological responses of Y. lipolytica to DO oscillations were different between batch and chemostat runs. Indeed, a yeast-to-mycelium transition was induced and progressively intensified during the batch fermentations (filamentous subpopulation reaching 74% (v/v)). While, in chemostat bioreactors, the culture consisted mainly of yeast-like cells (mean diameter not exceeding 5.7 μm) with a normal size distribution. During the continuous cultures, growth at low DO concentration did not induce any changes in Y. lipolytica morphology. Dimorphism (up to 80.5% (v/v) of filaments) was only detected under conditions of oxygen limitation in the presence of a residual glucose excess (more than 0.75 g L -1 ). These data suggest an impact of glucose levels on the signaling pathways regulating dimorphic responses in Y. lipolytica.

An evolutionary metabolic engineering approach for enhancing lipogenesis in Yarrowia lipolytica.

PubMed

Liu, Leqian; Pan, Anny; Spofford, Caitlin; Zhou, Nijia; Alper, Hal S

2015-05-01

Lipogenic organisms provide an ideal platform for biodiesel and oleochemical production. Through our previous rational metabolic engineering efforts, lipogenesis titers in Yarrowia lipolytica were significantly enhanced. However, the resulting strain still suffered from decreased biomass generation rates. Here, we employ a rapid evolutionary metabolic engineering approach linked with a floating cell enrichment process to improve lipogenesis rates, titers, and yields. Through this iterative process, we were able to ultimately improve yields from our prior strain by 55% to achieve production titers of 39.1g/L with upwards of 76% of the theoretical maximum yield of conversation. Isolated cells were saturated with up to 87% lipid content. An average specific productivity of 0.56g/L/h was achieved with a maximum instantaneous specific productivity of 0.89g/L/h during the lipid production phase in fermentation. Genomic sequencing of the evolved strains revealed a link between a decrease/loss of function mutation of succinate semialdehyde dehydrogenase, uga2, suggesting the importance of gamma-aminobutyric acid assimilation in lipogenesis. This linkage was validated through gene deletion experiments. This work presents an improved host strain that can serve as a platform for efficient oleochemical production. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Regulation of nitrogen metabolism by GATA zinc finger transcription factors in Yarrowia lipolytica

DOE PAGES

Pomraning, Kyle R.; Bredeweg, Erin L.; Baker, Scott E.; ...

2017-02-15

Here, fungi accumulate lipids in a manner dependent on the quantity and quality of the nitrogen source on which they are growing. In the oleaginous yeast Yarrowia lipolytica, growth on a complex source of nitrogen enables rapid growth and limited accumulation of neutral lipids, while growth on a simple nitrogen source promotes lipid accumulation in large lipid droplets. Here we examined the roles of nitrogen catabolite repression and its regulation by GATA zinc finger transcription factors on lipid metabolism in Y. lipolytica. Deletion of the GATA transcription factor genes gzf3 and gzf2 resulted in nitrogen source-specific growth defects and greatermore » accumulation of lipids when the cells were growing on a simple nitrogen source. Deletion of gzf1, which is most similar to activators of genes repressed by nitrogen catabolite repression in filamentous ascomycetes, did not affect growth on the nitrogen sources tested. We examined gene expression of wild-type and GATA transcription factor mutants on simple and complex nitrogen sources and found that expression of enzymes involved in malate metabolism, beta-oxidation, and ammonia utilization are strongly upregulated on a simple nitrogen source. Deletion of gzf3 results in overexpression of genes with GATAA sites in their promoters, suggesting that it acts as a repressor, while gzf2 is required for expression of ammonia utilization genes but does not grossly affect the transcription level of genes predicted to be controlled by nitrogen catabolite repression. Both GATA transcription factor mutants exhibit decreased expression of genes controlled by carbon catabolite repression via the repressor mig1, including genes for beta-oxidation, highlighting the complex interplay between regulation of carbon, nitrogen, and lipid metabolism.« less

Regulation of nitrogen metabolism by GATA zinc finger transcription factors in Yarrowia lipolytica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pomraning, Kyle R.; Bredeweg, Erin L.; Baker, Scott E.

Here, fungi accumulate lipids in a manner dependent on the quantity and quality of the nitrogen source on which they are growing. In the oleaginous yeast Yarrowia lipolytica, growth on a complex source of nitrogen enables rapid growth and limited accumulation of neutral lipids, while growth on a simple nitrogen source promotes lipid accumulation in large lipid droplets. Here we examined the roles of nitrogen catabolite repression and its regulation by GATA zinc finger transcription factors on lipid metabolism in Y. lipolytica. Deletion of the GATA transcription factor genes gzf3 and gzf2 resulted in nitrogen source-specific growth defects and greatermore » accumulation of lipids when the cells were growing on a simple nitrogen source. Deletion of gzf1, which is most similar to activators of genes repressed by nitrogen catabolite repression in filamentous ascomycetes, did not affect growth on the nitrogen sources tested. We examined gene expression of wild-type and GATA transcription factor mutants on simple and complex nitrogen sources and found that expression of enzymes involved in malate metabolism, beta-oxidation, and ammonia utilization are strongly upregulated on a simple nitrogen source. Deletion of gzf3 results in overexpression of genes with GATAA sites in their promoters, suggesting that it acts as a repressor, while gzf2 is required for expression of ammonia utilization genes but does not grossly affect the transcription level of genes predicted to be controlled by nitrogen catabolite repression. Both GATA transcription factor mutants exhibit decreased expression of genes controlled by carbon catabolite repression via the repressor mig1, including genes for beta-oxidation, highlighting the complex interplay between regulation of carbon, nitrogen, and lipid metabolism.« less

Production of Laccase by Recombinant Yarrowia lipolytica from Molasses: Bioprocess Development Using Statistical Modeling and Increase Productivity in Shake-Flask and Bioreactor Cultures.

PubMed

Darvishi, Farshad; Moradi, Marzieh; Madzak, Catherine; Jolivalt, Claude

2017-03-01

Laccases are used in numerous applications, from green degradation of various xenobiotic compounds, waste detoxification, textile dye bleaching, and delignification of lignocellulose materials to biofuel production. In this study, the recombinant Yarrowia lipolytica YL4 strain carrying the white-rot fungus Trametes versicolor laccase IIIb gene was used for laccase production from beet molasses as an agro-industrial residue. Response surface methodology was used to statistical optimization of the production of laccase by Y. lipolytica using an industrial medium containing molasses which allows a six times increase in laccase activity compared to primary medium contains glucose after 144 h. In bioreactor cultivation after 48 h, laccase production reached to 3.7- and 22.5-fold more than optimized and primary media in shake-flask cultures, respectively. Laccase productivity in bioreactor (0.0937 U/h) was higher than shake-flask culture (0.0084 U/h). The present study provides valuable information about statistical optimization of bioprocess development for cost-effective production of laccase and other heterologous proteins in Y. lipolytica from beet molasses as sole carbon source, thus allowing the valorization and decreasing environmental pollution of this agro-industrial waste.

The Strictly Aerobic Yeast Yarrowia lipolytica Tolerates Loss of a Mitochondrial DNA-Packaging Protein

PubMed Central

Bakkaiova, Jana; Arata, Kosuke; Matsunobu, Miki; Ono, Bungo; Aoki, Tomoyo; Lajdova, Dana; Nebohacova, Martina; Nosek, Jozef; Miyakawa, Isamu

2014-01-01

Mitochondrial DNA (mtDNA) is highly compacted into DNA-protein structures termed mitochondrial nucleoids (mt-nucleoids). The key mt-nucleoid components responsible for mtDNA condensation are HMG box-containing proteins such as mammalian mitochondrial transcription factor A (TFAM) and Abf2p of the yeast Saccharomyces cerevisiae. To gain insight into the function and organization of mt-nucleoids in strictly aerobic organisms, we initiated studies of these DNA-protein structures in Yarrowia lipolytica. We identified a principal component of mt-nucleoids in this yeast and termed it YlMhb1p (Y. lipolytica mitochondrial HMG box-containing protein 1). YlMhb1p contains two putative HMG boxes contributing both to DNA binding and to its ability to compact mtDNA in vitro. Phenotypic analysis of a Δmhb1 strain lacking YlMhb1p resulted in three interesting findings. First, although the mutant exhibits clear differences in mt-nucleoids accompanied by a large decrease in the mtDNA copy number and the number of mtDNA-derived transcripts, its respiratory characteristics and growth under most of the conditions tested are indistinguishable from those of the wild-type strain. Second, our results indicate that a potential imbalance between subunits of the respiratory chain encoded separately by nuclear DNA and mtDNA is prevented at a (post)translational level. Third, we found that mtDNA in the Δmhb1 strain is more prone to mutations, indicating that mtHMG box-containing proteins protect the mitochondrial genome against mutagenic events. PMID:24972935

Analysis of ATP-citrate lyase and malic enzyme mutants of Yarrowia lipolytica points out the importance of mannitol metabolism in fatty acid synthesis.

PubMed

Dulermo, Thierry; Lazar, Zbigniew; Dulermo, Rémi; Rakicka, Magdalena; Haddouche, Ramedane; Nicaud, Jean-Marc

2015-09-01

The role of the two key enzymes of fatty acid (FA) synthesis, ATP-citrate lyase (Acl) and malic enzyme (Mae), was analyzed in the oleaginous yeast Yarrowia lipolytica. In most oleaginous yeasts, Acl and Mae are proposed to provide, respectively, acetyl-CoA and NADPH for FA synthesis. Acl was mainly studied at the biochemical level but no strain depleted for this enzyme was analyzed in oleaginous microorganisms. On the other hand the role of Mae in FA synthesis in Y. lipolytica remains unclear since it was proposed to be a mitochondrial NAD(H)-dependent enzyme and not a cytosolic NADP(H)-dependent enzyme. In this study, we analyzed for the first time strains inactivated for corresponding genes. Inactivation of ACL1 decreases FA synthesis by 60 to 80%, confirming its essential role in FA synthesis in Y. lipolytica. Conversely, inactivation of MAE1 has no effects on FA synthesis, except in a FA overaccumulating strain where it improves FA synthesis by 35%. This result definitively excludes Mae as a major key enzyme for FA synthesis in Y. lipolytica. During the analysis of both mutants, we observed a negative correlation between FA and mannitol level. As mannitol and FA pathways may compete for carbon storage, we inactivated YlSDR, encoding a mannitol dehydrogenase converting fructose and NADPH into mannitol and NADP+. The FA content of the resulting mutant was improved by 60% during growth on fructose, demonstrating that mannitol metabolism may modulate FA synthesis in Y. lipolytica. Copyright © 2015. Published by Elsevier B.V.

Modeling and optimization of lipid accumulation by Yarrowia lipolytica from glucose under nitrogen depletion conditions.

PubMed

Robles-Rodríguez, Carlos E; Muñoz-Tamayo, Rafael; Bideaux, Carine; Gorret, Nathalie; Guillouet, Stéphane E; Molina-Jouve, Carole; Roux, Gilles; Aceves-Lara, César A

2018-05-01

Oleaginous yeasts have been seen as a feasible alternative to produce the precursors of biodiesel due to their capacity to accumulate lipids as triacylglycerol having profiles with high content of unsaturated fatty acids. The yeast Yarrowia lipolytica is a promising microorganism that can produce lipids under nitrogen depletion conditions and excess of the carbon source. However, under these conditions, this yeast also produces citric acid (overflow metabolism) decreasing lipid productivity. This work presents two mathematical models for lipid production by Y. lipolytica from glucose. The first model is based on Monod and inhibition kinetics, and the second one is based on the Droop quota model approach, which is extended to yeast. The two models showed good agreements with the experimental data used for calibration and validation. The quota based model presented a better description of the dynamics of nitrogen and glucose dynamics leading to a good management of N/C ratio which makes this model interesting for control purposes. Then, quota model was used to evaluate, by means of simulation, a scenario for optimizing lipid productivity and lipid content. For that, a control strategy was designed by approximating the flow rates of glucose and nitrogen with piecewise linear functions. Simulation results achieved productivity of 0.95 g L -1  hr -1 and lipid content fraction of 0.23 g g -1 , which indicates that this strategy is a promising alternative for the optimization of lipid production. © 2017 Wiley Periodicals, Inc.

Overproduction of Fatty Acid Ethyl Esters by the Oleaginous Yeast Yarrowia lipolytica through Metabolic Engineering and Process Optimization.

PubMed

Gao, Qi; Cao, Xuan; Huang, Yu-Ying; Yang, Jing-Lin; Chen, Jun; Wei, Liu-Jing; Hua, Qiang

2018-05-18

Recent advances in the production of biofuels by microbes have attracted attention due to increasingly limited fossil fuels. Biodiesels, especially fatty acid ethyl esters (FAEEs), are considered a potentially fully sustainable fuel in the near future due to similarities with petrodiesels and compatibility with existing infrastructure. However, biosynthesis of FAEEs is limited by the supply of precursor lipids and acetyl-CoA. In the present study, we explored the production potential of an engineered biosynthetic pathway coupled to the addition of ethanol in the oleaginous yeast Yarrowia lipolytica. This type of yeast is able to supply a greater amount of precursor lipids than species typically used. To construct the FAEEs synthesis pathway, WS genes that encode wax ester synthases (WSs) from different species were codon-optimized and heterologously expressed in Y. lipolytica. The most productive engineered strain was found to express a WS gene from Marinobacter hydrocarbonoclasticus strain DSM 8798. To stepwisely increase FAEEs production, we optimized the promoter of WS overexpression, eliminated β-oxidation by deleting the PEX10 gene in our engineered strains, and redirected metabolic flux toward acetyl-CoA. The new engineered strain, coupled with an optimized ethanol concentration, led to an approximate 5.5-fold increase in extracellular FAEEs levels compared to the wild-type strain and a maximum FAEEs titer of 1.18 g/L in shake flask cultures. In summary, the present study demonstrated that an engineered Y. lipolytica strain possessed a high capacity for FAEEs production and may serve as a platform for more efficient biodiesel production in the future.

Immobilization of Yarrowia lipolytica Lipase on Macroporous Resin Using Different Methods: Characterization of the Biocatalysts in Hydrolysis Reaction.

PubMed

Sun, Jingjing; Chen, Yiling; Sheng, Jun; Sun, Mi

2015-01-01

To improve the reusability and organic solvent tolerance of microbial lipase and expand the application of lipase (hydrolysis, esterification, and transesterification), we immobilized marine microbial lipase using different methods and determined the properties of immobilized lipases. Considering the activity and cost of immobilized lipase, the concentration of lipase was fixed at 2 mg/mL. The optimal temperature of immobilized lipases was 40°C and 5°C higher than free lipase. The activities of immobilized lipases were much higher than free lipase at alkaline pH (more than 50% at pH 12). The free lipase lost most activity (35.3%) and immobilized lipases retained more than 46.4% of their initial activity after 3 h heat treatment at 70°C. At alkaline pH, immobilized lipases were more stable than free lipase (more than 60% residue activity at pH 11 for 3 h). Immobilized lipases retained 80% of their activity after 5 cycles and increased enzyme activity (more than 108.7%) after 3 h treatment in tert-butanol. Immobilization of lipase which improved reusability of lipase and provided a chance to expand the application of marine microbial lipase in organic system expanded the application range of lipase to catalyze hydrolysis and esterification in harsh condition.

Inference and interrogation of a coregulatory network in the context of lipid accumulation in Yarrowia lipolytica.

PubMed

Trébulle, Pauline; Nicaud, Jean-Marc; Leplat, Christophe; Elati, Mohamed

2017-01-01

Complex phenotypes, such as lipid accumulation, result from cooperativity between regulators and the integration of multiscale information. However, the elucidation of such regulatory programs by experimental approaches may be challenging, particularly in context-specific conditions. In particular, we know very little about the regulators of lipid accumulation in the oleaginous yeast of industrial interest Yarrowia lipolytica . This lack of knowledge limits the development of this yeast as an industrial platform, due to the time-consuming and costly laboratory efforts required to design strains with the desired phenotypes. In this study, we aimed to identify context-specific regulators and mechanisms, to guide explorations of the regulation of lipid accumulation in Y. lipolytica . Using gene regulatory network inference, and considering the expression of 6539 genes over 26 time points from GSE35447 for biolipid production and a list of 151 transcription factors, we reconstructed a gene regulatory network comprising 111 transcription factors, 4451 target genes and 17048 regulatory interactions (YL-GRN-1) supported by evidence of protein-protein interactions. This study, based on network interrogation and wet laboratory validation (a) highlights the relevance of our proposed measure, the transcription factors influence, for identifying phases corresponding to changes in physiological state without prior knowledge (b) suggests new potential regulators and drivers of lipid accumulation and (c) experimentally validates the impact of six of the nine regulators identified on lipid accumulation, with variations in lipid content from +43.2% to -31.2% on glucose or glycerol.

A comparative study on glycerol metabolism to erythritol and citric acid in Yarrowia lipolytica yeast cells.

PubMed

Tomaszewska, Ludwika; Rakicka, Magdalena; Rymowicz, Waldemar; Rywińska, Anita

2014-09-01

Citric acid and erythritol biosynthesis from pure and crude glycerol by three acetate-negative mutants of Yarrowia lipolytica yeast was investigated in batch cultures in a wide pH range (3.0-6.5). Citric acid biosynthesis was the most effective at pH 5.0-5.5 in the case of Wratislavia 1.31 and Wratislavia AWG7. With a decreasing pH value, the direction of biosynthesis changed into erythritol synthesis accompanied by low production of citric acid. Pathways of glycerol conversion into erythritol and citric acid were investigated in Wratislavia K1 cells. Enzymatic activity was compared in cultures run at pH 3.0 and 4.5, that is, under conditions promoting the production of erythritol and citric acid, respectively. The effect of pH value (3.0 and 4.5) and NaCl presence on the extracellular production and intracellular accumulation of citric acid and erythritol was compared as well. Low pH and NaCl resulted in diminished activity of glycerol kinase, whereas such conditions stimulated the activity of glycerol-3-phosphate dehydrogenase. The presence of NaCl strongly influenced enzymes activity - the effective erythritol production was correlated with a high activity of transketolase and erythrose reductase. Therefore, presented results confirmed that transketolase and erythrose reductase are involved in the overproduction of erythritol in the cells of Y. lipolytica yeast. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Overlapping reading frames at the LYS5 locus in the yeast Yarrowia lipolytica.

PubMed Central

Xuan, J W; Fournier, P; Declerck, N; Chasles, M; Gaillardin, C

1990-01-01

Mutants affected at the LYS5 locus of Yarrowia lipolytica lack detectable dehydrogenase (SDH) activity. The LYS5 gene has previously been cloned, and we present here the sequence of the 2.5-kilobase-pair (kb) DNA fragment complementing the lys5 mutation. Two large antiparallel open reading frames (ORF1 and ORF2) were observed, flanked by potential transcription signals. Both ORFs appear to be transcribed, but several lines of evidence suggest that only ORF2 is translated and encodes SDH. (i) The global amino acid compositions of Saccharomyces cerevisiae SDH and of the putative ORF2 product are similar and that of ORF1 is dissimilar. (ii) An in-frame translational fusion of ORF2 with the Escherichia coli lacZ gene was introduced into yeast cells and resulted in a beta-galactosidase activity regulated similarly to SDH; no beta-galactosidase activity was obtained with an in-frame fusion of ORF1 with lacZ. (iii) The introduction of a stop codon at the beginning of ORF2 prevented SDH expression in yeast cells, whereas no phenotypic effect was observed when ORF1 translation was blocked. Images PMID:2388625

Leucine Biosynthesis Is Involved in Regulating High Lipid Accumulation in Yarrowia lipolytica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kerkhoven, Eduard J.; Kim, Young-Mo; Wei, Siwei

ABSTRACT The yeastYarrowia lipolyticais a potent accumulator of lipids, and lipogenesis in this organism can be influenced by a variety of factors, such as genetics and environmental conditions. Using a multifactorial study, we elucidated the effects of both genetic and environmental factors on regulation of lipogenesis inY. lipolyticaand identified how two opposite regulatory states both result in lipid accumulation. This study involved comparison of a strain overexpressing diacylglycerol acyltransferase (DGA1) with a control strain grown under either nitrogen or carbon limitation conditions. A strong correlation was observed between the responses on the transcript and protein levels. Combination ofDGA1overexpression with nitrogen limitationmore » resulted in a high level of lipid accumulation accompanied by downregulation of several amino acid biosynthetic pathways, including that of leucine in particular, and these changes were further correlated with a decrease in metabolic fluxes. This downregulation was supported by the measured decrease in the level of 2-isopropylmalate, an intermediate of leucine biosynthesis. Combining the multi-omics data with putative transcription factor binding motifs uncovered a contradictory role for TORC1 in controlling lipid accumulation, likely mediated through 2-isopropylmalate and a Leu3-like transcription factor. IMPORTANCEThe ubiquitous metabolism of lipids involves refined regulation, and an enriched understanding of this regulation would have wide implications. Various factors can influence lipid metabolism, including the environment and genetics. We demonstrated, using a multi-omics and multifactorial experimental setup, that multiple factors affect lipid accumulation in the yeastYarrowia lipolytica. Using integrative analysis, we identified novel interactions between nutrient restriction and genetic factors involving regulators that are highly conserved among eukaryotes. Given that lipid metabolism is involved in many

Functional overexpression and characterization of lipogenesis-related genes in the oleaginous yeast Yarrowia lipolytica.

PubMed

Silverman, Andrew M; Qiao, Kangjian; Xu, Peng; Stephanopoulos, Gregory

2016-04-01

Single cell oil (SCO) is an attractive energy source due to scalability, utilization of low-cost renewable feedstocks, and type of product(s) made. Engineering strains capable of producing high lipid titers and yields is crucial to the economic viability of these processes. However, lipid synthesis in cells is a complex phenomenon subject to multiple layers of regulation, making gene target identification a challenging task. In this study, we aimed to identify genes in the oleaginous yeast Yarrowia lipolytica whose overexpression enhances lipid production by this organism. To this end, we examined the effect of the overexpression of a set of 44 native genes on lipid production in Y. lipolytica, including those involved in glycerolipid synthesis, fatty acid synthesis, central carbon metabolism, NADPH generation, regulation, and metabolite transport and characterized each resulting strain's ability to produce lipids growing on both glucose and acetate as a sole carbon source. Our results suggest that a diverse subset of genes was effective at individually influencing lipid production in Y. lipolytica, sometimes in a substrate-dependent manner. The most productive strain on glucose overexpressed the diacylglycerol acyltransferase DGA2 gene, increasing lipid titer, cellular content, and yield by 236, 165, and 246 %, respectively, over our control strain. On acetate, our most productive strain overexpressed the acylglycerol-phosphate acyltransferase SLC1 gene, with a lipid titer, cellular content, and yield increase of 99, 91, and 151 %, respectively, over the control strain. Aside from genes encoding enzymes that directly catalyze the reactions of lipid synthesis, other ways by which lipogenesis was increased in these cells include overexpressing the glycerol-3-phosphate dehydrogenase (GPD1) gene to increase production of glycerol head groups and overexpressing the 6-phosphogluconolactonase (SOL3) gene from the oxidative pentose phosphate pathway to increase NADPH

Targeted mutations and MD simulations of a methanol-stable lipase YLIP9 from Yarrowia lipolytica MSR80 to develop a biodiesel enzyme.

PubMed

Syal, Poonam; Verma, Ved Vrat; Gupta, Rani

2017-11-01

Biodiesel, an environment friendly alternative for fuels, contains methyl esters of long-chain fatty acids. Our group has reported a methanol-stable YLIP9 from Yarrowia lipolytica MSR80 that shows poor catalysis of long-chain fatty acids. To shift its substrate specificity, residues within lid and binding pocket were identified for sequential mutations using YLIP2 as the template. Of the two point mutations (Glu116Leu and Ser119Val) introduced in the lid, the former mutation (YLIP9L1) increased the catalytic rate by ∼2-fold without any change in substrate specificity. In this mutant, six binding pocket residues (Bp2-Bp7) were further mutated to obtain six double mutants. YLIP9L1Bp3 showed significant shift in substrate specificity towards long-chain pNPesters with 11-fold increase in catalytic efficiency than YLIP9. Double mutations also led to increased thermostability and lowered activation energy of YLIP9L1Bp3 thereby shifting its optimum temperature from 60°C to 50°C. In silico molecular dynamics simulations revealed improved lid flexibility and increased catalytic triad volume in YLIP9L1Bp3. The enzyme YLIP9L1Bp3 was methanol-stable having selectivity for long-chain fatty acids with improved catalytic efficiency. Its application as a biodiesel enzyme was validated by transesterification of palm oil in presence of methanol, where it showed 8-fold increase in conversion of oil to methyl esters. Copyright © 2017 Elsevier B.V. All rights reserved.

A survey of yeast from the Yarrowia clade for lipid production in dilute-acid pretreated lignocellulosic biomass hydrolysate

USDA-ARS?s Scientific Manuscript database

Yarrowia lipolytica is an oleaginous yeast species that has attracted attention as a model organism for synthesis of single cell oil. Among over 50 isolates of Y. lipolytica identified, only a few of the strains have been studied extensively. Furthermore, 12 other yeast species were recently assigne...

Identification of the Transcription Factor Znc1p, which Regulates the Yeast-to-Hypha Transition in the Dimorphic Yeast Yarrowia lipolytica

PubMed Central

Martinez-Vazquez, Azul; Gonzalez-Hernandez, Angelica; Domínguez, Ángel; Rachubinski, Richard; Riquelme, Meritxell; Cuellar-Mata, Patricia; Guzman, Juan Carlos Torres

2013-01-01

The dimorphic yeast Yarrowia lipolytica is used as a model to study fungal differentiation because it grows as yeast-like cells or forms hyphal cells in response to changes in environmental conditions. Here, we report the isolation and characterization of a gene, ZNC1, involved in the dimorphic transition in Y. lipolytica. The ZNC1 gene encodes a 782 amino acid protein that contains a Zn(II)2C6 fungal-type zinc finger DNA-binding domain and a leucine zipper domain. ZNC1 transcription is elevated during yeast growth and decreases during the formation of mycelium. Cells in which ZNC1 has been deleted show increased hyphal cell formation. Znc1p-GFP localizes to the nucleus, but mutations within the leucine zipper domain of Znc1p, and to a lesser extent within the Zn(II)2C6 domain, result in a mislocalization of Znc1p to the cytoplasm. Microarrays comparing gene expression between znc1::URA3 and wild-type cells during both exponential growth and the induction of the yeast-to-hypha transition revealed 1,214 genes whose expression was changed by 2-fold or more under at least one of the conditions analyzed. Our results suggest that Znc1p acts as a transcription factor repressing hyphal cell formation and functions as part of a complex network regulating mycelial growth in Y. lipolytica. PMID:23826133

High-throughput fermentation screening for the yeast Yarrowia lipolytica with real-time monitoring of biomass and lipid production.

PubMed

Back, Alexandre; Rossignol, Tristan; Krier, François; Nicaud, Jean-Marc; Dhulster, Pascal

2016-08-23

Because the model yeast Yarrowia lipolytica can synthesize and store lipids in quantities up to 20 % of its dry weight, it is a promising microorganism for oil production at an industrial scale. Typically, optimization of the lipid production process is performed in the laboratory and later scaled up for industrial production. However, the scale-up process can be complicated by genetic modifications that are optimized for one set of growing conditions can confer a less-than-optimal phenotype in a different environment. To address this issue, small cultivation systems have been developed that mimic the conditions in benchtop bioreactors. In this work, we used one such microbioreactor system, the BioLector, to develop high-throughput fermentation procedures that optimize growth and lipid accumulation in Y. lipolytica. Using this system, we were able to monitor lipid and biomass production in real time throughout the culture duration. The BioLector can monitor the growth of Y. lipolytica in real time by evaluating scattered light; this produced accurate measurements until cultures reached an equivalent of OD600nm = 115 and a cell dry weight of 100 g L(-1). In addition, a lipid-specific fluorescent probe was applied which reliably monitored lipid production up to a concentration of 12 g L(-1). Through screening various growing conditions, we determined that a carbon/nitrogen ratio of 35 was the most efficient for lipid production. Further screening showed that ammonium chloride and glycerol were the most valuable nitrogen and carbon sources, respectively, for growth and lipid production. Moreover, a carbon concentration above 1 M appeared to impair growth and lipid accumulation. Finally, we used these optimized conditions to screen engineered strains of Y. lipolytica with high lipid-accumulation capability. The growth and lipid content of the strains cultivated in the BioLector were compared to those grown in benchtop bioreactors. To our knowledge, this is the

High performance microbiological transformation of L-tyrosine to L-dopa by Yarrowia lipolytica NRRL-143

PubMed Central

Ali, Sikander; Shultz, Jeffry L; Ikram-ul-Haq

2007-01-01

Background The 3,4-dihydroxy phenyl L-alanine (L-dopa) is a drug of choice for Parkinson's disease, controlling changes in energy metabolism enzymes of the myocardium following neurogenic injury. Aspergillus oryzae is commonly used for L-dopa production; however, potential improvements in ease of handling, growth rate and environmental impact have led to an interest in exploiting alternative yeasts. The two important elements required for L-dopa production are intracellular tyrosinases (thus pre-grown yeast cells are required for the transformation of L-tyrosine to L-dopa) and L-ascorbate, which acts as a reducing agent. Results Pre-grown cells of Yarrowia lipolytica NRRL-143 were used for the microbiological transformation of L-tyrosine to L-dopa. Different diatomite concentrations (0.5–3.0 mg/ml) were added to the acidic (pH 3.5) reaction mixture. Maximum L-dopa biosynthesis (2.96 mg/ml L-dopa from 2.68 mg/ml L-tyrosine) was obtained when 2.0 mg/ml diatomite was added 15 min after the start of the reaction. After optimizing reaction time (30 min), and yeast cell concentration (2.5 mg/ml), an overall 12.5 fold higher L-dopa production rate was observed when compared to the control. Significant enhancements in Yp/s, Qs and qs over the control were observed. Conclusion Diatomite (2.0 mg/ml) addition 15 min after reaction commencement improved microbiological transformation of L-tyrosine to L-dopa (3.48 mg/ml; p ≤ 0.05) by Y. lipolytica NRRL-143. A 35% higher substrate conversion rate was achieved when compared to the control. PMID:17705832

L-Phenylalanine catabolism and 2-phenylethanol synthesis in Yarrowia lipolytica--mapping molecular identities through whole-proteome quantitative mass spectrometry analysis.

PubMed

Celińska, Ewelina; Olkowicz, Mariola; Grajek, Włodzimierz

2015-08-01

A world-wide effort is now being pursued towards the development of flavors and fragrances (F&F) production independently from traditional sources, as well as autonomously from depleting fossil fuel supplies. Biotechnological production of F&F by microbes has emerged as a vivid solution to the current market limitations. Amongst a wide variety of fragrant chemicals, 2-PE is of significant interest to both scientific and industrial community. Although the general overview of the 2-PE synthesis pathway is commonly known, involvement of particular molecular identities in this pathway has not been elucidated in Yarrowia lipolytica to date. The aim of this study was mapping molecular identities involved in 2-PE synthesis in Y. lipolytica. To acquire a comprehensive landscape of the proteins that are directly and indirectly involved in L-Phe degradation and 2-PE synthesis, we took advantage of comprehensibility and sensitivity of high-throughput LC-MS/MS-quantitative analysis. Amongst a number of proteins involved in amino acid turnover and the central carbon metabolism, enzymes involved in L-Phe conversion to 2-PE have been identified. Results on yeast-to-hyphae transition in relation to the character of the provided nitrogen source have been presented. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Regulation of Nitrogen Metabolism by GATA Zinc Finger Transcription Factors in Yarrowia lipolytica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pomraning, Kyle R.; Bredeweg, Erin L.; Baker, Scott E.

ABSTRACT Fungi accumulate lipids in a manner dependent on the quantity and quality of the nitrogen source on which they are growing. In the oleaginous yeastYarrowia lipolytica, growth on a complex source of nitrogen enables rapid growth and limited accumulation of neutral lipids, while growth on a simple nitrogen source promotes lipid accumulation in large lipid droplets. Here we examined the roles of nitrogen catabolite repression and its regulation by GATA zinc finger transcription factors on lipid metabolism inY. lipolytica. Deletion of the GATA transcription factor genesgzf3andgzf2resulted in nitrogen source-specific growth defects and greater accumulation of lipids when the cells weremore » growing on a simple nitrogen source. Deletion ofgzf1, which is most similar to activators of genes repressed by nitrogen catabolite repression in filamentous ascomycetes, did not affect growth on the nitrogen sources tested. We examined gene expression of wild-type and GATA transcription factor mutants on simple and complex nitrogen sources and found that expression of enzymes involved in malate metabolism, beta-oxidation, and ammonia utilization are strongly upregulated on a simple nitrogen source. Deletion ofgzf3results in overexpression of genes with GATAA sites in their promoters, suggesting that it acts as a repressor, whilegzf2is required for expression of ammonia utilization genes but does not grossly affect the transcription level of genes predicted to be controlled by nitrogen catabolite repression. Both GATA transcription factor mutants exhibit decreased expression of genes controlled by carbon catabolite repression via the repressormig1, including genes for beta-oxidation, highlighting the complex interplay between regulation of carbon, nitrogen, and lipid metabolism. IMPORTANCENitrogen source is commonly used to control lipid production in industrial fungi. Here we identified regulators of nitrogen catabolite repression in the oleaginous yeast

Molecular Characterization of the Elaeis guineensis Medium-Chain Fatty Acid Diacylglycerol Acyltransferase DGAT1-1 by Heterologous Expression in Yarrowia lipolytica.

PubMed

Aymé, Laure; Jolivet, Pascale; Nicaud, Jean-Marc; Chardot, Thierry

2015-01-01

Diacylglycerol acyltransferases (DGAT) are involved in the acylation of sn-1,2-diacylglycerol. Palm kernel oil, extracted from Elaeis guineensis (oil palm) seeds, has a high content of medium-chain fatty acids mainly lauric acid (C12:0). A putative E. guineensis diacylglycerol acyltransferase gene (EgDGAT1-1) is expressed at the onset of lauric acid accumulation in the seed endosperm suggesting that it is a determinant of medium-chain triacylglycerol storage. To test this hypothesis, we thoroughly characterized EgDGAT1-1 activity through functional complementation of a Yarrowia lipolytica mutant strain devoid of neutral lipids. EgDGAT1-1 expression is sufficient to restore triacylglycerol accumulation in neosynthesized lipid droplets. A comparative functional study with Arabidopsis thaliana DGAT1 highlighted contrasting substrate specificities when the recombinant yeast was cultured in lauric acid supplemented medium. The EgDGAT1-1 expressing strain preferentially accumulated medium-chain triacylglycerols whereas AtDGAT1 expression induced long-chain triacylglycerol storage in Y. lipolytica. EgDGAT1-1 localized to the endoplasmic reticulum where TAG biosynthesis takes place. Reestablishing neutral lipid accumulation in the Y. lipolytica mutant strain did not induce major reorganization of the yeast microsomal proteome. Overall, our findings demonstrate that EgDGAT1-1 is an endoplasmic reticulum DGAT with preference for medium-chain fatty acid substrates, in line with its physiological role in palm kernel. The characterized EgDGAT1-1 could be used to promote medium-chain triacylglycerol accumulation in microbial-produced oil for industrial chemicals and cosmetics.

Molecular Characterization of the Elaeis guineensis Medium-Chain Fatty Acid Diacylglycerol Acyltransferase DGAT1-1 by Heterologous Expression in Yarrowia lipolytica

PubMed Central

Aymé, Laure; Jolivet, Pascale; Nicaud, Jean-Marc; Chardot, Thierry

2015-01-01

Diacylglycerol acyltransferases (DGAT) are involved in the acylation of sn-1,2-diacylglycerol. Palm kernel oil, extracted from Elaeis guineensis (oil palm) seeds, has a high content of medium-chain fatty acids mainly lauric acid (C12:0). A putative E. guineensis diacylglycerol acyltransferase gene (EgDGAT1-1) is expressed at the onset of lauric acid accumulation in the seed endosperm suggesting that it is a determinant of medium-chain triacylglycerol storage. To test this hypothesis, we thoroughly characterized EgDGAT1-1 activity through functional complementation of a Yarrowia lipolytica mutant strain devoid of neutral lipids. EgDGAT1-1 expression is sufficient to restore triacylglycerol accumulation in neosynthesized lipid droplets. A comparative functional study with Arabidopsis thaliana DGAT1 highlighted contrasting substrate specificities when the recombinant yeast was cultured in lauric acid supplemented medium. The EgDGAT1-1 expressing strain preferentially accumulated medium-chain triacylglycerols whereas AtDGAT1 expression induced long-chain triacylglycerol storage in Y. lipolytica. EgDGAT1-1 localized to the endoplasmic reticulum where TAG biosynthesis takes place. Reestablishing neutral lipid accumulation in the Y. lipolytica mutant strain did not induce major reorganization of the yeast microsomal proteome. Overall, our findings demonstrate that EgDGAT1-1 is an endoplasmic reticulum DGAT with preference for medium-chain fatty acid substrates, in line with its physiological role in palm kernel. The characterized EgDGAT1-1 could be used to promote medium-chain triacylglycerol accumulation in microbial-produced oil for industrial chemicals and cosmetics. PMID:26581109

Proteomic analysis of the response of α-ketoglutarate-producer Yarrowia lipolytica WSH-Z06 to environmental pH stimuli.

PubMed

Guo, Hongwei; Wan, Hui; Chen, Hongwen; Fang, Fang; Liu, Song; Zhou, Jingwen

2016-10-01

During bioproduction of short-chain carboxylates, a shift in pH is a common strategy for enhancing the biosynthesis of target products. Based on two-dimensional gel electrophoresis, comparative proteomics analysis of general and mitochondrial protein samples was used to investigate the cellular responses to environmental pH stimuli in the α-ketoglutarate overproducer Yarrowia lipolytica WSH-Z06. The lower environmental pH stimuli tensioned intracellular acidification and increased the level of reactive oxygen species (ROS). A total of 54 differentially expressed protein spots were detected, and 11 main cellular processes were identified to be involved in the cellular response to environmental pH stimuli. Slight decrease in cytoplasmic pH enhanced the cellular acidogenicity by elevating expression level of key enzymes in tricarboxylic acid cycle (TCA cycle). Enhanced energy biosynthesis, ROS elimination, and membrane potential homeostasis processes were also employed as cellular defense strategies to compete with environmental pH stimuli. Owing to its antioxidant role of α-ketoglutarate, metabolic flux shifted to α-ketoglutarate under lower pH by Y. lipolytica in response to acidic pH stimuli. The identified differentially expressed proteins provide clues for understanding the mechanisms of the cellular responses and for enhancing short-chain carboxylate production through metabolic engineering or process optimization strategies in combination with manipulation of environmental conditions.

Ylpex5 mutation partially suppresses the defective hyphal growth of a Yarrowia lipolytica ceramide synthase mutant, Yllac1, by recovering lipid raft polarization and vacuole morphogenesis.

PubMed

Bal, Jyotiranjan; Lee, Hye-Jeong; Cheon, Seon Ah; Lee, Kyung Jin; Oh, Doo-Byoung; Kim, Jeong-Yoon

2013-01-01

Sphingolipids are involved in cell differentiation and morphogenesis in eukaryotic cells. In this study, YlLac1p, a ceramide synthase required for glucosylceramide (GlcCer) synthesis, was found to be essential for hyphal growth in Yarrowia lipolytica. Y. lipolytica GlcCer was shown to be composed of a C16:0 fatty acid, which is hydroxylated at C2, and a C18:2 long chain base, which is unsaturated at both C4 and C8 and methylated at C9. Domain swapping analysis revealed that the entire TRAM/Lag1/CLN8 (TLC) domain, not the Lag1 motif, is crucial for the function of YlLac1p. YlDes1p, the C4 desaturase of the ceramide synthesized by YlLac1p, was also required for Y. lipolytica morphogenesis. Both Yllac1Δ and Yldes1Δ mutants neither polarize lipid rafts nor form normal vacuoles. Interestingly, mutation in YlPEX5, which encode a peroxisomal targeting signal receptor, partially suppressed the defective hyphal growth of Yllac1Δ. The Yllac1ΔYlpex5Δ mutant restored the ability to polarize lipid rafts and to form normal vacuoles, although it could not synthesize GlcCer. Taken together, our results suggest that GlcCer or GlcCer derivatives may be involved in hyphal morphogenesis in Y. lipolytica, at least in part, by affecting polarization of lipid rafts and vacuole morphogenesis. Copyright © 2012 Elsevier Inc. All rights reserved.

Dynamic behavior of Yarrowia lipolytica in response to pH perturbations: dependence of the stress response on the culture mode.

PubMed

Timoumi, Asma; Cléret, Mégane; Bideaux, Carine; Guillouet, Stéphane E; Allouche, Yohan; Molina-Jouve, Carole; Fillaudeau, Luc; Gorret, Nathalie

2017-01-01

Yarrowia lipolytica, a non-conventional yeast with a promising biotechnological potential, is able to undergo metabolic and morphological changes in response to environmental conditions. The effect of pH perturbations of different types (pulses, Heaviside) on the dynamic behavior of Y. lipolytica W29 strain was characterized under two modes of culture: batch and continuous. In batch cultures, different pH (4.5, 5.6 (optimal condition), and 7) were investigated in order to identify the pH inducing a stress response (metabolic and/or morphologic) in Y. lipolytica. Macroscopic behavior (kinetic parameters, yields, viability) of the yeast was slightly affected by pH. However, contrary to the culture at pH 5.6, a filamentous growth was induced in batch experiments at pH 4.5 and 7. Proportions of the filamentous subpopulation reached 84 and 93 % (v/v) under acidic and neutral conditions, respectively. Given the significant impact of neutral pH on morphology, pH perturbations from 5.6 to 7 were subsequently assayed in batch and continuous bioreactors. For both process modes, the growth dynamics remained fundamentally unaltered during exposure to stress. Nevertheless, morphological behavior of the yeast was dependent on the culture mode. Specifically, in batch bioreactors where cells proliferated at their maximum growth rate, mycelia were mainly formed. Whereas, in continuous cultures at controlled growth rates (from 0.03 to 0.20 h -1 ) even closed to the maximum growth rate of the stain (0.24 h -1 ), yeast-like forms predominated. This pointed out differences in the kinetic behavior of filamentous and yeast subpopulations, cell age distribution, and pH adaptive mechanisms between both modes of culture.

Optimization of a low-cost hyperosmotic medium and establishing the fermentation kinetics of erythritol production by Yarrowia lipolytica from crude glycerol.

PubMed

Yang, Li-Bo; Zhan, Xiao-Bei; Zhu, Li; Gao, Min-Jie; Lin, Chi-Chung

2016-05-18

The production of erythritol by Yarrowia lipolytica from low-cost substitutable substrates for high yield was investigated. Crude glycerol, urea, and NaCl related to osmotic pressure were the most significant factors affecting erythritol production. An artificial neural network model and genetic algorithm were used to search the optimal composition of the significant factors and locate the resulting erythritol yield. Medium with 232.39 g/L crude glycerol, 1.57 g/L urea, and 31.03 g/L NaCl led to predictive maximum erythritol concentration of 110.7 g/L. The erythritol concentration improved from 50.4 g/L to 109.2 g/L with the optimized medium, which was reproducible. Erythritol fermentation kinetics were investigated in a batch system. Multistep fermentation kinetic models with hyperosmotic inhibitory effects were developed. The resulting mathematical equations provided a good description of temporal variations such as microbial growth (X), substrate consumption (S), and product formation (P) in erythritol fermentation. The accordingly derived model is the first reported model for fermentative erythritol production from glycerol, providing useful information to optimize the growth of Y. lipolytica and contributing visual description for the erythritol fermentation process under high osmotic pressure, as well as improvement of productivity and efficiency.

Harnessing biodiesel-producing microbes: from genetic engineering of lipase to metabolic engineering of fatty acid biosynthetic pathway.

PubMed

Yan, Jinyong; Yan, Yunjun; Madzak, Catherine; Han, Bingnan

2017-02-01

Microbial production routes, notably whole-cell lipase-mediated biotransformation and fatty-acids-derived biosynthesis, offer new opportunities for synthesizing biodiesel. They compare favorably to immobilized lipase and chemically catalyzed processes. Genetically modified whole-cell lipase-mediated in vitro route, together with in vivo and ex vivo microbial biosynthesis routes, constitutes emerging and rapidly developing research areas for effective production of biodiesel. This review presents recent advances in customizing microorganisms for producing biodiesel, via genetic engineering of lipases and metabolic engineering (including system regulation) of fatty-acids-derived pathways. Microbial hosts used include Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris and Aspergillus oryzae. These microbial cells can be genetically modified to produce lipases under different forms: intracellularly expressed, secreted or surface-displayed. They can be metabolically redesigned and systematically regulated to obtain balanced biodiesel-producing cells, as highlighted in this study. Such genetically or metabolically modified microbial cells can support not only in vitro biotransformation of various common oil feedstocks to biodiesel, but also de novo biosynthesis of biodiesel from glucose, glycerol or even cellulosic biomass. We believe that the genetically tractable oleaginous yeast Yarrowia lipolytica could be developed to an effective biodiesel-producing microbial cell factory. For this purpose, we propose several engineered pathways, based on lipase and wax ester synthase, in this promising oleaginous host.

Improvement of erythrose reductase activity, deletion of by-products and statistical media optimization for enhanced erythritol production from Yarrowia lipolytica mutant 49.

PubMed

Ghezelbash, Gholam Reza; Nahvi, Iraj; Emamzadeh, Rahman

2014-08-01

The purpose of the present investigation was to produce erythritol by Yarrowia lipolytica mutant without any by-products. Mutants of Y. lipolytica were generated by ultra-violet for enhancing erythrose reductase (ER) activity and erythritol production. The mutants showing the highest ER activity were screened by triphenyl tetrazolium chloride agar plate assay. Productivity of samples was analyzed by thin-layer chromatography and high-performance liquid chromatography equipped with the refractive index detector. One of the mutants named as mutant 49 gave maximum erythritol production without any other by-products (particularly glycerol). Erythritol production and specific ER activity in mutant 49 increased to 1.65 and 1.47 times, respectively, in comparison with wild-type strain. The ER gene of wild and mutant strains was sequenced and analyzed. A general comparison of wild and mutant gene sequences showed the replacement of Asp(270) with Glu(270) in ER protein. In order to enhance erythritol production, we used a three component-three level-one response Box-Behnken of response surface methodology model. The optimum medium composition for erythritol production was found to be (g/l) glucose 279.49, ammonium sulfate 9.28, and pH 5.41 with 39.76 erythritol production.

Yarrowia lipolytica NCIM 3589, a tropical marine yeast, degrades bromoalkanes by an initial hydrolytic dehalogenation step.

PubMed

Vatsal, Aakanksha; Zinjarde, Smita S; Kumar, Ameeta Ravi

2015-04-01

The widespread industrial use of organobromines which are known persistent organic pollutants has led to their accumulation in sediments and water bodies causing harm to animals and humans. While degradation of organochlorines by bacteria is well documented, information regarding degradation pathways of these recalcitrant organobromines is scarce. Hence, their fates and effects on the environment are of concern. The present study shows that a tropical marine yeast, Yarrowia lipolytica NCIM 3589 aerobically degrades bromoalkanes differing in carbon chain length and position of halogen substitution viz., 2-bromopropane (2-BP), 1-bromobutane (1-BB), 1,5 dibromopentane (1,5-DBP) and 1-bromodecane (1-BD) as seen by an increase in cell mass, release of bromide and concomitant decrease in concentration of brominated compound. The amount of bromoalkane degraded was 27.3, 21.9, 18.0 and 38.3 % with degradation rates of 0.076, 0.058, 0.046 and 0.117/day for 2-BP, 1-BB, 1,5-DBP and 1-BD, respectively. The initial product formed respectively were alcohols viz., 2-propanol, 1-butanol, 1-bromo, 5-pentanol and 1-decanol as detected by GC-MS. These were further metabolized to fatty acids viz., 2-propionic, 1-butyric and 1-decanoic acid eventually leading to carbon dioxide formation. Neither higher chain nor brominated fatty acids were detected. An inducible extracellular dehalogenase responsible for removal of bromide was detected with activities of 21.07, 18.82, 18.96 and 26.67 U/ml for 2-BP, 1-BB, 1,5-DBP and 1-BD, respectively. We report here for the first time the proposed aerobic pathway of bromoalkane degradation by an eukaryotic microbe Y. lipolytica 3589, involving an initial hydrolytic dehalogenation step.

[Activation of the alternative oxidase of Yarrowia lipolytica by adenosine 5'-monophosphate].

PubMed

Medentsev, A G; Arinbasarova, A Iu; Smirnova, N M; Akimenko, V K

2004-01-01

The study of the effect of nucleoside phosphates on the activity of cyanide-resistant oxidase in the mitochondria and the submitochondrial particles of Yarrowia lipolytica showed that adenosine monophosphate (5'-AMP, AMP) did not stimulate the respiration of the intact mitochondria. The incubation of the mitochondria at room temperature (25 degrees C) for 3-5 h or their treatment with ultrasound, phospholipase A, and detergent Triton X-100 at a low temperature inactivated the cyanide-resistant alternative oxidase. The inactivated alternative oxidase could be reactivated by AMP. The reactivating effect of AMP was enhanced by azolectin. Some other nucleoside phosphates also showed reactivating ability in the following descending order. AMP = GMP > GDP > GTP > XMP > IMP. The apparent reaction rate constant Km for AMP upon the reactivation of the alternative oxidase of mitochondria treated with Triton X-100 or incubated at 25 degrees C was 12.5 and 20 microM, respectively. The Km for AMP upon the reactivation of the alternative oxidase of submitochondrial particles was 15 microM. During the incubation of yeast cells under conditions promoting the development of alternative oxidase, the content of adenine nucleotides (AMP, ADP, and ATP) in the cells and their respiration tended to decrease. The subsequent addition of cyanide to the cells activated their respiration, diminished the intracellular content of ATP three times, and augmented the content of AMP five times. These data suggest that the stimulation of cell respiration by cyanide may be due to the activation of alternative oxidase by AMP.

Design of an efficient medium for heterologous protein production in Yarrowia lipolytica: case of human interferon alpha 2b.

PubMed

Gasmi, Najla; Ayed, Atef; Nicaud, Jean-Marc; Kallel, Héla

2011-05-20

The non conventional yeast Yarrowia lipolytica has aroused a strong industrial interest for heterologous protein production. However most of the studies describing recombinant protein production by this yeast rely on the use of complex media, such media are not convenient for large scale production particularly for products intended for pharmaceutical applications. In addition medium composition can also affect the production yield. Hence it is necessary to design an efficient medium for therapeutic protein expression by this host. Five different media, including four minimal media and a complex medium, were assessed in shake flasks for the production of human interferon alpha 2b (hIFN α2b) by Y. lipolytica under the control of POX2 promoter inducible with oleic acid. The chemically defined medium SM4 formulated by Invitrogen for Pichia pastoris growth was the most suitable. Using statistical experimental design this medium was further optimized. The selected minimal medium consisting in SM4 supplemented with 10 mg/l FeCl₃, 1 g/l glutamate, 5 ml/l PTM1 (Pichia Trace Metals) solution and a vitamin solution composed of myo-inositol, thiamin and biotin was called GNY medium. Compared to shake flask, bioreactor culture in GNY medium resulted in 416-fold increase of hIFN α2b production and 2-fold increase of the biological activity. Furthermore, SM4 enrichment with 5 ml/l PTM1 solution contributed to protect hIFN α2b against the degradation by the 28 kDa protease identified by zymography gel in culture supernatant. The screening of the inhibitory effect of the trace elements present in PTM1 solution on the activity of this protease was achieved using a Box-Behnken design. Statistical data analysis showed that FeCl₃ and MnSO₄ had the most inhibitory effect. We have designed an efficient medium for large scale production of heterologous proteins by Y. lipolytica. The optimized medium GNY is suitable for the production of hIFN α2b with the advantage that no

MHY1 Encodes a C2H2-Type Zinc Finger Protein That Promotes Dimorphic Transition in the Yeast Yarrowia lipolytica

PubMed Central

Hurtado, Cleofe A. R.; Rachubinski, Richard A.

1999-01-01

The yeast-to-hypha morphological transition (dimorphism) is typical of many pathogenic fungi. Dimorphism has been attributed to changes in temperature and nutritional status and is believed to constitute a mechanism of response to adverse conditions. We have isolated and characterized a gene, MHY1, whose transcription is dramatically increased during the yeast-to-hypha transition in Yarrowia lipolytica. Deletion of MHY1 is viable and has no effect on mating, but it does result in a complete inability of cells to undergo mycelial growth. MHY1 encodes a C2H2-type zinc finger protein, Mhy1p, which can bind putative cis-acting DNA stress response elements, suggesting that Mhy1p may act as a transcription factor. Interestingly, Mhy1p tagged with a hemagglutinin epitope was concentrated in the nuclei of actively growing cells found at the hyphal tip. PMID:10322005

Harnessing the Effect of pH on Lipid Production in Batch Cultures of Yarrowia lipolytica SKY7.

PubMed

Kuttiraja, Mathiazhakan; Dhouha, Ayed; Tyagi, Rajeshwar Dayal

2018-04-01

The objective of this research was to investigate the kinetics of lipid production by Yarrowia lipolytica SKY7 in the crude glycerol-supplemented media with and without the control of pH. Lipid and citric acid production were improved with the pH control condition. There was no significant difference observed in the biomass concentration with or without the pH control. In the pH-controlled experiments, the biomass and lipid concentration reached 18 and 7.78 g/L, (45.5% w/w), respectively, with lipid yield (Yp/s) of 0.179 g/g at 60 h of fermentation. The lipid production was directly correlated with growth and the process was defined as growth associated. After 60 h of fermentation, the lipid degradation was noticed in the pH-controlled reactor whereas it occurred after 84 h in the pH-uncontrolled reactor. Apart from lipid, citric acid was produced as the major extracellular product in both fermentations but the much lower concentration in uncontrolled pH. Based on the experimental results, it is evident that controlling the pH will enhance the lipid production by 15% compared to pH-uncontrolled fermentation.

Production of oils and fats by oleaginous microorganisms with an emphasis given to the potential of the nonconventional yeast Yarrowia lipolytica.

PubMed

Carsanba, E; Papanikolaou, S; Erten, H

2018-05-15

Recently, there has been a great upsurge of interest in studies related to several aspects of microbial lipid production, which is one of the top topics in relevant research fields due to the high demand of these fatty materials in food, medical, oleochemical and biofuel industries. Lipid accumulation by the so-called "oleaginous microorganisms" can generate more than 20% w/w of oil in dry biomass and is governed by a plethora of parameters, such as medium pH, incubation temperature, nutrient limitation and C/N (carbon/nitrogen) ratio, which drastically affect the lipid production bioprocess. Until now, considerable work has been undertaken to find the cheapest substrate to enable lipid fermentation by oleaginous microorganisms. This review principally details information regarding microbial lipids, suitable production conditions and focuses attention on using the yeast Yarrowia lipolytica to achieve these objectives. Lipid production by this yeast is discussed and the necessary conditions and suitable substrates are reviewed.

Metrological aspects of enzyme production

NASA Astrophysics Data System (ADS)

Kerber, T. M.; Dellamora-Ortiz, G. M.; Pereira-Meirelles, F. V.

2010-05-01

Enzymes are frequently used in biotechnology to carry out specific biological reactions, either in industrial processes or for the production of bioproducts and drugs. Microbial lipases are an important group of biotechnologically valuable enzymes that present widely diversified applications. Lipase production by microorganisms is described in several published papers; however, none of them refer to metrological evaluation and the estimation of the uncertainty in measurement. Moreover, few of them refer to process optimization through experimental design. The objectives of this work were to enhance lipase production in shaken-flasks with Yarrowia lipolytica cells employing experimental design and to evaluate the uncertainty in measurement of lipase activity. The highest lipolytic activity obtained was about three- and fivefold higher than the reported activities of CRMs BCR-693 and BCR-694, respectively. Lipase production by Y. lipolytica cells aiming the classification as certified reference material is recommended after further purification and stability studies.

Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates.

PubMed

Lindquist, Mitch R; López-Núñez, Juan Carlos; Jones, Marjorie A; Cox, Elby J; Pinkelman, Rebecca J; Bang, Sookie S; Moser, Bryan R; Jackson, Michael A; Iten, Loren B; Kurtzman, Cletus P; Bischoff, Kenneth M; Liu, Siqing; Qureshi, Nasib; Tasaki, Kenneth; Rich, Joseph O; Cotta, Michael A; Saha, Badal C; Hughes, Stephen R

2015-11-01

Increased interest in sustainable production of renewable diesel and other valuable bioproducts is redoubling efforts to improve economic feasibility of microbial-based oil production. Yarrowia lipolytica is capable of employing a wide variety of substrates to produce oil and valuable co-products. We irradiated Y. lipolytica NRRL YB-567 with UV-C to enhance ammonia (for fertilizer) and lipid (for biodiesel) production on low-cost protein and carbohydrate substrates. The resulting strains were screened for ammonia and oil production using color intensity of indicators on plate assays. Seven mutant strains were selected (based on ammonia assay) and further evaluated for growth rate, ammonia and oil production, soluble protein content, and morphology when grown on liver infusion medium (without sugars), and for growth on various substrates. Strains were identified among these mutants that had a faster doubling time, produced higher maximum ammonia levels (enzyme assay) and more oil (Sudan Black assay), and had higher maximum soluble protein levels (Bradford assay) than wild type. When grown on plates with substrates of interest, all mutant strains showed similar results aerobically to wild-type strain. The mutant strain with the highest oil production and the fastest doubling time was evaluated on coffee waste medium. On this medium, the strain produced 0.12 g/L ammonia and 0.20 g/L 2-phenylethanol, a valuable fragrance/flavoring, in addition to acylglycerols (oil) containing predominantly C16 and C18 residues. These mutant strains will be investigated further for potential application in commercial biodiesel production.

D-stat culture for studying the metabolic shifts from oxidative metabolism to lipid accumulation and citric acid production in Yarrowia lipolytica.

