Differences in hydrolytic abilities of two crude lipases from Geotrichum candidum 4013

The fungus Geotrichum candidum 4013 produces two types of lipases (extracellular and cell‐bound). Both enzymes were tested for their hydrolytic ability to p‐nitrophenyl esters and compounds having a structure similar to the original substrate (triacylglycerols). Higher lipolytic activity of extracellular lipase was observed when triacylglycerols of medium‐ (C12) and long‐ (C18) chain fatty acids were used as substrates. Cell‐bound lipase preferentially hydrolysed trimyristate (C14). The differences in the abilities of these two enzymes to hydrolyse p‐nitrophenyl esters were observed as well. The order of extracellular lipase hydrolysis relation velocity was as follows: p‐nitrophenyl decanoate > p‐nitrophenyl caprylate > p‐nitrophenyl laurate > p‐nitrophenyl palmitate > p‐nitrophenyl stearate. The cell‐bound lipase indicates preference for p‐nitrophenyl palmitate. The most striking differences in the ratios between the activity of both lipases (extracellular : cell‐bound) towards different fatty acid methyl esters were 2.2 towards methyl hexanoate and 0.46 towards methyl stearate (C18). The Michaelis constant (K_m) and maximum reaction rate (V_max) for p‐nitrophenyl palmitate hydrolysis of cell‐bound lipase were significantly higher (K_m 2.462 mM and V_max 0.210 U/g/min) than those of extracellular lipase (K_m 0.406 mM and V_max 0.006 U/g/min). Copyright © 2010 John Wiley & Sons, Ltd.     

Cloning and sequencing of the URA3 gene of Kluyveromyces marxianus CBS 6556

The URA3 gene, coding for orotidine-5′-phosphate decarboxylase, from Kluyveromyces marxianus CBS 6556, was isolated from a genomic DNA library. The K. marxianus URA3 gene encodes a protein of 267 amino acids with a calculated molecular weight of 29·3 kDa. Comparison of the K. marxianus protein with the corresponding enzymes of Saccharomyces cerevisiae and Kluyveromyces lactis showed amino acid sequence identifies of 81% and 88%, respectively. Using contour-clamped homogeneous electric field gel electrophoresis, the genomic copy was found to be located on chromosome VI. We have used the cloned gene for the construction of a K. marxianus leu2 mutant. This mutant contains no heterologous sequences, which is essential to make it acceptable for application in the food industry.     

Lipase-catalysed production of triacylglycerols enriched in pinolenic acid at the sn-2 position from pine nut oil

BACKGROUND: The purpose of this study was to produce triacylglycerols (TAGs) enriched in pinolenic acid (PLA) at the sn‐2 position using the principle of acyl migration, from the pine nut oil containing PLA esterified exclusively at the sn‐3 position. RESULTS: Two types of lipase‐catalysed reactions, i.e. redistribution and reesterification of fatty acids, were successively performed using seven commercially available lipases as biocatalysts. Of the lipases tested, Novozym 435 and Lipozyme TL IM were effective biocatalysts for positioning PLA at the sn‐2 location. These biocatalysts were selected for further evaluation of the effects of reaction parameters, such as temperature and water content on the migration of PLA residues to the sn‐2 position and TAG content. For both lipases, a significant decrease in TAG content was observed after the lipase‐catalysed redistribution of fatty acids for both lipases. The reduced TAG content could be enhanced up to approx. 92%, through lipase‐catalysed re‐esterification of the hydrolysed fatty acids under vacuum. CONCLUSION: TAG enriched in PLA at the sn‐2 position was synthesised from pine nut oil via lipase‐catalysed redistribution and re‐esterification of fatty acid residues using Lipozyme TL IM and Novozym 435 as biocatalysts. Copyright © 2011 Society of Chemical Industry     

Concentration of n‐3 polyunsaturated fatty acids in cholesterol‐reduced cod‐liver oil by lipases

