Lipase Induction in Mucor hiemalis

The influence on lipase induction in Mucor hiemalis of different types of triglycerides containing mainly oleic acid (olive oil), erucic acid (mustard oil), or saturated fatty acids of 8 to 16 carbons (coconut oil) was studied. The fungus was grown in shake flasks in a fermentation medium containing peptone, minerals, and glucose or one of the oils as the carbon source. Maximum lipase was produced when the initial pH of the fermentation medium was kept at 4.0. Addition of Ca²⁺ to the medium did not increase lipase production. The optimum pH for activity of both the mycelial and extracellular lipases was found to be 7.0. The fungus produced a significant amount of lipase in the presence of glucose, but the lipase activity increased markedly when olive oil was added to the medium at the beginning of the fermentation. Addition of olive oil at a later stage did not induce as much enzyme. Studies with washed mycelia showed that a greater amount of lipase was released when olive oil was present than when glucose was present. Among the various types of triglycerides used as the carbon source, olive oil was found to be most effective in inducing the lipase. Olive oil and mustard oil fatty acids inhibited the lipase more than those of coconut oil. The lipase induced by a particular type of triglyceride did not seem to be specific for the same triglyceride, nor was it inhibited specifically by it. Irrespective of the triglyceride used in the fermentation medium, the lipase produced was most active against coconut oil triglyceride, and this specificity, as shown by lipase activities in an n-heptane system, was not found to be due to a better emulsification of this oil. The lipase of M. hiemalis can be considered to be both constitutive and inducible.     

Alkaline lipase activity from the marine protists,thraustochytrids

Two thraustochytrid protists of the genus Thraustochytrium isolated from coastal and mangrove habitats of Goa, India were studied for extracellular alkaline lipase production. Maximum lipase production was supported by a combination of peptone and yeast extract in the growth medium while strong inhibition of enzyme production was observed in presence of glucose. The inducible nature of the enzyme production was evidenced by the requirement of olive oil in the medium. Lipase production was salt-dependent and optimum production required 3.4% (w/v) crude sea salt. Ideal conditions for maximum production of lipases were therefore adopted as incubation at 30 ± 2°C for 168 h at an initial pH of 6.0 in a medium consisting of 0.5% peptone, 0.01% yeast extract, 0.5% olive oil and 3.4% crude salt. Extracellular lipase production by the two thraustochytrid isolates [designated TZ (ATCC #PRA-295) and AH-2 (ATCC #PRA-296)] was increased threefold under these optimized culture conditions. This appears to be the first report on optimization of cultivation conditions for the production of alkaline lipases by thraustochytrids.     

Lipase production by Trichosporon fermentans WU-C12, a newly isolated yeast

From the soil samples of various locations, 245 strains of microorganisms were isolated by the enrichment culture method using olive oil as a carbon source. Of these microorganisms one deuteromycotinous yeast was the best producer of extracellular lipase, and the strain WU-C12 was identified as Trichosporon fermentans from the morphological and taxonomical properties. When cultivated at 30°C for 4 d in the medium containing 8% (w/v) corn steep and 3% (v/v) olive oil as sources of nitrogen and carbon, T. fermentans WU-C12 produced 126 U/ml of extracellular lipase. When 3% (v/v) tung oil was used instead of 3% (v/v) olive oil, 146 U/ml of the lipase was produced. Although lipase production decreased to 40 U/ml by the addition of 2% (w/v) glucose to the corn steep-olive oil medium, the strain WU-C12 produced 34 U/ml of lipase in the medium containing 2% (w/v) glucose instead of 3% (v/v) olive oil. On the other hand, T. fermentans WU-C12 could grow and produce lipase in the medium containing n-paraffin as a carbon source.     

Production of lipase free of citrinin by Penicillium citrinum

Lipase (Glycerol ester hydrolase E.G. 3.1.1.3) from a Brazilian strain of Penicillium citrinum free of the mycotoxin citrinin has been investigated. Citrinin production was inhibited by using culture medium containing olive oil, soybean oil and corn oil as carbon sources. Potassium concentration and pH play an important role in citrinin production. Potassium concentration lower than 30 mM and pH below 4.5 inhibited the mycotoxin production. P. citrinum produced lipase free of extraneous proteins and citrinin when cultured using, as nitrogen source, ammonium sulphate (lipase activity of 7.88 U/mg) and yeast extract (lipase activity of 4.95 U/mg) with olive oil as carbon source. This data is relevant to the larger scale production of lipases for food technology applications, from Penicillium citrinum.     