PubMed

Ochoa-Estopier, Abril; Guillouet, Stéphane E

2014-01-20

Lipid accumulation in oleaginous yeasts is triggered by nutrient imbalance in the culture medium between the carbon source in excess and the nitrogen source in limiting concentration. However Yarrowia lipolytica when cultivated on glucose as the sole carbon source, mainly produces citric acid upon nitrogen limitation over lipid accumulation (only 5-10% triacylglycerol). Therefore for developing bioprocess for the production of triacylglycerol from renewable carbon source as glucose it is of first importance to control this imbalance in order to avoid citric acid production during TAG accumulation. Using D-stat cultivation system, where the N/C was linearly decreased using a constant change rate we were able to identify the N/C ratio inducing TAG accumulation (0.085NmolCmol(-1)) and citric acid (0.021NmolCmol(-1)). We therefore demonstrated that it was possible to accumulate lipids without excretion citric acid as long as the N/C was within this indicated range. Moreover enzyme specific activities measurement during the D-stat indicated that ATP-citrate lyase, malic enzyme and acetyl-coA carboxylase were strongly induced at the onset of lipid accumulation and showed different patterns when citric acid was excreted. Our results give relevant information for future industrial bioprocess development concerning the production of lipids using renewable carbohydrate substrates as an alternative way to produce synthons for fuel or chemical industry. By controlling the N/C over the fermentation process on glucose Y. lipolytica can accumulate lipids without excreting citric acid. Copyright © 2013 Elsevier B.V. All rights reserved.

Integrated Approach To Producing High-Purity Trehalose from Maltose by the Yeast Yarrowia lipolytica Displaying Trehalose Synthase (TreS) on the Cell Surface.

PubMed

Li, Ning; Wang, Hengwei; Li, Lijuan; Cheng, Huiling; Liu, Dawen; Cheng, Hairong; Deng, Zixin

2016-08-10

An alternative strategy that integrated enzyme production, trehalose biotransformation, and bioremoval in one bioreactor was developed in this study, thus simplifying the traditional procedures used for trehalose production. The trehalose synthase gene from a thermophilic archaea, Picrophilus torridus, was first fused to the YlPir1 anchor gene and then inserted into the genome of Yarrowia lipolytica, thus yielding an engineered yeast strain. The trehalose yield reached 73% under optimal conditions. The thermal and pH stabilities of the displayed enzyme were improved compared to those of its free form purified from recombinant Escherichia coli. After biotransformation, the glucose byproduct and residual maltose were directly fermented to ethanol by a Saccharomyces cerevisiae strain. Ethanol can be separated by distillation, and high-purity trehalose can easily be obtained from the fermentation broth. The results show that this one-pot procedure is an efficient approach to the economical production of trehalose from maltose.

Green and sustainable succinic acid production from crude glycerol by engineered Yarrowia lipolytica via agricultural residue based in situ fibrous bed bioreactor.

PubMed

Li, Chong; Gao, Shi; Yang, Xiaofeng; Lin, Carol Sze Ki

2018-02-01

In situ fibrous bed bioreactor (isFBB) for efficient succinic acid (SA) production by Yarrowia lipolytica was firstly developed in our former study. In this study, agricultural residues including wheat straw, corn stalk and sugarcane bagasse were investigated for the improvement of isFBB, and sugarcane bagasse was demonstrated to be the best immobilization material. With crude glycerol as the sole carbon source, optimization for isFBB batch fermentation was carried out. Under the optimal conditions of 20g sugarcane bagasse as immobilization material, 120gL -1 crude glycerol as carbon source and 4Lmin -1 of aeration rate, the resultant SA concentration was 53.6gL -1 with an average productivity of 1.45gL -1 h -1 and a SA yield of 0.45gg -1 . By feeding crude glycerol, SA titer up to 209.7gL -1 was obtained from fed batch fermentation, which was the highest value that ever reported. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mitochondria from Dipodascus (Endomyces) magnusii and Yarrowia lipolytica yeasts did not undergo a Ca²⁺-dependent permeability transition even under anaerobic conditions.

PubMed

Trendeleva, Tat'yana; Sukhanova, Evgeniya; Ural'skaya, Ludmila; Saris, Nils-Erik; Zvyagilskaya, Renata

2011-12-01

In this study we used tightly-coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts. The two yeast strains are good alternatives to Saccharomyces cerevisiae, being aerobes containing well structured mitochondria (thus ensuring less structural limitation to observe their appreciable swelling) and fully competent respiratory chain with three invariantly functioning energy conservation points, including Complex I, that can be involved in induction of the canonical Ca²⁺/P(i)-dependent mitochondrial permeability transition (mPTP pore) with an increased open probability when electron flux increases(Fontaine et al. J Biol Chem 273: 25734–25740, 1998; Bernardi et al. FEBS J 273:2077–2099, 2006). High amplitude swelling and collapse of the membrane potential were used as parameters for demonstrating pore opening. Previously (Kovaleva et al. J Bioenerg Biomembr 41:239–249, 2009; Kovaleva et al. Biochemistry (Moscow) 75: 297–303, 2010) we have shown that mitochondria from Y.lipolytica and D. magnusii were very resistant to the Ca²⁺overload combined with varying concentrations of P(i),palmitic acid, SH-reagents, carboxyatractyloside (an inhibitor of ADP/ATP translocator), as well as depletion of intramitochondrial adenine nucleotide pools, deenergization of mitochondria, and shifting to acidic pH values in the presence of high [P(i)]. Here we subjected yeast mitochondria to other conditions known to induce an mPTP in animal and plant mitochondria, namely to Ca²⁺ overload under hypoxic conditions (anaerobiosis). We were unable to observe Ca²⁺-induced high permeability of the inner membrane of D. magnusii and Y. lipolytica yeast mitochondria under anaerobic conditions, thus suggesting that an mPTP-like pore, if it ever occurs in yeast mitochondria, is not coupled with the Ca²⁺ uptake. The results provide the first demonstration of ATP-dependent energization of yeast mitochondria under conditions of anaerobiosis.

Integrating Cellular and Bioprocess Engineering in the Non-Conventional Yeast Yarrowia lipolytica for Biodiesel Production: A Review

PubMed Central

Xie, Dongming

2017-01-01

As one of the major biofuels to replace fossil fuel, biodiesel has now attracted more and more attention due to its advantages in higher energy density and overall less greenhouse gas generation. Biodiesel (fatty acid alkyl esters) is produced by chemically or enzymatically catalyzed transesterification of lipids from microbial cells, microalgae, oil crops, or animal fats. Currently, plant oils or waste cooking oils/fats remain the major source for biodiesel production via enzymatic route, but the production capacity is limited either by the uncertain supplement of plant oils or by the low or inconsistent quality of waste oils/fats. In the past decades, significant progresses have been made on synthesis of microalgae oils directly from CO2 via a photosynthesis process, but the production cost from any current technologies is still too high to be commercialized due to microalgae’s slow growth rate on CO2, inefficiency in photo-bioreactors, lack of efficient contamination control methods, and high cost in downstream recovery. At the same time, many oleaginous microorganisms have been studied to produce lipids via the fatty acid synthesis pathway under aerobic fermentation conditions, among them one of the most studied is the non-conventional yeast, Yarrowia lipolytica, which is able to produce fatty acids at very high titer, rate, and yield from various economical substrates. This review summarizes the recent research progresses in both cellular and bioprocess engineering in Y. lipolytica to produce lipids at a low cost that may lead to commercial-scale biodiesel production. Specific technologies include the strain engineering for using various substrates, metabolic engineering in high-yield lipid synthesis, cell morphology study for efficient substrate uptake and product formation, free fatty acid formation and secretion for improved downstream recovery, and fermentation engineering for higher productivities and less operating cost. To further improve the

Deciphering how LIP2 and POX2 promoters can optimally regulate recombinant protein production in the yeast Yarrowia lipolytica.

PubMed

Sassi, Hosni; Delvigne, Frank; Kar, Tambi; Nicaud, Jean-Marc; Coq, Anne-Marie Crutz-Le; Steels, Sebastien; Fickers, Patrick

2016-09-20

In recent years, the non-conventional model yeast species Yarrowia lipolytica has received much attention because it is a useful cell factory for producing recombinant proteins. In this species, expression vectors involving LIP2 and POX2 promoters have been developed and used successfully for protein production at yields similar to or even higher than those of other cell factories, such as Pichia pastoris. However, production processes involving these promoters can be difficult to manage, especially if carried out at large scales in fed-batch bioreactors, because they require hydrophobic inducers, such as oleic acid or methyl oleate. Thus, the challenge has become to reduce loads of hydrophobic substrates while simultaneously promoting recombinant protein production. One possible solution is to replace a portion of the inducer with a co-substrate that can serve as an alternative energy source. However, implementing such an approach would require detailed knowledge of how carbon sources impact promoter regulation, which is surprisingly still lacking for the LIP2 and POX2 promoters. This study's aim was thus to better characterize promoter regulation and cell metabolism in Y. lipolytica cultures grown in media supplemented with different carbon sources. pPOX2 induction could be detected when glucose or glycerol was used as sole carbon source, which meant these carbon source could not prevent promoter induction. In addition, when a mixture of glucose and oleic acid was used in complex medium, pPOX2 induction level was lower that that of pLIP2. In contrast, pLIP2 induction was absent when glucose was present in the culture medium, which meant that cell growth could occur without any recombinant gene expression. When a 40/60 mixture of glucose and oleic acid (w/w) was used, a tenfold increase in promoter induction, as compared to when an oleic-acid-only medium was observed. It was also clear that individual cells were adapting metabolically to use both glucose and oleic

An ultraviolet spectrophotometric assay for the screening of sn-2-specific lipases using 1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol as substrate

PubMed Central

Mendoza, Lilia D.; Rodriguez, Jorge A.; Leclaire, Julien; Buono, Gerard; Fotiadu, Frédéric; Carrière, Frédéric; Abousalham, Abdelkarim

2012-01-01

In the present study, we propose a continuous assay for the screening of sn-2 lipases by using triacylglycerols (TAGs) from Aleurites fordii seed (tung oil) and a synthetic TAG containing the α-eleostearic acid at the sn-2 position and the oleic acid (OA) at the sn-1 and sn-3 positions [1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol (sn-OEO)]. Each TAG was coated into a microplate well, and the lipase activity was measured by optical density increase at 272 nm due to transition of α-eleostearic acid from the adsorbed to the soluble state. The sn-1,3-regioselective lipases human pancreatic lipase (HPL), LIP2 lipase from Yarrowia lipolytica (YLLIP2), and a known sn-2 lipase, Candida antarctica lipase A (CALA) were used to validate this method. TLC analysis of lipolysis products showed that the lipases tested were able to hydrolyze the sn-OEO and the tung oil TAGs, but only CALA hydrolyzed the sn-2 position. The ratio of initial velocities on sn-OEO and tung oil TAGs was used to estimate the sn-2 preference of lipases. CALA was the enzyme with the highest ratio (0.22 ± 0.015), whereas HPL and YLLIP2 showed much lower ratios (0.072 ± 0.026 and 0.038 ± 0.016, respectively). This continuous sn-2 lipase assay is compatible with a high sample throughput and thus can be applied to the screening of sn-2 lipases. PMID:22114038

Citric acid production from partly deproteinized whey under non-sterile culture conditions using immobilized cells of lactose-positive and cold-adapted Yarrowia lipolytica B9.

PubMed

Arslan, Nazli Pinar; Aydogan, Mehmet Nuri; Taskin, Mesut

2016-08-10

The present study was performed to produce citric acid (CA) from partly deproteinized cheese whey (DPCW) under non-sterile culture conditions using immobilized cells of the cold-adapted and lactose-positive yeast Yarrowia lipolytica B9. DPCW was prepared using the temperature treatment of 90°C for 15min. Sodium alginate was used as entrapping agent for cell immobilization. Optimum conditions for the maximum CA production (33.3g/L) in non-sterile DPCW medium were the temperature of 20°C, pH 5.5, additional lactose concentration of 20g/L, sodium alginate concentration of 2%, number of 150 beads/100mL and incubation time of 120h. Similarly, maximum citric acid/isocitric acid (CA/ICA) ratio (6.79) could be reached under these optimal conditions. Additional nitrogen and phosphorus sources decreased CA concentration and CA/ICA ratio. Immobilized cells were reused in three continuous reaction cycles without any loss in the maximum CA concentration. The unique combination of low pH and temperature values as well as cell immobilization procedure could prevent undesired microbial contaminants during CA production. This is the first work on CA production by cold-adapted microorganisms under non-sterile culture conditions. Besides, CA production using a lactose-positive strain of the yeast Y. lipolytica was investigated for the first time in the present study. Copyright © 2016 Elsevier B.V. All rights reserved.

Induction of a non-specific permeability transition in mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts.

PubMed

Kovaleva, Mariya V; Sukhanova, Evgeniya I; Trendeleva, Tatyana A; Zyl'kova, Marina V; Ural'skaya, Ludmila A; Popova, Kristina M; Saris, Nils-Erik L; Zvyagilskaya, Renata A

2009-06-01

In this study we used tightly-coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, possessing a respiratory chain with the usual three points of energy conservation. High-amplitude swelling and collapse of the membrane potential were used as parameters for demonstrating induction of the mitochondrial permeability transition due to opening of a pore (mPTP). Mitochondria from Y. lipolytica, lacking a natural mitochondrial Ca(2+) uptake pathway, and from D. magnusii, harboring a high-capacitive, regulated mitochondrial Ca(2+) transport system (Bazhenova et al. J Biol Chem 273:4372-4377, 1998a; Bazhenova et al. Biochim Biophys Acta 1371:96-100, 1998b; Deryabina and Zvyagilskaya Biochemistry (Moscow) 65:1352-1356, 2000; Deryabina et al. J Biol Chem 276:47801-47806, 2001) were very resistant to Ca(2+) overload. However, exposure of yeast mitochondria to 50-100 microM Ca(2+) in the presence of the Ca(2+) ionophore ETH129 induced collapse of the membrane potential, possibly due to activation of the fatty acid-dependent Ca(2+)/nH(+)-antiporter, with no classical mPTP induction. The absence of response in yeast mitochondria was not simply due to structural limitations, since large-amplitude swelling occurred in the presence of alamethicin, a hydrophobic, helical peptide, forming voltage-sensitive ion channels in lipid membranes. Ca(2+)- ETH129-induced activation of the Ca(2+)/H(+)-antiport system was inhibited and prevented by bovine serum albumin, and partially by inorganic phosphate and ATP. We subjected yeast mitochondria to other conditions known to induce the permeability transition in animal mitochondria, i.e., Ca(2+) overload (in the presence of ETH129) combined with palmitic acid (Mironova et al. J Bioenerg Biomembr 33:319-331, 2001; Sultan and Sokolove Arch Biochem Biophys 386:37-51, 2001), SH-reagents, carboxyatractyloside (an inhibitor of the ADP/ATP translocator), depletion of intramitochondrial adenine nucleotide pools

Storage lipids of yeasts: a survey of nonpolar lipid metabolism in Saccharomyces cerevisiae, Pichia pastoris, and Yarrowia lipolytica.

PubMed

Koch, Barbara; Schmidt, Claudia; Daum, Günther

2014-09-01

Biosynthesis and storage of nonpolar lipids, such as triacylglycerols (TG) and steryl esters (SE), have gained much interest during the last decades because defects in these processes are related to severe human diseases. The baker's yeast Saccharomyces cerevisiae has become a valuable tool to study eukaryotic lipid metabolism because this single-cell microorganism harbors many enzymes and pathways with counterparts in mammalian cells. In this article, we will review aspects of TG and SE metabolism and turnover in the yeast that have been known for a long time and combine them with new perceptions of nonpolar lipid research. We will provide a detailed insight into the mechanisms of nonpolar lipid synthesis, storage, mobilization, and degradation in the yeast S. cerevisiae. The central role of lipid droplets (LD) in these processes will be addressed with emphasis on the prevailing view that this compartment is more than only a depot for TG and SE. Dynamic and interactive aspects of LD with other organelles will be discussed. Results obtained with S. cerevisiae will be complemented by recent investigations of nonpolar lipid research with Yarrowia lipolytica and Pichia pastoris. Altogether, this review article provides a comprehensive view of nonpolar lipid research in yeast. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Citric acid production from extract of Jerusalem artichoke tubers by the genetically engineered yeast Yarrowia lipolytica strain 30 and purification of citric acid.

PubMed

Wang, Ling-Fei; Wang, Zhi-Peng; Liu, Xiao-Yan; Chi, Zhen-Ming

2013-11-01

In this study, citric acid production from extract of Jerusalem artichoke tubers by the genetically engineered yeast Yarrowia lipolytica strain 30 was investigated. After the compositions of the extract of Jerusalem artichoke tubers for citric acid production were optimized, the results showed that natural components of extract of Jerusalem artichoke tubers without addition of any other components were suitable for citric acid production by the yeast strain. During 10 L fermentation using the extract containing 84.3 g L(-1) total sugars, 68.3 g L(-1) citric acid was produced and the yield of citric acid was 0.91 g g(-1) within 336 h. At the end of the fermentation, 9.2 g L(-1) of residual total sugar and 2.1 g L(-1) of reducing sugar were left in the fermented medium. At the same time, citric acid in the supernatant of the culture was purified. It was found that 67.2 % of the citric acid in the supernatant of the culture was recovered and purity of citric acid in the crystal was 96 %.

Cloning, expression and characterization of a lipase gene from marine bacterium Pseudoalteromonas lipolytica SCSIO 04301

NASA Astrophysics Data System (ADS)

Su, Hongfei; Mai, Zhimao; Zhang, Si

2016-12-01

A lipase gene, lip1233, isolated from Pseudoalteromonas lipolytica SCSIO 04301, was cloned and expressed in E. coli. The enzyme comprised 810 amino acid residues with a deduced molecular weight of 80 kDa. Lip1233 was grouped into the lipase family X because it contained a highly conserved motif GHSLG. The recombinant enzyme was purified with Ni-NTA affinity chromatography. The optimal temperature and pH value of Lip1233 were 45°C and 8.0, respectively. It retained more than 70% of original activity after being incubated in pH ranging from 6.0 to 9.5 for 30 min. It was stable when the temperature was below 45°C, but was unstable when the temperature was above 55°C. Most metal ions tested had no significant effect on the activity of Lip1233. Lip1233 remained more than original activity in some organic solvents at the concentration of 30% (v/v). It retained more than 30% activity after incubated in pure organic solvents for 12 h, while in hexane the activity was nearly 100%. Additionally, Lip1233 exhibited typical halotolerant characteristic as it was active under 4M NaCl. Lip1233 powder could catalyze efficiently the synthesis of fructose esters in hexane at 40°C. These characteristics demonstrated that Lip1233 is applicable to elaborate food processing and organic synthesis.

Multi-responses optimization of simultaneous biosorption of cationic dyes by live yeast Yarrowia lipolytica 70562 from binary solution: Application of first order derivative spectrophotometry.

PubMed

Dil, Ebrahim Alipanahpour; Ghaedi, Mehrorang; Ghezelbash, Gholam Reza; Asfaram, Arash

2017-05-01

Present study is based on application of live yeast Yarrowia lipolytica 70562 as new biosorbent was investigated for the simultaneous biosorption of Crystal Violet (CV) and Brilliant Green (BG) from wastewater. The effect of operating parameters such as initial dye concentrations (6-14mgL -1 ), solution pH (4.0-8.0) and contact time (4-20h) was investigated by response surface methodology (RSM) for modeling and optimization of biosorption process and accordingly the best operational conditions was set as: initial CV and BG concentration of 8.0, and 10mgL -1 , pH of 7.0 and contact time of 16h. Above specified conditions lead to achievement of maximum biosorption of 98.823% and 99.927% for CV and BG dyes, respectively. The experimental equilibrium data well explained according to Langmuir isotherm model with maximum biosorption capacity of 65.359 and 56.497mgg -1 for BG and CV, respectively. The second order and intraparticle diffusion models as cooperative mechanism has high efficiency and performance for interpretation of real data. Copyright © 2017. Published by Elsevier Inc.

Comparative evaluation of 13 yeast species in the Yarrowia clade on lignocellulosic biomass hydrolysate and genetic engineering of inhibitor tolerant strains for lipid and biofuel production

USDA-ARS?s Scientific Manuscript database

Yarrowia lipolytica is an oleaginous yeast that has garnered interest for commercial production of single cell oil and other fatty acid-derived chemicals because of its GRAS status and genetic tractability. Three recent peer-reviewed studies have highlighted the possibility of lipid production by th...

Mutants of Yarrowia lipolytica NCIM 3589 grown on waste cooking oil as a biofactory for biodiesel production.

PubMed

Katre, Gouri; Ajmera, Namasvi; Zinjarde, Smita; RaviKumar, Ameeta

2017-10-24

Oleaginous yeasts are fast emerging as a possible feedstock for biodiesel production. Yarrowia lipolytica, a model oleaginous yeast is known to utilize a variety of hydrophobic substrates for lipid accumulation including waste cooking oil (WCO). Approaches to increase lipid content in this yeast include metabolic engineering which requires manipulation of multiple genes in the lipid biosynthesis pathway. A classical and cost-effective approach, namely, random chemical mutagenesis on the yeast can lead to increased production of biodiesel as is explored here. In this study, chemical mutagenesis using the alkylating agent, N- methyl-N'-nitro-N-nitrosoguanidine (MNNG) as well as an additional treatment with cerulenin, a fatty acid synthase inhibitor generated 800 mutants of Y. lipolytica NCIM 3589 (761 MNNG treated and 39 MNNG + cerulenin treated). A three-stage screening using Sudan Black B plate technique, Nile red fluorimetry and total lipid extraction using solvent was performed, which enabled selection of ten high lipid yielding mutants. Time course studies of all the ten mutants were further undertaken in terms of biomass, lipid yield and lipid content to select three stable mutants (YlB6, YlC7 and YlE1) capable of growing and accumulating lipid on WCO, with lipid contents of 55, 60 and 67% as compared to 45% for the wild type. The mutants demonstrated increased volumetric lipid productivities (0.062, 0.044 and 0.041 g L -1  h -1 ) as compared to the wild type (0.033 g L -1  h -1 ). The fatty acid profile of the three mutants consisted of a high content of C16 and C18 saturated and monounsaturated fatty acids and was found to be suitable for biodiesel production. The fuel properties, namely, density, kinematic viscosity, total acid number, iodine value of the three mutants were evaluated and found to lie within the limits specified by internationally accepted standards. Additionally, it was noted that the mutants demonstrated better cetane numbers and

Novel Strategy of Using Methyl Esters as Slow Release Methanol Source during Lipase Expression by mut+ Pichia pastoris X33

PubMed Central

Kumari, Arti; Gupta, Rani

2014-01-01

One of the major issues with heterologous production of proteins in Pichia pastoris X33 under AOX1 promoter is repeated methanol induction. To obviate repeated methanol induction, methyl esters were used as a slow release source of methanol in lipase expressing mut+ recombinant. Experimental design was based on the strategy that in presence of lipase, methyl esters can be hydrolysed to release their products as methanol and fatty acid. Hence, upon break down of methyl esters by lipase, first methanol will be used as a carbon source and inducer. Then P. pastoris can switch over to fatty acid as a carbon source for multiplication and biomass maintenance till further induction by methyl esters. We validated this strategy using recombinant P. pastoris expressing Lip A, Lip C from Trichosporon asahii and Lip11 from Yarrowia lipolytica. We found that the optimum lipase yield under repeated methanol induction after 120 h was 32866 U/L, 28271 U/L and 21978 U/L for Lip C, Lip A and Lip 11 respectively. In addition, we found that a single dose of methyl ester supported higher production than repeated methanol induction. Among various methyl esters tested, methyl oleate (0.5%) caused 1.2 fold higher yield for LipA and LipC and 1.4 fold for Lip11 after 120 h of induction. Sequential utilization of methanol and oleic acid by P. pastoris was observed and was supported by differential peroxisome proliferation studies by transmission electron microscopy. Our study identifies a novel strategy of using methyl esters as slow release methanol source during lipase expression. PMID:25170843

Phenol Is the Initial Product Formed during Growth and Degradation of Bromobenzene by Tropical Marine Yeast, Yarrowia lipolytica NCIM 3589 via an Early Dehalogenation Step.

PubMed

Vatsal, Aakanksha A; Zinjarde, Smita S; RaviKumar, Ameeta

2017-01-01

Bromobenzene (BrB), a hydrophobic, recalcitrant organic compound, is listed by the environmental protection agencies as an environmental and marine pollutant having hepatotoxic, mutagenic, teratogenic, and carcinogenic effects. The tropical marine yeast Yarrowia lipolytica 3589 was seen to grow aerobically on BrB and displayed a maximum growth rate (μ max ) of 0.04 h -1 . Furthermore, we also observed an increase in cell size and sedimentation velocity for the cells grown on BrB as compared to the glucose grown cells. The cells attached to the hydrophobic bromobenzene droplets through its hydrophobic and acid-base interactions. The BrB (0.5%, 47.6 mM) was utilized by the cells with the release of a corresponding amount of bromide (12.87 mM) and yielded a cell mass of 1.86 g/L after showing 34% degradation in 96 h. Maximum dehalogenase activity of 16.16 U/mL was seen in the cell free supernatant after 24 h of growth. Identification of metabolites formed as a result of BrB degradation, namely, phenol, catechol, cis, cis muconic acid, and carbon dioxide were determined by LC-MS and GC-MS. The initial attack on bromobenzene by Y. lipolytica cells lead to the transient accumulation of phenol as an early intermediate which is being reported for the first time. Degradation of phenol led to catechol which was degraded by the ortho- cleavage pathway forming cis, cis muconic acid and then to Krebs cycle intermediates eventually leading to CO 2 production. The study shows that dehalogenation via an extracellular dehalogenase occurs prior to ring cleavage with phenol as the preliminary degradative compound being produced. The yeast was also able to grow on the degradative products, i.e., phenol and catechol, to varying degrees which would be of potential relevance in the degradation and remediation of xenobiotic environmental bromoaromatic pollutants such as bromobenzene.

Protease and lipase activities of fungal and bacterial strains derived from an artisanal raw ewe's milk cheese.

PubMed

Ozturkoglu-Budak, Sebnem; Wiebenga, Ad; Bron, Peter A; de Vries, Ronald P

2016-11-21

We previously identified the microbiota present during cheese ripening and observed high protease and lipase activity in Divle Cave cheese. To determine the contribution of individual isolates to enzyme activities, we investigated a range of species representing this microbiota for their proteolytic and lipolytic ability. In total, 17 fungal, 5 yeast and 18 bacterial strains, previously isolated from Divle Cave cheese, were assessed. Qualitative protease and lipase activities were performed on skim-milk agar and spirit-blue lipase agar, respectively, and resulted in a selection of strains for quantitative assays. For the quantitative assays, the strains were grown on minimal medium containing irradiated Divle Cave cheese, obtained from the first day of ripening. Out of 16 selected filamentous fungi, Penicillium brevicompactum, Penicillium cavernicola and Penicillium olsonii showed the highest protease activity, while Mucor racemosus was the best lipase producer. Yarrowia lipolytica was the best performing yeast with respect to protease and lipase activity. From the 18 bacterial strains, 14 and 11 strains, respectively showed protease and lipase activity in agar plates. Micrococcus luteus, Bacillus stratosphericus, Brevibacterium antiquum, Psychrobacter glacincola and Pseudomonas proteolytica displayed the highest protease and lipase activity. The proteases of yeast and filamentous fungi were identified as mainly aspartic protease by specific inhibition with Pepstatin A, whereas inhibition by PMSF (phenylmethylsulfonyl fluoride) indicated that most bacterial enzymes belong to serine type protease. Our results demonstrate that aspartic proteases, which usually have high milk clotting activity, are predominantly derived from fungal strains, and therefore fungal enzymes appear to be more suitable for use in the cheese industry. Microbial enzymes studied in this research might be alternatives for rennin (chymosin) from animal source because of their low cost and stable

Gene repression via multiplex gRNA strategy in Y. lipolytica.

PubMed

Zhang, Jin-Lai; Peng, Yang-Zi; Liu, Duo; Liu, Hong; Cao, Ying-Xiu; Li, Bing-Zhi; Li, Chun; Yuan, Ying-Jin

2018-04-20

The oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory due to their biochemical characteristics and native capacity to accumulate lipid-based chemicals. To create heterogenous biosynthesis pathway and manipulate metabolic flux in Y. lipolytica, numerous studies have been done for developing synthetic biology tools for gene regulation. CRISPR interference (CRISPRi), as an emerging technology, has been applied for specifically repressing genes of interest. In this study, we established CRISPRi systems in Y. lipolytica based on four different repressors, that was DNase-deactivated Cpf1 (dCpf1) from Francisella novicida, deactivated Cas9 (dCas9) from Streptococcus pyogenes, and two fusion proteins (dCpf1-KRAB and dCas9-KRAB). Ten gRNAs that bound to different regions of gfp gene were designed and the results indicated that there was no clear correlation between the repression efficiency and targeting sites no matter which repressor protein was used. In order to rapidly yield strong gene repression, a multiplex gRNAs strategy based on one-step Golden-brick assembly technology was developed. High repression efficiency 85% (dCpf1) and 92% (dCas9) were achieved in a short time by making three different gRNAs towards gfp gene simultaneously, which avoided the need of screening effective gRNA loci in advance. Moreover, two genes interference including gfp and vioE and three genes repression including vioA, vioB and vioE in protodeoxy-violaceinic acid pathway were also realized. Taken together, successful CRISPRi-mediated regulation of gene expression via four different repressors dCpf1, dCas9, dCpf1-KRAB and dCas9-KRAB in Y. lipolytica is achieved. And we demonstrate a multiplexed gRNA targeting strategy can efficiently achieve transcriptional simultaneous repression of several targeted genes and different sites of one gene using the one-step Golden-brick assembly. This timesaving method promised to be a potent transformative tool valuable for

Oleaginous yeast Yarrowia lipolytica culture with synthetic and food waste-derived volatile fatty acids for lipid production.

PubMed

Gao, Ruiling; Li, Zifu; Zhou, Xiaoqin; Cheng, Shikun; Zheng, Lei

2017-01-01

The sustainability of microbial lipids production from traditional carbon sources, such as glucose or glycerol, is problematic given the high price of raw materials. Considerable efforts have been directed to minimize the cost and find new alternative carbon sources. Volatile fatty acids (VFAs) are especially attractive raw materials, because they can be produced from a variety of organic wastes fermentation. Therefore, the use of volatile fatty acids as carbon sources seems to be a feasible strategy for cost-effective microbial lipid production. Lipid accumulation in Y. lipolytica using synthetic and food waste-derived VFAs as substrates was systematically compared and evaluated in batch cultures. The highest lipid content obtained with acetic, butyric, and propionic acids reached 31.62 ± 0.91, 28.36 ± 0.74, and 28.91 ± 0.66%, respectively. High concentrations of VFA inhibited cell growth in the following order: butyric acid > propionic acid > acetic acid. Within a 30-day experimental period, Y. lipolytica could adapt up to 20 g/L acetic acid, whereas the corresponding concentration of propionic acid and butyric acid were 10 and 5 g/L, respectively. Cultures on a VFA mixture showed that the utilization of different types of VFA by Y. lipolytica was not synchronized but rather performed in a step-wise manner. Although yeast fermentation is an exothermic process, and the addition of VFA will directly affect the pH of the system by increasing environmental acidity, cultures at a cultivation temperature of 38 °C and uncontrolled pH demonstrated that Y. lipolytica had high tolerance in the high temperature and acidic environment when a low concentration (2.5 g/L) of either synthetic or food waste-derived VFA was used. However, batch cultures fed with food fermentate yielded lower lipid content (18.23 ± 1.12%) and lipid productivity (0.12 ± 0.02 g/L/day). The lipid composition obtained with synthetic and food waste-derived VFA was similar to

A comparative analysis of single cell and droplet-based FACS for improving production phenotypes: Riboflavin overproduction in Yarrowia lipolytica.

PubMed

Wagner, James M; Liu, Leqian; Yuan, Shuo-Fu; Venkataraman, Maya V; Abate, Adam R; Alper, Hal S

2018-04-23

Evolutionary approaches to strain engineering inherently require the identification of suitable selection techniques for the product and phenotype of interest. In this work, we undertake a comparative analysis of two related but functionally distinct methods of high-throughput screening: traditional single cell fluorescence activated cell sorting (single cell FACS) and microdroplet-enabled FACS (droplet FACS) using water/oil/water (w/o/w) emulsions. To do so, we first engineer and evolve the non-conventional yeast Yarrowia lipolytica for high extracellular production of riboflavin (vitamin B2), an innately fluorescent product. Following mutagenesis and adaptive evolution, a direct parity-matched comparison of these two selection strategies was conducted. Both single cell FACS and droplet FACS led to significant increases in total riboflavin titer (32 and 54 fold relative to the parental PO1f strain, respectively). However, single cell FACS favored intracellular riboflavin accumulation (with only 70% of total riboflavin secreted) compared with droplet FACS that favored extracellular product accumulation (with 90% of total riboflavin secreted). We find that for the test case of riboflavin, the extent of secretion and total production were highly correlated. The resulting differences in production modes and levels clearly demonstrate the significant impact that selection approaches can exert on final evolutionary outcomes in strain engineering. Moreover, we note that these results provide a cautionary tale when intracellular read-outs of product concentration (including signals from biosensors) are used as surrogates for total production of potentially secreted products. In this regard, these results demonstrate that extracellular production is best assayed through an encapsulation technique when performing high throughput screening. Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Phenol Is the Initial Product Formed during Growth and Degradation of Bromobenzene by Tropical Marine Yeast, Yarrowia lipolytica NCIM 3589 via an Early Dehalogenation Step

PubMed Central

Vatsal, Aakanksha A.; Zinjarde, Smita S.; RaviKumar, Ameeta

2017-01-01

Bromobenzene (BrB), a hydrophobic, recalcitrant organic compound, is listed by the environmental protection agencies as an environmental and marine pollutant having hepatotoxic, mutagenic, teratogenic, and carcinogenic effects. The tropical marine yeast Yarrowia lipolytica 3589 was seen to grow aerobically on BrB and displayed a maximum growth rate (μmax) of 0.04 h-1. Furthermore, we also observed an increase in cell size and sedimentation velocity for the cells grown on BrB as compared to the glucose grown cells. The cells attached to the hydrophobic bromobenzene droplets through its hydrophobic and acid–base interactions. The BrB (0.5%, 47.6 mM) was utilized by the cells with the release of a corresponding amount of bromide (12.87 mM) and yielded a cell mass of 1.86 g/L after showing 34% degradation in 96 h. Maximum dehalogenase activity of 16.16 U/mL was seen in the cell free supernatant after 24 h of growth. Identification of metabolites formed as a result of BrB degradation, namely, phenol, catechol, cis, cis muconic acid, and carbon dioxide were determined by LC–MS and GC–MS. The initial attack on bromobenzene by Y. lipolytica cells lead to the transient accumulation of phenol as an early intermediate which is being reported for the first time. Degradation of phenol led to catechol which was degraded by the ortho- cleavage pathway forming cis, cis muconic acid and then to Krebs cycle intermediates eventually leading to CO2 production. The study shows that dehalogenation via an extracellular dehalogenase occurs prior to ring cleavage with phenol as the preliminary degradative compound being produced. The yeast was also able to grow on the degradative products, i.e., phenol and catechol, to varying degrees which would be of potential relevance in the degradation and remediation of xenobiotic environmental bromoaromatic pollutants such as bromobenzene. PMID:28690604

Co-expression of Exo-inulinase and Endo-inulinase Genes in the Oleaginous Yeast Yarrowia lipolytica for Efficient Single Cell Oil Production from Inulin.

PubMed

Shi, Nianci; Mao, Weian; He, Xiaoxia; Chi, Zhe; Chi, Zhenming; Liu, Guanglei

2018-05-01

Yarrowia lipolytica is a promising platform for the single cell oil (SCO) production. In this study, a transformant X+N8 in which exo- and endo-inulinase genes were co-expressed could produce an inulinase activity of 124.33 U/mL within 72 h. However, the inulinase activity of a transformant X2 carrying a single exo-inulinase gene was only 47.33 U/mL within 72 h. Moreover, the transformant X+N8 could accumulate 48.13% (w/w) SCO from inulin and the cell dry weight reached 13.63 g/L within 78 h, which were significantly higher than those of the transformant X2 (41.87% (w/w) and 11.23 g/L) under the same conditions. In addition, inulin hydrolysis and utilization of the transformant X+N8 were also more efficient than those of the transformant X2 during the fermentation process. These results demonstrated that the co-expression of the exo- and endo-inulinase genes significantly enhanced the SCO production from inulin due to the improvement of the inulinase activity and the synergistic action of exo- and endo-inulinase. Besides, over 95.01% of the fatty acids from the transformant X+N8 were C16-C18, especially C18:1 (53.10%), suggesting that the fatty acids could be used as feedstock for biodiesel production.

Development of New Genetic Manipulation Tools for Metabolic Engineering of Diatoms

DTIC Science & Technology

2008-08-28

protein L41 has been shown in a variety of yeasts to be involved in resistance to anisomycin and cycloheximide 7𔄂. A conserved mutation from proline...atipitis Yarrowia lipolytica Candida troplcalis S. cerevisiae A. nidulans Oryza sativa Homo sapiens P. tricornutum T. pseudonana...Pichia stipitis Yarrowia lipolytica Candida troplcalis S. cerevisiae A. nidulans Oryza sativa Homo sapiens P

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Wei; Wei, Hui; Alahuhta, Markus

In order to develop a direct microbial sugar conversion platform for the production of lipids, drop-in fuels and chemicals from cellulosic biomass substrate, we chose Yarrowia lipolytica as a viable demonstration strain. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing sugars to produce lipids; however, it lacks the lignocellulose-degrading enzymes needed to break down biomass directly. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. The XynII and XlnD expressing Yarrowia strains exhibited an abilitymore » to grow on xylan mineral plates. This was shown by Congo Red staining of halo zones on xylan mineral plates. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action in converting xylan to xylose was observed when XlnD acted in concert with XynII. Finally, the successful expression of these xylanases in Yarrowia further advances us toward our goal to develop a direct microbial conversion process using this organism.« less

Production of Eight Different Hydride Complexes and Nitrite Release from 2,4,6-Trinitrotoluene by Yarrowia lipolytica▿ †

PubMed Central

Ziganshin, Ayrat M.; Gerlach, Robin; Borch, Thomas; Naumov, Anatoly V.; Naumova, Rimma P.

2007-01-01

2,4,6-Trinitrotoluene (TNT) transformation by the yeast strain Yarrowia lipolytica AN-L15 was shown to occur via two different pathways. Direct aromatic ring reduction was the predominant mechanism of TNT transformation, while nitro group reduction was observed to be a minor pathway. Although growth of Y. lipolytica AN-L15 was inhibited initially in the presence of TNT, TNT transformation was observed, indicating that the enzymes necessary for TNT reduction were present initially. Aromatic ring reduction resulted in the transient accumulation of eight different TNT-hydride complexes, which were characterized using high-performance liquid chromatography, UV-visible diode array detection, and negative-mode atmospheric pressure chemical ionization mass spectrometry (APCI-MS). APCI-MS analysis revealed three different groups of TNT-hydride complexes with molecular ions at m/z 227, 228, and 230, which correspond to TNT-mono- and dihydride complexes and protonated dihydride isomers, respectively. One of the three protonated dihydride complex isomers detected appears to release nitrite in the presence of strain AN-L15. This release of nitrite is of particular interest since it can provide a pathway towards complete degradation and detoxification of TNT. PMID:17933928

The Gene YALI0E20207g from Yarrowia lipolytica Encodes an N-Acetylglucosamine Kinase Implicated in the Regulated Expression of the Genes from the N-Acetylglucosamine Assimilatory Pathway

PubMed Central

Flores, Carmen-Lisset; Gancedo, Carlos

2015-01-01

The non-conventional yeast Yarrowia lipolytica possesses an ORF, YALI0E20207g, which encodes a protein with an amino acid sequence similar to hexokinases from different organisms. We have cloned that gene and determined several enzymatic properties of its encoded protein showing that it is an N-acetylglucosamine (NAGA) kinase. This conclusion was supported by the lack of growth in NAGA of a strain carrying a YALI0E20207g deletion. We named this gene YlNAG5. Expression of YlNAG5 as well as that of the genes encoding the enzymes of the NAGA catabolic pathway—identified by a BLAST search—was induced by this sugar. Deletion of YlNAG5 rendered that expression independent of the presence of NAGA in the medium and reintroduction of the gene restored the inducibility, indicating that YlNag5 participates in the transcriptional regulation of the NAGA assimilatory pathway genes. Expression of YlNAG5 was increased during sporulation and homozygous Ylnag5/Ylnag5 diploid strains sporulated very poorly as compared with a wild type isogenic control strain pointing to a participation of the protein in the process. Overexpression of YlNAG5 allowed growth in glucose of an Ylhxk1glk1 double mutant and produced, in a wild type background, aberrant morphologies in different media. Expression of the gene in a Saccharomyces cerevisiae hxk1 hxk2 glk1 triple mutant restored ability to grow in glucose. PMID:25816199

Evaluation of the Composition of Culture Medium for Yeast Biomass Production Using Raw Glycerol from Biodiesel Synthesis

PubMed Central

dos Santos, Elisane Odriosolla; Michelon, Mariano; Furlong, Eliana Badiale; Burkert, Janaína Fernandes de Medeiros; Kalil, Susana Juliano; Burkert, Carlos André Veiga

2012-01-01

The work herewith investigated the production of yeast biomass as a source of protein, using Yarrowia lipolytica NRRL YB-423 and raw glycerol from biodiesel synthesis as the main carbon source. A significant influence of glycerol concentration, initial pH and yeast extract concentration on biomass and protein content was observed according to the 2v5-1 fractional design. These factors were further evaluated using a central composite design and response surface methodology, and an empirical model for protein content was established and validated. The biomass of Yarrowia lipolytica NRRL YB-423 reached 19.5 ± 1.0 g/L in shaken flasks cultivation, with a protein content of 20.1 ± 0.6% (w/w). PMID:24031849

21 CFR 173.165 - Candida lipolytica.

Code of Federal Regulations, 2010 CFR

2010-04-01

... description for Candida lipolytica variety lipolytica listed in “The Yeasts—A Toxonomic Study,” 2d Ed. (1970... equivalent). Activate as follows: Slurry 900 grams of silica gel reagent with 2 liters of purified water in a 3-liter beaker. Cool the mixture and pour into a 80 × 900 chromatographic column with coarse fritted...

21 CFR 173.165 - Candida lipolytica.

Code of Federal Regulations, 2011 CFR

2011-04-01

... description for Candida lipolytica variety lipolytica listed in “The Yeasts—A Toxonomic Study,” 2d Ed. (1970... equivalent). Activate as follows: Slurry 900 grams of silica gel reagent with 2 liters of purified water in a 3-liter beaker. Cool the mixture and pour into a 80 × 900 chromatographic column with coarse fritted...

Yeast cell surface display for lipase whole cell catalyst and its applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Yun; Zhang, Rui; Lian, Zhongshuai

The cell surface display technique allows for the expression of target proteins or peptides on the microbial cell surface by fusing an appropriate protein as an anchoring motif. Yeast display systems, such as Pichia pastoris, Yarowia lipolytica and Saccharomyces cerevisiae, are ideal, alternative and extensive display systems with the advantage of simple genetic manipulation and post-translational modification of expressed heterologous proteins. Engineered yeasts show high performance characteristics and variant utilizations. Herein, we comprehensively summarize the variant factors affecting lipase whole cell catalyst activity and display efficiency, including the structure and size of target proteins, screening anchor proteins, type and chainmore » length of linkers, and the appropriate matching rules among the above-mentioned display units. Furthermore, we also address novel approaches to enhance stability and activity of recombinant lipases, such as VHb gene co-expression, multi-enzyme co-display technique, and the micro-environmental interference and self-assembly techniques. Finally, we represent the variety of applications of whole cell surface displayed lipases on yeast cells in non-aqueous phases, including synthesis of esters, PUFA enrichment, resolution of chiral drugs, organic synthesis and biofuels. We demonstrate that the lipase surface display technique is a powerful tool for functionalizing yeasts to serve as whole cell catalysts, and increasing interest is providing an impetus for broad application of this technique.« less

Origins of cell-to-cell bioprocessing diversity and implications of the extracellular environment revealed at the single-cell level

DOE PAGES

Vasdekis, A. E.; Silverman, A. M.; Stephanopoulos, G.

2015-12-14

We probed the lipid expression dynamics of the oleaginous yeast Yarrowia Lipolytica. We observed that neutral lipid expression is sporadic. By performing single-cell analysis, we found that such noise emanates from the metabolic reaction level. Our results provide an alternative insight into the regulation and phenotypic variability of lipogenesis.

Complementation and Genetic Recombination in Candida lipolytica

PubMed Central

Bassel, John; Warfei, Jean; Mortimer, Robert

1971-01-01

Nutritional requirements were introduced into wild-type, heterothallic strains of Candida lipolytica by exposing the cells to X rays. Complementing hybrids were recovered from mixtures of the auxotrophic strains, and genetic recombination was observed in individually isolated ascospores from the hybrid strains. PMID:5122814

Gene cloning and characterization of a novel esterase from activated sludge metagenome

PubMed Central

2009-01-01

A metagenomic library was prepared using pCC2FOS vector containing about 3.0 Gbp of community DNA from the microbial assemblage of activated sludge. Screening of a part of the un-amplified library resulted in the finding of 1 unique lipolytic clone capable of hydrolyzing tributyrin, in which an esterase gene was identified. This esterase/lipase gene consists of 834 bp and encodes a polypeptide (designated EstAS) of 277 amino acid residuals with a molecular mass of 31 kDa. Sequence analysis indicated that it showed 33% and 31% amino acid identity to esterase/lipase from Gemmata obscuriglobus UQM 2246 (ZP_02733109) and Yarrowia lipolytica CLIB122 (XP_504639), respectively; and several conserved regions were identified, including the putative active site, HSMGG, a catalytic triad (Ser92, His125 and Asp216) and a LHYFRG conserved motif. The EstAS was overexpressed, purified and shown to hydrolyse p-nitrophenyl (NP) esters of fatty acids with short chain lengths (≤ C8). This EstAS had optimal temperature and pH at 35°C and 9.0, respectively, by hydrolysis of p-NP hexanoate. It also exhibited the same level of stability over wide temperature and pH ranges and in the presence of metal ions or detergents. The high level of stability of esterase EstAS with its unique substrate specificities make itself highly useful for biotechnological applications. PMID:20028524

Facile preparation of magnetic carbon nanotubes-immobilized lipase for highly efficient synthesis of 1,3-dioleoyl-2-palmitoylglycerol-rich human milk fat substitutes.

PubMed

Zheng, Mingming; Wang, Shi; Xiang, Xia; Shi, Jie; Huang, Juan; Deng, Qianchun; Huang, Fenghong; Xiao, Junyong

2017-08-01

In this study, Candida lipolytica lipase (CLL) was immobilized on magnetic multi-walled carbon nanotubes (mMWCNTs) via hydrophobic and cation-exchange interaction. The resultant immobilized CLL showed much better thermal stability, biocatalyst activity and easier recycling than the free form. A method for efficient enzymatic acidolysis of tripalmitin (PPP) with oleic acid (OA), to produce OPO-rich TAGs, was developed, using the immobilized CLL as the biocatalyst. Under optimized conditions (2% water, 20mg/ml enzyme, 1:6 PPP/OA, 50°C, 2h), the content of OPO in the final product reached 46.5%. CLL@mMWCNTs had a better activity and manipulative stability than commercial lipases. More importantly, the feasibility of CLL@mMWCNTs was also validated in the practical production of OPO-rich TAGs, using lard and restructured palm oil as the raw material. These results suggest that CLL@mMWCNTs is a promising biocatalyst for the OPO-rich TAGs production and will be helpful for the infant formula industry. Copyright © 2017 Elsevier Ltd. All rights reserved.

Adhesion of Bacillus subtilis and Pseudoalteromonas lipolytica to steel in a seawater environment and their effects on corrosion.

PubMed

Guo, Zhangwei; Liu, Tao; Cheng, Y Frank; Guo, Na; Yin, Yansheng

2017-09-01

In a marine environment, Bacillus subtilis and Pseudoalteromonas lipolytica are commonly found in the biofilms adherent to low-alloy engineering steel, and they have distinct effects on corrosion. In the present work, this phenomenon was investigated through the study of various materials characterization methods, electrochemical techniques, and contact angle measurements. It was found that the surface film formed on the steel in the presence of B. subtilis was compact, uniform, free of cracks, and hydrophobic. However, the film formed in the presence of P. lipolytica was loose, rough, heterogeneous, and hydrophilic. The main components of the films formed in the presence of B. subtilis and P. lipolytica were polysaccharides/TasA amyloid fibers and proteins/carboxylic acid, respectively. The composition, structure, and properties of the surface films formed on the steel were associated with different effects on corrosion. The presence of B. subtilis enhances the steel's resistance to corrosion, whereas corrosion was increased by the presence of P. lipolytica. In short, the compact and hydrophobic biofilm of B. subtilis appears to inhibit the corrosion of steel, while the loose, hydrophilic film of P. lipolytica tends to induce pitting corrosion. Copyright © 2017 Elsevier B.V. All rights reserved.

Occurrence and diversity of marine yeasts in Antarctica environments

NASA Astrophysics Data System (ADS)

Zhang, Xue; Hua, Mingxia; Song, Chunli; Chi, Zhenming

2012-03-01

A total of 28 yeast strains were obtained from the sea sediment of Antarctica. According to the results of routine identification and molecular characterization, the strains belonged to species of Yarrowia lipolytica, Debaryomyces hansenii, Rhodotorula slooffiae, Rhodotorula mucilaginosa, Sporidiobolus salmonicolor, Aureobasidium pullulans, Mrakia frigida and Guehomyces pullulans, respectively. The Antarctica yeasts have wide potential applications in biotechnology, for some of them can produce β-galactosidase and killer toxins.

Clinical, microbiological, and experimental animal studies of Candida lipolytica.

PubMed Central

Walsh, T J; Salkin, I F; Dixon, D M; Hurd, N J

1989-01-01

Candida lipolytica was recovered from six patients in three different clinical centers. The index isolate caused a persistent fungemia with catheter-associated Candida thrombophlebitis, the second isolate was from a polymicrobial sinusitis, and the remaining four isolates were involved in tissue colonization. These and 20 other isolates were consistent in their morphological and physiological characteristics. All formed true hyphae and blastoconidia on cornmeal-Tween 80 agar and all assimilated glucose, glycerol, and erythritol. In a murine model of disseminated candidiasis, the index isolate that caused clinical fungemia caused no mortality and produced only two lesions on a kidney, as determined at necropsy. The nine isolates selected for in vitro antifungal susceptibility studies had intermediate susceptibilities to amphotericin B but were susceptible to ketoconazole. We conclude that C. lipolytica is a weakly virulent pathogen which may require an intravascular foreign body to cause fungemia. Images PMID:2745702

Mechanisms of lipase maturation

PubMed Central

Péterfy, Miklós

2010-01-01

Lipases are acyl hydrolases that represent a diverse group of enzymes present in organisms ranging from prokaryotes to humans. This article focuses on an evolutionarily related family of extracellular lipases that include lipoprotein lipase, hepatic lipase and endothelial lipase. As newly synthesized proteins, these lipases undergo a series of co- and post-translational maturation steps occurring in the endoplasmic reticulum, including glycosylation and glycan processing, and protein folding and subunit assembly. This article identifies and discusses mechanisms that direct early and late events in lipase folding and assembly. Lipase maturation employs the two general chaperone systems operating in the endoplasmic reticulum, as well as a recently identified lipase-specific chaperone termed lipase maturation factor 1. We propose that the two general chaperone systems act in a coordinated manner early in lipase maturation in order to help create partially folded monomers; lipase maturation factor 1 then facilitates final monomer folding and subunit assembly into fully functional homodimers. Once maturation is complete, the lipases exit the endoplasmic reticulum and are secreted to extracellular sites, where they carry out a number of functions related to lipoprotein and lipid metabolism. PMID:20543905

Lipase-specific foldases.

PubMed

Rosenau, Frank; Tommassen, Jan; Jaeger, Karl-Erich

2004-02-06

Lipases represent the most important class of enzymes used in biotechnology. Many bacteria produce and secrete lipases but the enzymes originating from Pseudomonas and Burkholderia species seem to be particularly useful for a wide variety of different biocatalytic applications. These enzymes are usually encoded in an operon together with a second gene which codes for a lipase-specific foldase, Lif, which is necessary to obtain enzymatically active lipase. A detailed analysis based on amino acid homology has suggested the classification of Lif proteins into four different families and also revealed the presence of a conserved motif, Rx1x2FDY(F/C)L(S/T)A. Recent experimental evidence suggests that Lifs are so-called steric chaperones, which exert their physiological function by lowering energetic barriers during the folding of their cognate lipases, thereby providing essential steric information needed to fold lipases into their enzymatically active conformation.

The growth, properties and interactions of yeasts and bacteria associated with the maturation of Camembert and blue-veined cheeses.

PubMed

Addis, E; Fleet, G H; Cox, J M; Kolak, D; Leung, T

2001-09-19

The growth of yeasts and bacteria were monitored during the maturation of Camembert and blue-veined cheese produced in Australia. Yeasts were prominent throughout maturation, growing to 10(5)-10(9)/g, depending on the manufacturer. Debaryomyces hansenii predominated, but there were lesser, inconsistent contributions from Yarrowia lipolytica. Of the non-lactic acid bacteria, Acinetobacter species were significant during the maturation of Camembert but not blue-veined cheeses, and grew to 10(6)-10(8) cfu/g. Staphylococcus and Micrococcus species were consistently isolated from the cheeses with Staphylococcus xylosus growing to 10(5)-10(9) cfu/g, depending on the product. Lactic acid bacteria (10(7)-10(9) cfu/g) were present throughout maturation but were not identified. Interactions between the various yeasts and bacterial isolates were examined. Several strains of D. hansenii exhibited killer activity but not against Y. lipolytica. None of the yeasts were antagonistic towards the bacteria but some strains of D. hansenii enhanced the growth of Y. lipolytica and S. xylosus. The yeast and bacterial isolates exhibited various degrees of extracellular proteolytic and lipolytic activities.

Evaluation of cellulose-binding domain fused to a lipase for the lipase immobilization.

PubMed

Hwang, Sangpill; Ahn, Jungoh; Lee, Sumin; Lee, Tai Gyu; Haam, Seungjoo; Lee, Kangtaek; Ahn, Ik-Sung; Jung, Joon-Ki

2004-04-01

A cellulose-binding domain (CBD) fragment of a cellulase gene of Trichoderma hazianum was fused to a lipase gene of Bacillus stearothermophilus L1 to make a gene cluster for CBD-BSL lipase. The specific activity of CBD-BSL lipase for oil hydrolysis increased by 33% after being immobilized on Avicel (microcrystalline cellulose), whereas those of CBD-BSL lipase and BSL lipase decreased by 16% and 54%, respectively, after being immobilized on silica gel. Although the loss of activity of an enzyme immobilized by adsorption has been reported previously, the loss of activity of the CBD-BSL lipase immobilized on Avicel was less than 3% after 12 h due to the irreversible binding of CBD to Avicel.

Lipase Maturation Factor 1 (Lmf1): Structure and Role in Lipase Folding and Assembly

PubMed Central

Ehrhardt, Nicole

2014-01-01

Purpose of review Lipase maturation factor 1 (LMF1) is a membrane-bound protein located in the endoplasmic reticulum (ER). It is essential to the folding and assembly (i.e., maturation) of a select group of lipases that include lipoprotein lipase (LPL), hepatic lipase (HL) and endothelial lipase (EL). The purpose of this review is to examine recent studies that have begun to elucidate the structure and function of LMF1, and to place it in the context of lipase folding and assembly. Recent findings Recent studies identified mutations in LMF1 that cause combined lipase deficiency and hypertriglyceridemia in humans. These mutations result in the truncation of a large, evolutionarily conserved domain called DUF1222, which is essential for interaction with lipases and their attainment of enzymatic activity. The structural complexity of LMF1 has been further characterized by solving its topology in the ER membrane. Recent studies indicate that in addition to LPL and HL, the maturation of EL is also dependent on LMF1. Based on its apparent specificity for dimeric lipases, LMF1 is proposed to play an essential role in the assembly and/or stabilization of head-to-tail lipase homodimers. Summary LMF1 functions in the maturation of a select group of secreted lipases that assemble into homodimers in the ER. These dimeric lipases include LPL, HL and EL, all of which contribute significantly to plasma triglyceride and HDL cholesterol levels in human populations. Future studies involving genetically engineered mouse models will be required to fully elucidate the role of LMF1 in normal physiology and disease. PMID:20224398

Influence of selected factors on browning of Camembert cheese.

PubMed

Carreira, Alexandra; Dillinger, Klaus; Eliskases-Lechner, Frieda; Loureiro, Virgílio; Ginzinger, Wolfgang; Rohm, Harald

2002-05-01

Experimental Camembert cheeses were made to investigate the effects on browning of the following factors: inoculation with Yarrowia lipolytica, the use of Penicillium candidum strains with different proteolytic activity, the addition of tyrosine, and the addition of Mn2+ thus leading to 16 different variants of cheese. Two physical colour parameters were used to describe browning, depending on the location in the cheeses: a whiteness index for the outside browning (mould mycelium), and a brownness index for the inside browning (surface of the cheese body). Mn2+ promoted a significant increase of browning at both locations, whereas Yar. lipolytica had the opposite effect. Outside browning was significantly more intense when using the Pen. candidum strain with higher proteolytic activity. A significant interaction was found between Yar. lipolytica and Pen. candidum. The yeast had no effect in combination with a low proteolytic strain of Pen. candidum, but significantly reduced proteolysis and browning in combination with a high proteolytic strain of Pen. candidum. We further confirmed that both strains of Pen. candidum were able to produce brown pigments from tyrosine and thus both are presumably responsible for the browning activity in this type of cheese.

Metabolic Engineering of Oleaginous Yeasts for Fatty Alcohol Production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Wei; Wei, Hui; Knoshaug, Eric

To develop pathways for advanced biological upgrading of sugars to hydrocarbons, we are seeking biological approaches to produce high carbon efficiency intermediates amenable to separations and catalytic upgrading to hydrocarbon fuels. In this study, we successfully demonstrated fatty alcohol production by oleaginous yeasts Yarrowia lipolytica and Lipomyces starkeyi by expressing a bacteria-derived fatty acyl-CoA reductase (FAR). Moreover, we find higher extracellular distribution of fatty alcohols produced by FAR-expressing L. starkeyi strain as compared to Y. lipolytica strain, which would benefit the downstream product recovery process. In both oleaginous yeasts, long chain length saturated fatty alcohols were predominant, accounting for moremore » than 85% of the total fatty alcohols produced. To the best of our knowledge, this is the first report of fatty alcohol production in L. starkeyi. Taken together, our work demonstrates that in addition to Y. lipolytica, L. starkeyi can also serve as a platform organism for production of fatty acid-derived biofuels and bioproducts via metabolic engineering. We believe strain and process development both will significantly contribute to our goal of producing scalable and cost-effective fatty alcohols from renewable biomass.« less

Pancreatic lipase-related protein 2 digests fats in human milk and formula in concert with gastric lipase and carboxyl ester lipase

PubMed Central

Johnson, Karin; Ross, Leah; Miller, Rita; Xiao, Xunjun; Lowe, Mark E.