This study was conducted to elucidate the effects of different lipases originated from Candida rugosa (CR), porcine pancreas (PP) and Aspergillus niger (AN) on the degree of hydrolysis (DH) in cholesterol‐reduced cod‐liver oil (87.5%) and evaluate the changes in n‐3 polyunsaturated fatty acid concentrations in the oil hydrolysed by the lipases. The lipase‐catalysed hydrolysis of cholesterol‐reduced cod‐liver oil was performed at 37 °C for 8 h. Among all the lipase samples studied, DH in the oil after lipase‐catalysed hydrolysis followed the decreasing order: CR lipase (70.01%) > PP lipase (26.18%) > AN lipase (18.57%). Triacylglycerol levels in the oil hydrolysed by all the lipases studied decreased, while mono‐ and diacylglycerol levels increased during lipase‐catalysed hydrolysis. The eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) concentrations in cholesterol‐reduced cod‐liver oil hydrolysed by the CR lipase were remarkably higher than those by the PP or AN lipase. Thus, it is suggested in this study that the CR lipase appears to be most suitable for producing and n‐3 polyunsaturated fatty acids including EPA and DHA concentrates from cholesterol‐reduced cod‐liver oil.     

Enrichment of gamma‐linolenic acid from fungal oil by lipases

Abstract

Two microbial lipases from Geotrichum spp and Rhizopus spp isolated previously in this laboratory and a commercial lipase from Candida cylindracea were evaluated as biocatalysts for the enrichment of gamma‐linolenic acid (GLA, 18:3 n‐6) from a fungal oil derived from Mucor spp. Lipase from Geotrichum spp, as compared to lipases from Candida cylindracea and Rhizopus spp was found to be most effective in the enrichment of GLA in unesterified fatty acids upon esterification of the fungal oil fatty acids with n‐butanol. The level of GLA in the unesterified fatty acid could be raised from 10.9 % in the starting material to about 30 % in the unesterified fatty acids after a reaction period of 4 h. Selective hydrolysis of the fungal oil using lipase from Rhizopus spp resulted in about 1.5‐fold enrichment of gamma‐linolenic acid in the unhydrolysed acylglycerols. The other Upases tested were ineffective in the enrichment of GLA by selective hydrolysis of the fungal oil triacylglycerols.     

SUBCUTANEOUS ADIPOSE TISSUE LIPOLYSIS IN THE PROCESSING OF DRY-CURED HAM

ABSTRACT The activities of subcutaneous adipose tissue lipases and esterases were assayed at different stages (0 to 15 months) in the processing of dry-cured ham. The formation of free fatty acids during the process was also determined. Maximal generation of free fatty acids occurred during the first 10 months. Simultaneously, the triglyceride content decreased while the diglycerides increased during the aging period. Neutral and basic lipases showed maximal activity at the beginning of the process but only neutral lipase remained as the main enzyme responsible for the reported lipolysis during the drying ripening stages. Adipose tissue esterases showed excellent stability but the generation of volatile free fatty acids was negligible, suggesting a minor role of these enzymes.     

Hydrolysis of Milk Fat By Lipase in Solvent-free Phospholipid Reverse Micellar Media

Butterfat was hydrolyzed with lipases contained in lecithin reverse micelles. The influence of pH, temperature, molar ratio of surfactant to water (R) and surfactant concentration on the hydrolytic reaction indicated linear, quadratic, and interactive effects for reaction systems mediated by R. javanicus and C. rugosa lipases. The initial reaction rate was dependent on reaction parameters; however, the degree of hydrolysis was independent of pH. Both enzymes exhibited high thermal stability. The content and composition of milk fat hydrolysates prepared by R. javanicus lipase were most influenced by reaction temperature and R. The optimum conditions for production of free fatty acids, monoacyl-glycerols, diacylglycerols and specific regio-isomers were defined.     

Kinetic Properties and Enantioselectivity of The Lipases Produced by Four Aspergillus Species

Four high lipase-producing Aspergillus species, selected in our laboratory, were compared in terms of their stability and reactivity for enantioselective esterification between (R, S)-2-octanol with octanoic acid in n-hexane. We determined the pH and temperature reactions dependences of lipases activities, and we found that these enzymes exhibited various pH sensitivities. The optimum pH observed for Aspergillus terreus lipase was 5.5, for A. niger and A. oryzae lipases in the range of 6.0 to 6.5 and pH 7.0 for A. flavus lipase. Good stability was observed at pH ranging from 5.0 to 8.5 after 24 hours at 40° C, and the optimum activity was observed at 35–40° C for all lipases tested. The lipases from A. terreus and A. niger were highly thermostable, retaining 60% and 50% activity at 60° C after 1 hour, respectively. The lipases from A. niger and A. terreus lipases provided the best results in terms of enantioselectivity (E) in the esterification of (R, S)-2-octanol with octanoic acid in n-hexane (E = 4.9 and E = 4.5, respectively). These properties make these lipases good candidates for biocatalysis in organic media.     