Influence of carbon and nitrogen sources on lipase production by a newly isolated Candida viswanathii strain

Microorganisms can produce lipases with different biochemical characteristics making necessary the screening of new lipase-producing strains for different industrial applications. In this study, 90 microbial strains were screened as potential lipase producers using a sensitive agar plate method with a suitable medium supplemented with Tween 20 and also a liquid culture supplemented with olive oil. The highest cell growth and lipase production for Candida viswanathii were observed in triolein and oleic acid when used as the only pure carbon source. Renewable low-cost triacylglycerols supported the best cell growth, and olive oil was found to be the best inducer for lipase production (19.50 g/L and 58.50 U). The selected conditions for enzyme production were found with yeast extract as nitrogen source and 1.5 % (w/v) olive oil (85.70 U) that resulted in a good cell growth yield (Y_X/S?=?1.234 g/g) and lipase productivity (1.204 U/h) after 72 h of shake-flask cultivation. C. viswanathii lipase presented high hydrolytic activity on esters bonds of triacylglycerols of long-chain, and this strain can be considered an important candidate for future applications in chemical industries.     

Quantitative study of lipase secretion, extracellular lipolysis, and lipid storage in the yeast Yarrowia lipolytica grown in the presence of olive oil: analogies with lipolysis in humans

Lipase secretion, extracellular lipolysis, and fatty acid uptake were quantified in the yeast Yarrowia lipolytica grown in the presence of olive oil and/or glucose. Specific lipase assays, Western blot analysis, and ELISA indicated that most of the lipase activity measured in Y. lipolytica cultures resulted from the YLLIP2 lipase. Lipase production was triggered by olive oil and, during the first hours of culture, most of the lipase activity and YLLIP2 immunodetection remained associated with the yeast cells. YLLIP2 was then released in the culture medium before it was totally degraded by proteases. Olive oil triglycerides were largely degraded when the lipase was still attached to the cell wall. The fate of lipolysis products in the culture medium and inside the yeast cell, as well as lipid storage, was investigated simultaneously by quantitative TLC–FID and GC analysis. The intracellular levels of free fatty acids (FFA) and triglycerides increased transiently and were dependent on the carbon sources. A maximum fat storage of 37.8% w/w of yeast dry mass was observed with olive oil alone. A transient accumulation of saturated FFA was observed whereas intracellular triglycerides became enriched in unsaturated fatty acids. So far, yeasts have been mainly used for studying the intracellular synthesis, storage, and mobilization of neutral lipids. The present study shows that yeasts are also interesting models for studying extracellular lipolysis and fat uptake by the cell. The quantitative data obtained here allow for the first time to establish interesting analogies with gastrointestinal and vascular lipolysis in humans.     

Lipase location in Yarrowia lipolytica cells

Lipase production by Yarrowia lipolytica was growth associated and, at the beginning of cultivation, it was mainly cell-bound. Lipase release into the culture medium started when about 50% of the carbon source (olive oil or glucose) was consumed reaching its maximum concentration in the late stationary phase.     

Regioselective hydrolysis of acetates in the presence of different yeast strains

The model compound, hexane-1,2-diol diacetate, was hydrolyzed in the presence of supernatant obtained after cultivation of 4 yeast strains: Pichia jadinii, Rhodotorula glutinis and Yarrowia lipolytica KKP 379 and Saccharomyces cerevisiae 102 to evaluate the type of catalysis. The regioselectivity of extracellular enzymes as a function of hydrolysis towards primary and secondary acetic acid ester groups was monitored. The enzymes secreted by P. jadinii, R. glutinis and Y. lipolytica KKP 379 exhibited high regioselectivity towards primary position, while those from S. cerevisiae showed practically no discrimination between the ester groups.     

Investigation of lipase production by a new isolate of Aspergillus sp.

Fungi isolated from soil were screened for exogenous lipolytic activity. The highest lipase activity was found in a new soil isolate of Aspergillus sp. Some optimal cultural parameters influencing the growth and production of extracellular lipase from this Aspergillus sp. were investigated. The lipase yield was maximum on day 4 of incubation of the culture at pH 5.5 and 30 °C. When the medium was prepared using olive oil as carbon source and peptone as a nitrogen source, better lipase yields were obtained. Aeration enhanced growth and lipase production.     