2013-01-01

INTRODUCTION Dietary fats must be digested into fatty acids and monoacylglycerols prior to absorption. In adults, colipase-dependent pancreatic triglyceride lipase (PTL) contributes significantly to fat digestion. In newborn rodents and humans, the pancreas expresses low levels of PTL. In rodents, a homologue of PTL, pancreatic lipase related protein 2 (PLRP2) and carboxyl ester lipase (CEL) compensate for the lack of PTL. In human newborns, the role for PLRP2 in dietary fat digestion is unclear. To clarify the potential of human PLRP2 to influence dietary fat digestion in newborns, we determined PLRP2 activity against human milk and infant formula. METHODS The activity of purified recombinant PLRP2, gastric lipase and CEL against fats in human milk and formula was measured with each lipase alone and in combination with a standard pH-stat assay. RESULTS Colipase added to human milk stimulated fat digestion. PLRP2 and CEL had activity against human milk and formula. Pre-digestion with gastric lipase increased PLRP2 activity against both substrates. Together, CEL and PLRP2 activity was additive with formula and synergistic with human milk. CONCLUSIONS PLRP2 can digest fats in human milk and formula. PLRP2 acts in concert with CEL and gastric lipase to digest fats in human milk in vitro. PMID:23732775

Evolutionary relationships among pathogenic Candida species and relatives.

PubMed Central

Barns, S M; Lane, D J; Sogin, M L; Bibeau, C; Weisburg, W G

1991-01-01

Small subunit rRNA sequences have been determined for 10 of the most clinically important pathogenic species of the yeast genus Candida (including Torulopsis [Candida] glabrata and Yarrowia [Candida] lipolytica) and for Hansenula polymorpha. Phylogenetic analyses of these sequences and those of Saccharomyces cerevisiae, Kluyveromyces marxianus var. lactis, and Aspergillus fumigatus indicate that Candida albicans, C. tropicalis, C. parapsilosis, and C. viswanathii form a subgroup within the genus. The remaining significant pathogen, T. glabrata, falls into a second, distinct subgroup and is specifically related to S. cerevisiae and more distantly related to C. kefyr (psuedotropicalis) and K. marxianus var. lactis. The 18S rRNA sequence of Y. lipolytica has evolved rapidly in relation to the other Candida sequences examined and appears to be only distantly related to them. As anticipated, species of several other genera appear to bear specific relationships to members of the genus Candida. PMID:2007550

Acid Lipase Disease

MedlinePlus

... of Neurological Disorders and Stroke conducts and supports research to understand lipid storage diseases such as acid lipase deficiency and ... of Neurological Disorders and Stroke conducts and supports research to understand lipid storage diseases such as acid lipase deficiency and ...

[Industrial application of lipases].

PubMed

Bancerz, Renata

2017-01-01

The ability of lipases to perform specific reactions of transformation (biotransformation) makes these enzymes a useful tool used in many syntheses, for example: in the production of detergents, cosmetics, biosurfactants, in the oil-chemical, paper, dairy, food or pharmaceutical industries. Lipases are ubiquitous enzymes but only lipases produced by microorganisms are important for industrial applications due to their wide variety of properties such as stability in organic solvents, action under mild conditions, high substrate specificity and region- and enantioselectivity, as well as the relatively simple methods of their production in fermentors and recovery from the culture medium. This paper reviews the latest achievements in the production of lipases in the submerged fermentation and solid state fermentation using waste products from the agricultural industry. In addition, new applications of lipases were described, including those for the synthesis of biopolymers and biodiesel and for the production of enantiomeric pharmaceuticals, agrochemicals and flavoring compounds.

Identification and characterization of a triacylglycerol lipase in Arabidopsis homologous to mammalian acid lipases.

PubMed

El-Kouhen, Karim; Blangy, Stéphanie; Ortiz, Emilia; Gardies, Anne-Marie; Ferté, Natalie; Arondel, Vincent

2005-11-07

Triacylglycerol (TAG) lipases have been thoroughly characterized in mammals and microorganisms. By contrast, very little is known on plant TAG lipases. An Arabidopsis cDNA called AtLip1 (At2g15230), which exhibits strong homology to lysosomal acid lipase, was found to drive the synthesis of an active TAG lipase when expressed in the baculovirus system. The lipase had a maximal activity at pH 6 and the specific activity was estimated to be about 45 micromol min(-1) mg(-1) protein using triolein as a substrate. Knock-out mutant analysis showed no phenotype during germination indicating that this enzyme is fully dispensable for TAG storage breakdown during germination. Northern blot analyses indicated that the transcript is present in all tissues tested.

21 CFR 862.1465 - Lipase test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... Lipase test system. (a) Identification. A lipase test system is a device intended to measure the activity of the enzymes lipase in serum. Lipase measurements are used in diagnosis and treatment of diseases...

21 CFR 862.1465 - Lipase test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Lipase test system. (a) Identification. A lipase test system is a device intended to measure the activity of the enzymes lipase in serum. Lipase measurements are used in diagnosis and treatment of diseases...

Biodiesel production with immobilized lipase: A review.

PubMed

Tan, Tianwei; Lu, Jike; Nie, Kaili; Deng, Li; Wang, Fang

2010-01-01

Fatty acid alkyl esters, also called biodiesel, are environmentally friendly and show great potential as an alternative liquid fuel. Biodiesel is produced by transesterification of oils or fats with chemical catalysts or lipase. Immobilized lipase as the biocatalyst draws high attention because that process is "greener". This article reviews the current status of biodiesel production with immobilized lipase, including various lipases, immobilization methods, various feedstocks, lipase inactivation caused by short chain alcohols and large scale industrialization. Adsorption is still the most widely employed method for lipase immobilization. There are two kinds of lipase used most frequently especially for large scale industrialization. One is Candida antartica lipase immobilized on acrylic resin, and the other is Candida sp. 99-125 lipase immobilized on inexpensive textile membranes. However, to further reduce the cost of biodiesel production, new immobilization techniques with higher activity and stability still need to be explored. Copyright 2010 Elsevier Inc. All rights reserved.

Inhibition of Microbial Lipases by Fatty Acids

PubMed Central

Smith, J. L.; Alford, John A.

1966-01-01

Addition of lard or sodium oleate to the medium used for lipase production by Pseudomonas fragi resulted in a decreased accumulation of lipase in the culture supernatant fluid without affecting cell growth. The production and activity of lipase was inhibited by lard, sodium oleate, and the salts of other unsaturated fatty acids. Some divalent cations, Tweens, lecithin, and bovine serum prevented oleate inhibition, but did not reverse it. Similar inhibitory actions were observed with Geotrichum candidum lipase, but not with a staphylococcal lipase or pancreatic lipase. A protective effect by protein in crude enzyme preparations is indicated. The ability of oleate to lower surface tension does not appear to be related to its ability to inhibit lipase. PMID:5970458

High-oleate yeast oil without polyunsaturated fatty acids.

PubMed

Tsakraklides, Vasiliki; Kamineni, Annapurna; Consiglio, Andrew L; MacEwen, Kyle; Friedlander, Jonathan; Blitzblau, Hannah G; Hamilton, Maureen A; Crabtree, Donald V; Su, Austin; Afshar, Jonathan; Sullivan, John E; LaTouf, W Greg; South, Colin R; Greenhagen, Emily H; Shaw, A Joe; Brevnova, Elena E

2018-01-01

Oleate-enriched triacylglycerides are well-suited for lubricant applications that require high oxidative stability. Fatty acid carbon chain length and degree of desaturation are key determinants of triacylglyceride properties and the ability to manipulate fatty acid composition in living organisms is critical to developing a source of bio-based oil tailored to meet specific application requirements. We sought to engineer the oleaginous yeast Yarrowia lipolytica for production of high-oleate triacylglyceride oil. We studied the effect of deletions and overexpressions in the fatty acid and triacylglyceride synthesis pathways to identify modifications that increase oleate levels. Oleic acid accumulation in triacylglycerides was promoted by exchanging the native ∆9 fatty acid desaturase and glycerol-3-phosphate acyltransferase with heterologous enzymes, as well as deletion of the Δ12 fatty acid desaturase and expression of a fatty acid elongase. By combining these engineering steps, we eliminated polyunsaturated fatty acids and created a Y. lipolytica strain that accumulates triglycerides with > 90% oleate content. High-oleate content and lack of polyunsaturates distinguish this triacylglyceride oil from plant and algal derived oils. Its composition renders the oil suitable for applications that require high oxidative stability and further demonstrates the potential of Y. lipolytica as a producer of tailored lipid profiles.

Lipases in Medicine: An Overview.

PubMed

Loli, Heni; Narwal, Sunil Kumar; Saun, Nitin Kumar; Gupta, Reena

2015-01-01

Lipases are part of the family of hydrolases that act on carboxylic ester bonds. They are involved in catalyzing the hydrolysis of triglycerides (TG) into chylomicrons and very low density lipoprotein (VLDL) particles. Uses of lipases are evolving rapidly and currently they are reported to show high potential in medicine. Intensive study and investigations have led researchers to explore lipases for their use in substitution therapy, where in enzyme deficiency during diseased conditions is compensated by their external administration. In our body, they are used to break down fats present in food so that they can be absorbed in the intestine and deficiency of lipases leads to malabsorption of fats and fat-soluble vitamins. Lipases help a person who has cystic fibrosis, Alzheimer's disease, atherosclerosis and act as a candidate target for cancer prevention and therapy. They act as diagnostic tool and their presence or increasing levels can indicate certain infection or disease. Obesity causes metabolic disease and is a serious health problem around the world. Thus inhibiting digestive lipase to reduce fat absorption has become the main pharmacological approach to the treatment of obesity in recent years.

Immobilization of a Commercial Lipase from Penicillium camembertii (Lipase G) by Different Strategies

PubMed Central

Mendes, Adriano A.; Freitas, Larissa; de Carvalho, Ana Karine F.; de Oliveira, Pedro C.; de Castro, Heizir F.

2011-01-01

The objective of this work was to select the most suitable procedure to immobilize lipase from Penicillium camembertii (Lipase G). Different techniques and supports were evaluated, including physical adsorption on hydrophobic supports octyl-agarose, poly(hydroxybutyrate) and Amberlite resin XAD-4; ionic adsorption on the anionic exchange resin MANAE-agarose and covalent attachment on glyoxyl-agarose, MANAE-agarose cross-linked with glutaraldehyde, MANAE-agarose-glutaraldehyde, and epoxy-silica-polyvinyl alcohol composite. Among the tested protocols, the highest hydrolytic activity (128.2 ± 8.10 IU·g−1 of support) was achieved when the lipase was immobilized on epoxy-SiO2-PVA using hexane as coupling medium. Lipase immobilized by ionic adsorption on MANAE-agarose also gave satisfactory result, attaining 55.6 ± 2.60 IU·g−1 of support. In this procedure, the maximum loading of immobilized enzyme was 9.3 mg·g−1 of gel, and the highest activity (68.8 ± 2.70 IU·g−1 of support) was obtained when 20 mg of protein·g−1 was offered. Immobilization carried out in aqueous medium by physical adsorption on hydrophobic supports and covalent attachment on MANAE-agarose-glutaraldehyde and glyoxyl-agarose was shown to be unfeasible for Lipase G. Thermal stability tests revealed that the immobilized derivative on epoxy-SiO2-PVA composite using hexane as coupling medium had a slight higher thermal stability than the free lipase. PMID:21811674

Efficient biocatalyst by encapsulating lipase into nanoporous gold

PubMed Central

2013-01-01

Lipases are one of the most important biocatalysts for biotechnological applications. Immobilization is an efficient method to increase the stability and reusability of lipases. In this study, nanoporous gold (NPG), a new kind of nanoporous material with tunable porosity and excellent biocompatibility, was employed as an effective support for lipase immobilization. The pore size of NPG and adsorption time played key roles in the construction of lipase-NPG biocomposites. The morphology and composition of NPG before and after lipase loading are verified using a scanning electron microscope, equipped with an energy-dispersive X-ray spectrometer. The resulting lipase-NPG biocomposites exhibited excellent catalytic activity and remarkable reusability. The catalytic activity of the lipase-NPG biocomposite with a pore size of 35 nm had no decrease after ten recycles. Besides, the lipase-NPG biocomposite exhibited high catalytic activity in a broader pH range and higher temperature than that of free lipase. In addition, the leaching of lipase from NPG could be prevented by matching the protein’s diameter and pore size. Thus, the encapsulation of enzymes within NPG is quite useful for establishing new functions and will have wide applications for different chemical processes. PMID:23601503

21 CFR 184.1415 - Animal lipase.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Animal lipase. 184.1415 Section 184.1415 Food and....1415 Animal lipase. (a) Animal lipase (CAS Reg. No. 9001-62-1) is an enzyme preparation obtained from edible forestomach tissue of calves, kids, or lambs, or from animal pancreatic tissue. The enzyme...

Lipase Test: MedlinePlus Lab Test Information

MedlinePlus

... page: https://medlineplus.gov/labtests/lipasetest.html Lipase Test To use the sharing features on this page, please enable JavaScript. What is a lipase test? Lipase is a type of protein made by ...

Use of a fluorescent radiolabeled triacylglycerol as a substrate for lipoprotein lipase and hepatic triglyceride lipase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dousset, N.; Negre, A.; Salvayre, R.

1988-06-01

A fluorescent radiolabeled triacylglycerol has been synthesized by using a fluorescent fatty acid (pyrene decanoic acid) and a radiolabeled oleic acid. This analog of the natural substrate, 1(3)pyrene decanoic-2,3 (1,2)-dioleoyl-sn-glycerol, has been tested as substrate for determining lipoprotein lipase and hepatic triacylglycerol lipase activities in post-heparin plasma. Optimal conditions for the determination of the two post-heparin plasma lipases were similar to those using radiolabeled triolein. Using this substrate, both post-heparin lipases exhibited their characteristic properties (pH optimum and effect of inhibitors) and attacked external ester bonds (1 or 3) containing pyrene decanoic and oleic acids at a similar rate.

21 CFR 184.1415 - Animal lipase.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Animal lipase. 184.1415 Section 184.1415 Food and... Substances Affirmed as GRAS § 184.1415 Animal lipase. (a) Animal lipase (CAS Reg. No. 9001-62-1) is an enzyme preparation obtained from edible forestomach tissue of calves, kids, or lambs, or from animal pancreatic...

21 CFR 184.1415 - Animal lipase.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Animal lipase. 184.1415 Section 184.1415 Food and... Substances Affirmed as GRAS § 184.1415 Animal lipase. (a) Animal lipase (CAS Reg. No. 9001-62-1) is an enzyme preparation obtained from edible forestomach tissue of calves, kids, or lambs, or from animal pancreatic...

21 CFR 184.1415 - Animal lipase.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Animal lipase. 184.1415 Section 184.1415 Food and... Substances Affirmed as GRAS § 184.1415 Animal lipase. (a) Animal lipase (CAS Reg. No. 9001-62-1) is an enzyme preparation obtained from edible forestomach tissue of calves, kids, or lambs, or from animal pancreatic...

21 CFR 184.1415 - Animal lipase.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Animal lipase. 184.1415 Section 184.1415 Food and... Substances Affirmed as GRAS § 184.1415 Animal lipase. (a) Animal lipase (CAS Reg. No. 9001-62-1) is an enzyme preparation obtained from edible forestomach tissue of calves, kids, or lambs, or from animal pancreatic...

Neutrophil chemotaxis by Propionibacterium acnes lipase and its inhibition.

PubMed Central

Lee, W L; Shalita, A R; Suntharalingam, K; Fikrig, S M

1982-01-01

The chemoattraction of Propionibacterium acnes lipase for neutrophils and the effect of lipase inhibitor and two antibiotic agents on the chemotaxis were evaluated. Of the various fractions tested, partially purified lipase (fraction 2c) was the most active cytotaxin produced by P. acnes. Serum mediators were not required for the generation of chemotaxis by lipase in vitro. Diisopropyl phosphofluoridate at low concentration (10(-4) mM) completely inhibited lipase activity as well as polymorphonuclear leukocyte chemotaxis generated by lipase. Tetracycline hydrochloride and erythromycin base at concentrations of 10(-1) mM and 1 mM, respectively, caused 100% inhibition of PMN migration toward lipase or zymosan-activated serum. The inhibiting activity of the antibiotics was directed against cells independently of any effect on lipase. Chemotaxis by P. acnes lipase suggests a wider role for this enzyme in the inflammatory process and the pathogenesis of acne vulgaris. Images PMID:7054130

Multi-wavelength dye concentration determination for enzymatic assays: evaluation of chromogenic para-nitrophenol over a wide pH range.

PubMed

Max, Jean-Joseph; Meddeb-Mouelhi, Fatma; Beauregard, Marc; Chapados, Camille

2012-12-01

Enzymatic assays need robust, rapid colorimetric methods that can follow ongoing reactions. For this, we developed a highly accurate, multi-wavelength detection method that could be used for several systems. Here, it was applied to the detection of para-nitrophenol (pNP) in basic and acidic solutions. First, we confirmed by factor analysis that pNP has two forms, with unique spectral characteristics in the 240 to 600 nm range: Phenol in acidic conditions absorbs in the lower range, whereas phenolate in basic conditions absorbs in the higher range. Thereafter, the method was used for the determination of species concentration. For this, the intensity measurements were made at only two wavelengths with a microtiter plate reader. This yielded total dye concentration, species relative abundance, and solution pH value. The method was applied to an enzymatic assay. For this, a chromogenic substrate that generates pNP after hydrolysis catalyzed by a lipase from the fungus Yarrowia lipolytica was used. Over the pH range of 3-11, accurate amounts of acidic and basic pNP were determined at 340 and 405 nm, respectively. This method surpasses the commonly used single-wavelength assay at 405 nm, which does not detect pNP acidic species, leading to activity underestimations. Moreover, alleviation of this pH-related problem by neutralization is not necessary. On the whole, the method developed is readily applicable to rapid high-throughput of enzymatic activity measurements over a wide pH range.

Mechanism of acetaldehyde-induced deactivation of microbial lipases

PubMed Central

2011-01-01

Background Microbial lipases represent the most important class of biocatalysts used for a wealth of applications in organic synthesis. An often applied reaction is the lipase-catalyzed transesterification of vinyl esters and alcohols resulting in the formation of acetaldehyde which is known to deactivate microbial lipases, presumably by structural changes caused by initial Schiff-base formation at solvent accessible lysine residues. Previous studies showed that several lipases were sensitive toward acetaldehyde deactivation whereas others were insensitive; however, a general explanation of the acetaldehyde-induced inactivation mechanism is missing. Results Based on five microbial lipases from Candida rugosa, Rhizopus oryzae, Pseudomonas fluorescens and Bacillus subtilis we demonstrate that the protonation state of lysine ε-amino groups is decisive for their sensitivity toward acetaldehyde. Analysis of the diverse modification products of Bacillus subtilis lipases in the presence of acetaldehyde revealed several stable products such as α,β-unsaturated polyenals, which result from base and/or amino acid catalyzed aldol condensation of acetaldehyde. Our studies indicate that these products induce the formation of stable Michael-adducts at solvent-accessible amino acids and thus lead to enzyme deactivation. Further, our results indicate Schiff-base formation with acetaldehyde to be involved in crosslinking of lipase molecules. Conclusions Differences in stability observed with various commercially available microbial lipases most probably result from different purification procedures carried out by the respective manufacturers. We observed that the pH of the buffer used prior to lyophilization of the enzyme sample is of utmost importance. The mechanism of acetaldehyde-induced deactivation of microbial lipases involves the generation of α,β-unsaturated polyenals from acetaldehyde which subsequently form stable Michael-adducts with the enzymes. Lyophilization of

Biodegradable products by lipase biocatalysis.

PubMed

Linko, Y Y; Lämsä, M; Wu, X; Uosukainen, E; Seppälä, J; Linko, P

1998-11-18

The interest in the applications of biocatalysis in organic syntheses has rapidly increased. In this context, lipases have recently become one of the most studied groups of enzymes. We have demonstrated that lipases can be used as biocatalyst in the production of useful biodegradable compounds. A number of examples are given. 1-Butyl oleate was produced by direct esterification of butanol and oleic acid to decrease the viscosity of biodiesel in winter use. Enzymic alcoholysis of vegetable oils without additional organic solvent has been little investigated. We have shown that a mixture of 2-ethyl-1-hexyl esters can be obtained in a good yield by enzymic transesterification from rapeseed oil fatty acids for use as a solvent. Trimethylolpropane esters were also similarly synthesized as lubricants. Finally, the discovery that lipases can also catalyze ester syntheses and transesterification reactions in organic solvent systems has opened up the possibility of enzyme catalyzed production of biodegradable polyesters. In direct polyesterification of 1,4-butanediol and sebacic acid, polyesters with a mass average molar mass of the order of 56,000 g mol-1 or higher, and a maximum molar mass of about 130,000 g mol-1 were also obtained by using lipase as biocatalyst. Finally, we have demonstrated that also aromatic polyesters can be synthesized by lipase biocatalysis, a higher than 50,000 g mol-1 mass average molar mass of poly(1,6-hexanediyl isophthalate) as an example.

Lipase activity in stallion seminal plasma and the effect of lipase on stallion spermatozoa during storage at 5 degrees C.

PubMed

Carver, D A; Ball, B A

2002-11-01

Previous studies have demonstrated a detrimental effect of seminal plasma on the maintenance of motility of cooled equine spermatozoa; however, the mechanism for the adverse effect of seminal plasma during cooled storage remains undetermined. In goats, a glycoprotein component of bulbourethral gland secretion contains lipase activity that is detrimental to sperm motility when stored in skim milk-based extenders. The objective of the current study was to determine the amount of lipase activity in stallion seminal plasma and to determine the effect of added lipase on spermatozoal motility during cooled semen storage. In the first experiment, seminal plasma (1.0 ml) was assayed for lipase activity based upon hydrolysis of triglycerides (olive oil substrate) into free fatty acids and subsequent titration of pH change (SigmaDiagnostic Lipase Kit). Lipase activity in stallion seminal plasma was 0.36 +/- 0.02 Sigma units/ml, (mean + S.E.M.; n = 16 ejaculates from six stallions). In the second experiment, equine semen (three ejaculates from each of four stallions) was divided into five treatment aliquots. In Treatment 1, semen was extended 1:3 with nonfat dried skim milk extender (NFDSM). In treatment groups 2 through 5, spermatozoa were washed by centrifugation (300 x g for 15 min) and resuspended in NFDSM to a final concentration of 25 x 10(6) spermatozoa/ml. Porcine pancreatic lipase (pPL) was added to Treatment 3 (10 pPL units/ml), Treatment 4 (100 pPL units/ml) and Treatment 5 (100 pPL units/ml, heat inactivated at 100 degrees C for 5 min) while Treatment 2 had no pancreatic lipase added and served as the control. Samples were cooled slowly to 5 degrees C, and stored at 5 degrees C until evaluation. Sperm motility was evaluated at time 0, 24, 48 and 72 h by computerized semen analysis, and data were analyzed via repeated measures ANOVA. The addition of 100 units/ml but not 10 units/ml of pPL decreased (P < 0.01) total and progressive motility of stored sperm. Heat

Rat lingual lipase: partial purification, hydrolytic properties, and comparison with pancreatic lipase.

PubMed

Roberts, I M; Montgomery, R K; Carey, M C

1984-10-01

We have partially purified lingual lipase from the serous glands of rat tongue. With a combination of Triton X-100 extraction or Triton X-114 phase-separation techniques, Bio-Bead SM-2 treatment, dialysis, and gel filtration on Sephadex G-200 or Sephacryl S-300, we obtained a sparingly soluble lipid-free protein demonstrating hydrolytic activity against triglycerides and negligible phospholipase or cholesteryl esterase activities. Compared with homogenate, specific activities of the enzyme were enriched 3- to 5-fold prior to gel filtration and 10-fold after gel filtration. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration under denaturing conditions (6 M guanidine X HCl or 0.1% sodium dodecyl sulfate) revealed one major glycoprotein band with Mr approximately 50,000. Gel filtration of the active enzyme in 0.1% Triton X-100 gave an Mr approximately 270,000-300,000, suggesting extensive self-aggregation. With both tributyrin and triolein, the pH optimum of the purified enzyme was 4.0 and activity extended from pH 2.0 to 8.0. In contrast to purified human pancreatic lipase, lingual lipase hydrolyzed triglyceride emulsions and mixed micelles stabilized with both short-chain (dihexanoyl) and long-chain (egg) lecithin and were inhibited only slightly (18-25%) by micellar concentrations of two common bile salts, taurodeoxycholate and taurocholate. Our results suggest that the hydrolysis of dietary fat by lingual lipase may extend from the pharynx through the esophagus and stomach and into the upper small intestine.

Novel Metagenome-Derived, Cold-Adapted Alkaline Phospholipase with Superior Lipase Activity as an Intermediate between Phospholipase and Lipase

PubMed Central

Lee, Mi-Hwa; Oh, Ki-Hoon; Kang, Chul-Hyung; Kim, Ji-Hoon; Oh, Tae-Kwang; Ryu, Choong-Min

2012-01-01

A novel lipolytic enzyme was isolated from a metagenomic library obtained from tidal flat sediments on the Korean west coast. Its putative functional domain, designated MPlaG, showed the highest similarity to phospholipase A from Grimontia hollisae CIP 101886, though it was screened from an emulsified tricaprylin plate. Phylogenetic analysis showed that MPlaG is far from family I.6 lipases, including Staphylococcus hyicus lipase, a unique lipase which can hydrolyze phospholipids, and is more evolutionarily related to the bacterial phospholipase A1 family. The specific activities of MPlaG against olive oil and phosphatidylcholine were determined to be 2,957 ± 144 and 1,735 ± 147 U mg−1, respectively, which means that MPlaG is a lipid-preferred phospholipase. Among different synthetic esters, triglycerides, and phosphatidylcholine, purified MPlaG exhibited the highest activity toward p-nitrophenyl palmitate (C16), tributyrin (C4), and 1,2-dihexanoyl-phosphatidylcholine (C8). Finally, MPlaG was identified as a phospholipase A1 with lipase activity by cleavage of the sn-1 position of OPPC, interfacial activity, and triolein hydrolysis. These findings suggest that MPlaG is the first experimentally characterized phospholipase A1 with lipase activity obtained from a metagenomic library. Our study provides an opportunity to improve our insight into the evolution of lipases and phospholipases. PMID:22544255

Endothelial lipase is a major determinant of HDL level

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ishida, Tatsuro; Choi, Sungshin; Kundu, Ramendra K.

2003-01-30

For the past three decades, epidemiologic studies have consistently demonstrated an inverse relationship between plasma HDL cholesterol (HDL-C) concentrations and coronary heart disease (CHD). Population-based studies have provided compelling evidence that low HDL-C levels are a risk factor for CHD, and several clinical interventions that increased plasma levels of HDL-C were associated with a reduction in CHD risk. These findings have stimulated extensive investigation into the determinants of plasma HDL-C levels. Turnover studies using radiolabeled apolipoprotein A-I, the major protein component of HDL, suggest that plasma HDL-C concentrations are highly correlated with the rate of clearance of apolipoprotein AI. However,more » the metabolic mechanisms by which HDL are catabolized have not been fully defined. Previous studies in humans with genetic deficiency of cholesteryl ester transfer protein, and in mice lacking the scavenger receptor BI (SR-BI), have demonstrated that these proteins participate in the removal of cholesterol from HDL, while observations in individuals with mutations in hepatic lipase indicate that this enzyme hydrolyzes HDL triglycerides. In this issue of the JCI, reports from laboratories of Tom Quertermous and Dan Rader now indicate that endothelial lipase (LIPG), a newly identified member of the lipase family, catalyzes the hydrolysis of HDL phospholipids and facilitates the clearance of HDL from the circulation. Endothelial lipase was initially cloned by both of these laboratories using entirely different strategies. Quertermous and his colleagues identified endothelial lipase as a transcript that was upregulated in cultured human umbilical vein endothelial cells undergoing tube formation, whereas the Rader group cloned endothelial lipase as a transcript that was upregulated in the human macrophage-like cell line THP-1 exposed to oxidized LDL. Database searches revealed that endothelial lipase shows strong sequence similarity to

S5 Lipase: an organic solvent tolerant enzyme.

PubMed

Rahman, Raja Noor Zaliha Abdul; Baharum, Syarul Nataqain; Salleh, Abu Bakar; Basri, Mahiran

2006-12-01

In this study, an organic solvent tolerant bacterial strain was isolated. This strain was identified as Pseudomonas sp. strain S5, and was shown to degrade BTEX (Benzene, Toluene, Ethyl-Benzene, and Xylene). Strain S5 generates an organic solvent-tolerant lipase in the late logarithmic phase of growth. Maximum lipase production was exhibited when peptone was utilized as the sole nitrogen source. Addition of any of the selected carbon sources to the medium resulted in a significant reduction of enzyme production. Lower lipase generation was noted when an inorganic nitrogen source was used as the sole nitrogen source. This bacterium hydrolyzed all tested triglycerides and the highest levels of production were observed when olive oil was used as a natural triglyceride. Basal medium containing Tween 60 enhanced lipase production to the most significant degree. The absence of magnesium ions (Mg2+) in the basal medium was also shown to stimulate lipase production. Meanwhile, an alkaline earth metal ion, Na+, was found to stimulate the production of S5 lipase.

Isolation and analysis of lipase-overproducing mutants of Serratia marcescens.

PubMed

Kawai, E; Akatsuka, H; Sakurai, N; Idei, A; Matsumae, H; Shibatani, T; Komatsubara, S; Omori, K

2001-01-01

We have isolated a lipase-overproducing mutant, GE14, from Serratia marcescens 8000 after three rounds of N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The mutant GE14 produced 95 kU/ml of extracellular lipase in the lipase medium, which was about threefold higher than that of produced by the original strain 8000. Enzymatic characteristics including specific activity of purified lipases from culture supernatants of GE14 and 8000 were almost same. The lipase gene (lipA) of GE14 contained two base substitutions; one in the promoter region and another in the N-terminal region of the lipA gene without an amino acid substitution. Promoter analysis using lipA-lacZ fusion plasmids revealed that these substitutions were responsible for the increase in the lipA expression level, independently. In contrast, no base substitution was found in the genes encoding the lipase secretion device, the Lip system. In addition, the genes coding for metalloprotease and the cell surface layer protein which are both secreted through the Lip system and associated with extracellular lipase production, also contained no base substitution. The strain GE14 carrying a high-copy-number lipA plasmid produced a larger amount of the extracellular lipase than the recombinant strains of 8000 and other mutants also did, indicating that GE14 was not only a lipase-overproducing strain, but also an advantageous host strain for overproducing the lipase by a recombinant DNA technique. These results suggest that the lipase-overproducing mutant GE14 and its recombinant strains are promising candidates for the industrial production of the S. marcescens lipase.

[Immobilization of Candida sp. lipase on resin D301].

PubMed

Wang, Yanhua; Zhu, Kai; Liu, Hui; Han, Pingfang; Wei, Ping

2009-12-01

We immobilized Candida sp. lipase onto seven kinds of industrial adsorption and ion exchange resins. By determining the activity of each immobilized enzyme, the weakly basic anionic exchange resin of D301 showed the best results for the immobilization of Candida sp. lipase. Comparing the scanning electron micrographs of D301 with Novozym 435 (immobilized Candida antarctica lipase B from Novo Nordisk Corp.), we selected D301 as a carrier for the immobilization of Candida sp. lipase. And we pretreated the resin D301 with the bifunctional agent glutaraldehyde and crosslinked it with Candida sp. lipase. The optimal conditions for the immobilization of Candida sp. lipase were as follows: 8 mL of the amount of 5% glutaraldehyde solution, five hours of the time pretreated D301 with glutaraldehyde, 1.0 g/L the concentration of Candida sp. lipase used, pH of the phosphate buffered, 6.0 and 10 hours of time for immobilization, respectively. The activity of immobilized enzyme was over 35 U/mg and the efficiency of immobilization was around 3.5 Ul(mg x h).

Discrimination between closed and open forms of lipases using electrophoretic techniques.

PubMed

Miled, N; Riviere, M; Cavalier, J F; Buono, G; Berti, L; Verger, R

2005-03-15

The enhanced catalytic activity of lipases is often associated with structural changes. The three-dimensional (3D) structures showed that the covalently inhibited lipases exist under their open conformations, in contrast to their native closed forms. We studied the inhibition of various lipases--human and dog gastric lipases, human pancreatic lipase, and Humicola lanuginosa lipase--by the octyl-undecyl phosphonate inhibitor, and we measured the subsequent modifications of their respective electrophoretic mobility. Furthermore, the experimental values of the isoelectric points found for the native (closed) and inhibited (open) lipases are in agreement with theoretical calculations based on the electrostatic potential. We concluded that there is a significant difference in the isoelectric points between the closed (native) and open (inhibited) conformations of the four lipases investigated. Thus, analysis of the electrophoretic pattern is proposed as an easy experimental tool to differentiate between a closed and an open form of a given lipase.

Differential effect of combined lipase deficiency (cld/cld) on human hepatic lipase and lipoprotein lipase secretion.

PubMed

Boedeker, J C; Doolittle, M H; White, A L

2001-11-01

Combined lipase deficiency (cld) is a recessively inherited disorder in mice associated with a deficiency of LPL and hepatic lipase (HL) activity. LPL is synthesized in cld tissues but is retained in the endoplasmic reticulum (ER), whereas mouse HL (mHL) is secreted but inactive. In this study we investigated the effect of cld on the secretion of human HL (hHL) protein mass and activity. Differentiated liver cell lines were derived from cld mice and their normal heterozygous (het) littermates by transformation of hepatocytes with SV40 large T antigen. After transient transfection with lipase expression constructs, secretion of hLPL activity from cld cells was only 12% of that from het cells. In contrast, the rate of secretion of hHL activity and protein mass per unit of expressed hHL mRNA was identical for the two cell lines. An intermediate effect was observed for mHL, with a 46% reduction in secretion of activity from cld cells. The ER glucosidase inhibitor, castanospermine, decreased secretion of both hLPL and hHL from het cells by approximately 70%, but by only approximately 45% from cld cells. This is consistent with data suggesting that cld may result from a reduced concentration of the ER chaperone calnexin. In conclusion, our results demonstrate a differential effect of cld on hLPL, mHL, and hHL secretion, suggesting differential requirements for activation and exit of the enzymes from the ER.

Lipase or amylase for the diagnosis of acute pancreatitis?

PubMed

Ismail, Ola Z; Bhayana, Vipin

2017-12-01

Acute pancreatitis is a rapid onset of inflammation of the pancreas causing mild to severe life threatening conditions [1, 2]. In Canada, acute pancreatitis is the 5th most expensive digestive disease in Canada with a considerable economic burden on the health care system [3]. The diagnosis of acute pancreatitis is usually based on the presence of abdominal pain and elevated levels of serum amylase and/or lipase. Many health care centers use either serum amylase, lipase or both to diagnose acute pancreatitis without considering which one could provide a better diagnostic accuracy. The aim of this review is to investigate whether serum lipase alone is a sufficient biomarker for the diagnosis of acute pancreatitis. We have examined various studies looking at the utilization, sensitivity, specificity and cost associated savings of lipase and amylase in the diagnosis of acute pancreatitis. When comparing different studies, serum lipase offers a higher sensitivity than serum amylase in diagnosing acute pancreatitis. Lipase also offers a larger diagnostic window than amylase since it is elevated for a longer time, thus allowing it to be a useful diagnostic biomarker in early and late stages of acute pancreatitis. Several recent evidence-based guidelines recommend the use of lipase over amylase. Nevertheless, both lipase and amylase alone lack the ability to determine the severity and etiology of acute pancreatitis. The co-ordering of both tests has shown little to no increase in the diagnostic sensitivity and specificity. Thus, unnecessary testing and laboratory expenditures can be reduced by testing lipase alone. Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Inhibition of lipases from Chromobacterium viscosum and Rhizopus oryzae by tetrahydrolipstatin.

PubMed

Potthoff, A P; Haalck, L; Spener, F

1998-01-15

Tetrahydrolipstatin is known as an inhibitor for pancreatic lipase but not for microbial lipases. In this paper we demonstrate that in the presence of water-insoluble substrates like tributyrin or olive oil, tetrahydrolipstatin inhibits the lipases of Chromobacterium viscosum and Rhizopus oryzae, although with different potency. In contrast to porcine pancreatic lipase, which forms an irreversible and covalent enzyme-inhibitor complex with tetrahydrolipstatin, the inhibition of the microbial lipases is reversible as the inhibitor can be removed from the enzyme-inhibitor complex by solvent extraction. Moreover, after inhibition of Chromobacterium viscosum lipase tetrahydrolipstatin remains chemically unchanged.

Lipase activities in castor bean endosperm during germinaion. [Ricinus communis; glyoxysomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muto, S.; Beevers, H.

1974-01-01

Two lipases were found in extracts from castor bean (Ricinus communis L.) endosperm. One, with optimal activity at pH 5.0 (acid lipase), was present in dry seeds and displayed high activity during the first 2 days of germination. The second, with an alkaline pH optimum (alkaline lipase), was particularly active during days 3 to 5. When total homogenates of endosperm were fractionated into fat layer, supernatant, and particulate fractions, the acid lipase was recovered in the fat layer, and the alkaline lipase was located primarily in the particulate fraction. Sucrose density gradient centrifugation showed that the alkaline lipase was locatedmore » mainly in glyoxysomes, with some 30 percent of the activity in the endoplasmic reticulum. When glyoxysomes were broken by osmotic shock and exposed to KCl, which solubilizes most of the enzymes, the alkaline lipase remained particulate and was recovered with the glyoxysomal ''ghosts'' at equilibrium density 1.21 g/cm/sup 3/ on the sucrose gradient. Association of the lipase with the glyoxysomal membrane was supported by the responses to detergents and to butanol. The alkaline lipase hydrolyzed only monosubstituted glycerols. The roles of the two lipases in lipid utilization during germination of castor bean are discussed.« less

New lipase assay using Pomegranate oil coating in microtiter plates.

PubMed

Ülker, Serdar; Placidi, Camille; Point, Vanessa; Gadenne, Benoît; Serveau-Avesque, Carole; Canaan, Stéphane; Carrière, Frédéric; Cavalier, Jean-François

2016-01-01

Lipases play various roles in fat digestion, lipoprotein metabolism, and in the mobilization of fat stored in lipid bodies in animals, plants and microorganisms. In association with these physiological functions, there is an important field of research for discovering lipase inhibitors and developing new treatments of diseases such as obesity, atherosclerosis, diabetes and tuberculosis. In this context, the development of convenient, specific and sensitive analytical methods for the detection and assay of lipases and/or lipase inhibitors is of major importance. It is shown here that purified triacylglycerols (TAGs) from Punica granatum (Pomegranate) seed oil coated on microtiter plates can be used for the continuous assay of lipase activity by recording the variations with time of the UV absorption spectra at 275 nm. UV absorption is due the release of punicic acid (9Z,11E,13Z-octadeca-9,11,13-trienoic acid), a conjugated triene contained in Pomegranate oil. This new microtiter plate assay allows to accurately measure the activity of a wider range of lipases compared to the similar assay previously developed with Tung oil containing α-eleostearic acid (9Z,11E,13E-octadeca-9,11,13-trienoic acid), including the LipY lipase from Mycobacterium tuberculosis. Although punicic acid is a diastereoisomer of α-eleostearic acid, the Δ(13)cis double bound found in punicic acid gives a different structure to the acyl chain that probably favours the interaction of Pomegranate TAGs with the lipase active site. The microplate lipase assay using Pomegranate TAGs shows high sensitivity, reproducibility and remarkable relevance for the high-speed screening of lipases and/or lipase inhibitors directly from raw culture media without any purification step. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Improvement of soil characteristics and growth of Dorycnium pentaphyllum by amendment with agrowastes and inoculation with AM fungi and/or the yeast Yarowia lipolytica.

PubMed

Medina, A; Vassileva, M; Caravaca, F; Roldán, A; Azcón, R

2004-08-01

The effectiveness of two microbiologically treated agrowastes [dry olive cake (DOC) and/or sugar beet (SB)] on plant growth, soil enzymatic activities and other soil characteristics was determined in a natural soil from a desertified area. Dorycnium pentaphyllum, a legume plant adapted to stress situations, was the test plant to evaluate the effect of inoculation of native arbuscular mycorrhizal (AM) fungi and/or Yarowia lipolytica (a dry soil adapted yeast) on amended and non-amended soils. Plant growth and nutrition, symbiotic developments and soil enzymatic activities were limited in non-amended soil where microbial inoculations did not improve plant development. The lack of nodules formation and AM colonization can explain the limited plant growth in this natural soil. The effectiveness and performance of inocula applied was only evident in amended soils. AM colonization and spores number in natural soil were increased by amendments and the inoculation with Y. lipolytica promoted this value. The effect of the inoculations on plant N-acquisition was only important in AM-inoculated plants growing in SB medium. Enzymatic activities as urease and protease activities were particularly increased in DOC amended soil meanwhile dehydrogenase activity was greatest in treatments inoculated with Y. lipolytica in SB added soil. The biological activities in rhizosphere of agrowaste amended soil, used as indices of changes in soil properties and fertility, were affected not only by the nature of amendments but also by the inoculant applied. All these results show that the lignocellulosic agrowastes treated with a selected microorganism and its further interaction with beneficial microbial groups (native AM fungi and/or Y. lipolytica) is a useful tool to modify soil physico-chemical, biological and fertility properties that enhance the plant performance probably by making nutrients more available to plants.

Screening of a thiamine-auxotrophic yeast for alpha-ketoglutaric acid overproduction.

PubMed

Zhou, Jingwen; Zhou, Haiyan; Du, Guocheng; Liu, Liming; Chen, Jian

2010-09-01

To obtain a thiamine-auxotrophic yeast strain that overproduces alpha-ketoglutaric acid (alpha-KG) from glycerol and to investigate nutrient effects on alpha-KG production. Yeast strain WSH-Z06, a thiamine auxotroph that gave high yields of alpha-KG from glycerol, was obtained by screening for ampicillin/kanamycin resistance and thiamine auxotrophy. The strain was identified as Yarrowia lipolytica based on physiological, chemical, and phylogenetic analysis. The ability of the strain to convert glycerol to alpha-KG was analysed by investigating the effects of nutritional factors, including thiamine, riboflavin, nitrogen sources, and calcium ion. Thiamine and calcium ion concentration had the greatest effect on alpha-KG accumulation. Under optimal conditions, a yield of 39.2 g l(-1)alpha-KG was obtained from 100 g l(-1) glycerol, with 16.84 g l(-1) pyruvate as a by-product. The current work provides a method for screening for an alpha-KG overproducer. Nutrients have a significant impact on alpha-KG production in the yeast strain presented here. The alpha-KG-overproducing yeast strain Y. lipolytica WSH-Z06 is a promising parent strain for further metabolic engineering to lower by-product accumulation and accelerate glycerol utilization.

Bioreactors with immobilized lipases: state of the art.

PubMed

Balcão, V M; Paiva, A L; Malcata, F X

1996-05-01

This review attempts to provide an updated compilation of studies reported in the literature pertaining to reactors containing lipases in immobilized forms, in a way that helps the reader direct a bibliographic search and develop an integrated perspective of the subject. Highlights are given to industrial applications of lipases (including control and economic considerations), as well as to methods of immobilization and configurations of reactors in which lipases are used. Features associated with immobilized lipase kinetics such as enzyme activities, adsorption properties, optimum operating conditions, and estimates of the lumped parameters in classical kinetic formulations (Michaelis-Menten model for enzyme action and first-order model for enzyme decay) are presented in the text in a systematic tabular form.

21 CFR 184.1420 - Lipase enzyme preparation derived from Rhizopus niveus.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Lipase enzyme preparation derived from Rhizopus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1420 Lipase enzyme preparation derived from Rhizopus niveus. (a) Lipase enzyme preparation contains lipase enzyme (CAS Reg. No...

21 CFR 184.1420 - Lipase enzyme preparation derived from Rhizopus niveus.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Lipase enzyme preparation derived from Rhizopus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1420 Lipase enzyme preparation derived from Rhizopus niveus. (a) Lipase enzyme preparation contains lipase enzyme (CAS Reg. No...

21 CFR 184.1420 - Lipase enzyme preparation derived from Rhizopus niveus.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Lipase enzyme preparation derived from Rhizopus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1420 Lipase enzyme preparation derived from Rhizopus niveus. (a) Lipase enzyme preparation contains lipase enzyme (CAS Reg. No...

21 CFR 184.1420 - Lipase enzyme preparation derived from Rhizopus niveus.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Lipase enzyme preparation derived from Rhizopus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1420 Lipase enzyme preparation derived from Rhizopus niveus. (a) Lipase enzyme preparation contains lipase enzyme (CAS Reg. No...

Lingual lipase activity in the orosensory detection of fat by humans

PubMed Central

Kulkarni, Bhushan V.

2014-01-01

Lingual lipase generates nonesterified fatty acids (NEFA) from dietary fats during oral processing by lipolysis. Lingual lipase in rodents has strong lipolytic activity and plays a critical role in oral detection of fats. The functional activity of lingual lipase during oral processing of high-fat foods in humans remains poorly characterized. Five commonly consumed high-fat foods varying in physical states and fatty acid composition (almond, almond butter, olive oil, walnut, and coconut) were masticated by 15 healthy human subjects at the rate of one chew per second with and without lipase inhibitor orlistat. Salivary NEFA concentrations were measured. To determine the role of lingual lipase in oral fat detection, sensory ratings were obtained from the same 15 human subjects for almond butter with and without orlistat. Lingual lipase was active during oral processing of almond and coconut. No activity of lingual lipase was detected during processing of almond butter. There was only weak evidence lingual lipase is a determinant of oral fat detection. Lingual lipase may only contribute to NEFA generation and oral fat detection of fatty foods that require stronger oral processing effort. PMID:24694384

Stability studies of immobilized lipase on rice husk and eggshell membrane

NASA Astrophysics Data System (ADS)

Abdulla, R.; Sanny, S. A.; Derman, E.

2017-06-01

Lipase immobilization for biodiesel production is gaining importance day by day. In this study, lipase from Burkholderia cepacia was immobilized on activated support materials namely rice husk and egg shell membrane. Both rice husk and eggshell membrane are natural wastes that holds a lot of potential as immobilization matrix. Rice husk and eggshell membrane were activated with glutaraldehyde. Lipase was immobilized on the glutaraldehyde-activated support material through adsorption. Immobilization efficiency together with enzyme activity was observed to choose the highest enzyme loading for further stability studies. Immobilization efficiency of lipase on rice husk was 81 as compared to an immobilization efficiency of 87 on eggshell membrane. Immobilized lipase on eggshell membrane exhibited higher enzyme activity as compared to immobilized lipase on rice husk. Eggshell membrane also reported higher stability than rice husk as immobilization matrix. Both types of immobilized lipase retatined its activity after ten cycles of reuse. In short, eggshell membrane showed to be a better immobilization platform for lipase as compared to rice husk. However, with further improvement in technique of immobilization, the stability of both types of immobilized lipase can be improved to a greater extent.

Strain improvement of Aspergillus niger for enhanced lipase production.

PubMed

Sandana Mala, John Geraldine; Kamini, Numbi R.; Puvanakrishnan, Rengarajulu

2001-08-01

The enhancement of lipase production from Aspergillus niger was attempted by ultraviolet (UV) and nitrous acid mutagenesis, and the mutants were selected on media containing bile salts. Nitrous acid mutants exhibited increased efficiency for lipase production when compared with UV mutants in submerged fermentation. The hyperproducing UV and nitrous acid mutants were further subjected to a second step of mutagenesis to devise an economical and ecofriendly technique for lipase production by the effective use of hydrocarbons. One percent kerosene was found to be optimal for lipase production, and one of the mutant strains NAII exhibited 2.53 times more increased lipase activity than the parental strain did. This investigation indicates a possible role for the A. niger mutant strains in the biodegradation of oil-polluted environments for the development of ecofriendly technologies.

21 CFR 184.1420 - Lipase enzyme preparation derived from Rhizopus niveus.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Lipase enzyme preparation derived from Rhizopus... Specific Substances Affirmed as GRAS § 184.1420 Lipase enzyme preparation derived from Rhizopus niveus. (a) Lipase enzyme preparation contains lipase enzyme (CAS Reg. No. 9001-62-1), which is obtained from the...

Functional cell-surface display of a lipase-specific chaperone.

PubMed

Wilhelm, Susanne; Rosenau, Frank; Becker, Stefan; Buest, Sebastian; Hausmann, Sascha; Kolmar, Harald; Jaeger, Karl-Erich

2007-01-02

Lipases are important enzymes in biotechnology. Extracellular bacterial lipases from Pseudomonads and related species require the assistance of specific chaperones, designated "Lif" proteins (lipase specific foldases). Lifs, a unique family of steric chaperones, are anchored to the periplasmic side of the inner membrane where they convert lipases into their active conformation. We have previously shown that the autotransporter protein EstA from P. aeruginosa can be used to direct a variety of proteins to the cell surface of Escherichia coli. Here we demonstrate for the first time the functional cell-surface display of the Lif chaperone and FACS (fluorescence-activated cell sorting)-based analysis of bacterial cells that carried foldase-lipase complexes. The model Lif protein, LipH from P. aeruginosa, was displayed at the surface of E. coli cells. Surface exposed LipH was functional and efficiently refolded chemically denatured lipase. The foldase autodisplay system reported here can be used for a variety of applications including the ultrahigh-throughput screening of large libraries of foldase variants generated by directed evolution.

Engineering Lipases: walking the fine line between activity and stability

NASA Astrophysics Data System (ADS)

Dasetty, Siva; Blenner, Mark A.; Sarupria, Sapna

2017-11-01

Lipases are enzymes that hydrolyze lipids and have several industrial applications. There is a tremendous effort in engineering the activity, specificity and stability of lipases to render them functional in a variety of environmental conditions. In this review, we discuss the recent experimental and simulation studies focused on engineering lipases. Experimentally, mutagenesis studies have demonstrated that the activity, stability, and specificity of lipases can be modulated by mutations. It has been particularly challenging however, to elucidate the underlying mechanisms through which these mutations affect the lipase properties. We summarize results from experiments and molecular simulations highlighting the emerging picture to this end. We end the review with suggestions for future research which underscores the delicate balance of various facets in the lipase that affect their activity and stability necessitating the consideration of the enzyme as a network of interactions.

Bioremediation of cooking oil waste using lipases from wastes

PubMed Central

do Prado, Débora Zanoni; Facanali, Roselaine; Marques, Márcia Mayo Ortiz; Nascimento, Augusto Santana; Fernandes, Célio Junior da Costa; Zambuzzi, William Fernando

2017-01-01

Cooking oil waste leads to well-known environmental impacts and its bioremediation by lipase-based enzymatic activity can minimize the high cytotoxic potential. In addition, they are among the biocatalysts most commercialized worldwide due to the versatility of reactions and substrates. However, although lipases are able to process cooking oil wastes, the products generated from this process do not necessarily become less toxic. Thus, the aim of the current study is to analyze the bioremediation of lipase-catalyzed cooking oil wastes, as well as their effect on the cytotoxicity of both the oil and its waste before and after enzymatic treatment. Thus, assessed the post-frying modification in soybean oil and in its waste, which was caused by hydrolysis reaction catalyzed by commercial and home-made lipases. The presence of lipases in the extracts obtained from orange wastes was identified by zymography. The profile of the fatty acid esters formed after these reactions was detected and quantified through gas chromatography and fatty acids profile compared through multivariate statistical analyses. Finally, the soybean oil and its waste, with and without enzymatic treatment, were assessed for toxicity in cytotoxicity assays conducted in vitro using fibroblast cell culture. The soybean oil wastes treated with core and frit lipases through transesterification reaction were less toxic than the untreated oils, thus confirming that cooking oil wastes can be bioremediated using orange lipases. PMID:29073166

Bioremediation of cooking oil waste using lipases from wastes.

PubMed

Okino-Delgado, Clarissa Hamaio; Prado, Débora Zanoni do; Facanali, Roselaine; Marques, Márcia Mayo Ortiz; Nascimento, Augusto Santana; Fernandes, Célio Junior da Costa; Zambuzzi, William Fernando; Fleuri, Luciana Francisco

2017-01-01

Cooking oil waste leads to well-known environmental impacts and its bioremediation by lipase-based enzymatic activity can minimize the high cytotoxic potential. In addition, they are among the biocatalysts most commercialized worldwide due to the versatility of reactions and substrates. However, although lipases are able to process cooking oil wastes, the products generated from this process do not necessarily become less toxic. Thus, the aim of the current study is to analyze the bioremediation of lipase-catalyzed cooking oil wastes, as well as their effect on the cytotoxicity of both the oil and its waste before and after enzymatic treatment. Thus, assessed the post-frying modification in soybean oil and in its waste, which was caused by hydrolysis reaction catalyzed by commercial and home-made lipases. The presence of lipases in the extracts obtained from orange wastes was identified by zymography. The profile of the fatty acid esters formed after these reactions was detected and quantified through gas chromatography and fatty acids profile compared through multivariate statistical analyses. Finally, the soybean oil and its waste, with and without enzymatic treatment, were assessed for toxicity in cytotoxicity assays conducted in vitro using fibroblast cell culture. The soybean oil wastes treated with core and frit lipases through transesterification reaction were less toxic than the untreated oils, thus confirming that cooking oil wastes can be bioremediated using orange lipases.

Structural characterization of MAPLE deposited lipase biofilm

NASA Astrophysics Data System (ADS)

Aronne, Antonio; Ausanio, Giovanni; Bloisi, Francesco; Calabria, Raffaela; Califano, Valeria; Fanelli, Esther; Massoli, Patrizio; Vicari, Luciano R. M.

2014-11-01

Lipases (triacylglycerol ester hydrolases) are enzymes used in several industrial applications. Enzymes immobilization can be used to address key issues limiting widespread application at industrial level. Immobilization efficiency is related to the ability to preserve the native conformation of the enzyme. MAPLE (Matrix Assisted Pulsed Laser Evaporation) technique, a laser deposition procedure for treating organic/polymeric/biomaterials, was applied for the deposition of lipase enzyme in an ice matrix, using near infrared laser radiation. Microscopy analysis showed that the deposition occurred in micrometric and submicrometric clusters with a wide size distribution. AFM imaging showed that inter-cluster regions are uniformly covered with smaller aggregates of nanometric size. Fourier transform infrared spectroscopy was used for both recognizing the deposited material and analyzing its secondary structure. Results showed that the protein underwent reversible self-association during the deposition process. Actually, preliminary tests of MAPLE deposited lipase used for soybean oil transesterification with isopropyl alcohol followed by gas chromatography-mass spectrometry gave results consistent with undamaged deposition of lipase.

Biochemical characterization of a lipase from olive fruit (Olea europaea L.).

PubMed

Panzanaro, S; Nutricati, E; Miceli, A; De Bellis, L

2010-09-01

Lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is the first enzyme of the degradation path of stored triacylglycerols (TAGs). In olive fruits, lipase may determine the increase of free fatty acids (FFAs) which level is an important index of virgin olive oil quality. However, despite the importance of virgin olive oil for nutrition and human health, few studies have been realized on lipase activity in Olea europaea fruits. In order to characterize olive lipase, fruits of the cv. Ogliarola, widely diffused in Salento area (Puglia, Italy), were harvested at four stages of ripening according to their skin colour (green, spotted I, spotted II, purple). Lipase activity was detected in the fatty layer obtained after centrifugation of the olive mesocarp homogenate. The enzyme exhibited a maximum activity at pH 5.0. The addition of calcium in the lipase assay medium leads to an increment of activity, whereas in the presence of copper the activity was reduced by 75%. Furthermore, mesocarp lipase activity increases during olive development but declined at maturity (purple stage). The data represent the first contribution to the biochemical characterization of an olive fruit lipase associated to oil bodies. 2010 Elsevier Masson SAS. All rights reserved.

Acid Lipase from Candida viswanathii: Production, Biochemical Properties, and Potential Application

PubMed Central

de Almeida, Alex Fernando; Carmona, Eleonora Cano

2013-01-01

Influences of environmental variables and emulsifiers on lipase production of a Candida viswanathii strain were investigated. The highest lipase activity (101.1 U) was observed at 210 rpm, pH 6.0, and 27.5°C. Other fermentation parameters analyzed showed considerable rates of biomass yield (Y L/S = 1.381 g/g), lipase yield (Y L/S = 6.892 U/g), and biomass productivity (P X = 0.282 g/h). Addition of soybean lecithin increased lipase production in 1.45-fold, presenting lipase yield (Y L/S) of 10.061 U/g. Crude lipase presented optimal activity at acid pH of 3.5, suggesting a new lipolytic enzyme for this genus and yeast in general. In addition, crude lipase presented high stability in acid conditions and temperature between 40 and 45°C, after 24 h of incubation in these temperatures. Lipase remained active in the presence of organic solvents maintaining above 80% activity in DMSO, methanol, acetonitrile, ethanol, acetone, 1-propanol, isopropanol, and 2-propanol. Effectiveness for the hydrolysis of a wide range of natural triglycerides suggests that this new acid lipase has high potential application in the oleochemical and food industries for hydrolysis and/or modification of triacylglycerols to improve the nutritional properties. PMID:24350270

Realm of Thermoalkaline Lipases in Bioprocess Commodities

PubMed Central

2018-01-01

For decades, microbial lipases are notably used as biocatalysts and efficiently catalyze various processes in many important industries. Biocatalysts are less corrosive to industrial equipment and due to their substrate specificity and regioselectivity they produced less harmful waste which promotes environmental sustainability. At present, thermostable and alkaline tolerant lipases have gained enormous interest as biocatalyst due to their stability and robustness under high temperature and alkaline environment operation. Several characteristics of the thermostable and alkaline tolerant lipases are discussed. Their molecular weight and resistance towards a range of temperature, pH, metal, and surfactants are compared. Their industrial applications in biodiesel, biodetergents, biodegreasing, and other types of bioconversions are also described. This review also discusses the advance of fermentation process for thermostable and alkaline tolerant lipases production focusing on the process development in microorganism selection and strain improvement, culture medium optimization via several optimization techniques (i.e., one-factor-at-a-time, surface response methodology, and artificial neural network), and other fermentation parameters (i.e., inoculums size, temperature, pH, agitation rate, dissolved oxygen tension (DOT), and aeration rate). Two common fermentation techniques for thermostable and alkaline tolerant lipases production which are solid-state and submerged fermentation methods are compared and discussed. Recent optimization approaches using evolutionary algorithms (i.e., Genetic Algorithm, Differential Evolution, and Particle Swarm Optimization) are also highlighted in this article. PMID:29666707

Realm of Thermoalkaline Lipases in Bioprocess Commodities.