Isolation and characterization of Kluyveromyces marxianus mutants deficient in malate transport

In malic acid-grown cells of the strains ATCC 10022 and KMS3 of Kluyveromyces marxianus the transport of malic acid occurred by a malate-proton symport, which accepted l-malic, d-malic, succinic and fumaric acids, but not tartaric, malonic or maleic acids. The system was inducible and subjected to glucose repression. Mutants of the strain KMS3, unable to grow in a medium with malic acid, were isolated and checked for their capacity to utilize several carbon sources and to transport dicarboxylic acids by the malate-proton symport. Two distinct clones affected on malate transport were obtained. Both were able to grow on a medium with glycerol or ethanol but not with dl-malic, succinic, oxoglutaric and oxaloacetic acids as the sole carbon and energy sources. However, while one of the mutants (Mal7) displayed activity levels for the enzymes malate dehydrogenase, isocitrate lyase, and phosphoenolpyruvate carboxykinase similar to those of the wild strain, in the other mutant type (Mal6) the activities for the same enzymes were significantly reduced. Plasma membranes from derepressed cells of the wild strain and of the mutants Mal6 and Mal7 were isolated and the protein analysed by SDS–PAGE. The electrophoretic patterns of these preparations differed in a polypeptide with an apparent molecular mass of about 28 kDa, which was absent only in the mutant Mal7. The results indicated that Mal7 can be affected in a gene that encodes a malate carrier in K. marxianus. © 1998 John Wiley & Sons, Ltd.     

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non‐homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR‐amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour‐intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ‐mediated integrative transformation with PCR‐amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus. Copyright © 2013 John Wiley & Sons, Ltd.     

Synergistic effect of peppermint and garlic essential oil on fate of Kluyveromyces marxianus in Zucchini Bouranee

The purpose of the study was to evaluate the inhibitory effect of garlic (GEO) and peppermint (PEO) essential oils on Kluyveromyces marxianus (K. marxianus) in zucchini bouranee and also to assess the product's functional physicochemical properties during storage. The results showed that the combined use of the GEO and PEO caused a faster decrease in the yeast count. The growth rate of K. marxianus reduced significantly with increasing the EOs concentration (p < .05). No significant difference was found between amounts of the dry matter of the treatments until the 14th day. The syneresis percentages were significantly different between the treatments during storage. Meanwhile, the GEO treatments had a higher syneresis rate compared with the PEO treatments (p < .05). The organoleptic assays showed that the GEO 0.05% treatment obtained the highest score. As a result, the antimicrobial properties of the EOs, especially GEO, could increase the zucchini bouranee shelf life.     

Flavour development in dairy cream using fish digestive lipases from Chinook salmon (Oncorhynchustshawytscha) and New Zealand hoki (Macruronusnovaezealandiae)

Digestive lipases from Chinook salmon and New Zealand hoki were evaluated as flavour modifying agents in dairy products. Cream was incubated either with fish lipase or commercially available lipases used in dairy flavour development – calf pregastric esterase (Renco™ PGE) and microbial lipase (Palatase® 20,000 L). The fish enzymes were more similar to calf PGE in terms of the total amount and types of fatty acids released over the course of the reaction. Like the pregastric esterase, the fish enzymes released mainly short chain fatty acids. The highest specificity was towards the key dairy product flavour and odour compounds, butanoic and hexanoic acids. The odour intensity of hexanoic acid produced by the salmon lipase, as measured by SPME–GC–MS, was similar to that produced by both Palatase® and PGE. Free fatty acid composition, together with sensory characteristics of lipase-treated creams, demonstrated the potential for flavour enhancement in dairy products using fish lipases.     