Diversity of lipase-producing yeasts from marine environments and oil hydrolysis by their crude enzymes

Total 427 yeast strains from seawater, sediments, mud of salterns, guts of the marine fish and marine algae were obtained. After lipase activity of the yeast cultures was estimated, we found that nine yeast strains obtained in this study grown in the medium with olive oil could produce lipase. The results of routine identification and molecular methods show that they belonged toCandida intermedia YA01a,Pichia guilliermondii N12c,Candida parapsilosis 3eA2,Lodderomyces elongisporus YF12c,Candida quercitrusa JHSb,Candia rugosa wl8,Yarrowia lipolytica N9a,Rhodotorula mucilaginosa L10-2 andAureobasidium pullulans HN2.3, respectively. The optimal pHs and temperatures of lipases produced by them were between 6.0 and 8.5 and between 35 and 40°C, respectively. Majority of lipases from the yeast strains were cell-bound and only lipase fromA. pullulans HN2.3 was extracellular. Some lipases from the yeast strains could actively hydrolyse different oils, indicating that they may have potential applications in industry.     

Substrate specificity,regioselectivity and hydrolytic activity of lipases activated from Geotrichum sp.

Substrate specificity (typoselectivity), regioselectivity and hydrolytic activity of induced lipases from three strains (4012, 4013, 4166) of Geotrichum candidum and that of Geotrichum ludwigii (48) were investigated. The lipases were induced in two types of culture media, of which the medium containing peptone as nitrogen source was proved to give better results. Olive oil was employed as inductor for the lipase activity. Activated lipases represented mostly extracelullar lipases, which penetrated through cellular membrane into medium. The activity of cell-bound lipase was also determined. Most of lipases belong to the group of specific lipases able to hydrolyse ester bonds in the positions sn-1 and sn-3 ester of triacylglycerols (1,3-selective lipases) and display specificity to saturated fatty acids. All activated lipases from Geotrichum sp., extracellular and cell-bound, were used as biocatalyst in the blackcurrant oil hydrolysis.     

Enzymatic properties of lipase and characteristics production byLactobacillus delbrueckii subsp.bulgaricus

Some properties of an extracellular lipase produced byLactobacillus delbrueckii subsp.bulgaricus were studied. Maximum enzyme activity was found against olive and butter oil as enzyme substrates. Addition of 9% acacia gum, 0.1% Na-deoxycholate and 0.01 M CaCl₂ to the enzyme reaction mixture increased-lipase activity from 5.3 to 14.5 (FFA/mg protein/minute) at pH 6.0 and at 40° C. Maximum lipase production was reached in the presence of glucose as a sole source of carbon, wheat bran as nitrogen source, olive oil as a sole lipid source and butyric acid as fatty acid supporting the growth medium. An initial pH value of the culture medium of 6.0 and a temperature of 35° C gave the highest lipolytic activity.     

Production of lipase of Aspergillus foetidus in a batch stirred reactor

Summary Maximum lipase production byAspergillus foetidus was obtained from cultures grown in the medium of 2% olive oil and 0.5% sucrose. The optimal conditions for the production of lipases in the Multigen fermenters were found to be at 500rpm with an airflow of 1.5 liter per mimute. Immobilization of the fungal source was found to be infeasible in natural polymers.     

Optimization of extracellular lipase production by Geotrichum sp. using factorial design

Response surface methodology was employed to study the effects of carbon source (soy oil, olive oil and glucose) and nitrogen source concentrations (corn steep liquor and NH(4)NO(3)) on the lipase production by Geotrichum sp. The experiment included a 2(4) central composite rotatable design (CCRD) and four others 2(3) CCRD. According to the responses from the experimental designs, the effects of each variable were calculated and the interactions between them were determined. The response surface methodology was applied for the optimization of the nutrient concentrations in the culture medium for the enzyme production, at 30 degrees C. The optimum medium composition for lipase production by Geotrichum sp. was ammonium nitrate 2.1-2.5%, corn steep liquor 13-15% and soy oil 0.6% as carbon source, which lead to a lipase activity of about 20 U/ml. Using olive oil as carbon source, the optimum composition was ammonium nitrate 0.8-1%, corn steep liquor 13-15% and olive oil 0.6%, leading to an activity of 17 U/ml.     