PubMed

Lajis, Ahmad Firdaus B

2018-01-01

For decades, microbial lipases are notably used as biocatalysts and efficiently catalyze various processes in many important industries. Biocatalysts are less corrosive to industrial equipment and due to their substrate specificity and regioselectivity they produced less harmful waste which promotes environmental sustainability. At present, thermostable and alkaline tolerant lipases have gained enormous interest as biocatalyst due to their stability and robustness under high temperature and alkaline environment operation. Several characteristics of the thermostable and alkaline tolerant lipases are discussed. Their molecular weight and resistance towards a range of temperature, pH, metal, and surfactants are compared. Their industrial applications in biodiesel, biodetergents, biodegreasing, and other types of bioconversions are also described. This review also discusses the advance of fermentation process for thermostable and alkaline tolerant lipases production focusing on the process development in microorganism selection and strain improvement, culture medium optimization via several optimization techniques (i.e., one-factor-at-a-time, surface response methodology, and artificial neural network), and other fermentation parameters (i.e., inoculums size, temperature, pH, agitation rate, dissolved oxygen tension (DOT), and aeration rate). Two common fermentation techniques for thermostable and alkaline tolerant lipases production which are solid-state and submerged fermentation methods are compared and discussed. Recent optimization approaches using evolutionary algorithms (i.e., Genetic Algorithm, Differential Evolution, and Particle Swarm Optimization) are also highlighted in this article.

Lipase production in lipolytic yeast from Wonorejo mangrove area

NASA Astrophysics Data System (ADS)

Alami, Nur Hidayatul; Nasihah, Liziyatin; Umar, Rurin Luswidya Artaty; Kuswytasari, Nengah Dwianita; Zulaika, Enny; Shovitri, Maya

2017-06-01

Lipase is an enzyme that is often used in industry and become a commercial enzyme. One group of microorganisms capable of producing lipase is a yeast. This study aims to screen yeast from Wonorejo mangrove that potential to produce lipase and to optimize the production of these enzymes. Screening test include the measurement of lipolytic index and value of fatty acid. Yeast with the best value of fatty acid will be continued to the measurement of lipase activity. It is affected by several environmental factors, such as pH, temperature, and incubation time. This research was conducted to observe the optimization variation on environmental factors combination to produce lipase. Lipase activity was tested by using p-Nitrophenyl Palmitate (pNPP). Absorbency was measured by spectrofotometer on wavelength of 410 nm. Measurement of the enzyme activity was done by interpolating the absorbance values on the p-nitrophenol standard curve then calculated by the formula. All data were analyzed by using descriptive quantitative method. The results show that the highest lypolityc index was 2.08. The highest value of fatty acid was 0.49 that was reached on 168 hours of incubation. Candida W3.8 expressed the highest lypolylitic potential. The optimum environment to produce lipase by Candida W 3.8 was on 120 hours of incubation time, in temperature range of 27°C - 45°C and pH range of 4,5 - 7.

Liver lipase and high-density lipoprotein. Lipoprotein changes after incubation of human serum with rat liver lipase.

PubMed

Groot, P H; Scheek, L M; Jansen, H

1983-05-16

Human sera were incubated with rat liver lipase after inactivation of lecithin:cholesterol acyltransferase, and the changes in serum lipoprotein composition were measured. In the presence of liver lipase serum triacylglycerol and phosphatidylcholine were hydrolyzed. The main changes in the concentrations of these lipids were found in the high-density lipoprotein fraction. Subfractionation of high-density lipoprotein by rate-zonal ultracentrifugation showed a prominent decrease in all constituents of high-density lipoprotein2, a smaller decrease in the 'light' high-density lipoprotein3 and an increase in the 'heavy' high-density lipoprotein3. These data support a concept in which liver lipase is involved in high-density lipoprotein2 phospholipid and triacylglycerol catabolism and suggest that as a result of this action high-density lipoprotein2 is converted into high-density lipoprotein3.

Medium-chain versus long-chain triacylglycerol emulsion hydrolysis by lipoprotein lipase and hepatic lipase: Implications for the mechanisms of lipase action

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deckelbaum, R.J.; Hamilton, J.A.; Butbul, E.

1990-02-06

To explore how enzyme affinities and enzyme activities regulate hydrolysis of water-insoluble substrates, the authors compared hydrolysis of phospholipid-stabilized emulsions of medium-chain (MCT) versus long-chain triacylglycerols (LCT). Because substrate solubility at the emulsion surface might modulate rates of hydrolysis, the ability of egg yolk phosphatidylcholine to solubilize MCT was examined by NMR spectroscopy. Chemical shift measurements showed that 11 mol % of ({sup 13}C)carbonyl enriched trioctanoin was incorporated into phospholipid vesicles as a surface component. Line widths of trioctanoin surface peaks were half that of LCT, and relaxation times, T{sub 1}, were also shorter for trioctanoin, showing greater mobility formore » MCT in phospholipid. In assessing the effects of these differences in solubility on lipolysis, they found that both purified bovine milk lipoprotein lipase and human hepatic lipase hydrolyzed MCT at rates at least 2-fold higher than for LCT. Differences in affinity were also demonstrated in mixed incubations where increasing amounts of LCT emulsion resulted in decreased hydrolysis of MCT emulsions. These results suggest that despite lower enzyme affinity for MCT emulsions, shorter chain triacylglycerols are more readily hydrolyzed by lipoprotein and hepatic lipases than long-chain triacylglycerols because of greater MCT solubility and mobility at the emulsion-water interface.« less

Anti- and pro-lipase activity of selected medicinal, herbal and aquatic plants, and structure elucidation of an anti-lipase compound.

PubMed

Ado, Muhammad Abubakar; Abas, Faridah; Mohammed, Abdulkarim Sabo; Ghazali, Hasanah M

2013-11-26

Plants that help in slowing down the digestion of triacylglycerols (TAGs) in the pancreas and small intestine of humans play an important role in the reduction of obesity. On the other hand, there may be plants or plant parts that stimulate intestinal lipolytic activity, thus contributing to greater TAG assimilation. The aim of this study was to evaluate the aqueous methanolic extracts of ninety eight (98) medicinal, herbal and aquatic plant materials from Malaysia for their effect on porcine pancreatic lipase (PPL) activity and to identify the structure of an anti-lipase compound from one of the sources. The degree of inhibition was also quantified as relative to orlistat activity against PPL (orlistat equivalents). Results revealed that while 19.4% of the extracts were found to have anti-lipase activity ≥80%, 12% were actually found to promote PPL activity. Twenty two percent (22.4%) exhibited moderate inhibition (41%-80%) and 2% were neutral toward PPL activity. The ripe fruit of Averrhoa carambola and the leaves of Archidendron jiringa (Jack) I.C Nielsen L. (jering), Cynometra cauliflora (nam-nam) and Aleurites moluccana (L.) Willd (candle nut/buah keras) had the highest (100%) anti-lipase activity and are equivalent to 0.11 µg orlistat/mL. Plants that stimulated lipase activity included Pimpinella anisum L. (aniseed/jintan manis), activating the enzyme by 186.5%. Kaempferol 3-O-rhamnoside was isolated from the ethyl acetate fraction of C. cauliflora leaves and found to be an active lipase inhibitor. The structure was elucidated using 1H-NMR, 13C-NMR and 2D-NMR analyses.

Biotechnological applications of halophilic lipases and thioesterases.

PubMed

Schreck, Steven D; Grunden, Amy M

2014-02-01

Lipases and esterases are enzymes which hydrolyze ester bonds between a fatty acid moiety and an esterified conjugate, such as a glycerol or phosphate. These enzymes have a wide spectrum of use in industrial applications where their high activity, broad substrate specificity, and stability under harsh conditions have made them integral in biofuel production, textile processing, waste treatment, and as detergent additives. To date, these industrial applications have mainly leveraged enzymes from mesophilic and thermophilic organisms. However, increasingly, attention has turned to halophilic enzymes as catalysts in environments where high salt stability is desired. This review provides a brief overview of lipases and esterases and examines specific structural motifs and evolutionary adaptations of halophilic lipases. Finally, we examine the state of research involving these enzymes and provide an in-depth look at an exciting algal-based biofuel production system. This system uses a recombinant halophilic lipase to increase oil production efficiency by cleaving algal fatty acids from the acyl carrier protein, which eliminates feedback inhibition of fatty acid synthesis.

Serum Lipase as Clinical Laboratory Index for Chronic Renal Failure Diagnosis.

PubMed

Zhu, Ying; Dong, Jing; Wang, Ping; Huang, Huifang; Jin, Xiaohua; Zhou, Jingou; Shi, Jingfang; Gu, Guohao; Chen, Jun; Xu, Jun; Song, Yanhui

2016-07-01

Measuring the level of serum lipase has been used for the clinical diagnosis of acute pancreatitis. Reports showed that the serum lipase level increased in patients of clinical renal failure. In this study, we aimed to measure the change of serum lipase levels in chronic kidney diseases and determine whether it could serve as a clinical laboratory index for clinical renal failure diagnosis. Materials: The OLYMPUS AU5400 automatic biochemical analyzer was used to determine the serum levels of lipase and creatinine. The study included 120 cases in the clinical renal failure group, 76 cases in the nephrotic syndrome group, 81 cases in the chronic nephritis group, and 80 healthy controls from our hospital volunteers in the same period. We then compared the lipase levels and conducted statistical analyses among these groups. The serum lipase levels were 15.3 U/L, 79.8 U/L, 45.1 U/L, and 51.0 U/L in the normal control, clinical renal failure, nephrotic syndrome, and chronic nephritis groups, respectively. The lipase levels in the groups with diseases were significantly different compared with that of the normal control group (p < 0.01). The lipase level of the clinical renal failure group was significantly higher than that of the nephrotic syndrome group and chronic nephritis group (p < 0.01). However, no statistically significant difference between the nephrotic syndrome and chronic nephritis group (p > 0.05) was observed. Moreover, an association of the serum lipase with disease progression was observed in the study. Serum lipase is an effective serological index which can reflect the clinical changes in the clinical renal failure and tends to increase through the progression of renal dysfunction.

Effects of surfactants on lipase structure, activity, and inhibition.

PubMed

Delorme, Vincent; Dhouib, Rabeb; Canaan, Stéphane; Fotiadu, Frédéric; Carrière, Frédéric; Cavalier, Jean-François

2011-08-01

Lipase inhibitors are the main anti-obesity drugs prescribed these days, but the complexity of their mechanism of action is making it difficult to develop new molecules for this purpose. The efficacy of these drugs is known to depend closely on the physico-chemistry of the lipid-water interfaces involved and on the unconventional behavior of the lipases which are their target enzymes. The lipolysis reaction which occurs at an oil-water interface involves complex equilibria between adsorption-desorption processes, conformational changes and catalytic mechanisms. In this context, surfactants can induce significant changes in the partitioning of the enzyme and the inhibitor between the water phase and lipid-water interfaces. Surfactants can be found at the oil-water interface where they compete with lipases for adsorption, but also in solution in the form of micellar aggregates and monomers that may interact with hydrophobic parts of lipases in solution. These various interactions, combined with the emulsification and dispersion of insoluble substrates and inhibitors, can either promote or decrease the activity and the inhibition of lipases. Here, we review some examples of the various effects of surfactants on lipase structure, activity and inhibition, which show how complex the various equilibria involved in the lipolysis reaction tend to be.

Monoacylglycerol lipase – a target for drug development?

PubMed Central

Fowler, CJ

2012-01-01

The endocannabinoid (eCB) system is involved in processes as diverse as control of appetite, perception of pain and the limitation of cancer cell growth and invasion. The enzymes responsible for eCB breakdown are attractive pharmacological targets, and fatty acid amide hydrolase inhibitors, which potentiate the levels of the eCB anandamide, are now undergoing pharmaceutical development. ‘Drugable’ selective inhibitors of monoacylglycerol lipase, a key enzyme regulating the levels of the other main eCB, 2-arachidonoylglycerol, were however not identified until very recently. Their availability has resulted in a large expansion of our knowledge concerning the pharmacological consequences of monoacylglycerol lipase inhibition and hence the role(s) played by the enzyme in the body. In this review, the pharmacology of monoacylglycerol lipase will be discussed, together with an analysis of the therapeutic potential of monoacylglycerol lipase inhibitors as analgesics and anticancer agents. PMID:22428756

Yeast Kluyveromyces lactis as host for expression of the bacterial lipase: cloning and adaptation of the new lipase gene from Serratia sp.

PubMed

Šiekštelė, Rimantas; Veteikytė, Aušra; Tvaska, Bronius; Matijošytė, Inga

2015-10-01

Many microbial lipases have been successfully expressed in yeasts, but not in industrially attractive Kluyveromyces lactis, which among other benefits can be cultivated on a medium supplemented with whey--cheap and easily available industrial waste. A new bacterial lipase from Serratia sp. was isolated and for the first time expressed into the yeast Kluyveromyces lactis by heterologous protein expression system based on a strong promoter of Kluyveromyces marxianus triosephosphate isomerase gene and signal peptide of Kluyveromyces marxianus endopolygalacturonase gene. In addition, the bacterial lipase gene was synthesized de novo by taking into account a codon usage bias optimal for K. lactis and was expressed into the yeast K. lactis also. Both resulting strains were characterized by high output level of the target protein secreted extracellularly. Secreted lipases were characterized for activity and stability.

Enhancement of lipase catalyzed-fatty acid methyl esters production from waste activated bleaching earth by nullification of lipase inhibitors.

PubMed

Dwiarti, Lies; Ali, Ehsan; Park, Enoch Y

2010-01-01

This study sought to identify inhibitory factors of lipase catalyzed-fatty acid methyl esters (FAME) production from waste activated bleaching earth (wABE). During the vegetable oil refinery process, activated bleaching earth (ABE) is used for removing the impure compounds, but adsorbs vegetable oil up to 35-40% as on a weight basis, and then the wABE is discarded as waste material. The impurities were extracted from the wABE with methanol and evaluated by infra-red (IR) spectroscopy, which revealed that some were chlorophyll-plant pigments. The chlorophylls inhibited the lipase during FAME conversion from wABE. The inhibition by a mixture of chlorophyll a and b was found to be competitive. The inhibition of the enzymatic hydrolysis of waste vegetable oil contained in wABE by chlorophyll a alone was competitive, while the inhibition by chlorophyll b alone was non-competitive. Furthermore, the addition of a small amount of alkali nullified this inhibitory effect and accelerated the FAME production rate. When 0.9% KOH (w/w wABE) was added to the transesterification reaction with only 0.05% lipase (w/w wABE), the maximum FAME production rate improved 120-fold, as compared to that without the addition of KOH. The alkali-combined lipase significantly enhanced the FAME production rate from wABE, in spite of the presence of the plant pigments, and even when a lower amount of lipase was used as a catalyst.

Lipase and its modulator from Pseudomonas sp. strain KFCC 10818: proline-to-glutamine substitution at position 112 induces formation of enzymatically active lipase in the absence of the modulator.

PubMed

Kim, E K; Jang, W H; Ko, J H; Kang, J S; Noh, M J; Yoo, O J

2001-10-01

A lipase gene, lipK, and a lipase modulator gene, limK, of Pseudomonas sp. strain KFCC 10818 have been cloned, sequenced, and expressed in Escherichia coli. The limK gene is located immediately downstream of the lipK gene. Enzymatically active lipase was produced only in the presence of the limK gene. The effect of the lipase modulator LimK on the expression of active lipase was similar to those of the Pseudomonas subfamily I.1 and I.2 lipase-specific foldases (Lifs). The deduced amino acid sequence of LimK shares low homology (17 to 19%) with the known Pseudomonas Lifs, suggesting that Pseudomonas sp. strain KFCC 10818 is only distantly related to the subfamily I.1 and I.2 Pseudomonas species. Surprisingly, a lipase variant that does not require LimK for its correct folding was isolated in the study to investigate the functional interaction between LipK and LimK. When expressed in the absence of LimK, the P112Q variant of LipK formed an active enzyme and displayed 63% of the activity of wild-type LipK expressed in the presence of LimK. These results suggest that the Pro(112) residue of LipK is involved in a key step of lipase folding. We expect that the novel finding of this study may contribute to future research on efficient expression or refolding of industrially important lipases and on the mechanism of lipase folding.

Lipase and Its Modulator from Pseudomonas sp. Strain KFCC 10818: Proline-to-Glutamine Substitution at Position 112 Induces Formation of Enzymatically Active Lipase in the Absence of the Modulator

PubMed Central

Kim, Eun Kyung; Jang, Won Hee; Ko, Jung Ho; Kang, Jong Seok; Noh, Moon Jong; Yoo, Ook Joon

2001-01-01

A lipase gene, lipK, and a lipase modulator gene, limK, of Pseudomonas sp. strain KFCC 10818 have been cloned, sequenced, and expressed in Escherichia coli. The limK gene is located immediately downstream of the lipK gene. Enzymatically active lipase was produced only in the presence of the limK gene. The effect of the lipase modulator LimK on the expression of active lipase was similar to those of the Pseudomonas subfamily I.1 and I.2 lipase-specific foldases (Lifs). The deduced amino acid sequence of LimK shares low homology (17 to 19%) with the known Pseudomonas Lifs, suggesting that Pseudomonas sp. strain KFCC 10818 is only distantly related to the subfamily I.1 and I.2 Pseudomonas species. Surprisingly, a lipase variant that does not require LimK for its correct folding was isolated in the study to investigate the functional interaction between LipK and LimK. When expressed in the absence of LimK, the P112Q variant of LipK formed an active enzyme and displayed 63% of the activity of wild-type LipK expressed in the presence of LimK. These results suggest that the Pro112 residue of LipK is involved in a key step of lipase folding. We expect that the novel finding of this study may contribute to future research on efficient expression or refolding of industrially important lipases and on the mechanism of lipase folding. PMID:11566993

Glycerol Monolaurate Inhibits Lipase Production by Clinical Ocular Isolates Without Affecting Bacterial Cell Viability.

PubMed

Flanagan, Judith Louise; Khandekar, Neeta; Zhu, Hua; Watanabe, Keizo; Markoulli, Maria; Flanagan, John Terence; Papas, Eric

2016-02-01

We sought to determine the relative lipase production of a range of ocular bacterial isolates and to assess the efficacy of glycerol monolaurate (GML) in inhibiting this lipase production in high lipase-producing bacteria without affecting bacterial cell growth. Staphylococcus aureus,Staphylococcus epidermidis,Propionibacterium acnes, and Corynebacterium spp. were inoculated at a density of 10(6)/mL in varying concentrations of GML up to 25 μg/mL for 24 hours at 37 °C with constant shaking. Bacterial suspensions were centrifuged, bacterial cell density was determined, and production of bacterial lipase was quantified using a commercial lipase assay kit. Staphylococcus spp. produced high levels of lipase activity compared with P. acnes and Corynebacterium spp. GML inhibited lipase production by Staphylococcal spp. in a dose-dependent manner, with S. epidermidis lipase production consistently more sensitive to GML than S. aureus. Glycerol monolaurate showed significant (P < 0.05) lipase inhibition above concentrations of 15 μg/mL in S. aureus and was not cytotoxic up to 25 μg/mL. For S. epidermidis, GML showed significant (P < 0.05) lipase inhibition above 7.5 μg/mL. Lipase activity varied between species and between strains. Staphylococcal spp. produced higher lipase activity compared with P. acnes and Corynebacterium spp. Glycerol monolaurate inhibited lipase production by S. aureus and S. epidermidis at concentrations that did not adversely affect bacterial cell growth. GML can be used to inhibit ocular bacterial lipase production without proving detrimental to commensal bacteria viability.

Valorization of Palm Oil Industrial Waste as Feedstock for Lipase Production.

PubMed

Silveira, Erick A; Tardioli, Paulo W; Farinas, Cristiane S

2016-06-01

The use of residues from the industrial processing of palm oil as carbon source and inducer for microbial lipase production can be a way to add value to such residues and to contribute to reduced enzyme costs. The aim of this work was to investigate the feasibility of using palm oil industrial waste as feedstock for lipase production in different cultivation systems. Evaluation was made of lipase production by a selected strain of Aspergillus niger cultivated under solid-state (SSF) and submerged fermentation (SmF). Lipase activity levels up to 15.41 IU/mL were achieved under SSF. The effects of pH and temperature on the lipase activity of the SSF extract were evaluated using statistical design methodology, and maximum activities were obtained between pH 4.0 and 6.5 and at temperatures between 37 and 55 °C. This lipase presented good thermal stability up to 60 °C and higher specificity towards long carbon chain substrates. The results demonstrate the potential application of palm oil industrial residues for lipase production and contribute to the technological advances needed to develop processes for industrial enzymes production.

Evaluation of a New Lipase from Staphylococcus sp. for Detergent Additive Capability

PubMed Central

Chauhan, Mamta; Chauhan, Rajinder Singh; Garlapati, Vijay Kumar

2013-01-01

Lipases are the enzymes of choice for laundry detergent industries owing to their triglyceride removing ability from the soiled fabric which eventually reduces the usage of phosphate-based chemical cleansers in the detergent formulation. In the present study, a partially purified bacterial lipase from Staphylococcus arlettae JPBW-1 isolated from the rock salt mine has been assessed for its triglyceride removing ability by developing a presoak solution so as to use lipase as an additive in laundry detergent formulations. The effects of selected surfactants, commercial detergents, and oxidizing agents on lipase stability were studied in a preliminary evaluation for its further usage in the industrial environment. Partially purified lipase has shown good stability in presence of surfactants, commercial detergents, and oxidizing agents. Washing efficiency has been found to be enhanced while using lipase with 0.5% nonionic detergent than the anioinic detergent. The wash performance using 0.5% wheel with 40 U lipase at 40°C in 45 min results in maximum oil removal (62%) from the soiled cotton fabric. Hence, the present study opens the new era in enzyme-based detergent sector for formulation of chemical-free detergent using alkaline bacterial lipase. PMID:24106703

Evaluation of a new lipase from Staphylococcus sp. for detergent additive capability.

PubMed

Chauhan, Mamta; Chauhan, Rajinder Singh; Garlapati, Vijay Kumar

2013-01-01

Lipases are the enzymes of choice for laundry detergent industries owing to their triglyceride removing ability from the soiled fabric which eventually reduces the usage of phosphate-based chemical cleansers in the detergent formulation. In the present study, a partially purified bacterial lipase from Staphylococcus arlettae JPBW-1 isolated from the rock salt mine has been assessed for its triglyceride removing ability by developing a presoak solution so as to use lipase as an additive in laundry detergent formulations. The effects of selected surfactants, commercial detergents, and oxidizing agents on lipase stability were studied in a preliminary evaluation for its further usage in the industrial environment. Partially purified lipase has shown good stability in presence of surfactants, commercial detergents, and oxidizing agents. Washing efficiency has been found to be enhanced while using lipase with 0.5% nonionic detergent than the anioinic detergent. The wash performance using 0.5% wheel with 40 U lipase at 40°C in 45 min results in maximum oil removal (62%) from the soiled cotton fabric. Hence, the present study opens the new era in enzyme-based detergent sector for formulation of chemical-free detergent using alkaline bacterial lipase.

Immobilized lipase from Candida sp. 99-125 on hydrophobic silicate: characterization and applications.

PubMed

Zhao, Bin; Liu, Xinlong; Jiang, Yanjun; Zhou, Liya; He, Ying; Gao, Jing

2014-08-01

Lipase Candida sp. 99-125 has been proved to be quite effective in catalyzing organic synthesis reactions and is much cheaper than commercial lipases. Mesoporous silicates are attractive materials for the immobilization of enzymes due to their unique structures. The present research designed a hydrophobic silicate with uniform pore size suitable for the comfort of lipase Candida sp. 99-125 for improving its activity and stability. The resulting immobilized lipase (LP@PMO) by adsorption was employed to catalyze hydrolysis, esterification, and transesterification reactions, and the performances were compared with the lipase immobilized on hydrophilic silicate (LP@PMS) and native lipase. The LP@PMO showed as high activity as that of native lipase in hydrolysis and much increased catalytic activity and reusability in the reactions for biodiesel production. Besides, LP@PMO also possessed better organic stability. Such results demonstrate that immobilization of lipase onto hydrophobic supports is a promising strategy to fabricate highly active and stable biocatalysts for applications.

Immobilization of lipases in hydrophobic chitosan for selective hydrolysis of fish oil: The impact of support functionalization on lipase activity, selectivity and stability.

PubMed

Urrutia, P; Arrieta, R; Alvarez, L; Cardenas, C; Mesa, M; Wilson, L

2018-03-01

The objective of this paper was to carry out an integral study of the use of hydrophobic chitosan as a low-cost support for immobilizing lipases and their further application in the selective hydrolysis of fish oil. Chitosan functionalized with different alkyl chains (C4, C8, C12) were characterized by FTIR, TGA, SEM, and Rose Bengal adsorption. Lipase B from Candida antarctica (CalB) and lipase from Rhizomucor miehei (RML) were immobilized obtaining a higher expressed activity at a longer alkyl chain length of support. Biocatalyst thermal stability showed that the impact of the alkyl chain length on enzyme stabilization varied according to the lipase source. The biocatalysts were applied in menhaden oil hydrolysis. Total polyunsaturated fatty acids released after 30 h of reaction with lipases immobilized in butyl, octyl and dodecyl-chitosan was 60, 107, and 90 mM for CalB biocatalysts, and 560, 392, and 50 mM for RML biocatalysts, respectively. Selectivity of CalB was not affected by the alkyl chain, while in the case of RML, a higher selectivity to cis-4,7,10,13,16,19-docohexaenoic acid release was obtained with dodecyl-chitosan. In conclusion, the adequate functionalization of chitosan varied according to lipase source, affecting their activity, stability and performance in the hydrolysis of fish oil. Copyright © 2017 Elsevier B.V. All rights reserved.

Collagen-Immobilized Lipases Show Good Activity and Reusability for Butyl Butyrate Synthesis.

PubMed

Dewei, Song; Min, Chen; Haiming, Cheng

2016-11-01

Candida rugosa lipases were immobilized onto collagen fibers through glutaraldehyde cross-linking method. The immobilization process has been optimized. Under the optimal immobilization conditions, the activity of the collagen-immobilized lipase reached 340 U/g. The activity was recovered of 28.3 % by immobilization. The operational stability of the obtained collagen-immobilized lipase for hydrolysis of olive oil emulsion was determined. The collagen-immobilized lipase showed good tolerance to temperature and pH variations in comparison to free lipase. The collagen-immobilized lipase was also applied as biocatalyst for synthesis of butyl butyrate from butyric acid and 1-butanol in n-hexane. The conversion yield was 94 % at the optimal conditions. Of its initial activity, 64 % was retained after 5 cycles for synthesizing butyl butyrate in n-hexane.

Characterization of Purified Staphylococcal Lipase1

PubMed Central

Vadehra, D. V.; Harmon, L. G.

1967-01-01

Purified staphylococcal lipase had an optimal pH of 8.3 for activity at 37 C, and an optimal temperature of 45 C at pH 8.0. During storage, the enzyme lost less than 10% of the activity over a period of 21 days at 4 and -23 C. The enzyme retained 93% of the activity when heated for 30 min at 50 C and was 95% destroyed in 30 min at 70 C. The purified lipase was capable of hydrolyzing a variety of natural fats and oils. However, the enzyme was three times more active on nonhydrogenated soybean oil than on hydrogenated soybean oil with an iodine value of <3.0. The enzyme was also capable of hydrolyzing fatty acids on the α, β, and α′ positions of a synthetic mixed triglyceride. In general, the presence of oxidizing agents increased the activity and the presence of reducing agents decreased the activity of the lipase enzyme. PMID:6035042

Acid lipase inhibitor in chicken plasma identified as apolipoprotein A-I.

PubMed

Fujii, M; Higuchi, T; Mukai, S; Yonekura, M; Yano, T; Kawaguchi, H; Nonaka, K; Fukunaga, T; Sugimoto, Y; Yamada, S

1996-10-01

We have reported a inhibitor of acid lipases in liver lysosomes and erythrocytes from chickens [M. Fujii et al., Int. J. Biochem., 22, 895-898 (1990)]. In this paper, the properties of the inhibitor were described in comparison with those of apo A-I of chicken. The purified inhibitor migrated with the same mobility on SDS-PAGE as apo A-I, and had a molecular weight of 27,000. The peptide map from the lipase inhibitor was similar to that of apo A-I. Antibodies to the acid lipase inhibitor also reacted with apo A-I. Apo A-I inhibited the acid lipase activities of liver lysosomes and erythrocytes from chickens as strongly as the lipase inhibitor. The N-terminal amino acid sequence of lipase inhibitor was identical to that of apo A-I as far as residue 20. The amino acid sequence of peptides obtained from the inhibitor by cleavage with CNBr corresponded to internal sequence of apo A-I, and so the CNBr-peptides were derived by cleavage after the methionine residues in apo A-I. The findings showed that the inhibitor of the acid lipases in liver lysosomes and erythrocytes from chickens was identical to apo A-I.

Molecular modeling of lipase binding to a substrate-water interface.

PubMed

Gruber, Christian C; Pleiss, Jürgen

2012-01-01

Interactions of lipases with hydrophobic substrate-water interfaces are of great interest to design improved lipase variants and engineer reaction conditions. This chapter describes the necessary steps to carry out molecular dynamics simulations of Candida antarctica lipase B at tributyrin-water interface using the GROMACS simulation software. Special attention is drawn to the preparation of the protein and the substrate-water interface and to the analysis of the obtained trajectory.

Rhizomucor miehei triglyceride lipase is processed and secreted from transformed Aspergillus oryzae.

PubMed

Huge-Jensen, B; Andreasen, F; Christensen, T; Christensen, M; Thim, L; Boel, E

1989-09-01

The cDNA encoding the precursor of the Rhizomucor miehei triglyceride lipase was inserted in an Aspergillus oryzae expression vector. In this vector the expression of the lipase cDNA is under control of the Aspergillus oryzae alpha-amylase gene promoter and the Aspergillus niger glucoamylase gene terminator. The recombinant plasmid was introduced into Aspergillus oryzae, and transformed colonies were selected and screened for lipase expression. Lipase-positive transformants were grown in a small fermentor, and recombinant triglyceride lipase was purified from the culture broth. The purified enzymatically active recombinant lipase (rRML) secreted from A. oryzae was shown to have the same characteristics with respect to mobility on reducing SDS-gels and amino acid composition as the native enzyme. N-terminal amino acid sequencing indicated that approximately 70% of the secreted rRML had the same N-terminal sequence as the native Rhizomucor miehei enzyme, whereas 30% of the secreted rRML was one amino acid residue shorter in the N-terminal. The recombinant lipase precursor, which has a 70 amino acid propeptide, is thus processed in and secreted from Aspergillus oryzae. We have hereby demonstrated the utility of this organism as a host for the production of recombinant triglyceride lipases.

High cell density fed-batch fermentations for lipase production: feeding strategies and oxygen transfer.

PubMed

Salehmin, M N I; Annuar, M S M; Chisti, Y

2013-11-01

This review is focused on the production of microbial lipases by high cell density fermentation. Lipases are among the most widely used of the enzyme catalysts. Although lipases are produced by animals and plants, industrial lipases are sourced almost exclusively from microorganisms. Many of the commercial lipases are produced using recombinant species. Microbial lipases are mostly produced by batch and fed-batch fermentation. Lipases are generally secreted by the cell into the extracellular environment. Thus, a crude preparation of lipases can be obtained by removing the microbial cells from the fermentation broth. This crude cell-free broth may be further concentrated and used as is, or lipases may be purified from it to various levels. For many large volume applications, lipases must be produced at extremely low cost. High cell density fermentation is a promising method for low-cost production: it allows a high concentration of the biomass and the enzyme to be attained rapidly and this eases the downstream recovery of the enzyme. High density fermentation enhances enzyme productivity compared with the traditional submerged culture batch fermentation. In production of enzymes, a high cell density is generally achieved through fed-batch operation, not through perfusion culture which is cumbersome. The feeding strategies used in fed-batch fermentations for producing lipases and the implications of these strategies are discussed. Most lipase-producing microbial fermentations require oxygen. Oxygen transfer in such fermentations is discussed.

Physiological uncoupling of mitochondrial oxidative phosphorylation. Studies in different yeast species.

PubMed

Guerrero-Castillo, Sergio; Araiza-Olivera, Daniela; Cabrera-Orefice, Alfredo; Espinasa-Jaramillo, Juan; Gutiérrez-Aguilar, Manuel; Luévano-Martínez, Luís A; Zepeda-Bastida, Armando; Uribe-Carvajal, Salvador

2011-06-01

Under non-phosphorylating conditions a high proton transmembrane gradient inhibits the rate of oxygen consumption mediated by the mitochondrial respiratory chain (state IV). Slow electron transit leads to production of reactive oxygen species (ROS) capable of participating in deleterious side reactions. In order to avoid overproducing ROS, mitochondria maintain a high rate of O(2) consumption by activating different exquisitely controlled uncoupling pathways. Different yeast species possess one or more uncoupling systems that work through one of two possible mechanisms: i) Proton sinks and ii) Non-pumping redox enzymes. Proton sinks are exemplified by mitochondrial unspecific channels (MUC) and by uncoupling proteins (UCP). Saccharomyces. cerevisiae and Debaryomyces hansenii express highly regulated MUCs. Also, a UCP was described in Yarrowia lipolytica which promotes uncoupled O(2) consumption. Non-pumping alternative oxido-reductases may substitute for a pump, as in S. cerevisiae or may coexist with a complete set of pumps as in the branched respiratory chains from Y. lipolytica or D. hansenii. In addition, pumps may suffer intrinsic uncoupling (slipping). Promising models for study are unicellular parasites which can turn off their aerobic metabolism completely. The variety of energy dissipating systems in eukaryote species is probably designed to control ROS production in the different environments where each species lives.

Lipase polystyrene giant amphiphiles.

PubMed

Velonia, Kelly; Rowan, Alan E; Nolte, Roeland J M

2002-04-24

A new type of giant amphiphilic molecule has been synthesized by covalently connecting a lipase enzyme headgroup to a maleimide-functionalized polystyrene tail (40 repeat units). The resulting biohybrid forms catalytic micellar rods in water.

Hydrolysis of bovine and caprine milk fat globules by lipoprotein lipase. Effects of heparin and skim milk on lipase distribution and on lipolysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sundheim, G.; Bengtsson-Olivecrona, G.

1987-12-01

Heparin can dissociate lipoprotein lipase from casein micelles, and addition of heparin enhances lipolysis in bovine but not in caprine milk. Heparin shortened the lag-time for binding of lipoprotein lipase to milk fat globules and for lipolysis. Heparin counteracted the inhibitory effects of skim milk on binding of lipase and on lipolysis. Heparin stimulated lipolysis in all bovine milk samples when added before cooling and in spontaneously lipolytic milk samples also when added after cooling. Heparin enhanced lipolysis of isolated milk fat globules. Hence, its effect is not solely due to dissociation of lipoprotein lipase from the casein micelles. Coolingmore » of goat milk caused more marked changes in the distribution of lipase than cooling of bovine milk; the fraction of added /sup 125/I-labeled lipase that bound to cream increased from about 8 to 60%. In addition, caprine skim milk caused less inhibition of lipolysis than bovine skim milk. These observations provide an explanation for the high degree of cold storage lipolysis in goat milk. Heparin had only small effects on the distribution of lipoprotein lipase in caprine milk, which explains why heparin has so little effect on lipolysis in caprine milk. The distribution of /sup 35/S-labeled heparin in bovine milk was studied. In warm milk less than 10% bound to the cream fraction, but when milk was cooled, binding of heparin to cream increased to 45%. These results suggest that there exists in the skim fraction a relatively small amount of a heparin-binding protein, which on cooling of milk adsorbs to the milk fat, or suggests that cooling induces a conformational change in a membrane protein such that its affinity for heparin increases.« less

Improved catalytic properties of Penicillium notatum lipase immobilized in nanoscale silicone polymeric films.

PubMed

Rehman, Saima; Wang, Ping; Bhatti, Haq Nawaz; Bilal, Muhammad; Asgher, Muhammad

2017-04-01

Lipases are one of the most proficient biocatalysts having enormous biotechnological prospective. Immobilization offers a potential solution to improve the stability and recycling characteristics of lipases. An extracellular lipase from Penicillium notatum (PNL) was immobilized in silicon polymers (SiP) through entrapment, and subsequently coated this matrix on the network of fibers in the sponges. The silicone polymers-immobilized lipase (SiP-lipase) displayed highest apparent activity and entrapment efficiency of 1.19Ug -1 polymers and 92.3%, respectively. It also exhibited greater catalytic activity in broad-working pHs and higher temperature than equivalent free-state of enzyme. Immobilization caused an improvement in thermo-stability of the lipase with an increase in energy of activation. The recycling potential of SiP-lipase was investigated. After reusing the sponge pieces for ten reaction cycles, the SiP preserved its structure without leakage of enzyme, and retained around 90% of its original activity. The SiP surface analysis was envisaged by scanning electron microscopy that further confirmed the recycling efficiency of SiP-lipase. Overall, SiP-lipase displayed a number of useful properties that make it a promising candidate for future applications in different chemical processes. Copyright © 2017 Elsevier B.V. All rights reserved.

Gene organization and primary structure of human hormone-sensitive lipase: possible significance of a sequence homology with a lipase of Moraxella TA144, an antarctic bacterium.

PubMed Central

Langin, D; Laurell, H; Holst, L S; Belfrage, P; Holm, C

1993-01-01

The human hormone-sensitive lipase (HSL) gene encodes a 786-aa polypeptide (85.5 kDa). It is composed of nine exons spanning approximately 11 kb, with exons 2-5 clustered in a 1.1-kb region. The putative catalytic site (Ser423) and a possible lipid-binding region in the C-terminal part are encoded by exons 6 and 9, respectively. Exon 8 encodes the phosphorylation site (Ser551) that controls cAMP-mediated activity and a second site (Ser553) that is phosphorylated by 5'-AMP-activated protein kinase. Human HSL showed 83% identity with the rat enzyme and contained a 12-aa deletion immediately upstream of the phosphorylation sites with an unknown effect on the activity control. Besides the catalytic site motif (Gly-Xaa-Ser-Xaa-Gly) found in most lipases, HSL shows no homology with other known lipases or proteins, except for a recently reported unexpected homology between the region surrounding its catalytic site and that of the lipase 2 of Moraxella TA144, an antarctic psychrotrophic bacterium. The gene of lipase 2, which catalyses lipolysis below 4 degrees C, was absent in the genomic DNA of five other Moraxella strains living at 37 degrees C. The lipase 2-like sequence in HSL may reflect an evolutionarily conserved cold adaptability that might be of critical survival value when low-temperature-mobilized endogenous lipids are the primary energy source (e.g., in poikilotherms or hibernators). The finding that HSL at 10 degrees C retained 3- to 5-fold more of its 37 degrees C catalytic activity than lipoprotein lipase or carboxyl ester lipase is consistent with this hypothesis. Images Fig. 5 PMID:8506334

Lipase inhibition and antiobesity effect of Atractylodes lancea.

PubMed

Jiao, Ping; Tseng-Crank, Julie; Corneliusen, Brandon; Yimam, Mesfin; Hodges, Mandee; Hong, Mei; Maurseth, Catherine; Oh, Misun; Kim, Hyunjin; Chu, Min; Jia, Qi

2014-05-01

The ethanol extract of Atractylodes lancea rhizome displayed significant lipase inhibition with an IC50 value of 9.06 µg/mL in a human pancreatic lipase assay from high-throughput screening. Bioassay-guided isolation led to the identification of one new polyacetylene, syn-(5E,11E)-3-acetoxy-4-O-(3-methylbutanoyl)-1,5,11-tridecatriene-7,9-diyne-3,4-diol (7), along with six known compounds (1-6). The structure of compound 7 was determined based on the analysis of NMR and MS data. Among these seven lipase inhibitors, the major compound atractylodin (1) showed the highest lipase inhibitory activity (IC50 = 39.12 µM). The antiobesity effect of the ethanol extract of Atractylodes lancea rhizome was evaluated in a high-fat diet-induced obesity mice model at daily dosages of 250 mg/kg and 500 mg/kg body weight for 4 weeks, and treatment with this extract demonstrated a moderate efficacy at the 500 mg/kg dose level. Georg Thieme Verlag KG Stuttgart · New York.

Hydrolysis Activity of Virgin Coconut Oil Using Lipase from Different Sources.

PubMed

Nguyen, T A V; Le, Truong D; Phan, Hoa N; Tran, Lam B

2018-01-01

Two types of lipase, Candida rugosa lipase (CRL) and porcine pancreas lipase (PPL), were used to hydrolyze virgin coconut oil (VCO). The hydrolysis process was carried out under four parameters, VCO to buffer ratio, lipase concentration, pH, and temperature, which have a significant effect on hydrolysis of lipase. CRL obtained the best hydrolysis condition at 1 : 5 of VCO to buffer ratio, 1.5% of CRL concentration, pH 7, and temperature of 40°C. Meanwhile, PPL gave different results at 1 : 4 of VCO to buffer ratio, 2% of lipase concentration, pH 7.5, and 40°C. The highest hydrolysis degree of CRL and PPL was obtained after 16 hours and 26 hours, reaching 79.64% and 27.94%, respectively. Besides, the hydrolysis process was controlled at different time course (every half an hour) at the first 4 hours of reaction to compare the initial hydrolysis degree of these two lipase types. FFAs from hydrolyzed products were isolated and determined the percentage of each fatty acid which contributes to the FFAs mixture. As a result, medium chain fatty acids (MCFAs) made up the main contribution in composition of FFAs and lauric acid (C12) was the largest segment (47.23% for CRL and 44.23% for PPL).

Hydrolysis Activity of Virgin Coconut Oil Using Lipase from Different Sources

PubMed Central

Phan, Hoa N.; Tran, Lam B.

2018-01-01

Two types of lipase, Candida rugosa lipase (CRL) and porcine pancreas lipase (PPL), were used to hydrolyze virgin coconut oil (VCO). The hydrolysis process was carried out under four parameters, VCO to buffer ratio, lipase concentration, pH, and temperature, which have a significant effect on hydrolysis of lipase. CRL obtained the best hydrolysis condition at 1 : 5 of VCO to buffer ratio, 1.5% of CRL concentration, pH 7, and temperature of 40°C. Meanwhile, PPL gave different results at 1 : 4 of VCO to buffer ratio, 2% of lipase concentration, pH 7.5, and 40°C. The highest hydrolysis degree of CRL and PPL was obtained after 16 hours and 26 hours, reaching 79.64% and 27.94%, respectively. Besides, the hydrolysis process was controlled at different time course (every half an hour) at the first 4 hours of reaction to compare the initial hydrolysis degree of these two lipase types. FFAs from hydrolyzed products were isolated and determined the percentage of each fatty acid which contributes to the FFAs mixture. As a result, medium chain fatty acids (MCFAs) made up the main contribution in composition of FFAs and lauric acid (C12) was the largest segment (47.23% for CRL and 44.23% for PPL). PMID:29623233

Probing Conformational Changes and Interfacial Recognition Site of Lipases With Surfactants and Inhibitors.

PubMed

Mateos-Diaz, E; Amara, S; Roussel, A; Longhi, S; Cambillau, C; Carrière, F

2017-01-01

Structural studies on lipases by X-ray crystallography have revealed conformational changes occurring in the presence of surfactants/inhibitors and the pivotal role played by a molecular "lid" of variable size and structure depending on the enzyme. Besides controlling the access to the enzyme active site, the lid is involved in lipase activation, formation of the interfacial recognition site (IRS), and substrate docking within the active site. The combined use of surfactants and inhibitors has been critical for a better understanding of lipase structure-function relationships. An overview of crystal structures of lipases in complex with surfactants and inhibitors reveals common structural features and shows how surfactants monomers interact with the lid in its open conformation. The location of surfactants, inhibitors, and hydrophobic residues exposed upon lid opening provides insights into the IRS of lipases. The mechanism by which surfactants promote the lid opening can be further investigated in solution by site-directed spin labeling of lipase coupled to electron paramagnetic resonance spectroscopy. These experimental approaches are illustrated here by results obtained with mammalian digestive lipases, fungal lipases, and cutinases. © 2017 Elsevier Inc. All rights reserved.

Lipase immobilization on epoxy-activated poly(vinyl acetate-acrylamide) microspheres.

PubMed

Zhang, Dong-Hao; Peng, Li-Juan; Wang, Yun; Li, Ya-Qiong

2015-05-01

Poly(vinyl acetate-acrylamide) microspheres with an average diameter of 2-4μm were successfully prepared and characterized via SEM and FTIR. Then the microspheres were modified with epoxy groups through reacting with epichlorohydrin and used as carriers to covalently immobilize Candida rugosa lipase. The results revealed that agitation played an important role on epoxy activation and the immobilization ratio increased with the increase of the epoxy density. On the other hand, the specific activity of the immobilized lipase as well as the activity recovery declined gradually with the increase in the immobilization ratio from 72% to 93%, which were attributed to the steric hindrance effects caused by enzyme overloading. When epoxy density was 76μmol/g microsphere, the activity recovery reached the maximum at 47.5%, and the activity of the immobilized lipase was 261.3U/g microsphere. Moreover, the thermal stability of the immobilized lipase was much better than that of the free one, which indicated potential applications of the immobilized lipase. Copyright © 2015 Elsevier B.V. All rights reserved.

Secoiridoids from the stem barks of Fraxinus rhynchophylla with pancreatic lipase inhibitory activity.

PubMed

Ahn, Jong Hoon; Shin, Eunjin; Liu, Qing; Kim, Seon Beom; Choi, Kyeong-Mi; Yoo, Hwan-Soo; Hwang, Bang Yeon; Lee, Mi Kyeong

2013-01-01

Pancreatic lipase digests dietary fats by hydrolysis, which is a key enzyme for lipid absorption. Therefore, reduction of fat absorption by the inhibition of pancreatic lipase is suggested to be a therapeutic strategy for obesity. From the EtOAc-soluble fraction of the stem barks of Fraxinus rhynchophylla (Oleaceae), four secoiridoids such as ligstroside (1), oleuropein (2), 2"-hydroxyoleuropein (3) and hydroxyframoside B (4) were isolated. The inhibitory activity of these compounds on pancreatic lipase was assessed using porcine pancreatic lipase as an in vitro assay system. Compound 4 showed the strongest inhibition on pancreatic lipase, which followed by compounds 1-3. In addition, compound 4 exerted inhibitory effect on pancreatic lipase in a mixed mechanism of competitive and noncompetitive manner. Taken together, F. rhynchophylla and its constituents might be beneficial to obesity.

Evolution of the carboxylate Jen transporters in fungi.

PubMed

Lodi, Tiziana; Diffels, Julie; Goffeau, André; Baret, Philippe V

2007-08-01

Synteny analysis is combined with sequence similarity and motif identification to trace the evolution of the putative monocarboxylate (lactate/pyruvate) transporters Jen1p and the dicarboxylate (succinate/fumarate/malate) transporters Jen2p in Hemiascomycetes yeasts and Euascomycetes fungi. It is concluded that a precursor form of Jen1p, named here preJen1p, arose by the duplication of an ancestral Jen2p, during the speciation of Yarrowia lipolytica, which was transferred into a new syntenic context. The Jen1p transporters differentiated from preJen1p in Kluyveromyces lactis, before the Whole Genome Duplication (WGD), and are conserved as a single copy in the Saccharomyces species. In contrast, the ancestral Jen2p was definitively lost just prior to the WGD and is absent in Saccharomyces.

Biodegradation of micropollutant naproxen with a selected fungal strain and identification of metabolites.

PubMed

Aracagök, Y Doruk; Göker, Hakan; Cihangir, Nilüfer

2017-05-01

Pharmaceuticals are widely used for treating human and animal diseases. Naproxen [(S) 6-methoxy-α-methyl-2-naphthalene acetic acid] and its sodium salt are members of the α-arylpropionic acid group of nonsteroidal anti-inflammatory drugs. Due to excessive usage of naproxen, this drug has been determined even in drinking water. In this study, four fungal strains Phanerochaete chrysosporium, Funalia trogii, Aspergillus niger, and Yarrowia lipolytica were investigated in terms of naproxen removal abilities. According to LC/MS data, A. niger was found the most efficient strain with 98% removal rate. Two main by-products of fungal transformation, O-desmethylnaproxen and 7-hydroxynaproxen, were identified by using LC/MS, 1HNMR, and 13CNMR. Our results showed that O-demethylation and hydroxylation of naproxen is catalyzed by cytochrome P450 enzyme system.

Statistical optimization for lipase production from solid waste of vegetable oil industry.

PubMed

Sahoo, Rajesh Kumar; Kumar, Mohit; Mohanty, Swati; Sawyer, Matthew; Rahman, Pattanathu K S M; Sukla, Lala Behari; Subudhi, Enketeswara

2018-04-21

The production of biofuel using thermostable bacterial lipase from hot spring bacteria out of low-cost agricultural residue olive oil cake is reported in the present paper. Using a lipase enzyme from Bacillus licheniformis, a 66.5% yield of methyl esters was obtained. Optimum parameters were determined, with maximum production of lipase at a pH of 8.2, temperature 50.8°C, moisture content of 55.7%, and biosurfactant content of 1.693 mg. The contour plots and 3D surface responses depict the significant interaction of pH and moisture content with biosurfactant during lipase production. Chromatographic analysis of the lipase transesterification product was methyl esters, from kitchen waste oil under optimized conditions, generated methyl palmitate, methyl stearate, methyl oleate, and methyl linoleate.

Lipases as catalysts in synthesis of fine chemicals.

PubMed

Baldessari, Alicia

2012-01-01

The application of lipases as catalysts in the synthesis of an intermediate of alfuzosin and lapyrium chloride is described. In the first case, the one-pot procedure to obtain the intermediate involves the treatment of tetrahydrofuroic acid with ethanol in the presence of Candida antarctica lipase followed by the addition of N-methyl-1,3-diaminopropane. In the second part of the chapter, an efficient route for large-scale preparation of lapyrium chloride is developed from chloroacetic acid in four steps, three of them enzymatic. Due to the chemoselective behavior of the lipases, both products described in the present chapter were obtained in a high degree of purity and yield, applying mild reaction conditions, and following a low environmental impact methodology.

Identification and sequence analyses of novel lipase encoding novel thermophillic bacilli isolated from Armenian geothermal springs.

PubMed

Shahinyan, Grigor; Margaryan, Armine; Panosyan, Hovik; Trchounian, Armen

2017-05-02

Among the huge diversity of thermophilic bacteria mainly bacilli have been reported as active thermostable lipase producers. Geothermal springs serve as the main source for isolation of thermostable lipase producing bacilli. Thermostable lipolytic enzymes, functioning in the harsh conditions, have promising applications in processing of organic chemicals, detergent formulation, synthesis of biosurfactants, pharmaceutical processing etc. In order to study the distribution of lipase-producing thermophilic bacilli and their specific lipase protein primary structures, three lipase producers from different genera were isolated from mesothermal (27.5-70 °C) springs distributed on the territory of Armenia and Nagorno Karabakh. Based on phenotypic characteristics and 16S rRNA gene sequencing the isolates were identified as Geobacillus sp., Bacillus licheniformis and Anoxibacillus flavithermus strains. The lipase genes of isolates were sequenced by using initially designed primer sets. Multiple alignments generated from primary structures of the lipase proteins and annotated lipase protein sequences, conserved regions analysis and amino acid composition have illustrated the similarity (98-99%) of the lipases with true lipases (family I) and GDSL esterase family (family II). A conserved sequence block that determines the thermostability has been identified in the multiple alignments of the lipase proteins. The results are spreading light on the lipase producing bacilli distribution in geothermal springs in Armenia and Nagorno Karabakh. Newly isolated bacilli strains could be prospective source for thermostable lipases and their genes.

Purification and Characterization of a Thermostable Lipase from Geobacillus thermodenitrificans IBRL-nra

PubMed Central

Balan, Anuradha; Ibrahim, Darah; Abdul Rahim, Rashidah; Ahmad Rashid, Fatimah Azzahra

2012-01-01

Thermostable lipase from Geobacillus thermodenitrificans IBRL-nra was purified and characterized. The production of thermostable lipase from Geobacillus thermodenitrificans IBRL-nra was carried out in a shake-flask system at 65°C in cultivation medium containing; glucose 1.0% (w/v); yeast extract 1.25% (w/v); NaCl 0.45% (w/v) olive oil 0.1% (v/v) with agitation of 200 rpm for 24 hours. The extracted extracellular crude thermostable lipase was purified to homogeneity by using ultrafiltration, Heparin-affinity chromatography, and Sephadex G-100 gel-filtration chromatography by 34 times with a final yield of 9%. The molecular weight of the purified enzyme was estimated to be 30 kDa after SDS-PAGE analysis. The optimal temperature for thermostable lipase was 65°C and it retained its initial activity for 3 hours. Thermostable lipase activity was highest at pH 7.0 and stable for 16 hours at this pH at 65°C. Thermostable lipase showed elevated activity when pretreated with BaCl2, CaCl2, and KCl with 112%, 108%, and 106%, respectively. Lipase hydrolyzed tripalmitin (C16) and olive oil with optimal activity (100%) compared to other substrates. PMID:23198138

Cold-adapted organic solvent tolerant alkalophilic family I.3 lipase from an Antarctic Pseudomonas.

PubMed

Ganasen, Menega; Yaacob, Norhayati; Rahman, Raja Noor Zaliha Raja Abd; Leow, Adam Thean Chor; Basri, Mahiran; Salleh, Abu Bakar; Ali, Mohd Shukuri Mohamad

2016-11-01

Lipolytic enzymes with cold adaptation are gaining increasing interest due to their biotechnological prospective. Previously, a cold adapted family I.3 lipase (AMS8 lipase) was isolated from an Antarctic Pseudomonas. AMS8 lipase was largely expressed in insoluble form. The refolded His-tagged recombinant AMS8 lipase was purified with 23.0% total recovery and purification factor of 9.7. The purified AMS8 lipase migrated as a single band with a molecular weight approximately 65kDa via electrophoresis. AMS8 lipase was highly active at 30°C at pH 10. The half-life of AMS8 lipase was reported at 4 and 2h under the incubation of 30 and 40°C, respectively. The lipase was stable over a broad range of pH. It showed enhancement effect in its relative activity under the presence of Li + , Na + , K + , Rb + and Cs + after 30min treatment. Heavy metal ions such as Cu 2+ , Fe 3+ and Zn 2+ inhibited AMS8 activity. This cold adapted alkalophilic AMS lipase was also active in various organic solvent of different polarity. These unique properties of this biological macromolecule will provide considerable potential for many biotechnological applications and organic synthesis at low temperature. Copyright © 2016 Elsevier B.V. All rights reserved.

Strategies to characterize fungal lipases for applications in medicine and dairy industry.

PubMed

Gopinath, Subash C B; Anbu, Periasamy; Lakshmipriya, Thangavel; Hilda, Azariah

2013-01-01

Lipases are water-soluble enzymes that act on insoluble substrates and catalyze the hydrolysis of long-chain triglycerides. Lipases play a vital role in the food, detergent, chemical, and pharmaceutical industries. In the past, fungal lipases gained significant attention in the industries due to their substrate specificity and stability under varied chemical and physical conditions. Fungal enzymes are extracellular in nature, and they can be extracted easily, which significantly reduces the cost and makes this source preferable over bacteria. Soil contaminated with spillage from the products of oil and dairy harbors fungal species, which have the potential to secrete lipases to degrade fats and oils. Herein, the strategies involved in the characterization of fungal lipases, capable of degrading fatty substances, are narrated with a focus on further applications.

Strategies to Characterize Fungal Lipases for Applications in Medicine and Dairy Industry

PubMed Central

Gopinath, Subash C. B.; Anbu, Periasamy; Lakshmipriya, Thangavel; Hilda, Azariah

2013-01-01

Lipases are water-soluble enzymes that act on insoluble substrates and catalyze the hydrolysis of long-chain triglycerides. Lipases play a vital role in the food, detergent, chemical, and pharmaceutical industries. In the past, fungal lipases gained significant attention in the industries due to their substrate specificity and stability under varied chemical and physical conditions. Fungal enzymes are extracellular in nature, and they can be extracted easily, which significantly reduces the cost and makes this source preferable over bacteria. Soil contaminated with spillage from the products of oil and dairy harbors fungal species, which have the potential to secrete lipases to degrade fats and oils. Herein, the strategies involved in the characterization of fungal lipases, capable of degrading fatty substances, are narrated with a focus on further applications. PMID:23865040

Encapsulation of lipase within metal-organic framework (MOF) with enhanced activity intensified under ultrasound.

PubMed

Nadar, Shamraja S; Rathod, Virendra K

2018-01-01

The enzyme under lower-intensity ultrasonic irradiation leads to favorable conformational changes, thereby enhancing its activity. In this study, lipase activity was augmented upto 1.6-folds after ultrasonic treatment at 22kHz and 11.38Wcm -2 for 25min. This highly activated lipase was encapsulated within zeolite imidazolate framework-8 (ZIF-8) as a metal-organic framework (MOF) material via facile one-step biomineralization method by simply mixing aqueous solution of 2-methylimidazole (13.3mmol) and zinc acetate (1.33mmol) along with sonicated lipase within 10min at room temperature (28±2°C). The prepared lipase-MOF was characterized by using FT-IR, FT-Raman, XRD, BET, confocal scanning laser microscopy, TGA and SEM. Further, the thermal stability of lipase embedded MOF was evaluated in the range of 55-75°C on the basis of half-life which showed 3.2 folds increment as against free lipase. In Michaelis-Menten kinetics studies, sonicated lipase entrapped MOF showed nearly same K m and V max values as that of sonicated free lipase. Moreover, the immobilized lipase exhibited up to 54% of residual activity after seven successive cycles of reuse, whereas it retained 90% of residual activity till twenty-five days of storage. Finally, the conformational changes occurred in lipase after sonication treatment and encapsulation within MOF were analyzed by using FT-IR data analysis tools and fluorescent spectroscopy. Copyright © 2017 Elsevier Inc. All rights reserved.

Lipase-nanoporous gold biocomposite modified electrode for reliable detection of triglycerides.