Phytochemical Composition and in Vitro Analysis of Nopal (O. Ficus-Indica) Cladodes at Different Stages of Maturity

This study aimed to determine the phytochemical profile and nutraceutical properties of nopal cladodes (Opuntia ficus-indica) at different stages of maturity. Medium-age cladodes showed the highest total saponins, phytosterols, and indigestible fiber, as well as the highest in vitro antioxidant capacity and digestive enzymes inhibitory activity. Furthermore, these cladodes presented the highest content of p-hydroxybenzoic acid, p-coumaric acid, rutin, narcissin, nicotiflorin, β-sitosterol, and sitosteryl-3-β-glucopyranoside, as well as several amino acids, organic acids, and fatty acids. Whereas young cladodes contained the highest concentration of condensed and hydrolyzable tannins. These results demonstrated that maturity affects the nutritional and nutraceutical properties of nopal cladodes.     

Gel Matrix Influence on Hydrolysis of Triglycerides by Immobilized Lipases

The hydrolytic activities and specificities of gel-entrapped C. cylindracae lipase (CCL) and R. arrhizus lipase (RAL) toward olive oil and tributyrin were investigated. Lipases in hydrophobic gels with the longest chain lengths generally displayed highest activity. The optimal temperature was 30–35° for free and 37–40° for gel-entrapped lipases. The ratio of the activity on tributyrin to that of olive oil (expressed as T/O ratio), an indicator of substrate specificity, increased from 0.3 for free lipases to 12.3 ± 2.3 for CCL lipase in ENTP-2000-formed gel and 16.2 ± 0.3 for RAL lipase in ENTP-4000-formed gel.     

Lipase Mediated Synthesis of Low Molecular Weight Flavor Esters

Screening 27 commercial lipases showed that enzymes from Candida cylindracea, Pseudomonas fluorescens and Mucor miehei (immobilized) promoted synthesis of selected low molecular weight esters in nonaqueous systems. Maximum production after 24 hr incubation was obtained with substrate concentrations of 0.05 mol/L for isopentyl acetate, 0.2 mol/L for ethyl butyrate and 0.3 mol/L for isopentyl butyrate. Yield of butyl butryate was almost 100% at acid substrate greater than 0.2 mol/L. Substrate inhibition was observed with P. fluorescens lipase but not with C. cylindracea or M. miehei lipases, up to 1 mol/L. Hexane, octane and decane could be used as reaction media except for ethyl butyrate synthesis where hexane was the medium of choice. Poor synthesis was achieved when methylene chloride was used.     

THERMAL STABILIZATION OF FOUR MICROBIAL β-GALACTOSIDASES BY HISTIDINE, CASEIN AMINO ACIDS AND CASEIN

Thermal stability of four microbial β-galactosidases was evaluated in the presence of casein, casein amino acids and histidine. The enzymes from Esch-erichia coli, Kluyveromyces marxianus and Streptococcus thermophilus were stabilized up to 60 fold by these additives, but there was little effect on the enzyme from Aspergillus oryzae. Stabilization by casein amino acids and histidine was largely lactose dependent. Casein amino acids were not as effective as casein for any enzyme. For the K. marxianus enzyme, histidine alone was as effective as casein and the stabilizing effect was proportional to the logarithm of the histidine concentration.     

Encapsulated whey–native yeast Kluyveromyces marxianus as a feed additive for animal production

ABSTRACT

Whey is the main byproduct of the cheese industry. While the composition is variable, it retains up to 55% of milk nutrients. The beneficial features of whey indicates a promising source of new potentially probiotic strains for the development of food additives destined for animal production. The aim of this study was to identify Kluyveromyces spp. isolated from whey, to study some probiotic properties and to select the best strain to be encapsulated using derivatised chitosan. Kluyveromyces marxianus strains (VM003, VM004 and VM005) were isolated from whey and identified by phenotypic and molecular techniques. These three yeast strains were able to survive under gastrointestinal conditions. Moreover, they exhibited weak auto-aggregation and co-aggregation with pathogenic bacteria (Salmonella sp., Serratia sp., Escherichia coli and Salmonella typhimurium). In general the K. marxianus strains had a strong antimicrobial activity against pathogenic bacteria. The potential probiotic K. marxianus VM004 strain was selected for derivatised-chitosan encapsulation. Material treated with native chitosan exhibited a strong antimicrobial activity of K. marxianus, showing a total growth inhibition at 10 min exposure. However, derivatised-chitosan encapsulation showed a reduced antimicrobial activity. This is the first study to show some probiotic properties of whey–native K. marxianus, in vitro. An encapsulation strategy was applied using derivatised chitosan.     