Lipase production by Aspergillus terreus using mustard seed oil cake as a carbon source

An extracellular lipase-producing fungus was isolated from the garden soil of the Post Graduate Department of Botany, Utkal University, Bhubaneswar, Odisha, India and identified as Aspergillus terreus. The A. terreus strain isolated was found to be capable of producing lipase in both solid state culture and liquid static surface culture. Experiments aimed at evaluating and improving the production of lipase and at studying the culture conditions revealed that of the many different materials tested as substrates, mustard oil cake (MoC) was the best substrate for extracellular lipase production. A correlation was found between the lipase production profile and biomass development. In a study aimed at continuing this line of research, we have investigated the influence of various culture conditions, such as environmental (i.e. temperature and pH), nutritional (i.e. carbon, nitrogen, metal ions, vitamins, combined agro-wastes and growth regulators) and other factors (inoculum size and initial moisture content) on the production of lipase by A. terreus in solid state and liquid static surface cultures. We observed that optimum lipase biosynthesis occurred under the following conditions: initial pH of 6.0, 30 °C, a 96-h incubation, lactose and ammonium persulphate as the carbon and nitrogen source respectively and 80 % moisture content. Changes in the vitamins (vitamin C, riboflavin, folic acid and vitamin E) and growth regulators (gibberellic acid, kinetin, 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid) did not support enhanced lipase production. MoC and neem oil cake (NoC) added to the media at a ratio of 9:1 respectively, supported maximum lipase production. Based on these results, we concluded that controlling the various culture conditions, supplementing MoC as a substrate and nutrient source modification of the medium can spectacularly enhance lipase biosynthesis by A. terreus.     

Effect of culture conditions on batch growth ofPseudomonas fluorescens on olive oil

Summary Batch cultures ofPseudomonas fluores-cens were grown in minimal medium with olive oil as the sole carbon source. When olive oil me-dium was inoculated with cells from nutrient broth there was an initial lag phase followed by logarithmic growth. The duration of the lag phase was influenced by the incubation temperature and the growth phase of the inoculum. Both factors are known to affect lipase induction during growth in fat-free media. Maintenance of condi-tions reported to be conducive to lipase produc-tion in cultures used for inoculation ensured a minimal lag before logarithmic growth com-menced on olive oil. Growth on oil occurred when the culture was maintained at pH 6 or 7, but did not occur at pH 5 or 8.     

Physiology of lipase formation inPenicillium candidum

Penicillium candidum grew and produced lipase in a culture medium supplemented with 0.2% olive oil. Significant enzyme production required the presence of olive, oil and was prevented by cycloheximide. Polyacrylamide gel electrophoresis of filtrates from olive oil fermentations gave a single band of lipase activity (MW 80 KDa). Among the olive oil components only oleate allowed significant lipase production. Other carboxylic and saturated fatty acids containing similar or lower numbers of carbon atoms, did not cause derepression of lipase formation.     

Selection of new over-producing derivatives for the improvement of extracellular lipase production by the non-conventional yeast Yarrowia lipolytica

The non-conventional yeast Yarrowia lipolytica produces an extracellular lipase encoded by the LIP2 gene. Mutant strains with enhanced productivity were previously obtained either by chemical mutagenesis or genetic engineering. In this work, we used one of these mutants, named LgX64.81 to select new overproducing strains following by amplification of the LIP2 gene. We also developed a process for lipase production in bioreactors and compared lipase production levels in batch and fed-batch cultures. Batch culture led to a lipase production of 26450 U ml(-1) in a media containing olive oil and tryptone as carbon and nitrogen sources. Feeding of a combination of tryptone and olive oil at the end of the exponential growth phase yielded to lipase activity of 158246 U ml(-1) after 80 h of cultivation. In addition this production system developed for the extracellular lipase could also be applied for other heterologous protein production since we have demonstrated that LgX64.81 is an interesting alternative host strain.     

固液态发酵中橄榄油对Rhizopus chinensis全细胞脂肪酶的影响

主要对华根霉全细胞脂肪酶固态和液态两种发酵过程进行比较,并着重探讨不同培养方式下橄榄油对其合成活力和水解活力的影响。结果表明：液态培养较有利于菌体生长,对脂肪酶的生产也有一定的促进作用。橄榄油的加入不仅有利于菌体生长、提高脂肪酶水解活力,更可使脂肪酶的合成活力显著增加,液态发酵下的效果更为明显。橄榄油在整个发酵过程中可能既作为碳源又是脂肪酶的诱导物。另外,全细胞脂肪酶的水解活力和合成活力在固液态发酵条件下均存在不对应性,表明华根霉可能产性质不同的脂肪酶同功酶。