PubMed

Wu, Chao; Liu, Xueying; Li, Yufei; Du, Xiaoyu; Wang, Xia; Xu, Ping

2014-03-15

For triglycerides biosensor design, protein immobilization is necessary to create the interface between the enzyme and the electrode. In this study, a glassy carbon electrode (GCE) was modified with lipase-nanoporous gold (NPG) biocomposite (denoted as lipase/NPG/GCE). Due to highly conductive, porous, and biocompatible three-dimensional structure, NPG is suitable for enzyme immobilization. In cyclic voltammetry experiments, the lipase/NPG/GCE bioelectrode displayed surface-confined reaction in a phosphate buffer solution. Linear responses were obtained for tributyrin concentrations ranging from 50 to 250 mg dl(-1) and olive oil concentrations ranging from 10 to 200 mg dl(-1). The value of apparent Michaelis-Menten constant for tributyrin was 10.67 mg dl(-1) and the detection limit was 2.68 mg dl(-1). Further, the lipase/NPG/GCE bioelectrode had strong anti-interference ability against urea, glucose, cholesterol, and uric acid as well as a long shelf-life. For the detection of triglycerides in human serum, the values given by the lipase/NPG/GCE bioelectrode were in good agreement with those of an automatic biochemical analyzer. These properties along with a long self-life make the lipase/NPG/GCE bioelectrode an excellent choice for the construction of triglycerides biosensor. © 2013 Elsevier B.V. All rights reserved.

Biodiesel production by transesterification using immobilized lipase.

PubMed

Narwal, Sunil Kumar; Gupta, Reena

2013-04-01

Biodiesel can be produced by transesterification of vegetable or waste oil catalysed by lipases. Biodiesel is an alternative energy source to conventional fuel. It combines environmental friendliness with biodegradability, low toxicity and renewability. Biodiesel transesterification reactions can be broadly classified into two categories: chemical and enzymatic. The production of biodiesel using the enzymatic route eliminates the reactions catalysed under acid or alkali conditions by yielding product of very high purity. The modification of lipases can improve their stability, activity and tolerance to alcohol. The cost of lipases and the relatively slower reaction rate remain the major obstacles for enzymatic production of biodiesel. However, this problem can be solved by immobilizing the enzyme on a suitable matrix or support, which increases the chances of re-usability. The main factors affecting biodiesel production are composition of fatty acids, catalyst, solvents, molar ratio of alcohol and oil, temperature, water content, type of alcohol and reactor configuration. Optimization of these parameters is necessary to reduce the cost of biodiesel production.

Deciphering the toxicity of bisphenol a to Candida rugosa lipase through spectrophotometric methods.

PubMed

Zhang, Rui; Zhao, Lining; Liu, Rutao

2016-10-01

Bisphenol A is widely used in the manufacture of food packaging and beverage containers and can invade our food and cause contamination. Candida rugose lipase has been a versatile enzyme for biocatalysis and biotransformations to produce useful materials for food, pharmaceutical and flavor. The interactions between bisphenol A and Candida rugosa lipase in vitro were studied by UV-vis, steady-state fluorescence, circular dichroism, synchronous fluorescence, light scattering spectra, molecular docking and enzyme activity assay to better understand the toxicity and toxic mechanisms of bisphenol A. The intrinsic fluorescence of the tryptophan amino acid residue and the secondary structure of the globular protein candida rugose lipase were made use of to thoroughly investigate the structural changes caused by bisphenol A. The results of the fluorescence indicated that bisphenol A interacted with candida rugose lipase and made tryptophan be exposed to a hydrophobic environment. Multi-spectroscopic measurements showed that the addition of bisphenol A increased the intrinsic fluorescence of Candida rugosa lipase, loosened its skeleton structure and changed its secondary structure. Also, the increased activity of Candida rugosa lipase revealed that the position or the structure of the catalytic triad of Candida rugosa lipase may be changed. The molecular docking results showed that bisphenol A bound with the residue Serine 209 which could be another reason for the increased activity of Candida rugosa lipase. Moreover, as can be seen from the results of resonance light scattering and dynamic light scattering, the volume of the Candida rugosa lipase was decreased and the lid may be stripped. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of biotechnologically relevant extracellular lipase produced by Aspergillus terreus NCFT 4269.10

PubMed Central

Sethi, Bijay Kumar; Nanda, Prativa Kumari; Sahoo, Santilata

2016-01-01

Enzyme production by Aspergillus terreus NCFT 4269.10 was studied under liquid static surface and solid-state fermentation using mustard oil cake as a substrate. The maximum lipase biosynthesis was observed after incubation at 30 °C for 96 h. Among the domestic oils tested, the maximum lipase biosynthesis was achieved using palm oil. The crude lipase was purified 2.56-fold to electrophoretic homogeneity, with a yield of 8.44%, and the protein had a molecular weight of 46.3 kDa as determined by SDS-PAGE. Enzyme characterization confirmed that the purified lipase was most active at pH 6.0, temperature of 50 °C, and substrate concentration of 1.5%. The enzyme was thermostable at 60 °C for 1 h, and the optimum enzyme–substrate reaction time was 30 min. Sodium dodecyl sulfate and commercial detergents did not significantly affect lipase activity during 30-min incubation at 30 °C. Among the metal ions tested, the maximum lipase activity was attained in the presence of Zn2+, followed by Mg2+ and Fe2+. Lipase activity was not significantly affected in the presence of ethylenediaminetetraacetic acid, sodium lauryl sulfate and Triton X-100. Phenylmethylsulfonyl fluoride (1 mM) and the reducing, β-mercaptoethanol significantly inhibited lipase activity. The remarkable stability in the presence of detergents, additives, inhibitors and metal ions makes this lipase unique and a potential candidate for significant biotechnological exploitation. PMID:26887237

Fabrication of enzyme-immobilized halloysite nanotubes for affinity enrichment of lipase inhibitors from complex mixtures.

PubMed

Wang, Haibo; Zhao, Xiaoping; Wang, Shufang; Tao, Shan; Ai, Ni; Wang, Yi

2015-05-01

Lipase is the key enzyme for catalyzing triglyceride hydrolysis in vivo, and lipase inhibitors have been used in the management of obesity. We present the first report on the use of lipase-adsorbed halloysite nanotubes as an efficient medium for the selective enrichment of lipase inhibitors from natural products. A simple and rapid approach was proposed to fabricate lipase-adsorbed nanotubes through electrostatic interaction. Results showed that more than 85% lipase was adsorbed into nanotubes in 90 min, and approximately 80% of the catalytic activity was maintained compared with free lipase. The specificity and reproducibility of the proposed approach were validated by screening a known lipase inhibitor (i.e., orlistat) from a mixture that contains active and inactive compounds. Moreover, we applied this approach with high performance liquid chromatography-mass spectrometry technique to screen lipase inhibitors from the Magnoliae cortex extract, a medicinal plant used for treating obesity. Two novel biphenyl-type natural lipase inhibitors magnotriol A and magnaldehyde B were identified, and their IC50 values were determined as 213.03 and 96.96 μM, respectively. The ligand-enzyme interactions of magnaldehyde B were further investigated by molecular docking. Our findings proved that enzyme-adsorbed nanotube could be used as a feasible and selective affinity medium for the rapid screening of enzyme inhibitors from complex mixtures. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparison of immunoreactive serum trypsinogen and lipase in Cystic Fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lloyd-Still, J.D.; Weiss, S.; Wessel, H.

1984-01-01

The incidence of Cystic Fibrosis (CF) is 1 in 2,000. Early detection and treatment of CF may necessitate newborn screening with a reliable and cost-effective test. Serum immunoreactive trypsinogen (IRT) an enzyme produced by the pancreas, is detectable by radioimmunoassay (RIA) techniques. Recently, it has been shown that IRT is elevated in CF infants for the first few months of life and levels become subnormal as pancreatic insufficiency progresses. Other enzymes produced by the pancreas, such as lipase, are also elevated during this time. The author's earlier work confirmed previous reports of elevated IRT levels in CF infants. The developmentmore » of a new RIA for lipase (nuclipase) has enabled comparison of these 2 pancreatic enzymes in C.F. Serum IRT and lipase determinations were performed on 2 groups of CF patients; infants under 1 year of age, and children between 1 and 18 years of age. Control populations of the same age groups were included. The results showed that both trypsin (161 +- 92 ng/ml, range 20 to 400) and lipase (167 +- 151 ng/ml, range 29 to 500) are elevated in CF in the majority of infants. Control infants had values of IRT ranging from 20 to 29.5 ng/ml and lipase values ranging from 23 to 34 ng/ml. IRT becomes subnormal in most CF patients by 8 years of age as pancreatic function insufficiency increases. Lipase levels and IRT levels correlate well in infancy, but IRT is a more sensitive indicator of pancreatic insufficiency in older patients with CF.« less

Identification of a putative triacylglycerol lipase from papaya latex by functional proteomics.

PubMed

Dhouib, R; Laroche-Traineau, J; Shaha, R; Lapaillerie, D; Solier, E; Rualès, J; Pina, M; Villeneuve, P; Carrière, F; Bonneu, M; Arondel, V

2011-01-01

Latex from Caricaceae has been known since 1925 to contain strong lipase activity. However, attempts to purify and identify the enzyme were not successful, mainly because of the lack of solubility of the enzyme. Here, we describe the characterization of lipase activity of the latex of Vasconcellea heilbornii and the identification of a putative homologous lipase from Carica papaya. Triacylglycerol lipase activity was enriched 74-fold from crude latex of Vasconcellea heilbornii to a specific activity (SA) of 57 μmol·min(-1)·mg(-1) on long-chain triacylglycerol (olive oil). The extract was also active on trioctanoin (SA = 655 μmol·min(-1)·mg(-1) ), tributyrin (SA = 1107 μmol·min(-1)·mg(-1) ) and phosphatidylcholine (SA = 923 μmol·min(-1)·mg(-1) ). The optimum pH ranged from 8.0 to 9.0. The protein content of the insoluble fraction of latex was analyzed by electrophoresis followed by mass spectrometry, and 28 different proteins were identified. The protein fraction was incubated with the lipase inhibitor [(14) C]tetrahydrolipstatin, and a 45 kDa protein radiolabeled by the inhibitor was identified as being a putative lipase. A C. papaya cDNA encoding a 55 kDa protein was further cloned, and its deduced sequence had 83.7% similarity with peptides from the 45 kDa protein, with a coverage of 25.6%. The protein encoded by this cDNA had 35% sequence identity and 51% similarity to castor bean acid lipase, suggesting that it is the lipase responsible for the important lipolytic activities detected in papaya latex. © 2010 The Authors Journal compilation © 2010 FEBS.

The genotypic diversity and lipase production of some thermophilic bacilli from different genera

PubMed Central

Koc, Melih; Cokmus, Cumhur; Cihan, Arzu Coleri

2015-01-01

Abstract Thermophilic 32 isolates and 20 reference bacilli were subjected to Rep-PCR and ITS-PCR fingerprinting for determination of their genotypic diversity, before screening lipase activities. By these methods, all the isolates and references could easily be differentiated up to subspecies level from each other. In screening assay, 11 isolates and 7 references were found to be lipase producing. Their extracellular lipase activities were measured quantitatively by incubating in both tributyrin and olive oil broths at 60 °C and pH 7.0. During the 24, 48 and 72-h period of incubation, the changes in the lipase activities, culture absorbance, wet weight of biomass and pH were all measured. The activity was determined by using pNPB in 50 mM phosphate buffer at pH 7.0 at 60 °C. The lipase production of the isolates in olive oil broths varied between 0.008 and 0.052, whereas these values were found to be 0.002-0.019 (U/mL) in the case of tyributyrin. For comparison, an index was established by dividing the lipase activities to cell biomass (U/mg). The maximum thermostable lipase production was achieved by the isolates F84a, F84b, and G. thermodenitrificans DSM 465T (0.009, 0.008 and 0.008 U/mg) within olive oil broth, whereas G. stearothermophilus A113 displayed the highest lipase activity than its type strain in tyributyrin. Therefore, as some of these isolates displayed higher activities in comparison to references, new lipase producing bacilli were determined by presenting their genotypic diversity with DNA fingerprinting techniques. PMID:26691464

Adrenal and liver in normal and cld/cld mice synthesize and secrete hepatic lipase, but the lipase is inactive in cld/cld mice.

PubMed

Schultz, C J; Blanchette-Mackie, E J; Scow, R O

2000-02-01

Combined lipase deficiency (cld) is a recessive mutation in mice that causes a severe lack of lipoprotein lipase (LPL) and hepatic lipase (HL) activities, hyperlipemia, and death within 3 days after birth. Earlier studies showed that inactive LPL and HL were synthesized by cld/cld tissues and that LPL synthesized by cld/cld brown adipocytes was retained in their ER. We report here a study of HL in liver, adrenal, and plasma of normal newborn and cld/cld mice. Immunofluorescence studies showed HL was present in extracellular space, but not in cells, in liver and adrenal of both normal and cld/cld mice. When protein secretion was blocked with monensin, HL was retained intracellularly in liver cell cultures and in incubated adrenal tissues of both groups of mice. These findings demonstrated that HL was synthesized and secreted by liver and adrenal cells in normal newborn and cld/cld mice. HL activities in liver, adrenal, and plasma in cld/cld mice were very low, <8% of that in normal newborn mice, indicating that HL synthesized and secreted by cld/cld cells was inactive. Livers of both normal newborn and cld/cld mice synthesized LPL, but the level of LPL activity in cld/cld liver was very low, <9% of that in normal liver. Immunofluorescence studies showed that LPL was present intracellularly in liver of cld/cld mice, indicating that LPL was synthesized but not secreted by cld/cld liver cells. Immunofluorescent LPL was not found in normal newborn liver cells unless the cells were treated with monensin, thus demonstrating that normal liver cells synthesized and secreted LPL. Livers of both groups of mice contained an unidentified alkaline lipase activity which accounted for 34-54% of alkaline lipase activity in normal and 65% of that in cld/cld livers. Our findings indicate that liver and adrenal cells synthesized and secreted HL in both normal newborn and cld/cld mice, but the lipase was inactive in cld/cld mice. That cld/cld liver cells secreted inactive HL while

Characterization of an extracellular lipase by Pseudomonas koreensis BK-L07 isolated from soil.

PubMed

Anbu, Periasamy

2014-01-01

Screening using spirit blue agar revealed that strain BK-L07 had the highest lipase activity. Furthermore, the isolated strain was identified as Pseudomonas sp. based on morphological, physiological, biochemical, and molecular analyses. The 16S rRNA gene sequence of strain BK-L07 shared a high similarity with that of Pseudomonas koreensis (99%). The nutritional conditions and physicochemical properties were influenced by P. koreensis BK-L07. The maximum lipase production was obtained in tryptic soy broth medium at pH 8.0 and a temperature of 25°C after 36 hr of incubation. In addition, the lipase activity was determined using different carbon sources and lipase inducers. The lipase production was greatest when 1% maltose was used as the carbon source and olive oil was used as the lipase inducer. The lipase production was significantly increased approximately threefold in the optimized medium when compared with the original medium. Further, the lipase was purified by ammonium sulfate precipitation and gel filtration chromatography with a purification yield of 10.8%. The molecular mass of lipase was 45 kDa. The optimum temperature and pH were 40°C and 8.0, respectively. The enzyme was stable up to 50°C and at pH from 7 to 9. In addition, the enzyme activity was stimulated by MgSO4 and completely inhibited by ethylenediamine tetraacetic acid (EDTA), indicating the metalloenzyme type. The lipase activity was toward medium to long chain length of fatty acids (C10 to C18). Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.

21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

Code of Federal Regulations, 2014 CFR

2014-04-01

... (CONTINUED) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme... animals. (c) The enzyme is produced by a process which completely removes the organism Mucor miehei var...

Fat digestion by lingual lipase: mechanism of lipolysis in the stomach and upper small intestine.

PubMed

Liao, T H; Hamosh, P; Hamosh, M

1984-05-01

Ten to 30% of dietary fat is hydrolyzed in the stomach by lingual lipase, an enzyme secreted from lingual serous glands. We investigated the substrate specificity of this enzyme as well as the potential of lingual lipase to act in the upper small intestine i.e., in the presence of bile salts and lecithin. The data presented show that partially purified preparations of rat lingual lipase and the lipase in gastric aspirates of newborn infants have identical substrate specificity: medium-chain triglycerides were hydrolyzed at rates 5-8-fold higher than long-chain triglycerides; the rat and human enzymes do not hydrolyze the ester bond of lecithin or cholesteryl-ester. In contrast to pancreatic lipase, the hydrolysis of triglycerides by lingual lipase is not inhibited by lecithin. But, similar to pancreatic lipase the activity of lingual lipase is inhibited by bile salts, the extent of inhibition varying with its nature and concentration. This inactivation is not prevented by colipase but is partially averted by lipids and protein, suggesting that lingual lipase can remain active in the duodenum. The pH optimum of the enzyme (2.2-6.5 in the rat and 3.5-6.0 in human gastric aspirates) is compatible with continued activity in the upper small intestine, especially during the neonatal period, when the luminal pH is under 6.5. The marked variation in lipase activity levels in gastric aspirates of newborn infants is probably due to individual variations in enzyme amounts. The characteristics of the lipase are however identical in infants with low, intermediate or high activity levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipase genes in Mucor circinelloides: identification, sub-cellular location, phylogenetic analysis and expression profiling during growth and lipid accumulation.

PubMed

Zan, Xinyi; Tang, Xin; Chu, Linfang; Zhao, Lina; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Song, Yuanda

2016-10-01

Lipases or triacylglycerol hydrolases are widely spread in nature and are particularly common in the microbial world. The filamentous fungus Mucor circinelloides is a potential lipase producer, as it grows well in triacylglycerol-contained culture media. So far only one lipase from M. circinelloides has been characterized, while the majority of lipases remain unknown in this fungus. In the present study, 47 potential lipase genes in M. circinelloides WJ11 and 30 potential lipase genes in M. circinelloides CBS 277.49 were identified by extensive bioinformatics analysis. An overview of these lipases is presented, including several characteristics, sub-cellular location, phylogenetic analysis and expression profiling of the lipase genes during growth and lipid accumulation. All of these proteins contained the consensus sequence for a classical lipase (GXSXG motif) and were divided into four types including α/β-hydrolase_1, α/β-hydrolase_3, class_3 and GDSL lipase (GDSL) based on gene annotations. Phylogenetic analyses revealed that class_3 family and α/β-hydrolase_3 family were the conserved lipase family in M. circinelloides. Additionally, some lipases also contained a typical acyltransferase motif of H-(X) 4-D, and these lipases may play a dual role in lipid metabolism, catalyzing both lipid hydrolysis and transacylation reactions. The differential expression of all lipase genes were confirmed by quantitative real-time PCR, and the expression profiling were analyzed to predict the possible biological roles of these lipase genes in lipid metabolism in M. circinelloides. We preliminarily hypothesized that lipases may be involved in triacylglycerol degradation, phospholipid synthesis and beta-oxidation. Moreover, the results of sub-cellular localization, the presence of signal peptide and transcriptional analyses of lipase genes indicated that four lipase in WJ11 most likely belong to extracellular lipases with a signal peptide. These findings provide a platform

21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

Code of Federal Regulations, 2013 CFR

2013-04-01

... HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme derived from Mucor miehei var. Cooney et Emerson by... Emerson is nonpathogenic and nontoxic in man or other animals. (c) The enzyme is produced by a process...

21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

Code of Federal Regulations, 2012 CFR

2012-04-01

... HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme derived from Mucor miehei var. Cooney et Emerson by... Emerson is nonpathogenic and nontoxic in man or other animals. (c) The enzyme is produced by a process...

21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

Code of Federal Regulations, 2011 CFR

2011-04-01

... HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme derived from Mucor miehei var. Cooney et Emerson by... Emerson is nonpathogenic and nontoxic in man or other animals. (c) The enzyme is produced by a process...

21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

Code of Federal Regulations, 2010 CFR

2010-04-01

... HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme derived from Mucor miehei var. Cooney et Emerson by... Emerson is nonpathogenic and nontoxic in man or other animals. (c) The enzyme is produced by a process...

Dependence of PERT endpoint on endogenous lipase activity.

PubMed

Gao, Wen-Yi; Mulberg, Andrew E

2014-11-01

To clarify and to understand the potential for misinterpretation of change in fecal fat quantitation during pancreatic enzyme replacement therapy (PERT) trials for treatment of exocrine pancreatic insufficiency. Analysis of clinical trials submitted to the U.S. Food and Drug Administration (FDA) for approval of PERT that enrolled 123 cystic fibrosis adult and pediatric patients treated with Creon, Pertzye, Ultresa, and Zenpep. The CFA% defines lipase activity as a percentage of converting substrate of "Total Daily Dietary Fat Intake." PERT trials performed to date have modified the definition to converting the "Shared Daily Fat Intake," generating "Partial CFA" for the exogenous lipase: the higher the activity of coexisting endogenous lipase, the lower the "Partial CFA" of exogenous measured. This review shows that "Partial CFA" is not CFA. Enrollment of patients with low HPLA during treatment may improve the interpretability of "Partial CFA" measured by PERT trials.

Novel magnetic cross-linked lipase aggregates for improving the resolution of (R, S)-2-octanol.

PubMed

Liu, Ying; Guo, Chen; Liu, Chun-Zhao

2015-03-01

Novel magnetic cross-linked lipase aggregates were fabricated by immobilizing the cross-linked lipase aggregates onto magnetic particles with a high number of -NH2 terminal groups using p-benzoquinone as the cross-linking agent. At the optimal fabrication conditions, 100% of immobilization efficiency and 139% of activity recovery of the magnetic cross-linked lipase aggregates were achieved. The magnetic cross-linked lipase aggregates were able to efficiently resolve (R, S)-2-octanol, and retained 100% activity and 100% enantioselectivity after 10 cycles of reuse, whereas the cross-linked lipase aggregates only retained about 50% activity and 70% enantioselectivity due to insufficient cross-linking. These results provide a great potential for industrial applications of the magnetic cross-linked lipase aggregates. © 2014 Wiley Periodicals, Inc.

Acidolysis and glyceride synthesis reactions using fatty acids with two Pseudomonas lipases having different substrate specificities.

PubMed

Kojima, Yuzo; Sakuradani, Eiji; Shimizu, Sakayu

2006-09-01

Enzymatic acidolysis and glyceride synthesis using polyunsaturated fatty acids (PUFAs) with lipases from Pseudomonas fluorescens HU380 (HU-lipase), P. fluorescens AK102 (AK-lipase), and Candida rugosa (CR-lipase) were studied. The acidolysis of triolein with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) in n-hexane was evaluated with lipases immobilized on Celite 545. HU-lipase showed the highest incorporation rate at a low temperature (10 degrees C) with either EPA or DHA as the acyl donor, and the rate decreased with increasing reaction temperature. At 45 degrees C, the rates for EPA and DHA were 7.1 and 0.5 relative to those at 10 degrees C, respectively. The EPA incorporation rate was even higher at a low temperature (10 degrees C), and the DHA incorporation rate increased with decreasing temperature. Although AK-lipase showed the reverse tendency for incorporation rate, the DHA incorporation rate increased with increasing reaction temperature with both PUFAs. HU-lipase reacted well with PUFAs such as DHA, EPA, arachidonic acid (AA), mead acid (MA), and dihomo-gamma-linolenic acid (DGLA) on acidolysis and glyceride synthesis. The reactivities of AK-lipase toward these PUFAs except for DGLA, i.e., MA, AA, EPA, and DHA, were low for both reactions. The unique substrate specificities of the lipases from the Pseudomonas strains will enable us to use these lipases for the modification of fats and oils containing PUFAs such as fish oil.

Lipase-Secreting Bacillus Species in an Oil-Contaminated Habitat: Promising Strains to Alleviate Oil Pollution.

PubMed

Lee, Li Pin; Karbul, Hudzaifah Mohamed; Citartan, Marimuthu; Gopinath, Subash C B; Lakshmipriya, Thangavel; Tang, Thean-Hock

2015-01-01

Lipases are of great interest for different industrial applications due to their diversity and versatility. Among different lipases, microbial lipases are preferable due to their broad substrate specificity, and higher stability with lower production costs compared to the lipases from plants and animals. In the past, a vast number of bacterial species have been reported as potential lipases producers. In this study, the lipases-producing bacterial species were isolated from an oil spillage area in the conventional night market. Isolated species were identified as Bacillus species by biochemical tests which indicate their predominant establishment, and further screened on the agar solid surfaces using lipid and gelatin as the substrates. Out of the ten strains tested, four potential strains were subjected to comparison analysis of the lipolytic versus proteolytic activities. Strain 10 exhibited the highest lipolytic and proteolytic activity. In all the strains, the proteolytic activity is higher than the lipolytic activity except for strain 8, suggesting the possibility for substrate-based extracellular gene induction. The simultaneous secretion of both the lipase and protease is a mean of survival. The isolated bacterial species which harbour both lipase and protease enzymes could render potential industrial-based applications and solve environmental issues.

Influence of cosolvents on the hydrophobic surface immobilization topography of Candida antarctica lipase B

USDA-ARS?s Scientific Manuscript database

The presence of cosolvents and co-solutes during the immobilization of lipases on hydrophobic supports may influence the extent of lipase immobilization and the long-term catalytic stability of the biocatalyst. Candida antarctica B lipase immobilization was examined on a hydrophobic surface, i.e., ...

Effect of membranes with various hydrophobic/hydrophilic properties on lipase immobilized activity and stability.

PubMed

Chen, Guan-Jie; Kuo, Chia-Hung; Chen, Chih-I; Yu, Chung-Cheng; Shieh, Chwen-Jen; Liu, Yung-Chuan

2012-02-01

In this study, three membranes: regenerated cellulose (RC), glass fiber (GF) and polyvinylidene fluoride (PVDF), were grafted with 1,4-diaminobutane (DA) and activated with glutaraldehyde (GA) for lipase covalent immobilization. The efficiencies of lipases immobilized on these membranes with different hydrophobic/hydrophilic properties were compared. The lipase immobilized on hydrophobic PVDF-DA-GA membrane exhibited more than an 11-fold increase in activity compared to its immobilization on a hydrophilic RC-DA-GA membrane. The relationship between surface hydrophobicity and immobilized efficiencies was investigated using hydrophobic/hydrophilic GF membranes which were prepared by grafting a different ratio of n-butylamine/1,4-diaminobutane (BA/DA). The immobilized lipase activity on the GF membrane increased with the increased BA/DA ratio. This means that lipase activity was exhibited more on the hydrophobic surface. Moreover, the modified PVDF-DA membrane was grafted with GA, epichlorohydrin (EPI) and cyanuric chloride (CC), respectively. The lipase immobilized on the PVDF-DA-EPI membrane displayed the highest specific activity compared to other membranes. This immobilized lipase exhibited more significant stability on pH, thermal, reuse, and storage than did the free enzyme. The results exhibited that the EPI modified PVDF is a promising support for lipase immobilization. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Molecular and functional diversity of yeast and fungal lipases: their role in biotechnology and cellular physiology.

PubMed

Gupta, Rani; Kumari, Arti; Syal, Poonam; Singh, Yogesh

2015-01-01

Lipase catalyzes hydrolysis of fats in lipid water interphase and perform variety of biotransformation reactions under micro aqueous conditions. The major sources include microbial lipases; among these yeast and fungal lipases are of special interest because they can carry out various stereoselective reactions. These lipases are highly diverse and are categorized into three classes on the basis of oxyanion hole: GX, GGGX and Y. The detailed phylogenetic analysis showed that GX family is more diverse than GGGX and Y family. Sequence and structural comparisons revealed that lipases are conserved only in the signature sequence region. Their characteristic structural determinants viz. lid, binding pocket and oxyanion hole are hotspots for mutagenesis. Few examples are cited in this review to highlight the multidisciplinary approaches for designing novel enzyme variants with improved thermo stability and substrate specificity. In addition, we present a brief account on biotechnological applications of lipases. Lipases have also gained attention as virulence factors, therefore, we surveyed the role of lipases in yeast physiology related to colonization, adhesion, biofilm formation and pathogenesis. The new genomic era has opened numerous possibilities to genetically manipulate lipases for food, fuel and pharmaceuticals. Copyright © 2014 Elsevier Ltd. All rights reserved.

Role of lipase in the regulation of postprandial gastric acid secretion and emptying of fat in humans: a study with orlistat, a highly specific lipase inhibitor

PubMed Central

Borovicka, J; Schwizer, W; Guttmann, G; Hartmann, D; Kosinski, M; Wastiel, C; Bischof-Delaloye, A; Fried, M

2000-01-01

BACKGROUND AND AIMS—To investigate the importance of lipase on gastric functions, we studied the effects of orlistat, a potent and specific inhibitor of lipase, on postprandial gastric acidity and gastric emptying of fat.
METHODS—Fourteen healthy volunteers participated in a double blind, placebo controlled, randomised study. In a two way cross over study with two test periods of five days, separated by at least 14 days, orlistat 120 mg three times daily or placebo was given with standardised daily meals. In previous experiments we found that this dose almost completely inhibited postprandial duodenal lipase activity. Subjects underwent 28 hour intragastric pH-metry on day 4, and a gastric emptying study with a mixed meal (800 kcal) labelled with 999mTc sulphur colloid (solids) and 111Inthiocyanate (fat) on day 5. Gastric pH data were analysed for three postprandial hours and the interdigestive periods.
RESULTS—Orlistat inhibited almost completely (by 75%) lipase activity and accelerated gastric emptying of both the solid (by 52%) and fat (by 44%) phases of the mixed meal (p<0.03). Orlistat increased postprandial gastric acidity (from a median pH of 3.3 to 2.7; p<0.01). Postprandial cholecystokinin release was lower with orlistat (p<0.03).
CONCLUSION—Lipase has an important role in the regulation of postprandial gastric acid secretion and fat emptying in humans. These effects might be explained by lipolysis induced release of cholecystokinin.


Keywords: lipase; orlistat; gastric secretion; gastric emptying; pH-metry PMID:10807887

Investigation of the effect of biologically active threo-Ds-isocitric acid on oxidative stress in Paramecium caudatum.

PubMed

Morgunov, Igor G; Karpukhina, Olga V; Kamzolova, Svetlana V; Samoilenko, Vladimir A; Inozemtsev, Anatoly N

2018-01-02

The effect of biologically active form (threo-Ds-) of isocitric acid (ICA) on oxidative stress was studied using the infusorian Paramecium caudatum stressed by hydrogen peroxide and salts of some heavy metals (Cu, Pb, Zn, and Cd). ICA at concentrations between 0.5 and 10 mM favorably influenced the infusorian cells with oxidative stress induced by the toxicants studied. The maximal antioxidant effect of ICA was observed at its concentration 10 mM irrespective of the toxicant used (either H 2 O 2 or heavy metal ions). ICA was found to be a more active antioxidant than ascorbic acid. Biologically active pharmaceutically pure threo-Ds-ICA was produced through cultivation of the yeast Yarrowia lipolytica and isolated from the culture liquid in the form of crystalline monopotassium salt with a purity of 99.9%.

Antifungal activity of the lipopeptides produced by Bacillus amyloliquefaciens anti-CA against Candida albicans isolated from clinic.

PubMed

Song, Bo; Rong, Yan-Jun; Zhao, Ming-Xin; Chi, Zhen-Ming

2013-08-01

The bacterium Bacillus amyloliquefaciens anti-CA isolated from mangrove system was found to be able to actively kill Candida albicans isolated from clinic. The bacterial strain anti-CA could produce high level of bioactive substance, amylase and protease in the cheap medium containing 2.0 % soybean meal, 2.0 % wheat flour, pH 6.5 within 26 h. After purification, the main bioactive substance was confirmed to be a cyclic lipopeptide containing a heptapeptide, L-Asp→L-Leu→L-Leu→L-Val→L-Val→L-Glu→L-Leu and a 3-OH fatty acid (15 carbons). In addition to C. albicans, the purified lipopeptide can also kill many yeast strains including Metschnikowia bicuspidata, Candida tropicalis, Yarrowia lipolytica and Saccharomyces cerevisiae. After treated by the purified lipopeptide, both the whole cells and protoplasts of C. albicans were destroyed.

Metabolic engineering of microbial competitive advantage for industrial fermentation processes.

PubMed

Shaw, A Joe; Lam, Felix H; Hamilton, Maureen; Consiglio, Andrew; MacEwen, Kyle; Brevnova, Elena E; Greenhagen, Emily; LaTouf, W Greg; South, Colin R; van Dijken, Hans; Stephanopoulos, Gregory

2016-08-05

Microbial contamination is an obstacle to widespread production of advanced biofuels and chemicals. Current practices such as process sterilization or antibiotic dosage carry excess costs or encourage the development of antibiotic resistance. We engineered Escherichia coli to assimilate melamine, a xenobiotic compound containing nitrogen. After adaptive laboratory evolution to improve pathway efficiency, the engineered strain rapidly outcompeted a control strain when melamine was supplied as the nitrogen source. We additionally engineered the yeasts Saccharomyces cerevisiae and Yarrowia lipolytica to assimilate nitrogen from cyanamide and phosphorus from potassium phosphite, and they outcompeted contaminating strains in several low-cost feedstocks. Supplying essential growth nutrients through xenobiotic or ecologically rare chemicals provides microbial competitive advantage with minimal external risks, given that engineered biocatalysts only have improved fitness within the customized fermentation environment. Copyright © 2016, American Association for the Advancement of Science.

Cloning and characterization of newly isolated lipase from Enterobacter sp. Bn12.

PubMed

Farrokh, Parisa; Yakhchali, Bagher; Karkhane, Ali Asghar

2014-01-01

A mesophilic Enterobacter sp. Bn12 producing an alkaline thermostable lipase was isolated from soil in Tehran, Iran. The lipase gene (ELBn12) was identified from a genomic library. Sequence analysis of the DNA fragment revealed an open reading frame of 879 bp encoding a lipase with a molecular mass of 31.3 kDa. The deduced amino acid sequence showed 96% identity with a lipase of Enterobacter sp. Ag1 and the identity of their DNA sequences was 88.9%. ELBn12 belongs to the lipase subfamily I.1 and its catalytic triad consists of Ser82, Asp237 and His259. The lipase was expressed in Escherichia coli (BL21) pLysS and partially purified by anion exchange chromatography. The maximum activity of ELBn12 was obtained at temperature of 60 °C and pH 8.0 towards tricaprylin (C8) and its specific activity was around 2900 U/mg. ELBn12 was stable within a broad pH range from 6.0 to 11.0. The enzyme showed high stability in both polar and nonpolar organic solvents at 50% (v/v). The lipase activity was enhanced in the presence of 10 mM of Ca(2+), Mg(2+) and K(+), while heavy metals (Fe(3+) and Zn(2+)) had strong inhibitory effect. ELBn12 showed high activity in the presence of 1% (w/v) nonionic surfactants, however ionic surfactants inhibited the lipolytic activity. ELBn12 characteristics show that it has a potential to be used in various industrial processes.

Cloning and characterization of newly isolated lipase from Enterobacter sp. Bn12

PubMed Central

Farrokh, Parisa; Yakhchali, Bagher; Karkhane, Ali Asghar

2014-01-01

A mesophilic Enterobacter sp. Bn12 producing an alkaline thermostable lipase was isolated from soil in Tehran, Iran. The lipase gene (ELBn12) was identified from a genomic library. Sequence analysis of the DNA fragment revealed an open reading frame of 879 bp encoding a lipase with a molecular mass of 31.3 kDa. The deduced amino acid sequence showed 96% identity with a lipase of Enterobacter sp. Ag1 and the identity of their DNA sequences was 88.9%. ELBn12 belongs to the lipase subfamily I.1 and its catalytic triad consists of Ser82, Asp237 and His259. The lipase was expressed in Escherichia coli (BL21) pLysS and partially purified by anion exchange chromatography. The maximum activity of ELBn12 was obtained at temperature of 60 °C and pH 8.0 towards tricaprylin (C8) and its specific activity was around 2900 U/mg. ELBn12 was stable within a broad pH range from 6.0 to 11.0. The enzyme showed high stability in both polar and nonpolar organic solvents at 50% (v/v). The lipase activity was enhanced in the presence of 10 mM of Ca2+, Mg2+ and K+, while heavy metals (Fe3+ and Zn2+) had strong inhibitory effect. ELBn12 showed high activity in the presence of 1% (w/v) nonionic surfactants, however ionic surfactants inhibited the lipolytic activity. ELBn12 characteristics show that it has a potential to be used in various industrial processes. PMID:25242958

A newly high alkaline lipase: an ideal choice for application in detergent formulations

PubMed Central

2011-01-01

Background Bacterial lipases received much attention for their substrate specificity and their ability to function in extreme environments (pH, temperature...). Many staphylococci produced lipases which were released into the culture medium. Reports of thermostable lipases from Staphylococcus sp. and active in alkaline conditions are not previously described. Results A newly soil-isolated Staphylococcus sp. strain ESW secretes an induced lipase in the culture medium. The effects of temperature, pH and various components in a detergent on the activity and stability of Staphylococcus sp. lipase (SL1) were studied in a preliminary evaluation for use in detergent formulation solutions. The enzyme was highly active over a wide range of pH from 9.0 to 13.0, with an optimum at pH 12.0. The relative activity at pH 13.0 was about 60% of that obtained at pH 12.0. It exhibited maximal activity at 60°C. This novel lipase, showed extreme stability towards non-ionic and anionic surfactants after pre-incubation for 1 h at 40°C, and relative stability towards oxidizing agents. Additionally, the crude enzyme showed excellent stability and compatibility with various commercial solid and liquid detergents. Conclusions These properties added to the high activity in high alkaline pH make this novel lipase an ideal choice for application in detergent formulations. PMID:22123072

The immobilization of lipase on PVDF-co-HFP membrane

NASA Astrophysics Data System (ADS)

Kayhan, Naciye; Eyüpoǧlu, Volkan; Adem, Şevki

2016-04-01

Lipase is an enzyme having a lot of different industrial applications such as biodiesel production, biopolymer synthesis, enantiopure pharmaceutical productions, agrochemicals, etc. Its immobilized form on different substances is more conventional and useful than its free form. Supporting material was prepared using PVDF-co-HFP in laboratory conditions and attached 1,4-diaminobutane (DA) and epichlorohydrin (EPI) ligands to the membrane to immobilize lipase enzyme. The immobilization conditions such as enzyme amount, pH, the concentration of salt, thermal stability and activity were stabilized for our experimental setup. Then, biochemical characterizations were performed on immobilized lipase PVDF-co-HFP regarding optimal pH activity, temperature and thermal stability. Also, the desorption ratios of immobilized enzyme in two different pathway were investigated to confirm immobilization stability for 24 hours.

Fluorescent microplate assay method for high-throughput detection of lipase transesterification activity.

PubMed

Zheng, Jianyong; Wei, Wei; Lan, Xing; Zhang, Yinjun; Wang, Zhao

2018-05-15

This study describes a sensitive and fluorescent microplate assay method to detect lipase transesterification activity. Lipase-catalyzed transesterification between butyryl 4-methyl umbelliferone (Bu-4-Mu) and methanol in tert-butanol was selected as the model reaction. The release of 4-methylumbelliferone (4-Mu) in the reaction was determined by detecting the fluorescence intensity at λ ex 330 nm and λ em 390 nm. Several lipases were used to investigate the accuracy and efficiency of the proposed method. Apparent Michaelis constant (Km) was calculated for transesterification between Bu-4-Mu and methanol by the lipases. The main advantages of the assay method include high sensitivity, inexpensive reagents, and simple detection process. Copyright © 2018 Elsevier Inc. All rights reserved.

Effect of the physicochemical properties of binary ionic liquids on lipase activity and stability.

PubMed

Yao, Peipei; Yu, Xinxin; Huang, Xirong

2015-01-01

In the present study, the lipase-catalyzed hydrolysis of p-nitrophenyl butyrate is used as a model reaction to determine the activity and stability of Candida rugosa lipase in binary ionic liquids (ILs). The binary ILs consist of hydrophobic 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim]PF6) and a small amount of hydrophilic 1-butyl-3-methylimidazolium nitrate ([Bmim]NO3) or 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([Bmim]CF3SO3) or 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF4). The activity and the stability of lipase are first correlated with the physicochemical properties of the binary ILs. In the three binary IL systems, both the hydrophilicity and the polarity of the systems increase with the increase of the content of hydrophilic ILs (HILs). At a fixed concentration of HIL, they vary in a descending order of [Bmim]PF6/[Bmim]NO3>[Bmim]PF6/[Bmim]CF3SO3>[Bmim]PF6/[Bmim]BF4. This order is in contrast with the order of the lipase conformation stability, i.e., the higher the polarity of ILs, the more unstable the lipase conformation. However, both the activity and the stability of lipase depend on the type and the content of the HIL in binary ILs, showing a complex dependency. Analysis shows that the catalytic performance of lipase in the binary ILs is affected not only by the direct influence of the ILs on lipase conformation, but also through their indirect influence on the physicochemical properties of water. The present study helps to explore binary IL mixtures suitable for lipase-based biocatalysis. Copyright © 2015 Elsevier B.V. All rights reserved.

Oral lipase activities and fat-taste receptors for fat-taste sensing in chickens.

PubMed

Kawabata, Yuko; Kawabata, Fuminori; Nishimura, Shotaro; Tabata, Shoji

2018-01-01

It has been reported that a functional fat-taste receptor, GPR120, is present in chicken oral tissues, and that chickens can detect fat taste in a behavioral test. However, although triglycerides need to be digested to free fatty acids to be recognized by fat-taste receptors such as GPR120, it remains unknown whether lipase activities exist in chicken oral tissues. To examine this question, we first cloned another fat-taste receptor candidate gene, CD36, from the chicken palate. Then, using RT-PCR, we determined that GPR120 and CD36 were broadly expressed in chicken oral and gastrointestinal tissues. Also by RT-PCR, we confirmed that several lipase genes were expressed in both oral and gastrointestinal tissues. Finally, we analyzed the lipase activities of oral tissues by using a fluorogenic triglyceride analog as a lipase substrate. We found there are functional lipases in oral tissues as well as in the stomach and pancreas. These results suggested that chickens have a basic fat-taste reception system that incorporates a triglycerides/oral-lipases/free fatty acids/GPR120 axis and CD36 axis. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification and characterization of lipases from Malassezia restricta, a causative agent of dandruff.

PubMed

Sommer, Bettina; Overy, David P; Kerr, Russell G

2015-11-01

Dandruff, a skin disorder affecting 50% of the world population, is linked with proliferation of lipophilic yeasts of the genus Malassezia (particularly Malassezia globosa and M. restricta). Most Malassezia species show a unique lipid dependency and require external lipids for growth. Genome mining of the incomplete M. restricta genome led to the identification of eight lipase sequences. Sequences representing the class 3 and LIP lipase families were used to clone the lipases MrLip1, MrLip2 and MrLip3, recombinantly expressed in Pichia pastoris, and tested for their activity using mono-, di- and triacylglycerol substrates. Hydrolysis by the M. restricta lipase MrLip1 and MrLip2 (family class 3) was limited to the mono- and diacylglycerol, while MrLip3 (family LIP) hydrolyzed all three substrates. This result confirms that Malassezia family LIP lipases are responsible for the hydrolysis of triacylglycerols, the main component of human sebum. Furthermore, the information regarding lipases from M. restricta presented here might aid in the search for anti-dandruff agents. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Canine pancreatic lipase immunoreactivity concentrations associated with intervertebral disc disease in 84 dogs.

PubMed

Schueler, R O; White, G; Schueler, R L; Steiner, J M; Wassef, A

2018-05-01

To determine the differences in serum canine pancreatic lipase immunoreactivity between dogs with intervertebral disc herniation and healthy control dogs. Eighty-four client-owned dogs with intervertebral disc herniation, diagnosed by neurologic examination and imaging, and 18 healthy control dogs. Samples of whole blood were collected within 90 minutes of admission. Serum canine pancreatic lipase immunoreactivity concentrations were measured by a commercial immunoassay and evaluated for association with intervertebral disc herniation, signalment, neurolocalisation and the preadmission administration of glucocorticosteriods or non-steroidal anti-inflammatory drugs. Serum canine pancreatic lipase immunoreactivity concentrations were statistically increased in dogs with intervertebral disc herniation (P<0·01, n=38). A subgroup of dogs (19/38) with elevated canine pancreatic lipase immunoreactivity concentrations was re-evaluated between 2 and 4 weeks later, and 15 had resolution of clinical signs and values less than 200 μg/L. Serum canine pancreatic lipase immunoreactivity concentrations were not significantly correlated with clinical gastrointestinal disease, neurolocalisation or the preadmission administration of corticosteroids or non-steroidal anti-inflammatory drugs. These results suggest that serum canine pancreatic lipase immunoreactivity concentrations are significantly elevated in dogs with intervertebral disc herniation. © 2018 British Small Animal Veterinary Association.

Lipase-Secreting Bacillus Species in an Oil-Contaminated Habitat: Promising Strains to Alleviate Oil Pollution

PubMed Central

Lee, Li Pin; Karbul, Hudzaifah Mohamed; Citartan, Marimuthu; Gopinath, Subash C. B.; Lakshmipriya, Thangavel; Tang, Thean-Hock

2015-01-01

Lipases are of great interest for different industrial applications due to their diversity and versatility. Among different lipases, microbial lipases are preferable due to their broad substrate specificity, and higher stability with lower production costs compared to the lipases from plants and animals. In the past, a vast number of bacterial species have been reported as potential lipases producers. In this study, the lipases-producing bacterial species were isolated from an oil spillage area in the conventional night market. Isolated species were identified as Bacillus species by biochemical tests which indicate their predominant establishment, and further screened on the agar solid surfaces using lipid and gelatin as the substrates. Out of the ten strains tested, four potential strains were subjected to comparison analysis of the lipolytic versus proteolytic activities. Strain 10 exhibited the highest lipolytic and proteolytic activity. In all the strains, the proteolytic activity is higher than the lipolytic activity except for strain 8, suggesting the possibility for substrate-based extracellular gene induction. The simultaneous secretion of both the lipase and protease is a mean of survival. The isolated bacterial species which harbour both lipase and protease enzymes could render potential industrial-based applications and solve environmental issues. PMID:26180812

Zinc Finger Transcription Factors Displaced SREBP Proteins as the Major Sterol Regulators during Saccharomycotina Evolution

PubMed Central

Maguire, Sarah L.; Wang, Can; Holland, Linda M.; Brunel, François; Neuvéglise, Cécile; Nicaud, Jean-Marc; Zavrel, Martin; White, Theodore C.; Wolfe, Kenneth H.; Butler, Geraldine

2014-01-01

In most eukaryotes, including the majority of fungi, expression of sterol biosynthesis genes is regulated by Sterol-Regulatory Element Binding Proteins (SREBPs), which are basic helix-loop-helix transcription activators. However, in yeasts such as Saccharomyces cerevisiae and Candida albicans sterol synthesis is instead regulated by Upc2, an unrelated transcription factor with a Gal4-type zinc finger. The SREBPs in S. cerevisiae (Hms1) and C. albicans (Cph2) have lost a domain, are not major regulators of sterol synthesis, and instead regulate filamentous growth. We report here that rewiring of the sterol regulon, with Upc2 taking over from SREBP, likely occurred in the common ancestor of all Saccharomycotina. Yarrowia lipolytica, a deep-branching species, is the only genome known to contain intact and full-length orthologs of both SREBP (Sre1) and Upc2. Deleting YlUPC2, but not YlSRE1, confers susceptibility to azole drugs. Sterol levels are significantly reduced in the YlUPC2 deletion. RNA-seq analysis shows that hypoxic regulation of sterol synthesis genes in Y. lipolytica is predominantly mediated by Upc2. However, YlSre1 still retains a role in hypoxic regulation; growth of Y. lipolytica in hypoxic conditions is reduced in a Ylupc2 deletion and is abolished in a Ylsre1/Ylupc2 double deletion, and YlSre1 regulates sterol gene expression during hypoxia adaptation. We show that YlSRE1, and to a lesser extent YlUPC2, are required for switching from yeast to filamentous growth in hypoxia. Sre1 appears to have an ancestral role in the regulation of filamentation, which became decoupled from its role in sterol gene regulation by the arrival of Upc2 in the Saccharomycotina. PMID:24453983

Presence and changes in populations of yeasts on raw and processed poultry products stored at refrigeration temperature.

PubMed

Ismail, S A; Deak, T; El-Rahman, H A; Yassien, M A; Beuchat, L R

2000-12-05

A study was undertaken to determine populations and profiles of yeast species on fresh and processed poultry products upon purchase from retail supermarkets and after storage at 5 degrees C until shelf life expiration, and to assess the potential role of these yeasts in product spoilage. Fifty samples representing 15 commercial raw, marinated, smoked, or roasted chicken and turkey products were analyzed. Yeast populations were determined by plating on dichloran rose bengal chloramphenicol (DRBC) agar and tryptone glucose yeast extract (TGY) agar. Proteolytic activity was determined using caseinate and gelatin agars and lipolytic activity was determined on plate count agar supplemented with tributyrin. Populations of aerobic microorganisms were also determined. Initial populations of yeasts (log10 cfu/g) ranged from less than 1 (detection limit) to 2.89, and increased by the expiration date to 0.37-5.06, indicating the presence of psychrotrophic species. Highest initial populations were detected in raw chicken breast, wings, and ground chicken, as well as in turkey necks and legs, whereas roasted chicken and turkey products contained less than 1 log10 cfu/g. During storage, yeast populations increased significantly (P < or = 0.05) in whole chicken, ground chicken, liver, heart and gizzard, and in ground turkey and turkey sausage. Isolates (152 strains) of yeasts from poultry products consisted of 12 species. Yarrowia lipolytica and Candida zeylanoides were predominant, making up 39 and 26% of the isolates, respectively. Six different species of basidiomycetous yeasts representing 24% of the isolates were identified. Most Y. lipolytica strains showed strong proteolytic and lipolytic activities, whereas C. zeylanoides was weakly lipolytic. Results suggest that yeasts, particularly Y. lipolytica, may play a more prominent role than previously recognized in the spoilage of fresh and processed poultry stored at 5 degrees C.

Amplification of thermostable lipase genes fragment from thermogenic phase of domestic waste composting process

NASA Astrophysics Data System (ADS)

Nurhasanah, Nurbaiti, Santi; Madayanti, Fida; Akhmaloka

2015-09-01

Lipases are lipolytic enzymes, catalyze the hydrolysis of fatty acid ester bonds of triglycerides to produce free fatty acids and glycerol. The enzyme is widely used in various fields of biotechnological industry. Hence, lipases with unique properties (e.g.thermostable lipase) are still being explored by variation methods. One of the strategy is by using metagenomic approach to amplify the gene directly from environmental sample. This research was focused on amplification of lipase gene fragment directly from the thermogenic phase of domestic waste composting in aerated trenches. We used domestic waste compost from waste treatment at SABUGA, ITB for the sample. Total chromosomal DNA were directly extracted from several stages at thermogenic phase of compost. The DNA was then directly used as a template for amplification of thermostable lipase gene fragments using a set of internal primers namely Flip-1a and Rlip-1a that has been affixed with a GC clamp in reverse primer. The results showed that the primers amplified the gene from four stages of thermogenic phase with the size of lipase gene fragment of approximately 570 base pairs (bp). These results were further used for Denaturing Gradient Gel Electrophoresis (DGGE) analysis to determine diversity of thermostable lipase gene fragments.

New ether-functionalized ionic liquids for lipase-catalyzed synthesis of biodiesel.

PubMed

Zhao, Hua; Song, Zhiyan; Olubajo, Olarongbe; Cowins, Janet V

2010-09-01

Ionic liquids (ILs) are being explored as solvents for the enzymatic methanolysis of triglycerides. However, most available ILs (especially hydrophobic ones) have poor capability in dissolving lipids, while hydrophilic ILs tend to cause enzyme inactivation. Recently, we synthesized a new type of ether-functionalized ionic liquids (ILs) carrying anions of acetate or formate; they are capable of dissolving a variety of substrates and are also lipase-compatible (Green Chem., 2008, 10, 696-705). In the present study, we carried out the lipase-catalyzed transesterifications of Miglyol oil 812 and soybean oil in these novel ILs. These ILs are capable of dissolving oils at the reaction temperature (50 degrees C); meanwhile, lipases maintained high catalytic activities in these media even in high concentrations of methanol (up to 50% v/v). High conversions of Miglyol oil were observed in mixtures of IL and methanol (70/30, v/v) when the reaction was catalyzed by a variety of lipases and different enzyme preparations (free and immobilized), especially with the use of two alkylammonium ILs 2 and 3. The preliminary study on the transesterification of soybean oil in IL/methanol mixtures further confirms the potential of using oil-dissolving and lipase-stabilizing ILs in the efficient production of biodiesels.

A quantitative assay measuring the function of lipase maturation factor 1

PubMed Central

Yin, Fen; Doolittle, Mark H.; Péterfy, Miklós

2009-01-01

Newly synthesized lipoprotein lipase (LPL) and related members of the lipase gene family require an endoplasmic reticulum maturation factor for attainment of enzyme activity. This factor has been identified as lipase maturation factor 1 (Lmf1), and mutations affecting its function and/or expression result in combined lipase deficiency (cld) and hypertriglyceridemia. To assess the functional impact of Lmf1 sequence variations, both naturally occurring and induced, we report the development of a cell-based assay using LPL activity as a quantitative reporter of Lmf1 function. The assay uses a cell line homozygous for the cld mutation, which renders endogenous Lmf1 nonfunctional. LPL transfected into the mutant cld cell line fails to attain activity; however, cotransfection of LPL with wild-type Lmf1 restores its ability to support normal lipase maturation. In this report, we describe optimized conditions that ensure the detection of a complete range of Lmf1 function (full, partial, or complete loss of function) using LPL activity as the quantitative reporter. To illustrate the dynamic range of the assay, we tested several novel mutations in mouse Lmf1. Our results demonstrate the ability of the assay to detect and analyze Lmf1 mutations having a wide range of effects on Lmf1 function and protein expression. PMID:19471043

Stereoselectivity of Mucorales lipases toward triradylglycerols--a simple solution to a complex problem.

PubMed Central

Scheib, H.; Pleiss, J.; Kovac, A.; Paltauf, F.; Schmid, R. D.

1999-01-01

The lipases from Rhizopus and Rhizomucor are members of the family of Mucorales lipases. Although they display high sequence homology, their stereoselectivity toward triradylglycerols (sn-2 substituted triacylglycerols) varies. Four different triradylglycerols were investigated, which were classified into two groups: flexible substrates with rotatable O'-C1' ether or ester bonds adjacent to C2 of glycerol and rigid substrates with a rigid N'-C1' amide bond or a phenyl ring in sn-2. Although Rhizopus lipase shows opposite stereopreference for flexible and rigid substrates (hydrolysis in sn-1 and sn-3, respectively), Rhizomucor lipase hydrolyzes both groups of triradylglycerols preferably in sn-1. To explain these experimental observations, computer-aided molecular modeling was applied to study the molecular basis of stereoselectivity. A generalized model for both lipases of the Mucorales family highlights the residues mediating stereoselectivity: (1) L258, the C-terminal neighbor of the catalytic histidine, and (2) G266, which is located in a loop contacting the glycerol backbone of a bound substrate. Interactions with triradylglycerol substrates are dominated by van der Waals contacts. Stereoselectivity can be predicted by analyzing the value of a single substrate torsion angle that discriminates between sn-1 and sn-3 stereopreference for all substrates and lipases investigated here. This simple model can be easily applied in enzyme and substrate engineering to predict Mucorales lipase variants and synthetic substrates with desired stereoselectivity. PMID:10210199

Human pancreatic lipase-related protein 2 is a galactolipase.

PubMed

Sias, Barbara; Ferrato, Francine; Grandval, Philippe; Lafont, Dominique; Boullanger, Paul; De Caro, Alain; Leboeuf, Bernard; Verger, Robert; Carrière, Frédéric

2004-08-10

Human pancreatic lipase-related protein 2 (HPLRP2) was found to be expressed in the pancreas, but its biochemical properties were not investigated in detail. A recombinant HPLRP2 was produced in insect cells and the yeast Pichia pastoris and purified by cation exchange chromatography. Its substrate specificity was investigated using pH-stat and monomolecular film techniques and various lipid substrates (triglycerides, diglycerides, phospholipids, and galactolipids). Lipase activity of HPLRP2 on trioctanoin was inhibited by bile salts and poorly restored by adding colipase. In vivo, HPLRP2 therefore seems unlikely to show any lipase activity on dietary fat. In human pancreatic lipase (HPL), residues R256, D257, Y267, and K268 are involved in the stabilization of the open conformation of the lid domain, which interacts with colipase. These residues are not conserved in HPLRP2. When the corresponding mutations (R256G, D257G, Y267F, and K268E) are introduced into HPL, the effects of colipase are drastically reduced in the presence of bile salts. This may explain why colipase has such weak effects on HPLRP2. HPLRP2 displayed a very low level of activity on phospholipid micelles and monomolecular films. Its activity on monogalactosyldiglyceride monomolecular film, which was much higher, was similar to the activity of guinea pig pancreatic lipase related-protein 2, which shows the highest galactolipase activity ever measured. The physiological role of HPLRP2 suggested by the present results is the digestion of galactolipids, the most abundant lipids occurring in plant cells, and therefore, in the vegetables that are part of the human diet.

High-level expression and characterization of a chimeric lipase from Rhizopus oryzae for biodiesel production

PubMed Central

2013-01-01

Background Production of biodiesel from non-edible oils is receiving increasing attention. Tung oil, called “China wood oil” is one kind of promising non-edible biodiesel oil in China. To our knowledge, tung oil has not been used to produce biodiesel by enzymatic method. The enzymatic production of biodiesel has been investigated extensively by using Rhizopus oryzae lipase as catalyst. However, the high cost of R. oryzae lipase remains a barrier for its industrial applications. Through different heterologous expression strategies and fermentation techniques, the highest expression level of the lipase from R. oryzae reached 1334 U/mL in Pichia pastoris, which is still not optimistic for industry applications. Results The prosequence of lipases from Rhizopus sp. is very important for the folding and secretion of an active lipase. A chimeric lipase from R. oryzae was constructed by replacing the prosequence with that from the R. chinensis lipase and expressed in P. pastoris. The maximum activity of the chimera reached 4050 U/mL, which was 11 fold higher than that of the parent. The properties of the chimera were studied. The immobilized chimera was used successfully for biodiesel production from tung oil, which achieved higher FAME yield compared with the free chimeric lipase, non-chimeric lipase and mature lipase. By response surface methodology, three variables, water content, methanol to tung oil molar ratio and enzyme dosage were proved to be crucial parameters for biosynthesis of FAME and the FAME yield reached 91.9±2.5% at the optimized conditions by adding 5.66 wt.% of the initial water based on oil weight, 3.88 of methanol to tung oil molar ratio and 13.24 wt.% of enzyme concentration based on oil weight at 40°C. Conclusions This is the first report on improving the expression level of the lipase from R. oryzae by replacing prosequences. The immobilized chimera was used successfully for biodiesel production from tung oil. Using tung oil as non-edible raw

High-level expression and characterization of a chimeric lipase from Rhizopus oryzae for biodiesel production.