A novel esterase from Saccharomyces carlsbergensis,a possible function for the yeast TIP1 gene

An extracellular esterase was isolated from the brewer's yeast, Saccharomyces carlsbergensis. Inhibition by diisopropyl fluorophosphate shows that the enzyme has a serine active site. By mass spectrometry, the molecular weight of the enzyme was 16·9 kDa. The optimal pH for activity was in the range of four to five. Esterase activity was found in beer before pasteurization, and a low level of activity was still present after pasteurization. Caprylic acid, which is present in beer, competitively inhibited the esterase. The substrate preference towards esters of p-nitrophenol indicated that the enzyme prefers esters of fatty acids from four to 16 carbon atoms. The esterase has lipolytical activity; olive oil (C-18:1), which is a classical substrate for lipase, was hydrolysed. N-terminal sequence analysis of the esterase yielded a sequence which was identical to the deduced amino acid sequence of the S. cerevisiae TIP1 gene. The esterase preparation did not appear to contain significant amounts of other proteins than Tip1p, indicating that the TIP1 gene is the structural gene for the esterase. © 1998 John Wiley & Sons, Ltd.     

Cloning and Sequence Analysis of the estA gene encoding enzyme for producing (R)-β-acetylmercaptoisobutyric acid from Pseudomonas aeruginosa 1001

The estA gene encoding the enzyme that catalyzes the production of (R)-β-acetylmercaptoisobutyric acid from (R,S)-ester from Pseudomonas aeruginosa 1001, was cloned in Escherichia coli and its nucleotide sequence was determined, revealing the presumed open reading frame encoding a polypeptide of 316 amino acid residues (948 nucleotides). The overall A+T and C+G compositions were 32.59% and 67.41%, respectively. The amino acid sequence of the estA gene product showed a significant similarity with that of the triacylglycerol lipase from Psychrobacter immobilis (38% identity), triacylglycerol lipase from Moraxella sp. (36% identity), and two forms of carboxyl esterases from Acinetobacter calcoaceticus (17% and 17% identities). The deduced amino acid sequences have a pentapeptide consensus sequence, G-X-S-X-G, having an active serine residue, and another active site, dipeptides H-G, located at 70–100 amino acids upstream of the G-X-S-X-G consensus sequence.     

Ileal digestibility of defatted soybean,lupin and chickpea seed meals in cannulated Iberian pigs: I. Proteins

Five castrated male Iberian pigs (100 ± 2 kg body weight) fitted with T‐shaped cannulas at the terminal ileum were used to determine ileal digestibility of legume seed meals. The diets were based on defatted soybean, lupin or chickpea seed meals and contained similar levels of digestible energy (14.2–15.1 kJ g^?1) and protein (107 g kg^?1). Protein‐free and a hydrolysed casein diets were used to study endogenous ileal amino acid flows. Chromium oxide (10 g kg^?1 diet) was added to the diets as an indigestible marker. Ileal flows in pigs fed the hydrolysed casein diet were different (p < 0.05) in amino acid contents and composition from those on the protein‐free diet. Ileal sialic acid flows in pigs fed lupin‐ or chickpea‐based diets were higher (p < 0.05) than those of animals fed soybean or casein diets. Among essential amino acids, only the apparent ileal digestibilities of phenylalanine and valine in lupin meal were lower (p < 0.05) than those in soybean. Apparent ileal digestibilities of lupin aspartate and proline, together with chickpea aspartate, were also lower (p < 0.05) than those of soybean. True ileal digestibility of nitrogen in pigs fed lupin or chickpea meals, calculated according to values from animals fed the protein‐free diet, was lower (p < 0.05) than that for soybean or casein. Among individual essential amino acids, only the true ileal digestibility of phenylalanine in lupin was lower (p < 0.05) than that in soybean. True ileal digestibility of nitrogen calculated according to values obtained with pigs fed a hydrolysed casein diet was not different among soybean, lupin or chickpea meals. Among essential amino acids, only the true ileal digestibilities of isoleucine and lysine in chickpea were lower (p < 0.05) than those of soybean. It is concluded that true ileal nitrogen and amino acid digestibilities of lupin and chickpea meals are comparable to those of defatted soybean in Iberian pigs. The results with protein‐free diets tended to underestimate endogenous protein secretion in pigs fed on diets containing protein. Copyright © 2005 Society of Chemical Industry