PubMed

Yu, Xiao-Wei; Sha, Chong; Guo, Yong-Liang; Xiao, Rong; Xu, Yan

2013-02-21

Production of biodiesel from non-edible oils is receiving increasing attention. Tung oil, called "China wood oil" is one kind of promising non-edible biodiesel oil in China. To our knowledge, tung oil has not been used to produce biodiesel by enzymatic method. The enzymatic production of biodiesel has been investigated extensively by using Rhizopus oryzae lipase as catalyst. However, the high cost of R. oryzae lipase remains a barrier for its industrial applications. Through different heterologous expression strategies and fermentation techniques, the highest expression level of the lipase from R. oryzae reached 1334 U/mL in Pichia pastoris, which is still not optimistic for industry applications. The prosequence of lipases from Rhizopus sp. is very important for the folding and secretion of an active lipase. A chimeric lipase from R. oryzae was constructed by replacing the prosequence with that from the R. chinensis lipase and expressed in P. pastoris. The maximum activity of the chimera reached 4050 U/mL, which was 11 fold higher than that of the parent. The properties of the chimera were studied. The immobilized chimera was used successfully for biodiesel production from tung oil, which achieved higher FAME yield compared with the free chimeric lipase, non-chimeric lipase and mature lipase. By response surface methodology, three variables, water content, methanol to tung oil molar ratio and enzyme dosage were proved to be crucial parameters for biosynthesis of FAME and the FAME yield reached 91.9±2.5% at the optimized conditions by adding 5.66 wt.% of the initial water based on oil weight, 3.88 of methanol to tung oil molar ratio and 13.24 wt.% of enzyme concentration based on oil weight at 40°C. This is the first report on improving the expression level of the lipase from R. oryzae by replacing prosequences. The immobilized chimera was used successfully for biodiesel production from tung oil. Using tung oil as non-edible raw material and a chimeric lipase

Synthesis of structured triacylglycerols enriched in n-3 fatty acids by immobilized microbial lipase.

PubMed

Araújo, Maria Elisa Melo Branco de; Campos, Paula Renata Bueno; Alberto, Thiago Grando; Contesini, Fabiano Jares; Carvalho, Patrícia de Oliveira

The search for new biocatalysts has aroused great interest due to the variety of micro-organisms and their role as enzyme producers. Native lipases from Aspergillus niger and Rhizopus javanicus were used to enrich the n-3 long-chain polyunsaturated fatty acids content in the triacylglycerols of soybean oil by acidolysis with free fatty acids from sardine oil in solvent-free media. For the immobilization process, the best lipase/support ratios were 1:3 (w/w) for Aspergillus niger lipase and 1:5 (w/w) for Rhizopus javanicus lipase using Amberlite MB-1. Both lipases maintained constant activity for 6 months at 4°C. Reaction time, sardine-free fatty acids:soybean oil mole ratio and initial water content of the lipase were investigated to determine their effects on n-3 long-chain polyunsaturated fatty acids incorporation into soybean oil. Structured triacylglycerols with 11.7 and 7.2% of eicosapentaenoic acid+docosahexaenoic acid were obtained using Aspergillus niger lipase and Rhizopus javanicus lipase, decreasing the n-6/n-3 fatty acids ratio of soybean oil (11:1 to 3.5:1 and 4.7:1, respectively). The best reaction conditions were: initial water content of lipase of 0.86% (w/w), sardine-free faty acids:soybean oil mole ratio of 3:1 and reaction time of 36h, at 40°C. The significant factors for the acidolysis reaction were the sardine-free fatty acids:soybean oil mole ratio and reaction time. The characterization of structured triacylglycerols was obtained using easy ambient sonic-spray ionization mass spectrometry. The enzymatic reaction led to the formation of many structured triacylglycerols containing eicosapentaenoic acid, docosahexaenoic acid or both polyunsaturated fatty acids. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Inhibition of pancreatic lipase and amylase by extracts of different spices and plants.

PubMed

Sellami, Mohamed; Louati, Hanen; Kamoun, Jannet; Kchaou, Ali; Damak, Mohamed; Gargouri, Youssef

2017-05-01

The aim of this study is to search new anti-obesity and anti-diabetic agents from plant and spices crude extracts as alternative to synthetic drugs. The inhibitory effect of 72 extracts was evaluated, in vitro, on lipase and amylase activities. Aqueous extracts of cinnamon and black tea exhibited an appreciable inhibitory effect on pancreatic amylase with IC 50 values of 18 and 87 μg, respectively. Aqueous extracts of cinnamon and mint showed strong inhibitory effects against pancreatic lipase with IC 50 of 45 and 62 μg, respectively. The presence of bile salts and colipase or an excess of interface failed to restore the lipase activity. Therefore, the inhibition of pancreatic lipase, by extracts of spices and plants, belongs to an irreversible inhibition. Crude extract of cinnamon showed the strongest anti-lipase and anti-amylase activities which offer a prospective therapeutic approach for the management of diabetes and obesity.

Enzymatic Synthesis of Structured Lipids using a Novel Cold-Active Lipase from Pichia lynferdii NRRL Y-7723

USDA-ARS?s Scientific Manuscript database

Structured lipids (SL) were synthesized by the acidolysis of borage oil with caprylic acid using lipases. Six commercial lipases from different sources and a novel lipase from Pichia lynferdii NRRL Y-7723 were screened for their acidolysis activities and Lipozyme RM IM and NRRL Y-7723 lipase were s...

Fat digestion in the stomach: stability of lingual lipase in the gastric environment.

PubMed

Fink, C S; Hamosh, P; Hamosh, M

1984-03-01

Digestion of dietary fat starts in the stomach, where lingual lipase hydrolyzes triglycerides to free fatty acids and partial glycerides at pH 3.0-6.0. Lingual lipase is secreted continuously from lingual serous glands and accumulates in the stomach between meals, when gastric pH is less than 3.0. We have, therefore, examined the resistance of lingual lipase to low pH and its possible protection by dietary components present in the stomach contents. Partially purified rat lingual lipase (7-15 micrograms enzyme protein) was preincubated at 37 degrees C for 10-60 min at pH 1.0-6.0 before incubation for assay of lipolytic activity, hydrolysis of tri-[3H]olein at pH 5.4. The data show that partially purified rat lingual lipase preparations are stable at 37 degrees C in the pH range of 2.5-6.0. Enzyme activity, however, is rapidly and irreversibly lost during preincubation at pH 1.0-2.4 for 10-30 min. Protein (gelatin 1% or albumin 1% or 2.5%) cannot prevent the inactivation of lingual lipase at low pH. The large molecular species (molecular weight greater than 500,000) of lingual lipase (thought to be an aggregate of enzyme with lipids) is slightly more resistant to inactivation than the 46,000 dalton preparation, suggesting that lipids might protect the enzyme from inactivation. Indeed, about 60% of the initial lipase activity is preserved during incubation at pH 2.0 in the presence of 50 mM lecithin or 10 mM triolein. The data indicate that triglycerides which are hydrolyzed by this enzyme as well as phospholipids that are not hydrolyzed can prevent the inactivation of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipase-catalyzed ring-opening polymerization of lactones to polyesters and its mechanistic aspects.

PubMed

Namekawa, S; Suda, S; Uyama, H; Kobayashi, S

1999-01-01

Lipase catalysis induced a ring-opening polymerization of lactones with different ring-sizes. Small-size (four-membered) and medium-size lactones (six- and seven-membered) as well as macrolides (12-, 13-, 16-, and 17-membered) were subjected to lipase-catalyzed polymerization. The polymerization behaviors depended primarily on the lipase origin and the monomer structure. The macrolides showing much lower anionic polymerizability were enzymatically polymerized faster than epsilon-caprolactone. The granular immobilized lipase derived from Candida antartica showed extremely efficient catalysis in the polymerization of epsilon-caprolactone. Single-step terminal functionalization of the polyester was achieved by initiator and terminator methods. The enzymatic polymerizability of lactones was quantitatively evaluated by Michaelis-Menten kinetics.

Coconut oil induced production of a surfactant-compatible lipase from Aspergillus tamarii under submerged fermentation.

PubMed

Das, Arijit; Bhattacharya, Sourav; Shivakumar, Srividya; Shakya, Sujina; Sogane, Swathi Shankar

2017-02-01

Filamentous fungi are efficient producers of lipases. The present study focuses on identification of a potent lipolytic fungus and enhancement of lipase production through optimization of nutritional and cultural conditions under submerged fermentation. Molecular characterization of the fungus by 18S rDNA sequencing revealed its identity as Aspergillus tamarii with 98% homology. Maximum lipase production was noted in mineral salts medium supplemented with coconut oil (2.5%, v/v). A combination of ammonium chloride (2%, w/v) and tryptone (2%, w/v) facilitated maximum lipase production at pH 5 of the production medium. A carbon: nitrogen ratio of 1:4 led to significant (p < 0.00008) increase in the enzyme production in the presence of surfactant cetyltrimethylammonium bromide (0.5%, w/v). Maximum lipase activity (2,32,500 ± 192 U/ml/min) was recorded after 7 days of incubation at 25 °C on a rotary shaker at 120 rpm. A 9.8-fold increase in lipase activity was recorded after optimization of the process parameters. Addition of crude lipase enhanced the oil stain removal activity of a commercially available detergent by 2.2-fold. The current findings suggest the potentiality of this fungal lipase to be used in detergent formulation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genetics Home Reference: hepatic lipase deficiency

MedlinePlus

... affects the body's ability to break down fats (lipids). People with this disorder have increased amounts of ... It is unclear what effect this change in lipid levels has on people with hepatic lipase deficiency . ...

Pancreatic lipase inhibitory constituents from Morus alba leaves and optimization for extraction conditions.

PubMed

Jeong, Ji Yeon; Jo, Yang Hee; Kim, Seon Beom; Liu, Qing; Lee, Jin Woo; Mo, Eun Jin; Lee, Ki Yong; Hwang, Bang Yeon; Lee, Mi Kyeong

2015-06-01

The leaves of Morus alba (Moraceae) have been traditionally used for the treatment of metabolic diseases including diabetes and hyperlipidemia. Thus, inhibitory effect of M. alba leaves on pancreatic lipase and their active constituents were investigated in this study. Twenty phenolic compounds including ten flavonoids, eight benzofurans, one stilbene and one chalcones were isolated from the leaves of M. alba. Among the isolated compounds, morachalcone A (20) exerted strong pancreatic lipase inhibition with IC50 value of 6.2 μM. Other phenolic compounds containing a prenyl group showed moderate pancreatic lipase inhibition with IC50 value of <50 μM. Next, extraction conditions with maximum pancreatic lipase inhibition and phenolic content were optimized using response surface methodology with three-level-three-factor Box-Behnken design. Our results suggested the optimized extraction condition for maximum pancreatic lipase inhibition and phenolic content as ethanol concentration of 74.9%; temperature 57.4 °C and sample/solvent ratio, 1/10. The pancreatic lipase inhibition and total phenolic content under optimized condition were found to be 58.5% and 26.2 μg GAE (gallic acid equivalent)/mg extract, respectively, which were well matched with the predicted value. Copyright © 2015 Elsevier Ltd. All rights reserved.

Interfacial Activation of Candida antarctica Lipase B: Combined Evidence from Experiment and Simulation.

PubMed

Zisis, Themistoklis; Freddolino, Peter L; Turunen, Petri; van Teeseling, Muriel C F; Rowan, Alan E; Blank, Kerstin G

2015-09-29

Lipase immobilization is frequently used for altering the catalytic properties of these industrially used enzymes. Many lipases bind strongly to hydrophobic surfaces where they undergo interfacial activation. Candida antarctica lipase B (CalB), one of the most commonly used biocatalysts, is frequently discussed as an atypical lipase lacking interfacial activation. Here we show that CalB displays an enhanced catalytic rate for large, bulky substrates when adsorbed to a hydrophobic interface composed of densely packed alkyl chains. We attribute this increased activity of more than 7-fold to a conformational change that yields a more open active site. This hypothesis is supported by molecular dynamics simulations that show a high mobility for a small "lid" (helix α5) close to the active site. Molecular docking calculations confirm that a highly open conformation of this helix is required for binding large, bulky substrates and that this conformation is favored in a hydrophobic environment. Taken together, our combined approach provides clear evidence for the interfacial activation of CalB on highly hydrophobic surfaces. In contrast to other lipases, however, the conformational change only affects large, bulky substrates, leading to the conclusion that CalB acts like an esterase for small substrates and as a lipase for substrates with large alcohol substituents.

Molecular and enzymatic characterization of alkaline lipase from Bacillus amyloliquefaciens E1PA isolated from lipid-rich food waste.

PubMed

Saengsanga, Thanakorn; Siripornadulsil, Wilailak; Siripornadulsil, Surasak

2016-01-01

Bacillus amyloliquefaciens E1PA is a lipase-producing strain that was originally isolated from lipid-rich food waste, and the production of its lipase was found to be induced by vegetable oils. The E1PA lipase was successfully expressed and secreted in a heterologous Escherichia coli host and was ultimately purified. The conserved pentapeptide motif Ala-His-Ser-Met-Gly was observed at positions 108-112. The purified recombinant lipase was stable over a pH range of 4.0-11.0 at 40 °C and exhibited maximal activity at pH 10. The recombinant E1PA lipase hydrolyzed a wide range of acyl esters (C4-C18). However, the highest activity (3.5 units mg(-1)) was observed when the p-nitrophenyl ester of myristate (C14) was used as a substrate. Compared to the lipases produced by Bacillus spp., the E1PA lipase displayed a structural molecular mass excluding the leader sequence (19.22 kDa) and a pI (9.82) that were similar to those reported for B. amyloliquefaciens lipases and lipase subfamily I.4 but that were quite distinct from those of lipase subfamily I.5 (approximately 43 kDa, pI 6). These results suggested that Bacillus lipases are closely related. Although the recombinant E1PA lipase digested only certain oils, the wild-type E1PA lipase degraded a variety of oils, including blended and re-used cooking oils. The recombinant and wild-type forms of the E1PA lipase were able to digest heterogeneous lipid-rich food waste at similar levels; this result suggests that this lipase can function even when it solely consists of its structural enzyme component. The enzyme exhibited lipid hydrolysis ability as either an intracellular domain of the recombinant protein or an extracellular domain secreted by the E1PA strain. However, the recombinant lipase showed higher activity than the wild-type E1PA lipase, indicating that the recombinant protein from E. coli possessed effective lipase activity. Thus, the inducible alkaline E1PA lipase exhibited the ability to act on a broad spectrum

Relevant pH and lipase for in vitro models of gastric digestion.

PubMed

Sams, Laura; Paume, Julie; Giallo, Jacqueline; Carrière, Frédéric

2016-01-01

The development of in vitro digestion models relies on the availability of in vivo data such as digestive enzyme levels and pH values recorded in the course of meal digestion. The variations of these parameters along the GI tract are important for designing dynamic digestion models but also static models for which the choice of representative conditions of the gastric and intestinal conditions is critical. Simulating gastric digestion with a static model and a single set of parameters is particularly challenging because the variations in pH and enzyme concentration occurring in the stomach are much broader than those occurring in the small intestine. A review of the literature on this topic reveals that most models of gastric digestion use very low pH values that are not representative of the fed conditions. This is illustrated here by showing the variations in gastric pH as a function of meal gastric emptying instead of time. This representation highlights those pH values that are the most relevant for testing meal digestion in the stomach. Gastric lipolysis is still largely ignored or is performed with microbial lipases. In vivo data on gastric lipase and lipolysis have however been collected in humans and dogs during test meals. The biochemical characterization of gastric lipase has shown that this enzyme is rather unique among lipases: (i) stability and activity in the pH range 2 to 7 with an optimum at pH 4-5.4; (ii) high tensioactivity that allows resistance to bile salts and penetration into phospholipid layers covering TAG droplets; (iii) sn-3 stereospecificity for TAG hydrolysis; and (iv) resistance to pepsin. Most of these properties have been known for more than two decades and should provide a rational basis for the replacement of gastric lipase by other lipases when gastric lipase is not available.

Purification and characterization of extracellular lipase from a new strain: Pseudomonas aeruginosa SRT 9

PubMed Central

Borkar, Prita S.; Bodade, Ragini G.; Rao, Srinivasa R.; Khobragade, C.N.

2009-01-01

An extra cellular lipase was isolated and purified from the culture broth of Pseudomonas aeruginosa SRT 9 to apparent homogeneity using ammonium sulfate precipitation followed by chromatographic techniques on phenyl Sepharose CL- 4B and Mono Q HR 5/5 column, resulting in a purification factor of 98 fold with specific activity of 12307.8 U/mg. The molecular weight of the purified lipase was estimated by SDS-PAGE to be 29 kDa with isoelectric point of 4.5. Maximum lipase activity was observed in a wide range of temperature and pH values with optimum temperature of 55ºC and pH 6.9. The lipase preferably acted on triacylglycerols of long chain (C14-C16) fatty acids. The lipase was inhibited strongly by EDTA suggesting the enzyme might be metalloprotein. SDS and metal ions such as Hg2+, Zn2+, Cu2+, Ag2+ and Fe2+ decreased the lipase activity remarkedly. Its marked stability and activity in organic solvents suggest that this lipase is highly suitable as a biotechnological tool with a variety of applications including organo synthetic reactions and preparation of enantiomerically pure pharmaceuticals. The Km and Vmax value of the purified enzyme for triolein hydrolysis were calculated to be 1.11 mmol/L and 0.05 mmol/L/min respectively. PMID:24031373

Modeling of lipase catalyzed ring-opening polymerization of epsilon-caprolactone.

PubMed

Sivalingam, G; Madras, Giridhar

2004-01-01

Enzymatic ring-opening polymerization of epsilon-caprolactone by various lipases was investigated in toluene at various temperatures. The determination of molecular weight and structural identification was carried out with gel permeation chromatography and proton NMR, respectively. Among the various lipases employed, an immobilized lipase from Candida antartica B (Novozym 435) showed the highest catalytic activity. The polymerization of epsilon-caprolactone by Novozym 435 showed an optimal temperature of 65 degrees C and an optimum toluene content of 50/50 v/v of toluene and epsilon-caprolactone. As lipases can degrade polyesters, a maximum in the molecular weight with time was obtained due to the competition of ring opening polymerization and degradation by specific chain end scission. The optimum temperature, toluene content, and the variation of molecular weight with time are consistent with earlier observations. A comprehensive model based on continuous distribution kinetics was developed to model these phenomena. The model accounts for simultaneous polymerization, degradation and enzyme deactivation and provides a technique to determine the rate coefficients for these processes. The dependence of these rate coefficients with temperature and monomer concentration is also discussed.

GDSL lipases modulate immunity through lipid homeostasis in rice

PubMed Central

Lam, Sin Man; Tong, Xiaohong; Liu, Jiyun; Wang, Xin; Shui, Guanghou

2017-01-01

Lipids and lipid metabolites play important roles in plant-microbe interactions. Despite the extensive studies of lipases in lipid homeostasis and seed oil biosynthesis, the involvement of lipases in plant immunity remains largely unknown. In particular, GDSL esterases/lipases, characterized by the conserved GDSL motif, are a subfamily of lipolytic enzymes with broad substrate specificity. Here, we functionally identified two GDSL lipases, OsGLIP1 and OsGLIP2, in rice immune responses. Expression of OsGLIP1 and OsGLIP2 was suppressed by pathogen infection and salicylic acid (SA) treatment. OsGLIP1 was mainly expressed in leaf and leaf sheath, while OsGLIP2 showed high expression in elongating internodes. Biochemical assay demonstrated that OsGLIP1 and OsGLIP2 are functional lipases that could hydrolyze lipid substrates. Simultaneous down-regulation of OsGLIP1 and OsGLIP2 increased plant resistance to both bacterial and fungal pathogens, whereas disease resistance in OsGLIP1 and OsGLIP2 overexpression plants was significantly compromised, suggesting that both genes act as negative regulators of disease resistance. OsGLIP1 and OsGLIP2 proteins mainly localize to lipid droplets and the endoplasmic reticulum (ER) membrane. The proper cellular localization of OsGLIP proteins is indispensable for their functions in immunity. Comprehensive lipid profiling analysis indicated that the alteration of OsGLIP gene expression was associated with substantial changes of the levels of lipid species including monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). We show that MGDG and DGDG feeding could attenuate disease resistance. Taken together, our study indicates that OsGLIP1 and OsGLIP2 negatively regulate rice defense by modulating lipid metabolism, thus providing new insights into the function of lipids in plant immunity. PMID:29131851

Different Covalent Immobilizations Modulate Lipase Activities of Hypocrea pseudokoningii.

PubMed

Pereira, Marita G; Velasco-Lozano, Susana; Moreno-Perez, Sonia; Polizeli, Aline M; Heinen, Paulo R; Facchini, Fernanda D A; Vici, Ana C; Cereia, Mariana; Pessela, Benevides C; Fernandez-Lorente, Gloria; Guisan, Jose M; Jorge, João A; Polizeli, Maria de Lourdes T M

2017-09-04

Enzyme immobilization can promote several advantages for their industrial application. In this work, a lipase from Hypocrea pseudokoningii was efficiently linked to four chemical supports: agarose activated with cyanogen bromide (CNBr), glyoxyl-agarose (GX), MANAE-agarose activated with glutaraldehyde (GA) and GA-crosslinked with glutaraldehyde. Results showed a more stable lipase with both the GA-crosslinked and GA derivatives, compared to the control (CNBr), at 50 °C, 60 °C and 70 °C. Moreover, all derivatives were stabilized when incubated with organic solvents at 50%, such as ethanol, methanol, n -propanol and cyclohexane. Furthermore, lipase was highly activated (4-fold) in the presence of cyclohexane. GA-crosslinked and GA derivatives were more stable than the CNBr one in the presence of organic solvents. All derivatives were able to hydrolyze sardine, açaí ( Euterpe oleracea ), cotton seed and grape seed oils. However, during the hydrolysis of sardine oil, GX derivative showed to be 2.3-fold more selectivity (eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) ratio) than the control. Additionally, the types of immobilization interfered with the lipase enantiomeric preference. Unlike the control, the other three derivatives preferably hydrolyzed the R -isomer of 2-hydroxy-4-phenylbutanoic acid ethyl ester and the S -isomer of 1-phenylethanol acetate racemic mixtures. On the other hand, GX and CNBr derivatives preferably hydrolyzed the S -isomer of butyryl-2-phenylacetic acid racemic mixture while the GA and GA-crosslink derivatives preferably hydrolyzed the R -isomer. However, all derivatives, including the control, preferably hydrolyzed the methyl mandelate S -isomer. Moreover, the derivatives could be used for eight consecutive cycles retaining more than 50% of their residual activity. This work shows the importance of immobilization as a tool to increase the lipase stability to temperature and organic solvents, thus enabling the possibility of

Liquid lipases for enzymatic concentration of n-3 polyunsaturated fatty acids in monoacylglycerols via ethanolysis: Catalytic specificity and parameterization.

PubMed

He, Yongjin; Li, Jingbo; Kodali, Sitharam; Balle, Thomas; Chen, Bilian; Guo, Zheng

2017-01-01

This work examined catalytic specificity and fatty acid selectivity of five liquid lipases C. antarctica lipase A and B (CAL-A/B), and lipase TL (T. lanuginosus), Eversa Transfrom and NS in ethanolysis of fish oil with the aim to concentrate n-3 PUFAs into monoacylglycerols (MAGs) products. Lipase TL, Eversa Transform & NS entail a much faster reaction and produce higher MAGs yield (>30%); whereas CAL-A obtains the highest concentration of n-3 PUFAs/DHA/EPA into MAGs products (88.30%); followed by lipase NS (81.02%). 13 C NMR analysis indicates that CAL-B and lipase TL are sn-1,3 specific; but CAL-A and lipase Eversa Transform are non-regiospecific or weak sn-2 specific; which plausibly explains high enrichment effect of the latter two lipases. All liquid lipases are observed reusable for a certain times (lipase Eversa Transform up to 12 times), demonstrating their competitive advantage over immobilized form for industrial application because of their higher activity and cheaper operation cost. Copyright © 2016 Elsevier Ltd. All rights reserved.

Serial measurement of pancreatic lipase immunoreactivity concentration in dogs with immune-mediated disease treated with prednisolone.

PubMed

Ohta, H; Morita, T; Yokoyama, N; Osuga, T; Sasaki, N; Morishita, K; Nakamura, K; Takiguchi, M

2017-06-01

In this pilot study, serum canine pancreatic lipase immunoreactivity was measured repeatedly in dogs with various immune-mediated diseases that were treated with immunosuppressive doses of prednisolone. Ten client-owned dogs with newly diagnosed immune-mediated disease that had normal canine pancreatic lipase immunoreactivity concentrations (≤200 µg/l) were treated with 2 to 2.2 mg/kg prednisolone orally once daily as the initial treatment. Serum samples were obtained from each of the dogs prior to treatment and at 1- to 4-week intervals during immunosuppressive treatment. The highest canine pancreatic lipase immunoreactivity concentration detected during immunosuppressive treatment was defined as the peak canine pancreatic lipase immunoreactivity. Peak canine pancreatic lipase immunoreactivity concentrations were classified as normal in two dogs, questionable (201 to 399 µg/l) in three dogs, and abnormal (≥400 µg/l) in five dogs. Peak canine pancreatic lipase immunoreactivity concentrations were significantly higher than baseline canine pancreatic lipase immunoreactivity concentrations but there was no evidence of clinical pancreatitis. It remains unclear whether the five of 10 dogs with elevated canine pancreatic lipase immunoreactivity during prednisone treatment had subclinical pancreatitis or whether the abnormal results were a consequence of prednisolone administration. © 2017 British Small Animal Veterinary Association.

Microbial lipase mediated by health beneficial modification of cholesterol and flavors in food products: A review.

PubMed

Sharma, Ranjana; Sharma, Nivedita

2017-06-14

The tremendous need of lipase in varied applications in biotechnological increases its economical value in food and allied industries. Lipase has an impressive number of applications viz. enhancements of flavor in food products (Cheese, butter, alcoholic beverages, milk chocolate and diet control food stuffs), detergent industry in removing oil, grease stain, organic chemical processing, textile industry, oleochemical industry, cosmetic industry and also as therapeutic agents in pharmaceutical industries. This communication extends the frontier of lipase catalyzed benefits to human body by lowering serum cholesterol and enhancement of flavor in different food products. Among all, multiple innovations going on in the field of lipase applications are widening its scope in food industries consistently. Therefore in the present work an effort has been made to explore the utilization of lipase in the field of food product enhancement. Supplementation of food products with lipase results in modification of its physical, chemical and biochemical properties by enhancing its therapeutic activity. Lipases are the most important enzymes used in food industries. They are utilized as industrial catalysts for lipid hydrolysis. Because of lipases hydrolysis nature it is widely exploited to catalyze lipids or fats in different food products and enhancement of food flavors. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

PPARγ regulates exocrine pancreas lipase.

PubMed

Danino, Hila; Naor, Ronny Peri-; Fogel, Chen; Ben-Harosh, Yael; Kadir, Rotem; Salem, Hagit; Birk, Ruth

2016-12-01

Pancreatic lipase (triacylglycerol lipase EC 3.1.1.3) is an essential enzyme in hydrolysis of dietary fat. Dietary fat, especially polyunsaturated fatty acids (PUFA), regulate pancreatic lipase (PNLIP); however, the molecular mechanism underlying this regulation is mostly unknown. As PUFA are known to regulate expression of proliferator-activated receptor gamma (PPARγ), and as we identified in-silico putative PPARγ binding sites within the putative PNLIP promoter sequence, we hypothesized that PUFA regulation of PNLIP might be mediated by PPARγ. We used in silico bioinformatics tools, reporter luciferase assay, PPARγ agonists and antagonists, PPARγ overexpression in exocrine pancreas AR42J and primary cells to study PPARγ regulation of PNLIP. Using in silico bioinformatics tools we mapped PPARγ binding sites (PPRE) to the putative promoter region of PNLIP. Reporter luciferase assay in AR42J rat exocrine pancreas acinar cells transfected with various constructs of the putative PNLIP promoter showed that PNLIP transcription is significantly enhanced by PPARγ dose-dependently, reaching maximal levels with multi PPRE sites. This effect was significantly augmented in the presence of PPARγ agonists and reduced by PPARγ antagonists or mutagenesis abrogating PPRE sites. Over-expression of PPARγ significantly elevated PNLIP transcript and protein levels in AR42J cells and in primary pancreas cells. Moreover, PNLIP expression was up-regulated by PPARγ agonists (pioglitazone and 15dPGJ2) and significantly down-regulated by PPARγ antagonists in non-transfected rat exocrine pancreas AR42J cell line cells. PPARγ transcriptionally regulates PNLIP gene expression. This transcript regulation resolves part of the missing link between dietary PUFA direct regulation of PNLIP. Copyright © 2016 Elsevier B.V. All rights reserved.

Can the fatty acid selectivity of plant lipases be predicted from the composition of the seed triglyceride?

PubMed

Hellyer, S A; Chandler, I C; Bosley, J A

1999-09-22

To address the question can the fatty acid selectivity of plant lipases be predicted from the composition of the seed triglyceride, we have characterised the selectivity of lipases from a wide range of oilseeds with diverse fatty acid compositions. For this study, a novel hydrolysis assay using a fully randomised oil, was developed. From some seed sources (e.g. Cinnamomum camphora), lipases show high preference for particular fatty acids, whilst from others (e.g. Brassica napus, Theobroma cacao80% saturated or 'unusual' fatty acids may contain lipases which exhibit selectivity. It therefore follows that since the majority of seeds are composed of unsaturated fatty acids, that highly selective lipases will be unusual in nature. However lipases from some species of the Cuphea genera show exceptionally high preference for particular fatty acids. For example, lipase from seeds of Cuphea procumbans has over 20-fold selectivity for C10:0.

Thermosyntropha lipolytica gen. nov., sp. nov., a lipolytic, anaerobic, alkalitolerant, thermophilic bacterium utilizing short- and long-chain fatty acids in syntrophic coculture with a methanogenic archaeum.

PubMed

Svetlitshnyi, V; Rainey, F; Wiegel, J

1996-10-01

Three strains of an anaerobic thermophilic organoheterotrophic lipolytic alkalitolerant bacterium, Thermosyntropha lipolytica gen. nov., sp. nov. (type strain JW/VS-265T; DSM 11003), were isolated from alkaline hot springs of Lake Bogoria (Kenya). The cells were nonmotile, non-spore forming, straight or slightly curved rods. At 60 degrees C the pH range for growth determined at 25 degrees C [pH25 degrees C] was 7.15 to 9.5, with an optimum between 8.1 and 8.9 (pH60 degrees C of 7.6 and 8.1). At a pH25 degrees C of 8.5 the temperature range for growth was from 52 to 70 degrees C, with an optimum between 60 and 66 degrees C. The shortest doubling time was around 1 h. In pure culture the bacterium grew in a mineral base medium supplemented with yeast extract, tryptone, Casamino Acids, betaine, and crotonate as carbon sources, producing acetate as a major product and constitutively a lipase. During growth in the presence of olive oil, free long-chain fatty acids were accumulated in the medium but the pure culture could not utilize olive oil, triacylglycerols, short- and long-chain fatty acids, and glycerol for growth. In syntrophic coculture (Methanobacterium strain JW/VS-M29) the lipolytic bacteria grew on triacylglycerols and linear saturated and unsaturated fatty acids with 4 to 18 carbon atoms, but glycerol was not utilized. Fatty acids with even numbers of carbon atoms were degraded to acetate and methane, while from odd-numbered fatty acids 1 mol of propionate per mol of fatty acid was additionally formed. 16S rDNA sequence analysis identified Syntrophospora and Syntrophomonas spp. as closest phylogenetic neighbors.

[Lipase and phospholipase C from Staphylococcus aureus of different origin. I. Determination and occurrence (author's transl)].

PubMed

Berete, Y J; Schaeg, W; Brückler, J; Blobel, H

1980-11-01

Lipase and phospholipase C from Staphylococcus aureus of different origin were demonstrated qualitatively by agar diffusion on tributyrin- and lecithin agar. On test media with either 0,3% Na-azide or 0,3% KCN lipase-activity was not inhibited, phospholipase C, on the other hand, completely blocked (Table 1, Fig. 2). In this manner a tentative differentiation was possible between lipase and phospholipase C. For the quantitative determination of lipase the hydrolysis of p-nitrophenyl palmitate proved to be most useful (Fig. 1). S. aureus-cultures of human origin produced more often and more actively lipase and phospholipase C than those from cattle (Table 2).

Quantitative approach to track lipase producing Pseudomonas sp. S1 in nonsterilized solid state fermentation.

PubMed

Sahoo, R K; Subudhi, E; Kumar, M

2014-06-01

Proliferation of the inoculated Pseudomonas sp. S1 is quantitatively evaluated using ERIC-PCR during the production of lipase in nonsterile solid state fermentation an approach to reduce the cost of enzyme production. Under nonsterile solid state fermentation with olive oil cake, Pseudomonas sp. S1 produced 57·9 IU g(-1) of lipase. DNA fingerprints of unknown bacterial isolates obtained on Bushnell Haas agar (BHA) + tributyrin exactly matched with that of Pseudomonas sp. S1. Using PCR-based enumeration, population of Pseudomonas sp. S1 was proliferated from 7·6 × 10(4) CFU g(-1) after 24 h to 4·6 × 10(8) CFU g(-1) after 96 h, which tallied with the maximum lipase activity as compared to control. Under submerged fermentation (SmF), Pseudomonas sp. S1 produced maximum lipase (49 IU ml(-1) ) using olive oil as substrate, while lipase production was 9·754 IU ml(-1) when Pseudomonas sp. S1 was grown on tributyrin. Optimum pH and temperature of the crude lipase was 7·0 and 50°C. Crude enzyme activity was 71·2% stable at 50°C for 360 min. Pseudomonas sp. S1 lipase was also stable in methanol showing 91·6% activity in the presence of 15% methanol, whereas 75·5 and 51·1% of activity were retained in the presence of 20 and 30% methanol, respectively. Thus, lipase produced by Pseudomonas sp. S1 is suitable for the production of biodiesel as well as treatment of oily waste water. This study presents the first report on the production of thermophilic organic solvent tolerant lipase using agro-industry waste in nonsterile solid state fermentation. Positive correlation between survival of Pseudomonas sp. S1 and lipase production under nonsterile solid state fermentation was established, which may emphasize the need to combine molecular tools and solid state fermentation in future studies. Our study brings new insights into the lipase production in cost-effective manner, which is an industrially relevant approach. © 2014 The Society for Applied Microbiology.

Effect of bacterial or porcine lipase with low- or high-fat diets on nutrient absorption in pancreatic-insufficient dogs.

PubMed

Suzuki, A; Mizumoto, A; Rerknimitr, R; Sarr, M G; DiMango, E P

1999-02-01

Treatment of human exocrine pancreatic insufficiency is suboptimal. This study assessed the effects of bacterial lipase, porcine lipase, and diets on carbohydrate, fat, and protein absorption in pancreatic-insufficient dogs. Dogs were given bacterial or porcine lipase and 3 diets: a 48% carbohydrate, 27% fat, and 25% protein standard diet; a high-carbohydrate, low-fat, and low-protein diet; or a low-carbohydrate, high-fat, and high-protein diet (66%/18%/16% and 21%/43%/36% calories). With the standard diet, coefficient of fat absorption increased dose-dependently with both lipases (P < 0.05), but more fat was absorbed with porcine lipase (P < 0.05); 600, 000 IU of bacterial lipase (240 mg) and 300,000 IU of porcine lipase (18 g) nearly abolished steatorrhea. With 300,000 IU of bacterial lipase or 135,000 IU of porcine lipase, fat absorption was greater with the high-fat and -protein diet (P < 0.05 vs. low-fat and -protein diet). There were no interactions among carbohydrate, fat, and protein absorption. Correcting steatorrhea requires 75 times more porcine than bacterial lipase (18 vs. 240 mg). High-fat and high-protein diets optimize fat absorption with both enzymes. High-fat diets with bacterial or porcine lipase should be evaluated in humans with pancreatic steatorrhea.

Fast and economic immobilization methods described for non-commercial Pseudomonas lipases

PubMed Central

2014-01-01

Background There is an increasing interest to seek new enzyme preparations for the development of new products derived from bioprocesses to obtain alternative bio-based materials. In this context, four non-commercial lipases from Pseudomonas species were prepared, immobilized on different low-cost supports, and examined for potential biotechnological applications. Results To reduce costs of eventual scaling-up, the new lipases were obtained directly from crude cell extracts or from growth culture supernatants, and immobilized by simple adsorption on Accurel EP100, Accurel MP1000 and Celite®545. The enzymes evaluated were LipA and LipC from Pseudomonas sp. 42A2, a thermostable mutant of LipC, and LipI.3 from Pseudomonas CR611, which were produced in either homologous or heterologous hosts. Best immobilization results were obtained on Accurel EP100 for LipA and on Accurel MP1000 for LipC and its thermostable variant. Lip I.3, requiring a refolding step, was poorly immobilized on all supports tested (best results for Accurel MP1000). To test the behavior of immobilized lipases, they were assayed in triolein transesterification, where the best results were observed for lipases immobilized on Accurel MP1000. Conclusions The suggested protocol does not require protein purification and uses crude enzymes immobilized by a fast adsorption technique on low-cost supports, which makes the method suitable for an eventual scaling up aimed at biotechnological applications. Therefore, a fast, simple and economic method for lipase preparation and immobilization has been set up. The low price of the supports tested and the simplicity of the procedure, skipping the tedious and expensive purification steps, will contribute to cost reduction in biotechnological lipase-catalyzed processes. PMID:24755191

Utilization of agroindustrial residues for lipase production by solid-state fermentation

PubMed Central

Damaso, Mônica Caramez Triches; Passianoto, Moisés Augusto; de Freitas, Sidinéa Cordeiro; Freire, Denise Maria Guimarães; Lago, Regina Celi Araujo; Couri, Sonia

2008-01-01

The aim of this work was to produce lipases by solid-state fermentation (SSF) using, as substrate, agroindustrial residue supplemented with by-products from corn oil refining process or olive oil. For a group of ten fungi strains selected in the first steps, the lipase activity obtained by SSF varied from 7.7 to 58.6 U/g of dry substrate (gds). Among the evaluated strains, the Aspergillus niger mutant 11T53A14 was selected by presenting the best enzymatic production. For the fermentation tests, two substrates were also investigated: wheat bran and corn cob, both supplemented with olive oil. The best results were obtained with wheat bran. Additionally, three industrial by-products from corn oil refining (soapstock, stearin and fatty acids) were evaluated as substitutes to the olive oil in the function of lipases production inducer. Among them, soapstock and stearin were the best inducers, whereas fatty acids presented an inhibitor effect. The highest lipase activities using soapstock, stearin and fatty acids were 62.7 U/gds, 37.7 U/gds and 4.1 U/gds, respectively. PMID:24031288

Recombinant Lipases and Phospholipases and Their Use as Biocatalysts for Industrial Applications.

PubMed

Borrelli, Grazia M; Trono, Daniela

2015-09-01

Lipases and phospholipases are interfacial enzymes that hydrolyze hydrophobic ester linkages of triacylglycerols and phospholipids, respectively. In addition to their role as esterases, these enzymes catalyze a plethora of other reactions; indeed, lipases also catalyze esterification, transesterification and interesterification reactions, and phospholipases also show acyltransferase, transacylase and transphosphatidylation activities. Thus, lipases and phospholipases represent versatile biocatalysts that are widely used in various industrial applications, such as for biodiesels, food, nutraceuticals, oil degumming and detergents; minor applications also include bioremediation, agriculture, cosmetics, leather and paper industries. These enzymes are ubiquitous in most living organisms, across animals, plants, yeasts, fungi and bacteria. For their greater availability and their ease of production, microbial lipases and phospholipases are preferred to those derived from animals and plants. Nevertheless, traditional purification strategies from microbe cultures have a number of disadvantages, which include non-reproducibility and low yields. Moreover, native microbial enzymes are not always suitable for biocatalytic processes. The development of molecular techniques for the production of recombinant heterologous proteins in a host system has overcome these constraints, as this allows high-level protein expression and production of new redesigned enzymes with improved catalytic properties. These can meet the requirements of specific industrial process better than the native enzymes. The purpose of this review is to give an overview of the structural and functional features of lipases and phospholipases, to describe the recent advances in optimization of the production of recombinant lipases and phospholipases, and to summarize the information available relating to their major applications in industrial processes.

Recombinant Lipases and Phospholipases and Their Use as Biocatalysts for Industrial Applications

PubMed Central

Borrelli, Grazia M.; Trono, Daniela

2015-01-01

Lipases and phospholipases are interfacial enzymes that hydrolyze hydrophobic ester linkages of triacylglycerols and phospholipids, respectively. In addition to their role as esterases, these enzymes catalyze a plethora of other reactions; indeed, lipases also catalyze esterification, transesterification and interesterification reactions, and phospholipases also show acyltransferase, transacylase and transphosphatidylation activities. Thus, lipases and phospholipases represent versatile biocatalysts that are widely used in various industrial applications, such as for biodiesels, food, nutraceuticals, oil degumming and detergents; minor applications also include bioremediation, agriculture, cosmetics, leather and paper industries. These enzymes are ubiquitous in most living organisms, across animals, plants, yeasts, fungi and bacteria. For their greater availability and their ease of production, microbial lipases and phospholipases are preferred to those derived from animals and plants. Nevertheless, traditional purification strategies from microbe cultures have a number of disadvantages, which include non-reproducibility and low yields. Moreover, native microbial enzymes are not always suitable for biocatalytic processes. The development of molecular techniques for the production of recombinant heterologous proteins in a host system has overcome these constraints, as this allows high-level protein expression and production of new redesigned enzymes with improved catalytic properties. These can meet the requirements of specific industrial process better than the native enzymes. The purpose of this review is to give an overview of the structural and functional features of lipases and phospholipases, to describe the recent advances in optimization of the production of recombinant lipases and phospholipases, and to summarize the information available relating to their major applications in industrial processes. PMID:26340621

Properties of a membrane-bound triglyceride lipase of rapeseed (Brassica napus L.) cotyledons.

PubMed

Rosnitschek, I; Theimer, R R

1980-04-01

The properties of the alkaline lipase activity (EC 3.1.1.3) that was recovered almost completely from a microsomal membrane fraction of 4-d-old rapeseed (Brassica napus L.) cotyledons were studied employing a titrimetric test procedure. The apparent KM was 6.5 mmol l(-1), with emulgated sunflower oil as the substrate. The products of triglyceride hydrolysis in vitro were glycerol, free fatty acids, and minor amounts of mono- and diglycerides. Maximum lipase activity depended on the preincubation of the lipolytic membrane fraction in 0.15 mol l(-1) NaCl and on the presence of at least 0.1 mol l(-1) NaCl in the test mixture. Desoxycholate and up to 0.1 mol l(-1) CaCl2 also activated the enzyme while EDTA and detergents such as trito x-100, digitonin, tween 85, and sodium dodecylsulfate were inhibitory. The rapeseed lipase displayed a conspicuous substrate selectivity among different plant triglycerides; the activity was inversely correlated with the oleic acid content of the oils. Water-soluble triacetin and the phospholipid lecithin were not hydrolyzed. Increasing amounts of free fatty acids reduced lipase activity; erucic acid, a major component of rapeseed oil, exhibited the strongest effect, suggesting a possible role in the regulation of lipase activity in vivo. The data demonstrate that the lipolytic membrane fraction houses a triglyceride lipase with properties similar to other plant and animal lipases. It can both qualitatively and quantitatively account for the fat degradation in rapeseed cotyledons. The evidence that provides further reason to acknowledge the membranous appendices of the spherosomes as the intracellular site of lipolysis is discussed.

Hydrolysis of mixed monomolecular films of triglyceride/lecithin by pancreatic lipase.

PubMed

Pieroni, G; Verger, R

1979-10-25

The main purpose of this study was to describe the influence of lecithin upon lipolysis of mixed monomolecular films of trioctanoylglycerol/didodecanoylphosphatidycholine by pancreatic lipase in order to mimic some physiological situations. The quantity of enzyme adsorbed to the interface was simultaneously determined using 5-thio-2-nitro[14C]benzoyl lipase. Lipolytic activity was enhanced 3- to 4-fold in the presence of colipase, an effect which is attributed to increased enzyme turnover number. When a pure triglyceride film was progressively diluted with lecithin, the minimum specific activity of lipase exhibited a bell-shaped curve: a mixed film containing only 20% trioctanoylglycerol was hydrolyzed at the same rate as a monolayer of pure triglyceride.

Preliminary studies on immobilization of lipase using chicken eggshell

NASA Astrophysics Data System (ADS)

Salleh, S.; Serri, N. A.; Hena, S.; Tajarudin, H. A.

2016-06-01

A few advantages of enzyme immobilization are reusability of expensive enzyme, improvement of stability and activity compared to crude enzyme. Various organic components can be used as carrier for enzyme immobilization such as chicken eggshell. It can be used as a carrier for immobilization as its mineral component mostly contains of calcium carbonate. In the present study, Tributyrin method was used to test enzyme activity of Rhizomucour Miehei, Candida Antarctica and Candida Rugosa. Rhizomucour Miehei shows the highest enzyme activity (360.8 mol/min/mL lipase) and was used in further experiment. Experiment was continued to study incubation time for lipase immobilization on eggshell (1-4 hours) and reaction time of esterification of sugar ester (0-72 hours). Two hours incubation time for lipase immobilization was observed and gives the highest yield of sugar ester (78.13%). Fructose and stearic acid as substrate was used for the production of sugar ester. The highest percentage of sugar ester production was shown at 36 hours of reaction time.

New member of the hormone-sensitive lipase family from the permafrost microbial community.

PubMed

Petrovskaya, Lada E; Novototskaya-Vlasova, Ksenia A; Gapizov, Sultan Sh; Spirina, Elena V; Durdenko, Ekaterina V; Rivkina, Elizaveta M

2017-07-04

Siberian permafrost is a unique environment inhabited with diverse groups of microorganisms. Among them, there are numerous producers of biotechnologically relevant enzymes including lipases and esterases. Recently, we have constructed a metagenomic library from a permafrost sample and identified in it several genes coding for potential lipolytic enzymes. In the current work, properties of the recombinant esterases obtained from this library are compared with the previously characterized lipase from Psychrobacter cryohalolentis and other representatives of the hormone-sensitive lipase family.

Oral self-nanoemulsifying peptide drug delivery systems: impact of lipase on drug release.

PubMed

Mahjub, Reza; Dorkoosh, Farid Abedin; Rafiee-Tehrani, Morteza; Bernkop Schnürch, Andreas

2015-01-01

It was the aim of this study to evaluate the impact of lipases on the release behaviour of a peptide drug from oral self-nanoemulsifying drug delivery systems. Octreotide was ion paired with the anionic surfactants deoxycholate, decanoate, oleate and dodecylsulphate. The lipophilic character of these complexes was characterised by determining the n-octanol/buffer pH 7.4 partition coefficient. In the following the most hydrophilic complex was incorporated in a likely lipase degradable self-nanoemulsifying drug delivery systems (SNEDDS) formulation containing a triglyceride (olive oil; Pharm.Eur.) and in a likely not lipase degradable SNEDDS containing lipids and surfactants without any ester bonds. After 1:100 dilutions in artificial intestinal fluid (AIF), the lipid droplets were characterised regarding size distribution. With these SNEDDS, drug release studies were performed in AIF with and without lipase. Results showed that the most hydrophobic complex can be formed with deoxycholate in an octreotide:anionic surfactant ratio of 1:5. Even 73.1 ± 8.1% of it could be quantified in the n-octanol phase. SNEDDS containing octreotide | olive oil | cremophor EL | propylene glycol (2|57|38|3) and octreotide | liquid paraffin | Brij 35 | propylene glycol | ethanol (2|66.5|25|5|1.5) showed after dilution in AIF, a mean droplet size of 232 ± 53 nm and 235 ± 50 nm, respectively. Drug release studies showed a sustained release of octreotide out of these formulations for at least 24 h, whereas > 80% of the drug was released within 2 h in the presence of lipase in the case of the triglyceride containing SNEEDS. In contrast the release profile from ester-free SNEDDS was not significantly altered (p < 0.05) due to the addition of lipase providing evidence for the stability of this formulation towards lipases. According to these results, SNEDDS could be identified as a useful tool for sustained oral peptide delivery taking an enzymatic degradation by

Production of a Novel Cold-Active Lipase from Pichia lynferdii Y-7723

USDA-ARS?s Scientific Manuscript database

Lipase (triacylglycerol acylhydrolases, E.C. 3.1.1.3.) is one of the most important enzymes applied to the broad range of industrial application field. Especially, lipases with abnormal functionality such as thermo stability, alkaline, acidic, cold-activity gain special attention because of their a...

Hydrolysis products generated by lipoprotein lipase and endothelial lipase differentially impact THP-1 macrophage cell signalling pathways.

PubMed

Essaji, Yasmin; Yang, Yanbo; Albert, Carolyn J; Ford, David A; Brown, Robert J

2013-08-01

Macrophages express lipoprotein lipase (LPL) and endothelial lipase (EL) within atherosclerotic plaques; however, little is known about how lipoprotein hydrolysis products generated by these lipases might affect macrophage cell signalling pathways. We hypothesized that hydrolysis products affect macrophage cell signalling pathways associated with atherosclerosis. To test our hypothesis, we incubated differentiated THP-1 macrophages with products from total lipoprotein hydrolysis by recombinant LPL or EL. Using antibody arrays, we found that the phosphorylation of six receptor tyrosine kinases and three signalling nodes--most associated with atherosclerotic processes--was increased by LPL derived hydrolysis products. EL derived hydrolysis products only increased the phosphorylation of tropomyosin-related kinase A, which is also implicated in playing a role in atherosclerosis. Using electrospray ionization-mass spectrometry, we identified the species of triacylglycerols and phosphatidylcholines that were hydrolyzed by LPL and EL, and we identified the fatty acids liberated by gas chromatography-mass spectrometry. To determine if the total liberated fatty acids influenced signalling pathways, we incubated differentiated THP-1 macrophages with a mixture of the fatty acids that matched the concentrations of liberated fatty acids from total lipoproteins by LPL, and we subjected cell lysates to antibody array analyses. The analyses showed that only the phosphorylation of Akt was significantly increased in response to fatty acid treatment. Overall, our study shows that macrophages display potentially pro-atherogenic signalling responses following acute treatments with LPL and EL lipoprotein hydrolysis products.

Development of a high-throughput assay for measuring lipase activity using natural triacylglycerols coated on microtiter plates.

PubMed

Serveau-Avesque, Carole; Verger, Robert; Rodriguez, Jorge A; Abousalham, Abdelkarim

2013-09-21

We have designed a convenient, specific, sensitive and continuous lipase assay based on the use of natural triacylglycerols (TAGs) from the Aleurites fordii seed oil which contains α-eleostearic acid (9,11,13,cis,trans,trans-octadecatrienoic acid) and which was coated in the wells of microtiter plates. The coated TAG film cannot be desorbed by the various buffers used during the lipase assay. Upon lipase action, α-eleostearic acid is liberated and desorbed from the interface and then solubilized into the micellar phase. Consequently, the UV absorbance of the α-eleostearic acid is considerably enhanced due to the transformation from an adsorbed to a water soluble state. The lipase activity can be measured continuously by recording the variations with time of the UV absorption spectra. The rate of lipolysis was monitored by measuring the increase of OD at 272 nm, which was found to be linear with time and directly proportional to the amount of added lipase. This microtiter plate lipase assay, based on coated TAGs, presents various advantages as compared to the classical systems: (i) coated TAGs on the microtiter plates could be stored for a long-time at 4 °C, (ii) higher sensitivity in lipase detection, (iii) good reproducibility, and (iv) increase of signal to noise ratio due to high UV absorption after transfer of α-eleostearic acid from an adsorbed to a soluble state. Low concentrations, down to 1 pg mL(-1) of pure Thermomyces lanuginosus or human pancreatic lipase, could be detected under standard assay conditions. The detection sensitivity of this coated method is around 1000 times higher as compared to those obtained with the classical emulsified systems. This continuous high throughput lipase assay could be used to screen new lipases and/or lipase inhibitors present in various biological samples.

Comparative genomic analysis of the Lipase3 gene family in five plant species reveals distinct evolutionary origins.

PubMed

Wang, Dan; Zhang, Lin; Hu, JunFeng; Gao, Dianshuai; Liu, Xin; Sha, Yan

2018-04-01

Lipases are physiologically important and ubiquitous enzymes that share a conserved domain and are classified into eight different families based on their amino acid sequences and fundamental biological properties. The Lipase3 family of lipases was reported to possess a canonical fold typical of α/β hydrolases and a typical catalytic triad, suggesting a distinct evolutionary origin for this family. Genes in the Lipase3 family do not have the same functions, but maintain the conserved Lipase3 domain. There have been extensive studies of Lipase3 structures and functions, but little is known about their evolutionary histories. In this study, all lipases within five plant species were identified, and their phylogenetic relationships and genetic properties were analyzed and used to group them into distinct evolutionary families. Each identified lipase family contained at least one dicot and monocot Lipase3 protein, indicating that the gene family was established before the split of dicots and monocots. Similar intron/exon numbers and predicted protein sequence lengths were found within individual groups. Twenty-four tandem Lipase3 gene duplications were identified, implying that the distinctive function of Lipase3 genes appears to be a consequence of translocation and neofunctionalization after gene duplication. The functional genes EDS1, PAD4, and SAG101 that are reportedly involved in pathogen response were all located in the same group. The nucleotide diversity (Dxy) and the ratio of nonsynonymous to synonymous nucleotide substitutions rates (Ka/Ks) of the three genes were significantly greater than the average across the genomes. We further observed evidence for selection maintaining diversity on three genes in the Toll-Interleukin-1 receptor type of nucleotide binding/leucine-rich repeat immune receptor (TIR-NBS LRR) immunity-response signaling pathway, indicating that they could be vulnerable to pathogen effectors.

Crystal structure of a triacylglycerol lipase from Penicillium expansum at 1.3 A determined by sulfur SAD

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bian, Chuanbing; Yuan, Cai; Chen, Liqing

2010-04-05

Triacylglycerol lipases (EC 3.1.1.3) are present in many different organisms including animals, plants, and microbes. Lipases catalyze the hydrolysis of long-chain triglycerides into fatty acids and glycerol at the interface between the water insoluble substrate and the aqueous phase. Lipases can also catalyze the reverse esterification reaction to form glycerides under certain conditions. Lipases of microbial origin are of considerable commercial interest for wide variety of biotechnological applications in industries, including detergent, food, cosmetic, pharmaceutical, fine chemicals, and biodiesel. Nowadays, microbial lipases have become one of the most important industrial enzymes. PEL (Penicillium expansum lipase) is a fungal lipase frommore » Penicillium expansum strain PF898 isolated from Chinese soil that has been subjected to several generations of mutagenesis to increase its enzymatic activity. PEL belongs to the triacylglycerol lipases family, and its catalytic characteristics have been studied. The enzyme has been used in Chinese laundry detergent industry for several years (http://www.leveking.com). However, the poor thermal stability of the enzyme limits its application. To further study and improve this enzyme, PEL was cloned and sequenced. Furthermore, it was overexpressed in Pichia pastoris. PEL contains GHSLG sequence, which is the lipase consensus sequence Gly-X1-Ser-X2-Gly, but has a low amino acid sequence identities to other lipases. The most similar lipases are Rhizomucor miehei (PML) and Rhizopus niveus (PNL) with a 21% and 20% sequence identities to PEL, respectively. Interestingly, the similarity of PEL with the known esterases is somewhat higher with 24% sequence identity to feruloyl esterase A. Here, we report the 1.3 {angstrom} resolution crystal structure of PEL determined by sulfur SAD phasing. This structure not only presents a new lipase structure at high resolution, but also provides a structural platform to analyze the

Interactions between yeasts and bacteria in the smear surface-ripened cheeses.

PubMed

Corsetti, A; Rossi, J; Gobbetti, M

2001-09-19

In the initial phase of ripening, the microflora of bacterial smear surface-ripened cheeses such as Limburger, Taleggio, Brick, Münster and Saint-Paulin and that of surface mould-ripened cheeses such as Camembert and Brie may be similar, but at the end of the ripening, bacteria such as Brevibacterium spp., Arthrobacter spp., Micrococcus spp., Corynebacterium spp. and moulds such as Penicillium camemberti are, respectively, the dominant microorganisms. Yeasts such as Candida spp., Cryptococcus spp., Debaryomyces spp., Geotrichum candidum, Pichia spp., Rhodotorula spp., Saccharomyces spp. and Yarrowia lipolytica are often and variably isolated from the smear surface-ripened cheeses. Although not dominant within the microorganisms of the smear surface-ripened cheeses, yeasts establish significant interactions with moulds and especially bacteria, including surface bacteria and lactic acid bacteria. Some aspects of the interactions between yeasts and bacteria in such type of cheeses are considered in this paper.

Solvent-dependent gating motions of an extremophilic lipase from Pseudomonas aeruginosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, Quentin R.; Nellas, Ricky B.; Shen, Tongye

2012-07-25

Understanding how organic solvent-stable proteins can function in anhydrous and often complex solutions is essential for the study of the interaction of protein and molecular immiscible interfaces and the design of efficient industrial enzymes in nonaqueous solvents. Using an extremophilic lipase from Pseudomonas aeruginosa as an example, we investigated the conformational dynamics of an organic solvent-tolerant enzyme in complex solvent milieux. Four 100-ns molecular dynamics simulations of the lipase were performed in solvent systems: water, hexane, and two mixtures of hexane and water, 5% and 95% (w/w) hexane. Our results show a solvent-dependent structural change of the protein, especially inmore » the region that regulates the admission of the substrate. We observed that the lipase is much less flexible in hexane than in aqueous solution or at the immiscible interface. Quantified by the size of the accessible channel, the lipase in water has a closed-gate conformation and no access to the active site, while in the hexane-containing systems, the lipase is at various degrees of open-gate state, with the immiscible interface setup being in the widely open conformation ensembles. Furthermore, the composition of explicit solvents in the access channel showed a significant influence on the conformational dynamics of the protein. Interestingly, the slowest step (bottleneck) of the hexane-induced conformational switch seems to be correlated with the slow dehydration dynamics of the channel.« less

Light harvesting amphiphiles boost the performance of lipase-based washing formulations.

PubMed

Díaz Blanco, Carlos; Trifonov, Anatoli; Georgiev, George; Tzanov, Tzanko

2012-08-10

One of the major industrial uses of lipases is as active agent in bio-based washing formulations. Current methods to improve lipase stability in detergent formulations usually entail a decrease in the enzymatic activity, thus lowering the general performance of the detergent. This work proposes an alternative approach for enzyme stabilization and activity enhancement based on the application of amphiphilic light-harvesting copolymers, called here photozymes. The biopolymer-based chitosan-Rose Bengal and the synthetic poly(SSS(0.75)-co-VBA(0.24)-co-VB/hematoporphyrin(0.01)) photozymes were used as boosting agents in washing formulations containing lipase. Organic stain removal from textiles was improved by 33% at 25°C. This cleaning enhancement was attributed to the increase of lipase activity due to interfacial activation in presence of photozymes, along with prevention of dirt re-deposition on the cleaned surfaces. Although both photozymes improved lipase activity, chitosan-Rose Bengal photozyme performed better at pHs above 9 while at pHs below this value SSS-VBA-VB/HP was the most effective. Dynamic light scattering, zeta-potential measurements, fluorescence spectroscopy and FRET experiments confirmed the pseudomicellar conformation and hosting capacity of the photozymes in aqueous media leading to improved dirt solubilization and emulsification. Moreover, the photocatalytic activity of the photozyme allowed for a de-coloration of the waste washing liquor upon UV irradiation. Copyright © 2012 Elsevier Inc. All rights reserved.

Exploring the specific features of interfacial enzymology based on lipase studies.

PubMed

Aloulou, Ahmed; Rodriguez, Jorge A; Fernandez, Sylvie; van Oosterhout, Dirk; Puccinelli, Delphine; Carrière, Frédéric

2006-09-01

Many enzymes are active at interfaces in the living world (such as in the signaling processes at the surface of cell membranes, digestion of dietary lipids, starch and cellulose degradation, etc.), but fundamental enzymology remains largely focused on the interactions between enzymes and soluble substrates. The biochemical and kinetic characterization of lipolytic enzymes has opened up new paths of research in the field of interfacial enzymology. Lipases are water-soluble enzymes hydrolyzing insoluble triglyceride substrates, and studies on these enzymes have led to the development of specific interfacial kinetic models. Structure-function studies on lipases have thrown light on the interfacial recognition sites present in the molecular structure of these enzymes, the conformational changes occurring in the presence of lipids and amphiphiles, and the stability of the enzymes present at interfaces. The pH-dependent activity, substrate specificity and inhibition of these enzymes can all result from both "classical" interactions between a substrate or inhibitor and the active site, as well as from the adsorption of the enzymes at the surface of aggregated substrate particles such as oil drops, lipid bilayers or monomolecular lipid films. The adsorption step can provide an alternative target for improving substrate specificity and developing specific enzyme inhibitors. Several data obtained with gastric lipase, classical pancreatic lipase, pancreatic lipase-related protein 2 and phosphatidylserine-specific phospholipase A1 were chosen here to illustrate these specific features of interfacial enzymology.

Design of Heterogeneous Hoveyda-Grubbs Second-Generation Catalyst-Lipase Conjugates.

PubMed

Neville, Anthony; Iniesta, Javier; Palomo, Jose M

2016-12-06

Heterogeneous catalysts have been synthesi zed by the conjugation of Hoveyda-Grubbs second-generation catalyst with a lipase. The catalytic properties of the organometallic compound in solution were firstly optimized, evaluating the activity of Ru in the ring-closing metathesis of diethyldiallymalonate at 25 °C at different solvents and in the presence of different additives. The best result was found using tetrahydrofuran as a solvent. Some additives such as phenylboronic acid or polyetheneglycol slightly improved the activity of the Ru catalyst whereas others, such as pyridine or dipeptides affected it negatively. The organometallic compound immobilized on functionalized-surface materials activated with boronic acid or epoxy groups (around 50-60 µg per mg support) and showed 50% conversion at 24 h in the ring-closing metathesis. Cross-linked enzyme aggregates (CLEA's) of the Hoveyda-Grubbs second-generation catalyst with Candida antarctica lipase (CAL-B) were prepared, although low Ru catalyst was found to be translated in low conversion. Therefore, a sol-gel preparation of the Hoveyda-Grubbs second-generation and CAL-B was performed. This catalyst exhibited good activity in the metathesis of diethyldiallymalonate in toluene and in aqueous media. Finally, a new sustainable approach was used by the conjugation lipase-Grubbs in solid phase in aqueous media. Two strategies were used: one using lipase previously covalently immobilized on an epoxy-Sepharose support (hydrophilic matrix) and then conjugated with grubbs; and in the second, the free lipase was incubated with organometallic in aqueous solution and then immobilized on epoxy-Sepharose. The different catalysts showed excellent conversion values in the ring-closing metathesis of diethyldiallymalonate in aqueous media at 25 °C.

A plasmonic nanosensor for lipase activity based on enzyme-controlled gold nanoparticles growth in situ

NASA Astrophysics Data System (ADS)

Tang, Yan; Zhang, Wei; Liu, Jia; Zhang, Lei; Huang, Wei; Huo, Fengwei; Tian, Danbi

2015-03-01

A plasmonic nanosensor for lipase activity was developed based on one-pot nanoparticle growth. Tween 80 was selected not only as the substrate for lipase recognition but also as the reducing and stabilizing agent for the sensor fabrication. The different molecular groups in Tween 80 could have different roles in the fabrication procedure; the H2O2 produced by the autoxidation of the ethylene oxide subunits in Tween 80 could reduce the AuCl4- ions to Au atoms, meanwhile, the lipase could hydrolyze its carboxyl ester bond, which could, in turn, control the rate of nucleation of the gold nanoparticles (AuNPs) and tailor the localized surface plasmon resonance (LSPR) of the AuNP transducers. The color changes, which depend on the absence or presence of the lipase, could be used to sense the lipase activity. A linear response ranging from 0.025 to 4 mg mL-1 and a detection limit of the lipase as low as 3.47 μg mL-1 were achieved. This strategy circumvents the problems encountered by general enzyme assays that require sophisticated instruments and complicated assembling steps. The methodology can benefit the assays of heterogeneous-catalyzed enzymes.A plasmonic nanosensor for lipase activity was developed based on one-pot nanoparticle growth. Tween 80 was selected not only as the substrate for lipase recognition but also as the reducing and stabilizing agent for the sensor fabrication. The different molecular groups in Tween 80 could have different roles in the fabrication procedure; the H2O2 produced by the autoxidation of the ethylene oxide subunits in Tween 80 could reduce the AuCl4- ions to Au atoms, meanwhile, the lipase could hydrolyze its carboxyl ester bond, which could, in turn, control the rate of nucleation of the gold nanoparticles (AuNPs) and tailor the localized surface plasmon resonance (LSPR) of the AuNP transducers. The color changes, which depend on the absence or presence of the lipase, could be used to sense the lipase activity. A linear response

Factors influencing production of lipase under metal supplementation by bacterial strain, Bacillus subtilis BDG-8.

PubMed

Dhevahi, B; Gurusamy, R

2014-11-01

Lipases are biocatalyst having wide applications in industries due to their versatile properties. In the present study, a lipolytic bacterial strain, Bacillus subtilis BDG-8 was isolated from an oil based industrial soil. The effect of selenium and nickel as a media supplement on enhancement of lipase production, was studied individually with the isolated strain by varying the concentration of selected metal. 60 μg l(-1) selenium enhanced lipase production to an enzyme activity measuring 7.8 U ml(-1) while 40 μgI(-1) nickel gave the maximum enzyme activity equivalent to 7.5 U ml(-1). However, nickel and selenium together at a range of concentration with an equal w/v ratio, at 60 μg l(-1) each, showed the maximum lipase activity of 8.5 U ml(-1). The effect of pH and temperature on lipase production showed maximum enzyme activity in the presence of each of the metals at pH 7 and 35°C among the other tested ranges. After optimisation of the parameters such as metal concentration, pH and temperature lipase production by Bacillus subtilis BDG-8 had increased several folds. This preliminary investigation may consequently lead as to various industrial applications such as treatment of wastewater contaminated with metal or oil with simultaneous lipase production.

Comparative study on digestive lipase activities on the self emulsifying excipient Labrasol, medium chain glycerides and PEG esters.

PubMed

Fernandez, Sylvie; Jannin, Vincent; Rodier, Jean-David; Ritter, Nicolas; Mahler, Bruno; Carrière, Frédéric

2007-05-01

Labrasol is a lipid-based self-emulsifying excipient used in the preparation of lipophilic drugs intended for oral delivery. It is mainly composed of PEG esters and glycerides with medium acyl chains, which are potential substrates for digestive lipases. The hydrolysis of Labrasol by porcine pancreatic extracts, human pancreatic juice and several purified digestive lipases was investigated in the present study. Classical human pancreatic lipase (HPL) and porcine pancreatic lipase, which are the main lipases involved in the digestion of dietary triglycerides, showed very low levels of activity on the entire Labrasol excipient as well as on separated fractions of glycerides and PEG esters. On the other hand, gastric lipase, pancreatic lipase-related protein 2 (PLRP2) and carboxyl ester hydrolase (CEH) showed high specific activities on Labrasol. These lipases were found to hydrolyze the main components of Labrasol (PEG esters and monoglycerides) used as individual substrates, whereas these esters were found to be poor substrates for HPL. The lipolytic activity of pancreatic extracts and human pancreatic juice on Labrasol(R) is therefore mainly due to the combined action of CEH and PLRP2. These two pancreatic enzymes, together with gastric lipase, are probably the main enzymes involved in the in vivo lipolysis of Labrasol taken orally.

Ionic liquids as a reaction medium for lipase-catalyzed methanolysis of sunflower oil.

PubMed

Sunitha, S; Kanjilal, S; Reddy, P S; Prasad, R B N

2007-12-01

Ionic liquids, 1-butyl-3-methyl imidazolium hexafluorophosphate ([BMIm][PF(6)]) and 1-ethyl-3-methyl imidazolium hexafluorophosphate ([EMIm][PF(6)]), were used for the methanolysis of sunflower oil using Candida antarctica lipase (Novozyme 435) and gave yields of fatty acid methyl esters at 98-99% within 10 h. The optimum conditions of methanolysis in hydrophobic ionic liquids are 2% (w/w) lipase, 1:1 (w/w) oil/ionic liquid and 1:8 (mol/mol) oil/methanol at 58-60 degrees C. Methanolysis using hydrophilic ionic liquids, 3-methyl imidazolium tetrafluoroborate ([HMIm][BF(4)]) and 1-butyl-3-methyl imidazolium tetrafluoroborate ([BMIm][BF(4)]), gave very poor yields. A hydrophobic ionic liquid thus protects the lipase from methanol. Recovered ionic liquids and lipase were used for four successive reaction cycles without any significant loss of activity.

Inhibition of phospholipase A1, lipase and galactolipase activities of pancreatic lipase-related protein 2 by methyl arachidonyl fluorophosphonate (MAFP).

PubMed

Amara, Sawsan; Delorme, Vincent; Record, Michel; Carrière, Frédéric

2012-11-01

Methyl arachidonyl fluorophosphonate (MAFP) is a known inhibitor of cytosolic phospholipase A2 and some other serine enzymes. MAFP was found here to be an irreversible inhibitor of human pancreatic lipase-related protein 2 (HPLRP2), an enzyme displaying lipase, phospholipase A1 and galactolipase activities. In the presence of MAFP, mass spectrometry analysis of HPLRP2 revealed a mass increase of 351Da, suggesting a covalent binding of MAFP to the active site serine residue. When HPLRP2 was pre-incubated with MAFP before measuring residual activity, a direct inhibition of HPLRP2 occurred, confirming that HPLRP2 has an active site freely accessible to solvent and differs from most lipases in solution. HPLRP2 activities on tributyrin (TC4), phosphatidylcholine (PC) and monogalactosyl dioctanoylglycerol (C8-MGDG) were equally inhibited under these conditions. Bile salts were not required to trigger the inhibition, but they significantly increased the rate of HPLRP2 inhibition, probably because of MAFP micellar solubilization. Since HPLRP2 is active on various substrates that self-organize differently in the presence of water, HPLRP2 inhibition by MAFP was tested in the presence of these substrates after adding MAFP in the course of the lipolysis reaction. In this case, the rates of inhibition of lipase, phospholipase A1 and galactolipase activities were not equivalent (triglycerides>PC>MGDG), suggesting different enzyme/inhibitor partitioning between the aqueous phase and lipid aggregates. The inhibition by MAFP of a well identified phospholipase A1 (HPLRP2), present in pancreatic juice and also in human monocytes, indicates that MAFP cannot be used for discriminating phospholipase A2 from A1 activities at the cellular level. Copyright © 2012 Elsevier B.V. All rights reserved.

The effect of diet on the expression of lipase genes in the midgut of the lightbrown apple moth (Epiphyas postvittana Walker; Tortricidae).

PubMed

Christeller, J T; Poulton, J; Markwick, N M; Simpson, R M

2010-02-01

We have identified lipase-like genes from an Epiphyas postvittana larval midgut EST library. Of the 10 pancreatic lipase family genes, six appear to encode active lipases and four encode inactive lipases, based on the presence/absence of essential catalytic residues. The four gastric lipase family genes appear to encode active proteins. Phylogenetic analysis of 54 lepidopteran pancreatic lipase proteins resolved the clade into five groups of midgut origin and a sixth of non-midgut lipases. The inactive proteins formed two separate groups with highly conserved mutations. The lepidopteran midgut lipases formed a ninth subfamily of pancreatic lipases. Eighteen insect and human gastric lipases were analysed phylogenetically with only very weak support for any groupings. Gene expression was measured in the larval midgut following feeding on five artificial diets and on apple leaves. The artificial diets contained different levels of triacylglycerol, linoleic acid and cholesterol. Significant changes in gene expression (more than 100-fold for active pancreatic lipases) were observed. All the inactive lipases were also highly expressed. The gastric lipase genes were expressed at lower levels and suppressed in larvae feeding on leaves. Together, protein motif analysis and the gene expression data suggest that, in phytophagous lepidopteran larvae, the pancreatic lipases may function in vivo as galactolipases and phospholipases whereas the gastric lipases may function as triacylglycerol hydrolases.

Immobilised lipases in the cosmetics industry.

PubMed

Ansorge-Schumacher, Marion B; Thum, Oliver

2013-08-07

Commercial products for personal care, generally perceived as cosmetics, have an important impact on everyday life worldwide. Accordingly, the market for both consumer products and specialty chemicals comprising their ingredients is considerable. Lipases have started to play a minor role as active ingredients in so-called 'functional cosmetics' as well as a major role as catalysts for the industrial production of various specialty esters, aroma compounds and active agents. Interestingly, both applications almost always require preparation by appropriate immobilisation techniques. In addition, for catalytic use special reactor concepts often have to be employed due to the mostly limited stability of these preparations. Nevertheless, these processes show distinct advantages based on process simplification, product quality and environmental footprint and are therefore apt to more and more replace traditional chemical processes. Here, for the first time a review on the various aspects of using immobilised lipases in the cosmetics industry is given.

Cell-bound lipases from Burkholderia sp. ZYB002: gene sequence analysis, expression, enzymatic characterization, and 3D structural model.

PubMed

Shu, Zhengyu; Lin, Hong; Shi, Shaolei; Mu, Xiangduo; Liu, Yanru; Huang, Jianzhong

2016-05-03

The whole-cell lipase from Burkholderia cepacia has been used as a biocatalyst in organic synthesis. However, there is no report in the literature on the component or the gene sequence of the cell-bound lipase from this species. Qualitative analysis of the cell-bound lipase would help to illuminate the regulation mechanism of gene expression and further improve the yield of the cell-bound lipase by gene engineering. Three predictive cell-bound lipases, lipA, lipC21 and lipC24, from Burkholderia sp. ZYB002 were cloned and expressed in E. coli. Both LipA and LipC24 displayed the lipase activity. LipC24 was a novel mesophilic enzyme and displayed preference for medium-chain-length acyl groups (C10-C14). The 3D structural model of LipC24 revealed the open Y-type active site. LipA displayed 96 % amino acid sequence identity with the known extracellular lipase. lipA-inactivation and lipC24-inactivation decreased the total cell-bound lipase activity of Burkholderia sp. ZYB002 by 42 % and 14 %, respectively. The cell-bound lipase activity from Burkholderia sp. ZYB002 originated from a multi-enzyme mixture with LipA as the main component. LipC24 was a novel lipase and displayed different enzymatic characteristics and structural model with LipA. Besides LipA and LipC24, other type of the cell-bound lipases (or esterases) should exist.

Preparation of Chiral Triacylglycerols, sn-POO and sn-OOP, via Lipase-mediated Acidolysis Reaction.

PubMed

Yamamoto, Yukihiro; Yoshida, Hiroki; Nagai, Toshiharu; Hara, Setsuko

2018-02-01

It is well known that lipases are useful tools for preparing various structured triacylglycerols (TAGs). However, the lipase-mediated preparation of chiral TAGs has never been reported. This study aimed to prepare chiral TAGs (viz., 1-palmitoyl-2,3-dioleoyl-sn-glycerol (sn-POO) or 1,2-dioleoyl-3-palmitoyl-sn-glycerol (sn-OOP)) via lipase mediated acidolysis, using triolein (TO) and palmitic acid (P) as substrates. Three commercially available lipases (viz., Lipozyme RM-IM ® , Lipozyme TL-IM ® , and Lipase OF ® ) were used. Lipozyme RM-IM ® resulted in an increase 1P-2O (sn-POO + sn-OOP + 1,3-dioleoyl-2-palmitoyl-sn-glycerol) content with reaction time, which plateaued at 2~24 h (max. yield 47.1% at 4 h). The highest sn-POO/sn-OOP ratio of ca. 9 was obtained at 0.25 h, and the rate got close to 1 with reaction time (sn-POO/sn-OOP = 1.3 at 24 h). Lipozyme TL-IM ® resulted in a lower 1P-2O synthesis rate than Lipozyme RM-IM ® , where its highest sn-POO/sn-OOP ratio of ca. 2 was obtained at 0.25 h and did not vary much further with reaction time. In the case of Lipase OF ® , its reaction rate for 1P-2O synthesis was lower than that of the other two lipases, and the highest sn-POO/sn-OOP ratio of ca. 1.4 was obtained at 0.5 h, reaching closer to 1 with a longer reaction time. Reaction solvents (viz., hexane, acetone, and benzene) also affected the 1P-2O preparation, where the highest 1P-2O content was obtained with the solvent-free system. Furthermore, the solvent-free system showed a higher reaction rate for 1P-2O synthesis than did the hexane system, with no effect on chiral specificity of the lipase for the TAG molecules. These results suggested that among three types of commercial lipase, Lipozyme RM-IM ® is the most useful for the preparation of chiral TAGs by acidolysis reaction.

Medicinal Plants and Their Inhibitory Activities against Pancreatic Lipase: A Review

PubMed Central

Seyedan, Atefehalsadat; Alshawsh, Mohammed Abdullah; Alshagga, Mustafa Ahmed; Koosha, Sanaz

2015-01-01

Obesity is recognized as a major life style disorder especially in developing countries and it is prevailing at an alarming speed in new world countries due to fast food intake, industrialization, and reduction of physical activity. Furthermore, it is associated with a vast number of chronic diseases and disabilities. To date, relatively effective drugs, from either natural or synthetic sources, are generally associated with serious side effects, often leading to cessation of clinical trials or even withdrawal from the market. In order to find new compounds which are more effective or with less adverse effects compared to orlistat, the drug that has been approved for obesity, new compounds isolated from natural products are being identified and screened for antiobesity effects, in particular, for their pancreatic lipase inhibitory effect. Pancreatic lipase inhibitory activity has been extensively used for the determination of potential efficacy of natural products as antiobesity agents. In attempts to identify natural products for overcoming obesity, more researches have been focused on the identification of newer pancreatic lipase inhibitors with less unpleasant adverse effects. In this review, we consider the potential role of plants that have been investigated for their pancreatic lipase inhibitory activity. PMID:26640503

Immobilization of lipase and keratinase on functionalized SBA-15 nanostructured materials

NASA Astrophysics Data System (ADS)

Le, Hy G.; Vu, Tuan A.; Tran, Hoa T. K.; Dang, Phuong T.

2013-12-01

SBA-15 nanostructured materials were synthesized via hydrothermal treatment and were functionalized with 3- aminopropyltriethoxysilane (APTES). The obtained samples were characterized by different techniques such as XRD, BET, TEM, IR and DTA. After functionalization, it showed that these nanostrucrured materials still maintained the hexagonal pore structure of the parent SBA-15. The model enzyms chosen in this study were lipase and keratinase. Lipase was a biocatalyst for hydrolyzation of long chain triglycerides or methyl esters of long chain alcohols and fatty acids; keratinase is a proteolytic enzyme that catalyzes the cleavage of keratin. The functionalized SBA-15 materials were used to immobilize lipase and keratinase, exhibiting higher activity than that of the unfunctionalized pure silica SBA-15 ones. This might be due to the enhancing of surface hydrophobicity upon functionalization. The surface functionalization of the nanostructured silicas with organic groups can favor the interaction between enzyme and the supports and consequently increasing the operational stability of the immobilized enzymes. The loading of lipase on functionalized SBA-15 materials was higher than that of keratinase. This might be rationalized by the difference in size of enzyms.

Screening Brazilian Macrophomina phaseolina isolates for alkaline lipases and other extracellular hydrolases.

PubMed

Schinke, Claudia; Germani, José C

2012-03-01

Macrophomina phaseolina, phylum Ascomycota, is a phytopathogenic fungus distributed worldwide in hot dry areas. There are few studies on its secreted lipases and none on its colony radial growth rate, an indicator of fungal ability to use nutrients for growth, on media other than potato-dextrose agar. In this study, 13 M. phaseolina isolates collected in different Brazilian regions were screened for fast-growth and the production of hydrolases of industrial interest, especially alkaline lipases. Hydrolase detection and growth rate determination were done on citric pectin, gelatin, casein, soluble starch, and olive oil as substrates. Ten isolates were found to be active on all substrates tested. The most commonly detected enzymes were pectinases, amylases, and lipases. The growth rate on pectin was significantly higher (P < 0.05), while the growth rates on the different media identified CMM 2105, CMM 1091, and PEL as the fastest-growing isolates. The lipase activity of four isolates grown on olive oil was followed for 4 days by measuring the activity in the cultivation broth. The specific lipolytic activity of isolate PEL was significantly higher at 96 h (130 mU mg protein(-1)). The broth was active at 37 °C, pH 8, indicating the potential utility of the lipases of this isolate in mild alkaline detergents. There was a strong and positive correlation (0.86) between radial growth rate and specific lipolytic activity.

Isolation and characterization of novel lipases/esterases from a bovine rumen metagenome.

PubMed

Privé, Florence; Newbold, C Jamie; Kaderbhai, Naheed N; Girdwood, Susan G; Golyshina, Olga V; Golyshin, Peter N; Scollan, Nigel D; Huws, Sharon A

2015-07-01

Improving the health beneficial fatty acid content of meat and milk is a major challenge requiring an increased understanding of rumen lipid metabolism. In this study, we isolated and characterized rumen bacterial lipases/esterases using functional metagenomics. Metagenomic libraries were constructed from DNA extracted from strained rumen fluid (SRF), solid-attached bacteria (SAB) and liquid-associated rumen bacteria (LAB), ligated into a fosmid vector and subsequently transformed into an Escherichia coli host. Fosmid libraries consisted of 7,744; 8,448; and 7,680 clones with an average insert size of 30 to 35 kbp for SRF, SAB and LAB, respectively. Transformants were screened on spirit blue agar plates containing tributyrin for lipase/esterase activity. Five SAB and four LAB clones exhibited lipolytic activity, and no positive clones were found in the SRF library. Fosmids from positive clones were pyrosequenced and twelve putative lipase/esterase genes and two phospholipase genes retrieved. Although the derived proteins clustered into diverse esterase and lipase families, a degree of novelty was seen, with homology ranging from 40 to 78% following BlastP searches. Isolated lipases/esterases exhibited activity against mostly short- to medium-chain substrates across a range of temperatures and pH. The function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses.

Electrospun polylactic acid and polyvinyl alcohol fibers as efficient and stable nanomaterials for immobilization of lipases.

PubMed

Sóti, Péter Lajos; Weiser, Diana; Vigh, Tamás; Nagy, Zsombor Kristóf; Poppe, László; Marosi, György

2016-03-01

Electrospinning was applied to create easy-to-handle and high-surface-area membranes from continuous nanofibers of polyvinyl alcohol (PVA) or polylactic acid (PLA). Lipase PS from Burkholderia cepacia and Lipase B from Candida antarctica (CaLB) could be immobilized effectively by adsorption onto the fibrous material as well as by entrapment within the electrospun nanofibers. The biocatalytic performance of the resulting membrane biocatalysts was evaluated in the kinetic resolution of racemic 1-phenylethanol (rac-1) and 1-phenylethyl acetate (rac-2). Fine dispersion of the enzymes in the polymer matrix and large surface area of the nanofibers resulted in an enormous increase in the activity of the membrane biocatalyst compared to the non-immobilized crude powder forms of the lipases. PLA as fiber-forming polymer for lipase immobilization performed better than PVA in all aspects. Recycling studies with the various forms of electrospun membrane biocatalysts in ten cycles of the acylation and hydrolysis reactions indicated excellent stability of this forms of immobilized lipases. PLA-entrapped lipases could preserve lipase activity and enantiomer selectivity much better than the PVA-entrapped forms. The electrospun membrane forms of CaLB showed high mechanical stability in the repeated acylations and hydrolyses than commercial forms of CaLB immobilized on polyacrylamide beads (Novozyme 435 and IMMCALB-T2-150).

A broad pH range indicator-based spectrophotometric assay for true lipases using tributyrin and tricaprylin.

PubMed

Camacho-Ruiz, María de Los Angeles; Mateos-Díaz, Juan Carlos; Carrière, Frédéric; Rodriguez, Jorge A

2015-05-01

A continuous assay is proposed for the screening of acidic, neutral, or alkaline lipases using microtiter plates, emulsified short- and medium-chain TGs, and a pH indicator. The lipase activity measurement is based on the decrease of the pH indicator optical density due to protonation which is caused by the release of FFAs during the hydrolysis of TGs and thus acidification. Purified lipases with distinct pH optima and an esterase were used to validate the method. The rate of lipolysis was found to be linear with time and proportional to the amount of enzyme added in each case. Specific activities measured with this microplate assay method were lower than those obtained by the pH-stat technique. Nevertheless, the pH-dependent profiles of enzymatic activity were similar with both assays. In addition, the substrate preference of each enzyme tested was not modified and this allowed discriminating lipase and esterase activities using tributyrin (low water solubility) and tricaprylin (not water soluble) as substrates. This continuous lipase assay is compatible with a high sample throughput and can be applied for the screening of lipases and lipase inhibitors from biological samples. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

Effect of metal ions on the hydrolytic and transesterification activities of Candida rugosa lipase.

PubMed

Katiyar, Madhu; Ali, Amjad

2013-01-01

In order to study the effect of metal ions on lipase activity, hydrolytic and transesterification activities of Candida rugosa lipase were investigated in presence of alkali (Na⁺ and K⁺), alkaline earth (Ca⁺² and Ba⁺²) and transition (Cr⁺³, Fe⁺³, Co⁺², Cu⁺² and Ni⁺²) metal ions. Maximum enhancement in hydrolytic activity of lipase was observed by Ca⁺², and in transesterification activity by Cr⁺³ and Co⁺². The kinetics of the lipase catalyzed transesterification (methanolysis and ethanolysis) reactions were also studied, and the activation energies of methanolysis and ethanolysis were reduced from 10.16 and 10.24 kcal mol⁻¹, respectively, to 5.41 and 7.55 kcal mol⁻¹, respectively, when reactions were performed in presence of Co⁺². Thus, in lipase catalyzed transesterification Cr⁺³ or Co⁺² could be added to the assay in order to produce the biodiesel in relatively shorter reaction duration.

Purification and biochemical characterization of a cold-active lipase from Antarctic sea ice bacteria Pseudoalteromonas sp. NJ 70.

PubMed

Wang, Quanfu; Hou, Yanhua; Ding, Yu; Yan, Peisheng

2012-09-01

An extracellular cold-active lipase from Antarctic sea ice bacteria Pseudoalteromonas sp. NJ 70 was purified and characterized. The overall purification based on lipase activity was 27.5-fold with a yield of 25.4 %. The purified lipase showed as a single band on SDS-PAGE with an apparent molecular weight of 37 kDa. The optimum temperature and pH were 35 °C and 7.0, respectively. The lipase activity was enhanced by Ca(2+) and Mg(2+), while was partially inhibited by other metals such as Cu(2+), Zn(2+), Ba(2+), Pb(2+), Fe(2+) and Mn(2+). The lipase had high tolerance to a wide range of NaCl concentrations (0-2 M NaCl). It exhibited high levels of activity in the presence of DTT, Thiourea, H(2)O(2) as well as in the presence of various detergents such as Span 60, Tween-80, Triton X-100. In addition, the lipase showed a preference for long-chain p-nitrophenyl esters (C(12)-C(18)). These results indicated that this lipase could be a novel cold-active lipase.

Inhibitors of pancreatic lipase: state of the art and clinical perspectives

PubMed Central

Lunagariya, Nitin A.; Patel, Neeraj K.; Jagtap, Sneha C.; Bhutani, Kamlesh K.

2014-01-01

Obesity is a disorder of lipid metabolism and continues to be a global problem, ranking fifth for deaths worldwide. It also leads to diabetes, cardiovascular disorders, musculoskeletal disorders and some types of cancer. Obesity is regarded as the output of a long-term imbalance between energy intake and energy expenditure. Digestion and absorption of dietary lipids by pancreatic lipase, a major source of excess calorie intake, can be targeted for development of anti-obesity agents. Being the major factor of concern, food materials and edible plants are most widely studied for the anti-obesity activity, so that they can be incorporated in the routine diet. In this review, an attempt was made to present a current scenario of the bioactive compounds from plant and microbial origin that have been investigated for their pancreatic lipase inhibition. Compounds belonging to various classes of natural products such as alkaloids, carotenoids, glycosides, polyphenols, polysaccharides, saponins and terpenoids are well studied while lipophilic compounds from microbial sources are the most active against the pancreatic lipase. Few studies on the synthetic analogues, structurally similar to the triglycerides have been described in the review. Despite of tremendous research on the finding of potential pancreatic lipase inhibitor, very few compounds have entered the clinical studies and no new molecule after orlistat has been marketed. Along with HTS based screening, detailed structure-activity relationship studies on semi-synthetic and synthetic derivatives might also provide a direction for the development of potential lead(s) or pharmacophore for pancreatic lipase inhibition in order to treat and/or prevent obesity and related disorders. PMID:26417311

Engineering of baker's yeasts, E. coli and Bacillus hosts for the production of Bacillus subtilis Lipase A.

PubMed

Sánchez, Marta; Prim, Núria; Rández-Gil, Francisca; Pastor, F I Javier; Diaz, Pilar

2002-05-05

Lipases are versatile biocatalists showing multiple applications in a wide range of biotechnological processes. The gene lipA coding for Lipase A from Bacillus subtilis was isolated by PCR amplification, cloned and expressed in Escherichia coli, Saccharomyces cerevisiae and Bacillus subtilis strains, using pBR322, YEplac112 and pUB110-derived vectors, respectively. Lipase activity analysis of the recombinant strains showed that the gene can be properly expressed in all hosts assayed, this being the first time a lipase from bacterial origin can be expressed in baker's S. cerevisiae strains. An important increase of lipase production was obtained in heterologous hosts with respect to that of parental strains, indicating that the described systems can represent a useful tool to enhance productivity of the enzyme for biotechnological applications, including the use of the lipase in bread making, or as a technological additive. Copyright 2002 Wiley Periodicals, Inc.

Production of Thermoalkaliphilic Lipase from Geobacillus thermoleovorans DA2 and Application in Leather Industry

PubMed Central

Abol Fotouh, Deyaa M.; Bayoumi, Reda A.; Hassan, Mohamed A.

2016-01-01

Thermophilic and alkaliphilic lipases are meeting a growing global attention as their increased importance in several industrial fields. Over 23 bacterial strains, novel strain with high lipolytic activity was isolated from Southern Sinai, Egypt, and it was identified as Geobacillus thermoleovorans DA2 using 16S rRNA as well as morphological and biochemical features. The lipase was produced in presence of fatty restaurant wastes as an inducing substrate. The optimized conditions for lipase production were recorded to be temperature 60°C, pH 10, and incubation time for 48 hrs. Enzymatic production increased when the organism was grown in a medium containing galactose as carbon source and ammonium phosphate as nitrogen source at concentrations of 1 and 0.5% (w/v), respectively. Moreover, the optimum conditions for lipase production such as substrate concentration, inoculum size, and agitation rate were found to be 10% (w/v), 4% (v/v), and 120 rpm, respectively. The TA lipase with Triton X-100 had the best degreasing agent by lowering the total lipid content to 2.6% as compared to kerosene (7.5%) or the sole crude enzyme (8.9%). It can be concluded that the chemical leather process can be substituted with TA lipase for boosting the quality of leather and reducing the environmental hazards. PMID:26881066

Production of Thermoalkaliphilic Lipase from Geobacillus thermoleovorans DA2 and Application in Leather Industry.

PubMed

Abol Fotouh, Deyaa M; Bayoumi, Reda A; Hassan, Mohamed A

2016-01-01

Thermophilic and alkaliphilic lipases are meeting a growing global attention as their increased importance in several industrial fields. Over 23 bacterial strains, novel strain with high lipolytic activity was isolated from Southern Sinai, Egypt, and it was identified as Geobacillus thermoleovorans DA2 using 16S rRNA as well as morphological and biochemical features. The lipase was produced in presence of fatty restaurant wastes as an inducing substrate. The optimized conditions for lipase production were recorded to be temperature 60°C, pH 10, and incubation time for 48 hrs. Enzymatic production increased when the organism was grown in a medium containing galactose as carbon source and ammonium phosphate as nitrogen source at concentrations of 1 and 0.5% (w/v), respectively. Moreover, the optimum conditions for lipase production such as substrate concentration, inoculum size, and agitation rate were found to be 10% (w/v), 4% (v/v), and 120 rpm, respectively. The TA lipase with Triton X-100 had the best degreasing agent by lowering the total lipid content to 2.6% as compared to kerosene (7.5%) or the sole crude enzyme (8.9%). It can be concluded that the chemical leather process can be substituted with TA lipase for boosting the quality of leather and reducing the environmental hazards.

Pseudomonas sp. BUP6 produces a thermotolerant alkaline lipase with trans-esterification efficiency in producing biodiesel.

PubMed

Priji, Prakasan; Sajith, Sreedharan; Faisal, Panichikkal Abdul; Benjamin, Sailas

2017-12-01

The present study describes the characteristics of a thermotolerant and alkaline lipase secreted by Pseudomonas sp. BUP6, a novel rumen bacterium isolated from Malabari goat, and its trans -esterification efficiency in producing biodiesel from used cooking oil (UCO). The extracellular lipase was purified to homogeneity (35.8 times purified with 14.8% yield) employing (NH 4 ) 2 SO 4 salt precipitation and Sephadex G-100 chromatography. The apparent molecular weight of this lipase on SDS-PAGE was 35 kDa, the identity of which was further confirmed by MALDI-TOF/MS. The purified lipase was found stable at a pH range of 7-9 with the maximum activity (707 U/ml) at pH 8.2; and was active at the temperature ranging from 35 to 50 °C with the optimum at 45 °C (891 U/ml). Triton X-100 and EDTA had no effect on the activity of lipase; whereas SDS, Tween-80 and β-mercaptoethanol inhibited its activity significantly. Moreover, Ca 2+ (1.0 mM) enhanced the activity of lipase (1428 U/ml) by 206% vis-à-vis initial activity; while Zn 2+ , Fe 2+ and Cu 2+ decreased the activity significantly. Using para -nitrophenyl palmitate as substrate, the K m (11.6 mM) and V max [668.9 μmol/(min/mg)] of the purified lipase were also determined. Crude lipase was used for analyzing its trans -esterification efficiency with used cooking oil and methanol which resulted in the worthy yield of fatty acid methyl esters, FAME (45%) at 37 °C, indicating its prospects in biodiesel industry. Thus, the lipase secreted by the rumen bacterium, Pseudomonas sp. BUP6, offers great potentials to be used in various industries including the production of biodiesel by trans -esterification.

Cloning and seasonal secretion of the pancreatic lipase-related protein 2 present in goat seminal plasma.

PubMed

Sias, Barbara; Ferrato, Francine; Pellicer-Rubio, Maria-Teresa; Forgerit, Yvonick; Guillouet, Philippe; Leboeuf, Bernard; Carrière, Frédéric

2005-01-05

The storage of frozen semen for artificial insemination is usually performed in the presence of egg yolk or skimmed milk as protective agents. In goats, the use of skimmed milk extenders requires, however, that most of the seminal plasma is removed before dilution of spermatozoa because it is deleterious for their survival. It has been previously demonstrated that a lipase (BUSgp60) secreted by the accessory bulbourethral gland was responsible for the cellular death of goat spermatozoa, through the lipolysis of residual milk lipids and the release of toxic free fatty acids. This lipase was purified from the whole seminal plasma of goat and was found to display both lipase and phospholipase A activities, this latter activity representing the main phospholipase activity detected in goat seminal plasma. Based on its N-terminal amino acid sequence, identical to that of BUSgP60 purified from bulbourethral gland secretion, and the design of degenerated oligonucleotides, the lipase was cloned from total mRNA isolated from bulbourethral gland. DNA sequencing confirmed it was the goat pancreatic-lipase-related protein 2 (GoPLRP2). The physiological role of GoPLRP2 is still unknown but this enzyme might be associated with the reproductive activity of goats. A significant increase in lipase secretion was observed every year in August and the level of lipase activity in the semen remained high till December, i.e., during the breeding season. A parallel increase in the plasmatic levels of testosterone suggested that GoPLRP2 expression might be regulated by sexual hormones. The lipase activity level measured in goat seminal plasma, which could reach 1000 U/ml during the breeding season, was one of the highest lipase activity measured in natural sources, including gastric and pancreatic juices.

Kinetic properties of dromedary pancreatic lipase: a comparative study on emulsified and monomolecular substrate.

PubMed

Jemel, Ikram; Fendri, Ahmed; Gargouri, Youssef; Bezzine, Sofiane

2009-05-01

Using the classical emulsified system and the monomolecular film technique, we compared several interfacial properties of dromedary pancreatic lipase (DrPL) with those of a mammal (human) and an avian (turkey) model. Like turkey pancreatic lipase (TPL) and unlike human pancreatic lipase (HPL), in the absence of colipase and bile salts, using tributyrin emulsion or monomolecular films of dicaprin at low surface pressure, DrPL hydrolyses pure tributyrin emulsion, as well as dicaprin films maintained at low surface pressures. DrPL was also able to hydrolyse triolein emulsion in the absence of any additive and despite the accumulation of long-chain free fatty acids at the interface. The difference of behaviours between the two mammal pancreatic lipases (DrPL and HPL) can be explained by the penetration capacity of each enzyme. DrPL presents a critical surface pressure value (21 m Nm(-1)) that is more important than this of HPL. Subsequently, the dromedary pancreatic lipase interacts efficiently with interfaces and it is not denaturated at high interfacial energy. A kinetic study on the surface pressure dependency, stereospecificity and regioselectivity of DrPL was performed using optically pure stereoisomers of either three dicaprin isomers containing a single hydrolysable decanoyl ester bond that were spread as monomolecular films at the air/water interface. Interestingly, in comparison with all the previously studied mammal pancreatic lipases, DrPL presents the highest preference for adjacent ester groups of dicaprin isomers (1,2-sn-dicaprin and 2,3-sn-dicaprin) at high surface pressure. Furthermore, DrPL forms a pancreatic lipase subgroup in which the stereopreference switches from sn-3 position to the sn-1 position when increasing the surface pressure.

A simplified method for active-site titration of lipases immobilised on hydrophobic supports.

PubMed

Nalder, Tim D; Kurtovic, Ivan; Barrow, Colin J; Marshall, Susan N

2018-06-01

The aim of this work was to develop a simple and accurate protocol to measure the functional active site concentration of lipases immobilised on highly hydrophobic supports. We used the potent lipase inhibitor methyl 4-methylumbelliferyl hexylphosphonate to titrate the active sites of Candida rugosa lipase (CrL) bound to three highly hydrophobic supports: octadecyl methacrylate (C18), divinylbenzene crosslinked methacrylate (DVB) and styrene. The method uses correction curves to take into account the binding of the fluorophore (4-methylumbelliferone, 4-MU) by the support materials. We showed that the uptake of the detection agent by the three supports is not linear relative to the weight of the resin, and that the uptake occurs in an equilibrium that is independent of the total fluorophore concentration. Furthermore, the percentage of bound fluorophore varied among the supports, with 50 mg of C18 and styrene resins binding approximately 64 and 94%, respectively. When the uptake of 4-MU was calculated and corrected for, the total 4-MU released via inhibition (i.e. the concentration of functional lipase active sites) could be determined via a linear relationship between immobilised lipase weight and total inhibition. It was found that the functional active site concentration of immobilised CrL varied greatly among different hydrophobic supports, with 56% for C18, compared with 14% for DVB. The described method is a simple and robust approach to measuring functional active site concentration in immobilised lipase samples. Copyright © 2018 Elsevier Inc. All rights reserved.

Adipose Triglyceride Lipase Is Implicated in Fuel- and Non-fuel-stimulated Insulin Secretion*

PubMed Central

Peyot, Marie-Line; Guay, Claudiane; Latour, Martin G.; Lamontagne, Julien; Lussier, Roxane; Pineda, Marco; Ruderman, Neil B.; Haemmerle, Guenter; Zechner, Rudolf; Joly, Érik; Madiraju, S. R. Murthy; Poitout, Vincent; Prentki, Marc

2009-01-01

Reduced lipolysis in hormone-sensitive lipase-deficient mice is associated with impaired glucose-stimulated insulin secretion (GSIS), suggesting that endogenous β-cell lipid stores provide signaling molecules for insulin release. Measurements of lipolysis and triglyceride (TG) lipase activity in islets from HSL−/− mice indicated the presence of other TG lipase(s) in the β-cell. Using real time-quantitative PCR, adipose triglyceride lipase (ATGL) was found to be the most abundant TG lipase in rat islets and INS832/13 cells. To assess its role in insulin secretion, ATGL expression was decreased in INS832/13 cells (ATGL-knockdown (KD)) by small hairpin RNA. ATGL-KD increased the esterification of free fatty acid (FFA) into TG. ATGL-KD cells showed decreased glucose- or Gln + Leu-induced insulin release, as well as reduced response to KCl or palmitate at high, but not low, glucose. The KATP-independent/amplification pathway of GSIS was considerably reduced in ATGL-KD cells. ATGL−/− mice were hypoinsulinemic and hypoglycemic and showed decreased plasma TG and FFAs. A hyperglycemic clamp revealed increased insulin sensitivity and decreased GSIS and arginine-induced insulin secretion in ATGL−/− mice. Accordingly, isolated islets from ATGL−/− mice showed reduced insulin secretion in response to glucose, glucose + palmitate, and KCl. Islet TG content and FFA esterification into TG were increased by 2-fold in ATGL−/− islets, but glucose usage and oxidation were unaltered. The results demonstrate the importance of ATGL and intracellular lipid signaling for fuel- and non-fuel-induced insulin secretion. PMID:19389712

Pancreatic lipase inhibitory activity of taraxacum officinale in vitro and in vivo

PubMed Central

Zhang, Jian; Kang, Min-Jung; Kim, Myung-Jin; Kim, Mi-Eun; Song, Ji-Hyun; Lee, Young-Min

2008-01-01

Obesity has become a worldwide health problem. Orlistat, an inhibitor of pancreatic lipase, is currently approved as an anti-obesity drug. However, gastrointestinal side effects caused by Orlistat may limit its use. In this study the inhibitory activities of dandelion (Taraxacum officinale) against pancreatic lipase in vitro and in vivo were measured to determine its possible use as a natural anti-obesity agent. The inhibitory activities of the 95% ethanol extract of T. officinale and Orlistat were measured using 4-methylumbelliferyl oleate (4-MU oleate) as a substrate at concentrations of 250, 125, 100, 25, 12.5 and 4 µg/ml. To determine pancreatic lipase inhibitory activity in vivo, mice (n=16) were orally administered with corn oil emulsion (5 ml/kg) alone or with the 95% ethanol extract of T. officinale (400 mg/kg) following an overnight fast. Plasma triglyceride levels were measured at 0, 90, 180, and 240 min after treatment and incremental areas under the response curves (AUC) were calculated. The 95% ethanol extract of T. officinale and Orlistat, inhibited, porcine pancreatic lipase activity by 86.3% and 95.7% at a concentration of 250 µg/ml, respectively. T. officinale extract showed dose-dependent inhibition with the IC50 of 78.2 µg/ml. A single oral dose of the extract significantly inhibited increases in plasma triglyceride levels at 90 and 180 min and reduced AUC of plasma triglyceride response curve (p<0.05). The results indicate that T. officinale exhibits inhibitory activities against pancreatic lipase in vitro and in vivo. Further studies to elucidate anti-obesity effects of chronic consumption of T. officinale and to identify the active components responsible for inhibitory activity against pancreatic lipase are necessary. PMID:20016719

Genetics Home Reference: lysosomal acid lipase deficiency

MedlinePlus

... Cegielska J, Whitley CB, Eckert S, Valayannopoulos V, Quinn AG. Clinical Features of Lysosomal Acid Lipase Deficiency. J ... qualified healthcare professional . About Selection Criteria for Links Data Files & API Site Map Subscribe Customer Support USA. ...

Synthesis of retinyl palmitate catalyzed by Candida sp.99-125 lipase immobilized on fiber-like SBA-15 mesoporous material.

PubMed

Kai, Zhu; Jianqiang, Wang; Yan-hua, Wang; Hui, Liu; Ping-fang, Han; Ping, Wei

2011-09-01

Candida sp.99-125 lipase was suitable for transesterification of fats and oils to produce fatty acid methyl ester. The adsorption of Candida sp.99-125 lipase onto the fiber-like SBA-15 mesoporous material has been studied. The unaltered structural order of the fiber-like SBA-15 before and after the adsorption has been confirmed by FT-IR, SEM and N2 adsorption. The amount of adsorbed Candida sp.99-125 lipase depends both on the solution pH and reaction time. Good adsorption capacity of Candida sp.99-125 lipase on fiber-like SBA-15 may be due to solution pH from 5.0 to 9.0 especially at 7.0 (93.99 mg enzyme per gram silica is obtained and the activity recovery is 281.05%). A high lipase loading (135.9 mg enzyme per gram silica) was obtained, but it did not produce a proportionate level of catalytic activity. The immobilized Candida sp.99-125 lipase showed increased adaptability in the hydrolysis of p-nitrophenyl acetate compared to free Candida sp.99-125 lipase at pH 5.0-9.0. Meanwhile, the immobilized Candida sp.99-125 lipase showed higher thermal stability than that of free Candida sp.99-125 lipase. And the synthesis of retinyl palmitate in organic solvent with the immobilized Candida sp.99-125 lipase was investigated. The influence factors, such as: the solvent used, the molar ratio and concentrations of substrates, the reaction time and the amount of lipase were studied and optimized. In the conditions of transesterificating 0.164 g retinyl acetate and 0.32 g palmitic acid, 10 mL of solvent hexane, 1:4 of mass ratio of lipase to retinyl acetate, and 6 hours of reaction time, 74.6% of retinyl acetate was converted into retinyl plamitate.

A broad pH range indicator-based spectrophotometric assay for true lipases using tributyrin and tricaprylin[S

PubMed Central

Camacho-Ruiz, María de los Angeles; Mateos-Díaz, Juan Carlos; Carrière, Frédéric; Rodriguez, Jorge A.

2015-01-01

A continuous assay is proposed for the screening of acidic, neutral, or alkaline lipases using microtiter plates, emulsified short- and medium-chain TGs, and a pH indicator. The lipase activity measurement is based on the decrease of the pH indicator optical density due to protonation which is caused by the release of FFAs during the hydrolysis of TGs and thus acidification. Purified lipases with distinct pH optima and an esterase were used to validate the method. The rate of lipolysis was found to be linear with time and proportional to the amount of enzyme added in each case. Specific activities measured with this microplate assay method were lower than those obtained by the pH-stat technique. Nevertheless, the pH-dependent profiles of enzymatic activity were similar with both assays. In addition, the substrate preference of each enzyme tested was not modified and this allowed discriminating lipase and esterase activities using tributyrin (low water solubility) and tricaprylin (not water soluble) as substrates. This continuous lipase assay is compatible with a high sample throughput and can be applied for the screening of lipases and lipase inhibitors from biological samples. PMID:25748441

Screening for hydrolytic enzymes reveals Ayr1p as a novel triacylglycerol lipase in Saccharomyces cerevisiae.

PubMed

Ploier, Birgit; Scharwey, Melanie; Koch, Barbara; Schmidt, Claudia; Schatte, Jessica; Rechberger, Gerald; Kollroser, Manfred; Hermetter, Albin; Daum, Günther

2013-12-13

Saccharomyces cerevisiae, as well as other eukaryotes, preserves fatty acids and sterols in a biologically inert form, as triacylglycerols and steryl esters. The major triacylglycerol lipases of the yeast S. cerevisiae identified so far are Tgl3p, Tgl4p, and Tgl5p (Athenstaedt, K., and Daum, G. (2003) YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae. J. Biol. Chem. 278, 23317-23323; Athenstaedt, K., and Daum, G. (2005) Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae, are localized to lipid particles. J. Biol. Chem. 280, 37301-37309). We observed that upon cultivation on oleic acid, triacylglycerol mobilization did not come to a halt in a yeast strain deficient in all currently known triacylglycerol lipases, indicating the presence of additional not yet characterized lipases/esterases. Functional proteome analysis using lipase and esterase inhibitors revealed a subset of candidate genes for yet unknown hydrolytic enzymes on peroxisomes and lipid droplets. Based on the conserved GXSXG lipase motif, putative functions, and subcellular localizations, a selected number of candidates were characterized by enzyme assays in vitro, gene expression analysis, non-polar lipid analysis, and in vivo triacylglycerol mobilization assays. These investigations led to the identification of Ayr1p as a novel triacylglycerol lipase of yeast lipid droplets and confirmed the hydrolytic potential of the peroxisomal Lpx1p in vivo. Based on these results, we discuss a possible link between lipid storage, lipid mobilization, and peroxisomal utilization of fatty acids as a carbon source.

Lipase immobilized on the hydrophobic polytetrafluoroethene membrane with nonwoven fabric and its application in intensifying synthesis of butyl oleate.

PubMed

Wang, Shu-Guang; Zhang, Wei-Dong; Li, Zheng; Ren, Zhong-Qi; Liu, Hong-Xia

2010-11-01

The synthesis of butyl oleate was studied in this paper with immobilized lipase. Five types of membrane were used as support to immobilize Rhizopus arrhizus lipase by following a procedure combining filtration and protein cross-linking. Results showed that hydrophobic polytetrafluoroethene membrane with nonwoven fabric (HO-PTFE-NF) was the favorite choice in terms of higher protein loading, activity, and specific activity of immobilized lipase. The factors including solvent polarity, lipase dosage, concentration, and molar ratio of substrate and temperature were found to have significant influence on conversion. Results showed that hexane (logP = 3.53) was a favorable solvent for the biosynthesis of butyl oleate in our studies. The optimal conditions were experimentally determined of 50 U immobilized lipase, molar ratio of oleic acid to butanol of 1.0, substrate concentration of 0.12 mol/L, temperature of 37 °C, and reaction time of 2 h. The conversion was beyond 91% and decreased slightly after 18 cycles. Lipase immobilization can improve the conversion and the repeated use of immobilized lipase relative to free lipase.

Production and Characterization of Lipases by Two New Isolates of Aspergillus through Solid-State and Submerged Fermentation

PubMed Central

Colla, Luciane Maria; Ficanha, Aline M. M.; Rizzardi, Juliana; Bertolin, Telma Elita; Reinehr, Christian Oliveira; Costa, Jorge Alberto Vieira

2015-01-01

Due to the numerous applications of lipases in industry, there is a need to study their characteristics, because lipases obtained from different sources may present different properties. The aim of this work was to accomplish the partial characterization of lipases obtained through submerged fermentation and solid-state fermentation by two species of Aspergillus. Fungal strains were isolated from a diesel-contaminated soil and selected as good lipases producers. Lipases obtained through submerged fermentation presented optimal activities at 37°C and pH 7.2 and those obtained through solid-state fermentation at 35°C and pH 6.0. The enzymes produced by submerged fermentation were more temperature-stable than those obtained by solid-state fermentation, presenting 72% of residual activity after one hour of exposition at 90°C. Lipases obtained through submerged fermentation had 80% of stability in acidic pH and those obtained through solid-state fermentation had stability greater than 60% in alkaline pH. PMID:26180809

Regioselective alcoholysis of silychristin acetates catalyzed by lipases.

PubMed

Vavříková, Eva; Gavezzotti, Paolo; Purchartová, Kateřina; Fuksová, Kateřina; Biedermann, David; Kuzma, Marek; Riva, Sergio; Křen, Vladimír

2015-05-26

A panel of lipases was screened for the selective acetylation and alcoholysis of silychristin and silychristin peracetate, respectively. Acetylation at primary alcoholic group (C-22) of silychristin was accomplished by lipase PS (Pseudomonas cepacia) immobilized on diatomite using vinyl acetate as an acetyl donor, whereas selective deacetylation of 22-O-acetyl silychristin was accomplished by Novozym 435 in methyl tert-butyl ether/ n-butanol. Both of these reactions occurred without diastereomeric discrimination of silychristin A and B. Both of these enzymes were found to be capable to regioselective deacetylation of hexaacetyl silychristin to afford penta-, tetra- and tri-acetyl derivatives, which could be obtained as pure synthons for further selective modifications of the parent molecule.

Cell-Bound Lipase and Esterase of Brevibacterium linens

PubMed Central

Sørhaug, Terje; Ordal, Z. John

1974-01-01

The activities of glycerol ester hydrolase, lipase (EC 3.1.1.3) and carboxylesterase, and esterase (EC 3.1.1.1) were determined for whole cell preparations of Brevibacterium linens by using the pH-stat assay. The culture growth liquors were inactive against the three substrates, tributyrin emulsion, triacetin, and methyl butyrate. Cells washed in water had less activity than cells washed in 5% NaCl; the ratio of activities was close to 1:2 for all strains using tributyrin emulsion as the substrate. For the esterase substrates, this relationship varied widely and was strain dependent. The ability to hydrolyze the two esterase substrates varied independently of the level of lipase activity. PMID:4824883

Application of Plackett-Burman Experimental Design for Lipase Production by Aspergillus niger Using Shea Butter Cake

PubMed Central

Salihu, Aliyu; Bala, Muntari; Bala, Shuaibu M.

2013-01-01

Plackett-Burman design was used to efficiently select important medium components affecting the lipase production by Aspergillus niger using shea butter cake as the main substrate. Out of the eleven medium components screened, six comprising of sucrose, (NH4)2SO4, Na2HPO4, MgSO4, Tween-80, and olive oil were found to contribute positively to the overall lipase production with a maximum production of 3.35 U/g. Influence of tween-80 on lipase production was investigated, and 1.0% (v/w) of tween-80 resulted in maximum lipase production of 6.10 U/g. Thus, the statistical approach employed in this study allows for rapid identification of important medium parameters affecting the lipase production, and further statistical optimization of medium and process parameters can be explored using response surface methodology. PMID:25937979

Application of Plackett-Burman Experimental Design for Lipase Production by Aspergillus niger Using Shea Butter Cake.

PubMed

Salihu, Aliyu; Bala, Muntari; Bala, Shuaibu M

2013-01-01

Plackett-Burman design was used to efficiently select important medium components affecting the lipase production by Aspergillus niger using shea butter cake as the main substrate. Out of the eleven medium components screened, six comprising of sucrose, (NH4)2SO4, Na2HPO4, MgSO4, Tween-80, and olive oil were found to contribute positively to the overall lipase production with a maximum production of 3.35 U/g. Influence of tween-80 on lipase production was investigated, and 1.0% (v/w) of tween-80 resulted in maximum lipase production of 6.10 U/g. Thus, the statistical approach employed in this study allows for rapid identification of important medium parameters affecting the lipase production, and further statistical optimization of medium and process parameters can be explored using response surface methodology.

Production and application of a thermostable lipase from Serratia marcescens in detergent formulation and biodiesel production.

PubMed

García-Silvera, Edgar Edurman; Martínez-Morales, Fernando; Bertrand, Brandt; Morales-Guzmán, Daniel; Rosas-Galván, Nashbly Sarela; León-Rodríguez, Renato; Trejo-Hernández, María R

2018-03-01

In this study, extracellular lipase was produced by Serratia marcescens wild type and three mutant strains. The maximum lipase activity (80 U/mL) was obtained with the SMRG4 mutant strain using soybean oil. Using a 2 2 factorial design, the lipase production increased 1.55-fold (124 U/mL) with 4% and 0.05% of soybean oil and Triton X-100, respectively. The optimum conditions for maximum lipase activity were 50 °C and pH 8. However, the enzyme was active in a broad range of pH (6-10) and temperatures (5-55 °C). This lipase was stable in organic solvents and in the presence of oxidizing agents. The enzyme also proved to be efficient for the removal of triacylglycerol from olive oil in cotton cloth. A Box-Behnken experimental design was used to evaluate the effects of the interactions between total lipase activity, buffer pH, and wash temperatures on oil removal. The model obtained suggested that all selected factors had a significant impact on oil removal, with optimum conditions of 550 U lipase, 45 °C, pH 9.5, with 79.45% removal. Biotransformation of waste frying oil using the enzyme and in presence of methanol resulted in the synthesis of methyl esters such as methyl oleate, methyl palmitate, and methyl stearate. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

Acetylsalicylic acid (aspirin) reduces damage to reconstituted human tissues infected with Candida species by inhibiting extracellular fungal lipases

PubMed Central

Trofa, David; Agovino, Mariangela; Stehr, Frank; Schäfer, Wilhelm; Rykunov, Dmitry; Fiser, András; Hamari, Zsuzsanna; Nosanchuk, Joshua D.; Gácser, Attila

2009-01-01

A reconstituted human tissue model was used to mimic Candida albicans and Candida parapsilosis infection in order to investigate the protective effects of acetylsalicylic acid (aspirin, ASA). We found that therapeutic concentrations of ASA reduced tissue damage in the in vitro infection model. We further evaluated the lipase inhibitory effects of ASA by investigating the growth of C. albicans, C. parapsilosis and C. parapsilosis lipase negative (Δcplip1-2/Δcplip1-2) mutants in a lipid rich minimal medium supplemented with olive oil and found that a therapeutic concentration of ASA inhibited the growth of wild type fungi. The lipase inhibitors quinine and ebelactone B were also shown to reduce growth and protect against tissue damage from Candida species, respectively. A lipolytic activity assay also showed that therapeutic concentrations of ASA inhibited C. antarctica and C. cylindracea purified lipases obtained through a commercial kit. The relationship between ASA and lipase was characterized through a computed structural model of the Lipase-2 protein from C. parapsilosis in complex with ASA. The results suggest that development of inhibitors of fungal lipases could result in broad-spectrum therapeutics, especially since fungal lipases are not homologous to their human analogues. PMID:19703582

Lipase assay in soils by copper soap colorimetry.

PubMed

Saisuburamaniyan, N; Krithika, L; Dileena, K P; Sivasubramanian, S; Puvanakrishnan, R

2004-07-01

A simple and sensitive method for the estimation of lipase activity in soils is reported. In this method, 50mg of soil is incubated with emulsified substrate, the fatty acids liberated are treated with cupric acetate-pyridine reagent, and the color developed is measured at 715 nm. Use of olive oil in this protocol leads to an estimation of true lipase activity in soils. The problem of released fatty acids getting adsorbed onto the soil colloids is obviated by the use of isooctane, and separate standards for different soils need not be developed. Among the various surfactants used for emulsification, polyvinyl alcohol is found to be the most effective. Incubation time of 20 min, soil concentration of 50 mg, pH 6.5, and incubation temperature of 37 degrees C were found to be the most suitable conditions for this assay. During the process of enrichment of the soils with oil, interference by the added oil is avoided by the maintenance of a suitable control, wherein 50 mg of soil is added after stopping the reaction. This assay is sensitive and it could be adopted to screen for lipase producers from enriched soils and oil-contaminated soils before resorting to isolation of the microbes by classical screening methods.

The Purification and Characterization of Lipases from Lasiodiplodia theobromae, and Their Immobilization and Use for Biodiesel Production from Coconut Oil.

PubMed

Venkatesagowda, Balaji; Ponugupaty, Ebenezer; Barbosa-Dekker, Aneli M; Dekker, Robert F H

2017-12-18

The coconut kernel-associated fungus, Lasiodiplodia theobromae VBE1, was grown on coconut cake with added coconut oil as lipase inducer under solid-state fermentation conditions. The extracellular-produced lipases were purified and resulted in two enzymes: lipase A (68,000 Da)-purified 25.41-fold, recovery of 47.1%-and lipase B (32,000 Da)-purified 18.47-fold, recovery of 8.2%. Both lipases showed optimal activity at pH 8.0 and 35 °C, were activated by Ca 2+ , exhibited highest specificity towards coconut oil and p-nitrophenyl palmitate, and were stable in iso-octane and hexane. Ethanol supported higher lipase activity than methanol, and n-butanol inactivated both lipases. Crude lipase immobilized by entrapment within 4% (w/v) calcium alginate beads was more stable than the crude-free lipase preparation within the range pH 2.5-10.0 and 20-80 °C. The immobilized lipase preparation was used to catalyze the transesterification/methanolysis of coconut oil to biodiesel (fatty acyl methyl esters (FAMEs)) and was quantified by gas chromatography. The principal FAMEs were laurate (46.1%), myristate (22.3%), palmitate (9.9%), and oleate (7.2%), with minor amounts of caprylate, caprate, and stearate also present. The FAME profile was comparatively similar to NaOH-mediated transesterified biodiesel from coconut oil, but distinctly different to petroleum-derived diesel. This study concluded that Lasiodiplodia theobromae VBE1 lipases have potential for biodiesel production from coconut oil.

Biodiesel production from Nannochloropsis gaditana lipids through transesterification catalyzed by Rhizopus oryzae lipase.

PubMed

Navarro López, Elvira; Robles Medina, Alfonso; González Moreno, Pedro Antonio; Esteban Cerdán, Luis; Martín Valverde, Lorena; Molina Grima, Emilio

2016-03-01

Biodiesel (fatty acid methyl esters, FAMEs) was produced from saponifiable lipids (SLs) extracted from wet Nannochloropsis gaditana biomass using methanolysis catalyzed by Rhizopus oryzae intracellular lipase. SLs were firstly extracted with ethanol to obtain 31 wt% pure SLs. But this low SL purity also gave a low biodiesel conversion (58%). This conversion increased up to 80% using SLs purified by crystallization in acetone (95 wt% purity). Polar lipids play an important role in decreasing the reaction velocity - using SLs extracted with hexane, which have lower polar lipid content (37.4% versus 49.0% using ethanol), we obtained higher reaction velocities and less FAME conversion decrease when the same lipase batch was reused. 83% of SLs were transformed to biodiesel using a 70 wt% lipase/SL ratio, 11:1 methanol/SL molar ratio, 10 mL t-butanol/g SLs after 72 h. The FAME conversion decreased to 71% after catalyzing three reactions with the same lipase batch. Copyright © 2015 Elsevier Ltd. All rights reserved.

Pancreatitis with normal lipase and amylase in setting of end-stage renal disease.

PubMed

Sharma, Anuj; Masood, Umair; Khan, Babar; Chawla, Kunal; Manocha, Divey

2017-09-01

Pancreatitis with normal lipase and amylase level is a rare phenomenon. This is especially true in patient with end-stage renal disease as lipase and amylase are renally excreted. Literature review reveals previous case report of pancreatitis with normal lipase and amylase level, however, none of them occurred in the setting of end-stage renal disease. Our case is the first such reported case of pancreatitis in such setting. Here we report a 30year old male with past medical history of end-stage renal disease who presented in emergency department with acute abdominal pain. Laboratory work up revealed normal lipase and amylase level. However, radiological work up was consistent with pancreatitis. This case report highlight the importance of taking the overall clinical picture rather than laboratory work up to rule in or rule out the diagnosis of pancreatitis. Furthermore, this should also serve an important reminder for clinicians to further investigate where clinical suspicion for pancreatitis is high. Copyright © 2017 Elsevier Inc. All rights reserved.

Olive pomace valorization by Aspergillus species: lipase production using solid-state fermentation.

PubMed

Oliveira, Felisbela; Moreira, Cláudia; Salgado, José Manuel; Abrunhosa, Luís; Venâncio, Armando; Belo, Isabel

2016-08-01

Pollution by olive mill wastes is an important problem in the Mediterranean area and novel solutions for their proper management and valorization are needed. The aim of this work was to optimize a solid-state fermentation (SSF) process to produce lipase using olive pomace (OP) as the main source of nutrients by several Aspergillus spp. Optimized variables in two different designs were: ratio between olive pomace and wheat bran (OP:WB), NaNO3 , Czapek nutrients, fermentation time, moisture content (MC) and temperature. Results showed that the mixture OP:WB and MC were the most significant factors affecting lipase production for all fungi strains tested. With MC and temperature optimization, a 4.4-fold increase in A. ibericus lipase was achieved (90.5 ± 1.5 U g(-1) ), using a mixture of OP and WB at 1:1 ratio, 0.02 g NaNO3 g(-1) dry substrate, absence of Czapek nutrients, 60% of MC and incubation at 30 °C for 7 days. For A. niger and A. tubingensis, highest lipase activity obtained was 56.6 ± 5.4 and 7.6 ± 0.6 U g(-1) , respectively. Aspergillus ibericus was found to be the most promising microorganism for lipase production using mixtures of OP and WB. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Covalent attachment of microbial lipase onto microporous styrene-divinylbenzene copolymer by means of polyglutaraldehyde.

PubMed

Dizge, Nadir; Keskinler, Bülent; Tanriseven, Aziz

2008-10-01

A novel method for immobilization of Thermomyces lanuginosus lipase onto polyglutaraldehyde-activated poly(styrene-divinylbenzene) (STY-DVB), which is a hydrophobic microporous support has been successfully developed. The copolymer was prepared by the polymerization of the continuous phase of a high internal phase emulsion (polyHIPE). The concentrated emulsion consists of a mixture of styrene and divinylbenzene containing a suitable surfactant and an initiator as the continuous phase and water as the dispersed phase. Lipase from T. lanuginosus was immobilized covalently with 85% yield on the internal surface of the hydrophobic microporous poly(styrene-divinylbenzene) copolymer and used as a biocatalyst for the transesterification reaction. The immobilized enzyme has been fully active 30 days in storage and retained the activity during the 15 repeated batch reactions. The properties of free and immobilized lipase were studied. The effects of protein concentration, pH, temperature, and time on the immobilization, activity, and stability of the immobilized lipase were also studied. The newly synthesized microporous poly(styrene-divinylbenzene) copolymer constitutes excellent support for lipase. It given rise to high immobilization yield, retains enzymatic activity for 30 days, stable in structure and allows for the immobilization of large amount of protein (11.4mg/g support). Since immobilization is simple yet effective, the newly immobilized lipase could be used in several application including oil hydrolysis, production of modified oils, biodiesel synthesis, and removal of fatty acids from oils.

[Influence of tobacco smoking on lipase activity in patients with pancreatitis].

PubMed

Sliwińska-Mossoń, Mariola; Milnerowicz, Halina

2005-01-01

The aim of this study is to prove the influence of tobacco smoking on lipase activity in the blood of smoking and non-smoking health persons and in smoking and non-smoking patients with diagnosed acute (AP), chronic exaggerated (CEP) and chronic pancreatitis (CP). The blood has been collected from 28 healthy persons and 55 patients with AP, CEP and CP. The enzyme activity has been determined using the colorimetric method with substrate 1,2-odilauryl-rac-glycero-3-glutaric acid -(6-methylresorufin) ester. The exposures to tobacco smoke have been examined on the basic of concentration of cotinine in the serum of patients. The highest lipase activity has been found in smoking patients with CEP. It has been noted that the serum lipase activity is significantly higher in smoking and healthy persons (p<0,05) then in non-smoking and healthy patients. However no significant differences have been found between the lipase activity in smoking patients with CP and non-smoking patients with CP. Smoking patients with AP and CEP have been found to have a significantly increased enzyme activity (p>0.01; p>0.05 respectively) when compared to non-smoking patients. Results of examination indicate that tobacco smoking has a significant influence on exocrine function of pancreas.

Biodiesel production using lipase immobilized on epoxychloropropane-modified Fe3O4 sub-microspheres.

PubMed

Zhang, Qian; Zheng, Zhong; Liu, Changxia; Liu, Chunqiao; Tan, Tianwei

2016-04-01

Superparamagnetic Fe3O4 sub-microspheres with diameters of approximately 200 nm were prepared via a solvothermal method, and then modified with epoxychloropropane. Lipase was immobilized on the modified sub-microspheres. The immobilized lipase was used in the production of biodiesel fatty acid methyl esters (FAMEs) from acidified waste cooking oil (AWCO). The effects of the reaction conditions on the biodiesel yield were investigated using a combination of response surface methodology and three-level/three-factor Box-Behnken design (BBD). The optimum synthetic conditions, which were identified using Ridge max analysis, were as follows: immobilized lipase:AWCO mass ratio 0.02:1, fatty acid:methanol molar ratio 1:1.10, hexane:AWCO ratio 1.33:1 (mL/g), and temperature 40 °C. A 97.11% yield was obtained under these conditions. The BBD and experimental data showed that the immobilized lipase could generate biodiesel over a wide temperature range, from 0 to 40 °C. Consistently high FAME yields, in excess of 80%, were obtained when the immobilized lipase was reused in six replicate trials at 10 and 20 °C. Copyright © 2016 Elsevier B.V. All rights reserved.

Screening for Hydrolytic Enzymes Reveals Ayr1p as a Novel Triacylglycerol Lipase in Saccharomyces cerevisiae*

PubMed Central

Ploier, Birgit; Scharwey, Melanie; Koch, Barbara; Schmidt, Claudia; Schatte, Jessica; Rechberger, Gerald; Kollroser, Manfred; Hermetter, Albin; Daum, Günther

2013-01-01

Saccharomyces cerevisiae, as well as other eukaryotes, preserves fatty acids and sterols in a biologically inert form, as triacylglycerols and steryl esters. The major triacylglycerol lipases of the yeast S. cerevisiae identified so far are Tgl3p, Tgl4p, and Tgl5p (Athenstaedt, K., and Daum, G. (2003) YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae. J. Biol. Chem. 278, 23317–23323; Athenstaedt, K., and Daum, G. (2005) Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae, are localized to lipid particles. J. Biol. Chem. 280, 37301–37309). We observed that upon cultivation on oleic acid, triacylglycerol mobilization did not come to a halt in a yeast strain deficient in all currently known triacylglycerol lipases, indicating the presence of additional not yet characterized lipases/esterases. Functional proteome analysis using lipase and esterase inhibitors revealed a subset of candidate genes for yet unknown hydrolytic enzymes on peroxisomes and lipid droplets. Based on the conserved GXSXG lipase motif, putative functions, and subcellular localizations, a selected number of candidates were characterized by enzyme assays in vitro, gene expression analysis, non-polar lipid analysis, and in vivo triacylglycerol mobilization assays. These investigations led to the identification of Ayr1p as a novel triacylglycerol lipase of yeast lipid droplets and confirmed the hydrolytic potential of the peroxisomal Lpx1p in vivo. Based on these results, we discuss a possible link between lipid storage, lipid mobilization, and peroxisomal utilization of fatty acids as a carbon source. PMID:24187129

A computational search for lipases that can preferentially hydrolyze long-chain omega-3 fatty acids from fish oil triacylglycerols.

PubMed

Kamal, Md Zahid; Barrow, Colin J; Rao, Nalam Madhusudhana

2015-04-15

Consumption of long-chain omega-3 fatty acids is known to decrease the risk of major cardiovascular events. Lipases, a class of triacylglycerol hydrolases, have been extensively tested to concentrate omega-3 fatty acids from fish oils, under mild enzymatic conditions. However, no lipases with preference for omega-3 fatty acids selectivity have yet been discovered or developed. In this study we performed an exhaustive computational study of substrate-lipase interactions by docking, both covalent and non-covalent, for 38 lipases with a large number of structured triacylglycerols containing omega-3 fatty acids. We identified some lipases that have potential to preferentially hydrolyze omega-3 fatty acids from structured triacylglycerols. However omega-3 fatty acid preferences were found to be modest. Our study provides an explanation for absence of reports of lipases with omega-3 fatty acid hydrolyzing ability and suggests methods for developing these selective lipases. Copyright © 2014 Elsevier Ltd. All rights reserved.

Lipase-Catalyzed Kinetic Resolution of Novel Antifungal N-Substituted Benzimidazole Derivatives.

PubMed

Łukowska-Chojnacka, Edyta; Staniszewska, Monika; Bondaryk, Małgorzata; Maurin, Jan K; Bretner, Maria

2016-04-01

A series of new N-substituted benzimidazole derivatives was synthesized and their antifungal activity against Candida albicans was evaluated. The chemical step included synthesis of appropriate ketones containing benzimidazole ring, reduction of ketones to the racemic alcohols, and acetylation of alcohols to the esters. All benzimidazole derivatives were obtained with satisfactory yields and in relatively short times. All synthesized compounds exhibit significant antifungal activity against Candida albicans 900028 ATCC (% cell inhibition at 0.25 μg concentration > 98%). Additionally, racemic mixtures of alcohols were separated by lipase-catalyzed kinetic resolution. In the enzymatic step a transesterification reaction was applied and the influence of a lipase type and solvent on the enantioselectivity of the reaction was studied. The most selective enzymes were Novozyme SP 435 and lipase Amano AK from Pseudomonas fluorescens (E > 100). © 2016 Wiley Periodicals, Inc.

Thermostable, alkaline and detergent-tolerant lipase from a newly isolated thermophilic Bacillus stearothermophilus.

PubMed

Ben Bacha, Abir; Moubayed, Nadine M S; Abid, Islam

2015-04-01

Lipases are the enzymes of choice for laundry detergent industries, owing to their triglyceride removing ability from the soiled fabric, which eventually reduces the usage of phosphate-based chemical cleansers in the detergent formulation. In this study, a novel thermo-alkaline lipase-producing strain identified as Bacillus stearothermophilus was isolated from the soil samples of olive oil mill. Enhanced lipase production was observed at 55 degrees C, pH 11 and after 48 h of incubation. Among the substrates tested, xylose (a carbon source), peptone (a nitrogen source) and olive oil at a concentration of 1% were suitable substrates for enhancing lipase production. MgSO4 and Tween-80 were suitable substrates for maximizing lipase production. The enzyme was purified to homogeneity by a single CM-Sephadex column chromatography and revealed molecular mass of 67 kDa. The enzyme (BL1) was active over a wide range of pH from 9.0 to 13.0, with an optimum at pH 11.0, exhibited maximal activity at 55 degreesC and retained more than 70% of its activity after incubation at 70 degrees C or pH 13 for 0.5 h or 24 h, respectively. The enzyme hydrolyzed both short and long-chain triacylglycerols at comparable rates. BL1 was studied in a preliminary evaluation for use in detergent formulation solutions. This novel lipase showed extreme stability towards non-ionic and anionic surfactants after pre-incubation for 1 h at 40 degrees C, and good stability towards oxidizing agents. Additionally, the enzyme showed excellent stability and compatibility with various commercial detergents, suggesting its potential as an additive in detergent formulations.

Lipase-catalyzed highly enantioselective kinetic resolution of boron-containing chiral alcohols.

PubMed

Andrade, Leandro H; Barcellos, Thiago

2009-07-16

The first application of enzymes as catalysts to obtain optically pure boron compounds is described. The kinetic resolution of boron-containing chiral alcohols via enantioselective transesterification catalyzed by lipases was studied. Aromatic, allylic, and aliphatic secondary alcohols containing a boronate ester or boronic acid group were resolved by lipase from Candida antartica (CALB), and excellent E values (E > 200) and high enantiomeric excesses (up to >99%) of both remaining substrates and acetylated product were obtained.

Factors Influencing the Digestibility of Solid Fats: Mammalian and Plant Lipases--Glyceride Structure and Solvent

DTIC Science & Technology

1993-05-01

butter 7 and l-paladtcyl-2-oleoyl-3-stearin 5. Pancreatic lipase digestion of the fats extracted from desert chocolate bars 9 6. Lipase digestion of...of cocoa butter (m.p. 36° C), which consists predominantly (50%) of a TC containing palmitic, stearic, and oleic acids and of 18% of oleoyl distearin...determined. Pancreatic lipase digestion of cocoa butter and palmitoyloleovlstearin Contrary to the results obtained in vivo by Apgar et al.2, only 7% of

Cloning, sequencing and characterization of lipase genes from a polyhydroxyalkanoate- (PHA-) synthesizing Pseudomonas resinovorans

USDA-ARS?s Scientific Manuscript database

Lipase (lip) and lipase-specific foldase (lif) genes of a biodegradable polyhydroxyalkanoate- (PHA-) synthesizing Pseudomonas resinovorans NRRL B-2649 were cloned using primers based on consensus sequences, followed by PCR-based genome walking. Sequence analyses showed a putative Lip gene-product (...

Active-site titration analysis of surface influence on immobilized Candida antarctica Lipase B activity

USDA-ARS?s Scientific Manuscript database

Matrix morphology and surface polarity effects were investigated for Candida antarctica lipase B immobilization. Measurements of the amount of lipase immobilized (bicinchoninic acid method) and the catalyst’s tributyrin hydrolysis activity, coupled with a determination of the lipase’s functional fr...

Rheology, microstructure and baking characteristics of frozen dough containing Rhizopus chinensis lipase and transglutaminase

USDA-ARS?s Scientific Manuscript database

The beneficial effects of a new recombinant lipase (Rhizopus chinensis lipase, RCL) and transglutaminase (TG) were investigated on frozen dough systems and their breadmaking quality. Rheological properties and microstructure of doughs were measured using a dynamic rheometer, rheofermentometer F3, an...

How gastric lipase, an interfacial enzyme with a Ser-His-Asp catalytic triad, acts optimally at acidic pH.

PubMed

Chahinian, Henri; Snabe, Torben; Attias, Coralie; Fojan, Peter; Petersen, Steffen B; Carrière, Frédéric

2006-01-24

Gastric lipase is active under acidic conditions and shows optimum activity on insoluble triglycerides at pH 4. The present results show that gastric lipase also acts in solution on vinyl butyrate, with an optimum activity above pH 7, which suggests that gastric lipase is able to hydrolyze ester bonds via the classical mechanism of serine hydrolases. These results support previous structural studies in which the catalytic triad of gastric lipase was reported to show no specific features. The optimum activity of gastric lipase shifted toward lower pH values, however, when the vinyl butyrate concentration was greater than the solubility limit. Experiments performed with long-chain triglycerides showed that gastric lipase binds optimally to the oil-water interface at low pH values. To study the effects of the pH on the adsorption step independently from substrate hydrolysis, gastric lipase adsorption on solid hydrophobic surfaces was monitored by total internal reflection fluorescence (TIRF), as well as using a quartz crystal microbalance. Both techniques showed a pH-dependent reversible gastric lipase adsorption process, which was optimum at pH 5 (Kd = 6.5 nM). Lipase adsorption and desorption constants (ka = 147,860 M(-1) s(-1) and kd = 139 x 10(-4) s(-1) at pH 6) were estimated from TIRF experiments. These results indicate that the optimum activity of gastric lipase at acidic pH is only "apparent" and results from the fact that lipase adsorption at lipid-water interfaces is the pH-dependent limiting step in the overall process of insoluble substrate hydrolysis. This specific kinetic feature of interfacial enzymology should be taken into account when studying any soluble enzyme acting on an insoluble substrate.

Characterization of Proteus vulgaris K80 lipase immobilized on amine-terminated magnetic microparticles.

PubMed

Natalia, Agnes; Kristiani, Lidya; Kim, Hyung Kwoun

2014-10-01

Proteus vulgaris K80 lipase was expressed in Escherichia coli BL21 (DE3) cells and immobilized on amine-terminated magnetic microparticles (Mag-MPs). The immobilization yield and activity retention were 84.15% and 7.87%, respectively. A homology model of lipase K80 was constructed using P. mirabilis lipase as the template. Many lysine residues were located on the protein surface, remote from active sites. The biochemical characteristics of immobilized lipase K80 were compared with the soluble free form of lipase K80. The optimum temperature of K80-Mag-MPs was 60°C, which was 20°C higher than that of the soluble form. K80-Mag-MPs also tended to be more stable than the soluble form at elevated temperatures and a broad range of pH. K80-Mag-MP maintained its stable form at up to 40°C and in a pH range of 5.0- 10.0, whereas soluble K80 maintained its activity up to 35°C and pH 6.0-10.0. K80-Mag-MPs had broader substrate specificity compared with that of soluble K80. K80-Mag-MPs showed about 80% residual relative activity after five recovery trials. These results indicate the potential benefit of K80-Mag-MPs as a biocatalyst in various industries.

Application of a statistically enhanced, novel, organic solvent stable lipase from Bacillus safensis DVL-43.

PubMed

Kumar, Davender; Parshad, Rajinder; Gupta, Vijay Kumar

2014-05-01

This paper presents the molecular identification of a newly isolated bacterial strain producing a novel and organic solvent stable lipase, statistical optimization of fermentation medium, and its application in the synthesis of ethyl laurate. On the basis of nucleotide homology and phylogenetic analysis of 16S rDNA sequence, the strain was identified as Bacillus safensis DVL-43 (Gen-bank accession number KC156603). Optimization of fermentation medium using Plackett-Burman design and response surface methodology led to 11.4-fold increase in lipase production. The lipase from B. safensis DVL-43 exhibited excellent stability in various organic solvents. The enzyme retained 100% activity after 24h incubation in xylene, DMSO and toluene, each solvent being used at a concentration of 25% (v/v). The use of partially purified DVL-43 lipase as catalyst in the synthesis of ethyl laurate, an esterification product of lauric acid and ethanol, resulted in 80% esterification in 12h under optimized conditions. The formation of ethyl laurate was confirmed using TLC and (1)H NMR. Organic solvent stable lipases exhibiting potential application in enzymatic esterification are in great demand in flavor, fine chemicals and pharma industries. We could not find any report on lipase production from B. safensis strain and its application in esterification. Copyright © 2014 Elsevier B.V. All rights reserved.

Lipolysis of the semi-solid self-emulsifying excipient Gelucire 44/14 by digestive lipases.

PubMed

Fernandez, Sylvie; Rodier, Jean-David; Ritter, Nicolas; Mahler, Bruno; Demarne, Frédéric; Carrière, Frédéric; Jannin, Vincent

2008-08-01

Gelucire 44/14 is a semi-solid self-emulsifying excipient used for the oral delivery of poorly water-soluble drugs. It is composed of C8-C18 acylglycerols and PEG-32 esters, all of which are potential substrates for digestive lipases. Here we studied the lipolysis of Gelucire 44/14 by porcine pancreatic extracts, human pancreatic juice and several purified digestive lipases. Human pancreatic lipase (HPL), the main lipase involved in the digestion of triacylglycerols, did not show any significant activity on Gelucire 44/14 or on either of its individual compounds, C8-C18 acylglycerols and PEG-32 esters. Other pancreatic lipases such as human pancreatic lipase-related protein 2 (HPLRP2) showed low activity on Gelucire 44/14 although the highest activity of HPLRP2 was that observed on the C8-C18 acylglycerol fraction, which accounts for 20% (w/w) of Gelucire 44/14. In addition, HPLRP2 showed low activities on the PEG-32 esters, whether these were tested individually or mixed together. Carboxyl ester hydrolase (CEH) showed high activity on Gelucire 44/14, and the highest activities of CEH were those recorded on the total PEG-32 ester fraction and on each individual PEG-32 ester, except for PEG-32 monostearate. The highest activity of all the enzymes tested was that of dog gastric lipase (DGL) on Gelucire 44/14, although DGL showed low activity on the PEG-32 ester fraction and on each individual PEG-32 ester. We compared the lipolysis of Gelucire 44/14 with that of Labrasol, another self-emulsifying excipient, which is liquid at room temperature. Human pancreatic juice showed similar rates of activity on both Gelucire 44/14 and Labrasol. This finding means that these excipients are hydrolyzed in vivo during pancreatic digestion, mainly by CEH in the case of Gelucire 44/14 and by both HPLRP2 and CEH in that of Labrasol, whereas HPL showed very low activities on each of these two excipients. This is the first time the effects of PEG and acyl chain length on the lipolytic

Efficient resource recycling from liquid digestate by microalgae-yeast mixed culture and the assessment of key gene transcription related to nitrogen assimilation in microalgae.

PubMed

Qin, Lei; Liu, Lu; Wang, Zhongming; Chen, Weining; Wei, Dong

2018-05-18

To determine the feasibility of microalgae-yeast mixed culture using the liquid digestate of dairy wastewater (LDDW) for biofuels and single cell protein (SCP) production, the cell growth, nutrient removal and outputs evaluation of the mono and mixed culture of Chlorella vulgaris and Yarrowia lipolytica in LDDW were investigated by adding glycerol as carbon source. The results showed that the mixed culture could enhance the biological utilization efficiency of nitrogen and phosphorus, and obtain higher yield of biomass (1.62 g/L), lipid (0.31 g/L), protein (0.51 g/L), and higher heating value (34.06 KJ/L). Compared with the mono culture of C. vulgaris, a decline of the transcription level in nitrate reductase and glutamine synthetase II genes in C. vulgaris was observed in the mixed culture when ammonia was sufficient. The results suggest the possibility of using the mixed culture for the efficient treatment of LDDW and resources recycling. Copyright © 2018. Published by Elsevier Ltd.

Molecular characterization and expression of microbial inulinase genes.

PubMed

Liu, Guang-Lei; Chi, Zhe; Chi, Zhen-Ming

2013-05-01

Many genes encoding exo- and endo-inulinases from bacteria, yeasts and filamentous fungi have been cloned and characterized. All the inulinases have several conserved motifs, such as WMND(E)PNGL, RDP, EC(V)P, SVEVF, Q and FS(T), which play an important role in inulinase catalysis and substrate binding. However, the exo-inulinases produced by yeasts has no conserved motif SVEVF and the yeasts do not produce any endo-inulinase. Exo- and endo-inulinases found in different microorganisms cluster separately at distant positions from each other. Most of the cloned inulinase genes have been expressed in Yarrowia lipolytica, Saccharomyces cerevisiae, Pichia pastoris, Klyuveromyces lactis and Escherichia coli, respectively. The recombinant inulinases produced and the engineered hosts using the cloned inulinase genes have many potential applications. Expression of most of the inulinase genes is repressed by glucose and fructose and induced by inulin and sucrose. However, the detailed mechanisms of the repression and induction are still unknown.

Metabolic engineering of yeast for lignocellulosic biofuel production.

PubMed

Jin, Yong-Su; Cate, Jamie Hd

2017-12-01

Production of biofuels from lignocellulosic biomass remains an unsolved challenge in industrial biotechnology. Efforts to use yeast for conversion face the question of which host organism to use, counterbalancing the ease of genetic manipulation with the promise of robust industrial phenotypes. Saccharomyces cerevisiae remains the premier host for metabolic engineering of biofuel pathways, due to its many genetic, systems and synthetic biology tools. Numerous engineering strategies for expanding substrate ranges and diversifying products of S. cerevisiae have been developed. Other yeasts generally lack these tools, yet harbor superior phenotypes that could be exploited in the harsh processes required for lignocellulosic biofuel production. These include thermotolerance, resistance to toxic compounds generated during plant biomass deconstruction, and wider carbon consumption capabilities. Although promising, these yeasts have yet to be widely exploited. By contrast, oleaginous yeasts such as Yarrowia lipolytica capable of producing high titers of lipids are rapidly advancing in terms of the tools available for their metabolic manipulation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Synthetic biology and molecular genetics in non-conventional yeasts: Current tools and future advances.

PubMed

Wagner, James M; Alper, Hal S

2016-04-01

Coupling the tools of synthetic biology with traditional molecular genetic techniques can enable the rapid prototyping and optimization of yeast strains. While the era of yeast synthetic biology began in the well-characterized model organism Saccharomyces cerevisiae, it is swiftly expanding to include non-conventional yeast production systems such as Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, and Yarrowia lipolytica. These yeasts already have roles in the manufacture of vaccines, therapeutic proteins, food additives, and biorenewable chemicals, but recent synthetic biology advances have the potential to greatly expand and diversify their impact on biotechnology. In this review, we summarize the development of synthetic biological tools (including promoters and terminators) and enabling molecular genetics approaches that have been applied in these four promising alternative biomanufacturing platforms. An emphasis is placed on synthetic parts and genome editing tools. Finally, we discuss examples of synthetic tools developed in other organisms that can be adapted or optimized for these hosts in the near future. Copyright © 2015 Elsevier Inc. All rights reserved.

The Effect of Storage at Three Different Temperatures on the Activity of Lipase Solution.

ERIC Educational Resources Information Center

Bradley, Karen; Mathewman, David

1984-01-01

Presented are procedures used to assay the activity of lipase during storage at three different temperatures. Since lipase solutions can decay even when refrigerated, it is recommended that the enzyme be freshly prepared prior to laboratory sessions in which they are used. (JN)

Production of a solvent, detergent, and thermotolerant lipase by a newly isolated Acinetobacter sp. in submerged and solid-state fermentations.

PubMed

Khoramnia, Anahita; Ebrahimpour, Afshin; Beh, Boon Kee; Lai, Oi Ming

2011-01-01

The lipase production ability of a newly isolated Acinetobacter sp. in submerged (SmF) and solid-state (SSF) fermentations was evaluated. The results demonstrated this strain as one of the rare bacterium, which is able to grow and produce lipase in SSF even more than SmF. Coconut oil cake as a cheap agroindustrial residue was employed as the solid substrate. The lipase production was optimized in both media using artificial neural network. Multilayer normal and full feed forward backpropagation networks were selected to build predictive models to optimize the culture parameters for lipase production in SmF and SSF systems, respectively. The produced models for both systems showed high predictive accuracy where the obtained conditions were close together. The produced enzyme was characterized as a thermotolerant lipase, although the organism was mesophile. The optimum temperature for the enzyme activity was 45°C where 63% of its activity remained at 70°C after 2 h. This lipase remained active after 24 h in a broad range of pH (6-11). The lipase demonstrated strong solvent and detergent tolerance potentials. Therefore, this inexpensive lipase production for such a potent and industrially valuable lipase is promising and of considerable commercial interest for biotechnological applications.

Using the reversible inhibition of gastric lipase by Orlistat for investigating simultaneously lipase adsorption and substrate hydrolysis at the lipid-water interface.

PubMed

Bénarouche, Anaïs; Point, Vanessa; Carrière, Frédéric; Cavalier, Jean-François

2014-06-01

The lipolysis reaction carried out by lipases at the water-lipid interface is a complex process including enzyme conformational changes, adsorption/desorption equilibrium and substrate hydrolysis. Mixed monomolecular films of the lipase inhibitor Orlistat and 1,2-dicaprin were used here to investigate the adsorption of dog gastric lipase (DGL) followed by the hydrolysis of 1,2-dicaprin. The combined study of these two essential catalysis steps was made possible thanks to the highest affinity of DGL for Orlistat than 1,2-dicaprin and the fact that the inhibition of DGL by Orlistat is reversible. Upon DGL binding to mixed 1,2-dicaprin/Orlistat monolayers, an increase in surface pressure reflecting lipase adsorption was first recorded. Limited amounts of Orlistat allowed to maintain DGL inactive on 1,2-dicaprin during a period of time that was sufficient to determine DGL adsorption and desorption rate constants. A decrease in surface pressure reflecting 1,2-dicaprin hydrolysis and product desorption was observed after the slow hydrolysis of the covalent DGL-Orlistat complex was complete. The rate of 1,2-dicaprin hydrolysis was recorded using the surface barostat technique. Based on a kinetic model describing the inhibition by Orlistat and the activity of DGL on a mixed 1,2-dicaprin/Orlistat monolayer spread at the air-water interface combined with surface pressure measurements, it was possible to monitor DGL adsorption at the lipid-water interface and substrate hydrolysis in the course of a single experiment. This allowed to assess the kcat/KM* ratio for DGL acting on 1,2-dicaprin monolayer, after showing that mixed monolayers containing a low fraction of Orlistat were similar to pure 1,2-dicaprin monolayers. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Implication of substrate-assisted catalysis on improving lipase activity or enantioselectivity in organic solvents.

PubMed

Tsai, Shau-Wei; Chen, Chun-Chi; Yang, Hung-Shien; Ng, I-Son; Chen, Teh-Liang

2006-08-01

In comparison with the biocatalyst engineering and medium engineering approaches, very few examples have been reported on using the substrate engineering approach such as substrate-assisted catalysis (SAC) for naturally occurring or engineered lipases and serine proteases to improve the enzyme activity and enantioselectivity. By employing lipase-catalyzed hydrolysis of (R,S)-naproxen esters in water-saturated isooctane as the model system, we demonstrate the proton shuttle device to the leaving alcohol of the substrate as a new means of SAC to effectively improve the lipase activity or enantioselectivity. The result cannot only provide a strong evidence for the rate-limiting proton transfer for the bond-breaking of tetrahedron intermediate of the acylation step, but also sheds light for performing the hydrolysis, transesterification or aminolysis in organic solvents for the ester substrate that originally lipases cannot catalyze, but now can after introducing the device.

Synthesis and kinetic evaluation of cyclophostin and cyclipostins phosphonate analogs as selective and potent inhibitors of microbial lipases.

PubMed

Point, Vanessa; Malla, Raj K; Diomande, Sadia; Martin, Benjamin P; Delorme, Vincent; Carriere, Frederic; Canaan, Stephane; Rath, Nigam P; Spilling, Christopher D; Cavalier, Jean-François

2012-11-26

A new series of customizable diastereomeric cis- and trans-monocyclic enol-phosphonate analogs to Cyclophostin and Cyclipostins were synthesized. Their potencies and mechanisms of inhibition toward six representative lipolytic enzymes belonging to distinct lipase families were examined. With mammalian gastric and pancreatic lipases no inhibition occurred with any of the compounds tested. Conversely, Fusarium solani Cutinase and lipases from Mycobacterium tuberculosis (Rv0183 and LipY) were all fully inactivated. The best inhibitors displayed a cis conformation (H and OMe) and exhibited higher inhibitory activities than the lipase inhibitor Orlistat toward the same enzymes. Our results have revealed that chemical group at the γ-carbon of the phosphonate ring strongly impacts the inhibitory efficiency, leading to a significant improvement in selectivity toward a target lipase over another. The powerful and selective inhibition of microbial (fungal and mycobacterial) lipases suggests that these seven-membered monocyclic enol-phosphonates should provide useful leads for the development of novel and highly selective antimicrobial agents.

Synthesis and kinetic evaluation of Cyclophostin and Cyclipostins phosphonate analogs as selective and potent inhibitors of microbial lipases

PubMed Central

Point, Vanessa; Malla, Raj K.; Diomande, Sadia; Martin, Benjamin P.; Delorme, Vincent; Carriere, Frederic; Canaan, Stephane; Rath, Nigam P.; Spilling, Christopher D.; Cavalier, Jean-François

2012-01-01

New series of customizable diastereomeric cis- and trans-monocyclic enol-phosphonate analogs to Cyclophostin and Cyclipostins were synthesized. Their potencies and mechanisms of inhibition toward six representative lipolytic enzymes belonging to distinct lipase families were examined. With mammalian gastric and pancreatic lipases no inhibition occurred with any of the compounds tested. Conversely, Fusarium solani Cutinase and lipases from Mycobacterium tuberculosis (Rv0183 and LipY) were all fully inactivated. Best inhibitors displayed a cis conformation (H and OMe) and exhibited higher inhibitory activities than the lipase inhibitor Orlistat towards same enzymes. Our results have revealed that chemical group at the γ-carbon of the phosphonate ring strongly impacts the inhibitory efficiency, leading to a significant improvement in selectivity toward a target lipase over another. The powerful and selective inhibition of microbial (fungal and mycobacterial) lipases suggests that these 7-membered monocyclic enol-phosphonates should provide useful leads for the development of novel and highly selective antimicrobial agents. PMID:23095026

Comparative study of thermostability and ester synthesis ability of free and immobilized lipases on cross linked silica gel.

PubMed

Kumari, Annapurna; Mahapatra, Paramita; Kumar, Garlapati Vijay; Banerjee, Rintu

2008-06-01

A novel support has been utilized for immobilization of lipase, which was prepared by amination of silica with ethanolamine followed by cross linking with glutaraldehyde. Lipases from Rhizopus oryzae 3562 and Enterobacter aerogenes were immobilized on activated silica gel, where they retained 60 and 50% of respective original activity. The thermal stability of the immobilized lipases was significantly improved in comparison to the free forms while the pH stability remained unchanged. E. aerogenes and R. oryzae 3562 lipases retained 75 and 97% of respective initial activity on incubation at 90 degrees C, whereas both the free forms became inactive at this temperature. The conversion yield of isoamyl acetate was found to be higher with the immobilized fungal (90 vs. 21%) and bacterial lipases (64 vs. 18%) than the respective free forms. Immobilized R. oryzae 3562 lipases retained 50% activity for isoamyl acetate synthesis up to ten cycles whereas it was eight cycles for E. aerogenes.

Recent Advances in Lipase-Mediated Preparation of Pharmaceuticals and Their Intermediates

PubMed Central

Carvalho, Ana Caroline Lustosa de Melo; Fonseca, Thiago de Sousa; de Mattos, Marcos Carlos; de Oliveira, Maria da Conceição Ferreira; de Lemos, Telma Leda Gomes; Molinari, Francesco; Romano, Diego; Serra, Immacolata

2015-01-01

Biocatalysis offers an alternative approach to conventional chemical processes for the production of single-isomer chiral drugs. Lipases are one of the most used enzymes in the synthesis of enantiomerically pure intermediates. The use of this type of enzyme is mainly due to the characteristics of their regio-, chemo- and enantioselectivity in the resolution process of racemates, without the use of cofactors. Moreover, this class of enzymes has generally excellent stability in the presence of organic solvents, facilitating the solubility of the organic substrate to be modified. Further improvements and new applications have been achieved in the syntheses of biologically active compounds catalyzed by lipases. This review critically reports and discusses examples from recent literature (2007 to mid-2015), concerning the synthesis of enantiomerically pure active pharmaceutical ingredients (APIs) and their intermediates in which the key step involves the action of a lipase. PMID:26690428

Utilization of coconut oil cake for the production of lipase using Bacillus coagulans VKL1.

PubMed

Gowthami, Palanisamy; Muthukumar, Karuppan; Velan, Manickam

2015-01-01

The overproduction of enzymes was performed by manipulating the medium components. In our study, solvent-tolerant thermophilic lipase-producing Bacillus coagulans was isolated from soil samples and a stepwise optimization strategy was employed to increase the lipase production using coconut oil cake basal medium. In the first step, the influence of pH, temperature, carbon source, nitrogen source and inducers on lipase activity was investigated by the One-Factor-At-A-Time (OFAT) method. In the second step, the three significant factors resulted from OFAT were optimized by the statistical approach (CCD).The optimum values of olive oil (0.5%), Tween 80 (0.6%) and FeSO4 (0.05%) was found to be responsible for a 3.2-fold increase in the lipase production identified by Central Composite Design.

Optimization of Lipase production from a novel strain Thalassospira permensis M35-15 using Response Surface Methodology

PubMed Central

Kai, Wang; Peisheng, Yan

2016-01-01

ABSTRACT Lipases can catalyze the hydrolysis of glycerol, esters and long chain fatty acids. A lipase producing isolate M35-15 was screened and identified as Thalassospira permensis using 16S rRNA gene sequence analysis. To our knowledge this is the first report on Thalassospira permensis producing lipases. In this paper the optimization of medium composition for the increase in bacterial lipase was achieved using statistical methods. Firstly the key ingredients were selected by Plackett-Burman experimental design, then the levels of the ingredients were optimized using central composite design of Response Surface Methodology. The predicted optimal lipase activity was 11.49 U under the conditions that medium composition were 5.15 g/l glucose, 11.74 g/l peptone, 6.74 g/l yeast powder and 22.90 g/l olive oil emulsifier. PMID:27285376

Optimization of Lipase production from a novel strain Thalassospira permensis M35-15 using Response Surface Methodology.

PubMed

Kai, Wang; Peisheng, Yan

2016-09-02

Lipases can catalyze the hydrolysis of glycerol, esters and long chain fatty acids. A lipase producing isolate M35-15 was screened and identified as Thalassospira permensis using 16S rRNA gene sequence analysis. To our knowledge this is the first report on Thalassospira permensis producing lipases. In this paper the optimization of medium composition for the increase in bacterial lipase was achieved using statistical methods. Firstly the key ingredients were selected by Plackett-Burman experimental design, then the levels of the ingredients were optimized using central composite design of Response Surface Methodology. The predicted optimal lipase activity was 11.49 U under the conditions that medium composition were 5.15 g/l glucose, 11.74 g/l peptone, 6.74 g/l yeast powder and 22.90 g/l olive oil emulsifier.

Optimization of lipase production by solid-state fermentation of olive pomace: from flask to laboratory-scale packed-bed bioreactor.

PubMed

Oliveira, Felisbela; Salgado, José Manuel; Abrunhosa, Luís; Pérez-Rodríguez, Noelia; Domínguez, José M; Venâncio, Armando; Belo, Isabel

2017-07-01

Lipases are versatile catalysts with many applications and can be produced by solid-state fermentation (SSF) using agro-industrial wastes. The aim of this work was to maximize the production of Aspergillus ibericus lipase under SSF of olive pomace (OP) and wheat bran (WB), evaluating the effect on lipase production of C/N ratio, lipids, phenols, content of sugars of substrates and nitrogen source addition. Moreover, the implementation of the SSF process in a packed-bed bioreactor and the improvement of lipase extraction conditions were assessed. Low C/N ratios and high content of lipids led to maximum lipase production. Optimum SSF conditions were achieved with a C/N mass ratio of 25.2 and 10.2% (w/w) lipids in substrate, by the mixture of OP:WB (1:1) and supplemented with 1.33% (w/w) (NH 4 ) 2 SO 4 . Studies in a packed-bed bioreactor showed that the lower aeration rates tested prevented substrate dehydration, improving lipase production. In this work, the important role of Triton X-100 on lipase extraction from the fermented solid substrate has been shown. A final lipase activity of 223 ± 5 U g -1 (dry basis) was obtained after 7 days of fermentation.

Studies on the substrate and stereo/regioselectivity of adipose triglyceride lipase, hormone-sensitive lipase, and diacylglycerol-O-acyltransferases.

PubMed

Eichmann, Thomas O; Kumari, Manju; Haas, Joel T; Farese, Robert V; Zimmermann, Robert; Lass, Achim; Zechner, Rudolf

2012-11-30

Adipose triglyceride lipase (ATGL) is rate-limiting for the initial step of triacylglycerol (TAG) hydrolysis, generating diacylglycerol (DAG) and fatty acids. DAG exists in three stereochemical isoforms. Here we show that ATGL exhibits a strong preference for the hydrolysis of long-chain fatty acid esters at the sn-2 position of the glycerol backbone. The selectivity of ATGL broadens to the sn-1 position upon stimulation of the enzyme by its co-activator CGI-58. sn-1,3 DAG is the preferred substrate for the consecutive hydrolysis by hormone-sensitive lipase. Interestingly, diacylglycerol-O-acyltransferase 2, present at the endoplasmic reticulum and on lipid droplets, preferentially esterifies sn-1,3 DAG. This suggests that ATGL and diacylglycerol-O-acyltransferase 2 act coordinately in the hydrolysis/re-esterification cycle of TAGs on lipid droplets. Because ATGL preferentially generates sn-1,3 and sn-2,3, it suggests that TAG-derived DAG cannot directly enter phospholipid synthesis or activate protein kinase C without prior isomerization.

Rapid screening natural-origin lipase inhibitors from hypolipidemic decoctions by ultrafiltration combined with liquid chromatography-mass spectrometry.

PubMed

Xiao, Shun; Yu, Runru; Ai, Ni; Fan, Xiaohui

2015-02-01

Lipase inhibitors generate hypolipidemic effect that is helpful to control or treat some obesity diseases by inactivating catalytic activity of human pancreatic lipase, a key enzyme involved in triglyceride hydrolysis in vivo. Many traditional Chinese medicine (TCM) formulae have been effectively used to treat obesity and other fat related diseases for centuries and modern biological experiments demonstrate therapeutic effect of these formulae can be linked to their lipid-lowering capability in blood. These observations suggest that these hypolipidemic decoctions (HDs) could be a promising resource of natural-origin lipase inhibitors. This work described a rapid approach for screening lipase inhibitors from four widely used HDs, including Wu-Ling-San (WLS), Ze-Xie decoction (ZX), Xiao-Xian-Xiong decoction (XXX) and Xiao-Chai-Hu decoction (XCH), by ultrafiltration combing with high performance liquid chromatography-mass spectrometry (HPLC-MS). Our results showed sixteen natural-origin lipase inhibitors were discovered and identified by high resolution and multistage mass spectrometry. Inhibitory activities of two compounds were confirmed by a functional assay of lipase, which validated the reliability of our approach. Molecular docking simulation was then performed to investigate potential mechanism of action for these compounds. Together we present an efficient method for rapid screening lipase inhibitors from complex natural products, which can be easily accommodated to other important enzymatic system with therapeutic values. Copyright © 2014 Elsevier B.V. All rights reserved.

Purification and substrate specificity of Staphylococcus hyicus lipase.

PubMed

van Oort, M G; Deveer, A M; Dijkman, R; Tjeenk, M L; Verheij, H M; de Haas, G H; Wenzig, E; Götz, F

1989-11-28

The Staphylococcus hyicus lipase gene has been cloned and expressed in Staphylococcus carnosus. From the latter organism the enzyme was secreted into the medium as a protein with an apparent molecular mass of 86 kDa. This protein was purified, and the amino-terminal sequence showed that the primary gene product was indeed cleaved at the proposed signal peptide cleavage site. The protein was purified from large-scale preparations after tryptic digestion. This limited proteolysis reduced the molecular mass to 46 kDa and increased the specific activity about 3-fold. Although the enzyme had a low specific activity in the absence of divalent cations, the activity increased about 40-fold in the presence of Sr2+ or Ca2+ ions. The purified lipase has a broad substrate specificity. The acyl chains were removed from the primary and secondary positions of natural neutral glycerides and from a variety of synthetic glyceride analogues. Thus triglycerides were fully hydrolyzed to free fatty acid and glycerol. The enzyme hydrolyzed naturally occurring phosphatidylcholines, their synthetic short-chain analogues, and lysophospholipids to free fatty acids and water-soluble products. The enzyme had a 2-fold higher activity on micelles of short-chain D-lecithins than on micelles composed of the L-isomers. Thus the enzyme from S. hyicus has lipase activity and also high phospholipase A and lysophospholipase activity.

Beauveria bassiana Lipase A expressed in Komagataella (Pichia) pastoris with potential for biodiesel catalysis.

PubMed

Vici, Ana C; da Cruz, Andrezza F; Facchini, Fernanda D A; de Carvalho, Caio C; Pereira, Marita G; Fonseca-Maldonado, Raquel; Ward, Richard J; Pessela, Benevides C; Fernandez-Lorente, Gloria; Torres, Fernando A G; Jorge, João A; Polizeli, Maria L T M

2015-01-01

Lipases (EC 3.1.1.3) comprise a biotechnologically important group of enzymes because they are able to catalyze both hydrolysis and synthesis reactions, depending on the amount of water in the system. One of the most interesting applications of lipase is in the biofuel industry for biodiesel production by oil and ethanol (or methanol) transesterification. Entomopathogenic fungi, which are potential source of lipases, are still poorly explored in biotechnological processes. The present work reports the heterologous expression and biochemical characterization of a novel Beauveria bassiana lipase with potential for biodiesel production. The His-tagged B. bassiana lipase A (BbLA) was produced in Komagataella pastoris in buffered methanol medium (BMM) induced with 1% methanol at 30°C. Purified BbLA was activated with 0.05% Triton X-100 and presented optimum activity at pH 6.0 and 50°C. N-glycosylation of the recombinant BbLA accounts for 31.5% of its molecular weight. Circular dichroism and molecular modeling confirmed a structure composed of α-helix and β-sheet, similar to α/β hydrolases. Immobilized BbLA was able to promote transesterification reactions in fish oil, demonstrating potential for biodiesel production. BbLA was successfully produced in K. pastoris and shows potential use for biodiesel production by the ethanolysis reaction.

Lipase-catalyzed transesterification of soybean oil and phytosterol in supercritical CO2.

PubMed

Hu, Lizhi; Llibin, Sun; Li, Jun; Qi, Liangjun; Zhang, Xu; Yu, Dianyu; Walid, Elfalleh; Jiang, Lianzhou

2015-12-01

The transesterification of phytosterol and soybean oil was performed using Novozym 435 in supercritical carbon dioxide (SC-CO2). The transesterification reaction was conducted in soybean oil containing 5-25% phytosterol at 55-95 °C and free-water solvent. The effects of temperature, reaction time, phytosterol concentration, lipase dosage and reaction pressure on the conversion rate of transesterification were investigated. The optimal reaction conditions were the reaction temperature (85 °C), reaction time (1 h), phytosterol concentration (5%), reaction pressure (8 Mpa) and lipase dosage (1%). The highest conversion rate of 92% could be achieved under the optimum conditions. Compared with the method of lipase-catalyzed transesterification of phytosterol and soybean oil at normal pressure, the transesterification in SC-CO2 reduced significantly the reaction temperature and reaction time.

Hydrolysis of diacylglycerols by lipoprotein lipase.

PubMed

Morley, N H; Kuksis, A; Buchnea, D; Myher, J J

1975-05-10

Enantiomeric diacylglycerols were emulsified, mole for mole, with lyso(1-acyl) lecithin and were hydrolyzed with lipoprotein lipase in NH4Cl-beef serum albumin buffer at pH 8.6 after a brief incubation with delipidated rat serum. The enzyme was prepared from lyophilized and dialyzed bovine skim milk in a 4 percent solution. The course of hydrolysis for each set of enantiomers was determined by gas-liquid chromatography of the masses of the diacylglycerols remaining or monoacylglycerols released in the medium between 0 and 15 min. The majority of sets of sn-1,2- and 2,3-diacylglycerols, including an isotope-labeled true enantiomeric set which was assessed by mass spectrometry, demonstrated preference by the enzyme for lipolysis at position 1 but with less specificity than previously was shown in sn-triacylglycerol hydrolysis. The results preclude the possibility that the predominance of sn-2,3-diacylglycerol intermediates during triacylglycerol hydrolysis is due solely to a preferential breakdown of the 1,2-isomers and reinforce the conclusion that lipoprotein lipase is specific for position 1.

Lysophosphatidylcholine synthesis by lipase-catalyzed ethanolysis.

PubMed

Yang, Guolong; Yang, Ruoxi; Hu, Jingbo

2015-01-01

Lysophosphatidylcholine (LPC) is amphiphilic substance, and possesses excellent physiological functions. In this study, LPC was prepared through ethanolysis of phosphatidylcholine (PC) in n-hexane or solvent free media catalyzed by Novozym 435 (from Candida antarctica), Lipozyme TLIM (from Thermomcyces lanuginosus) and Lipozyme RMIM (from Rhizomucor miehei). The results showed that three immobilized lipases from Candida Antarctica, Thermomcyces lanuginosus and Rhizomucor miehei could catalyze ethanolysis of PC efficiently. In n-hexane, the LPC conversions of ethanolysis of PC catalyzed by Novozyme 435, Lipozyme TLIM and Lipozyme RMIM could reach to 98.5 ± 1.6%, 94.6 ± 1.4% and 93.7 ± 1.8%, respectively. In solvent free media, the highest LPC conversions of ethanolysis of PC catalyzed by Novozyme 435, Lipozyme TL IM and Lipozyme RM IM were 97.7 ± 1.7%, 93.5 ± 1.2% and 93.8 ± 1.9%, respectively. The catalytic efficiencies of the three lipases were in the order of Novozyme 435 > Lipozyme TLIM > Lipozyme RMIM. Furthermore, their catalytic efficiencies in n-hexane were better than those in solvent free media.

Microwave-Assisted Resolution of α-Lipoic Acid Catalyzed by an Ionic Liquid Co-Lyophilized Lipase.

PubMed

Liu, Ning; Wang, Lei; Wang, Zhi; Jiang, Liyan; Wu, Zhuofu; Yue, Hong; Xie, Xiaona

2015-05-29

The combination of the ionic liquid co-lyophilized lipase and microwave irradiation was used to improve enzyme performance in enantioselective esterification of α-lipoic acid. Effects of various reaction conditions on enzyme activity and enantioselectivity were investigated. Under optimal condition, the highest enantioselectivity (E = 41.2) was observed with a high enzyme activity (178.1 μmol/h/mg) when using the ionic liquid co-lyophilized lipase with microwave assistance. Furthermore, the ionic liquid co-lyophilized lipase exhibited excellent reusability under low power microwave.

Effect of prolonged refrigeration on the lipid profile, lipase activity, and oxidative status of human milk.

PubMed

Bertino, Enrico; Giribaldi, Marzia; Baro, Cristina; Giancotti, Valeria; Pazzi, Marco; Peila, Chiara; Tonetto, Paola; Arslanoglu, Sertac; Moro, Guido E; Cavallarin, Laura; Gastaldi, Daniela

2013-04-01

The study was aimed at evaluating the effect of prolonged refrigeration of fresh human milk (HM) on its fatty acid profile, free fatty acid content, lipase activities, and oxidative status. HM from mothers of preterm newborns was collected, pooled, and placed in the neonatal intensive care unit (NICU) refrigerator. Pooled milk was aliquoted and analyzed within 3 hours of collection, and after 24, 48, 72, and 96 hours of storage. The milk samples were analyzed for pH, total and free fatty acid profile, lipase activity at room temperature and at 4°C, lipase activity at room temperature in presence of sodium cholate (bile salt-dependent lipase), total antioxidant capacity, thiobarbituric acid reactive species, malondialdehyde, and conjugated diene concentration. The experiment was replicated in 3 independent trials. Prolonged refrigeration did not affect the fatty acid composition of breast milk, and preserved both its overall oxidative status and the activity of HM lipolytic enzymes. In particular, bile salt-dependent lipase activity, long-chain polyunsaturated fatty acids, and medium-chain saturated fatty acid concentrations were unaffected for up to 96 hours of refrigerated storage. Prolonged refrigeration of fresh HM for 96 hours maintained its overall lipid composition. The limited lipolysis during storage should be ascribed to the activity of lipoprotein lipase, responsible for the decrease in pH. Our study demonstrates that infants who receive expressed milk stored for up to 96 hours receive essentially the same supply of fatty acids and active lipases as do infants fed directly at the breast.

Integration process of fermentation and liquid biphasic flotation for lipase separation from Burkholderia cepacia.

PubMed

Sankaran, Revathy; Show, Pau Loke; Lee, Sze Ying; Yap, Yee Jiun; Ling, Tau Chuan

2018-02-01

Liquid Biphasic Flotation (LBF) is an advanced recovery method that has been effectively applied for biomolecules extraction. The objective of this investigation is to incorporate the fermentation and extraction process of lipase from Burkholderia cepacia using flotation system. Initial study was conducted to compare the performance of bacteria growth and lipase production using flotation and shaker system. From the results obtained, bacteria shows quicker growth and high lipase yield via flotation system. Integration process for lipase separation was investigated and the result showed high efficiency reaching 92.29% and yield of 95.73%. Upscaling of the flotation system exhibited consistent result with the lab-scale which are 89.53% efficiency and 93.82% yield. The combination of upstream and downstream processes in a single system enables the acceleration of product formation, improves the product yield and facilitates downstream processing. This integration system demonstrated its potential for biomolecules fermentation and separation that possibly open new opportunities for industrial production. Copyright © 2017 Elsevier Ltd. All rights reserved.

Screening and characterization of a novel thermostable lipase with detergent-additive potential from the metagenomic library of a mangrove soil.