Lipase-producing Microorganisms Research Articles

Activation and inhibition of Candida rugosa and Bacillus-related lipases by saturated fatty acids, evaluated by a new colorimetric microassay.

Research on lipase inhibitors could help in the therapy of diseases caused by lipase-producing microorganisms and in the design of novel lipase substrate specificities for biotechnology. Here we report a fast and sensitive colorimetric microassay that is low-cost and suitable for high-throughput experiments for the evaluation of lipase activity and inhibition. Comparison of Candida rugosa activity and inhibition with previous HPLC results validated the method, and revealed the importance of the reaction mixture composition. The assay was used to evaluate the effect of saturated fatty acids on Bacillus-related lipases. Cell-bound esterases were strongly inhibited by fatty acids, suggesting a negative feedback regulation by product, and a role of these enzymes in cell membrane turnover. Bacillus subtilis LipA was moderately activated by low concentrations of fatty acids and was inhibited at greater concentrations. LipB-like esterases were highly activated by myristic and lauric acids and were only slightly inhibited by high capric acid concentrations. Such an activation, reported here for the first time in bacterial lipases, seems to be part of a regulatory system evolved to ensure a high use of carbon sources, and could be related to the successful adaptation of Bacillus strains to nutrient-rich environments with strong microbial competition.

Screening and catalytic activity in organic synthesis of novel fungal and yeast lipases

A total of 969 microbial strains were isolated from soil samples and tested to determine their lipolytic activity by employing screening techniques on solid and in liquid media. Ten lipase-producing microorganisms were selected and their taxonomic identification was carried out. From these strains Achremonium murorum, Monascus mucoroides, Arthroderma ciferri, Fusarium poae, Ovadendron sulphureo-ochraceum and Rhodotorula araucariae are described as lipase-producers for the first time. Hydrolysis activity of the crude lipases against both tributyrin and olive oil was measured. Heptyl oleate synthesis was carried out to test the activity of the selected lipases as biocatalysts in organic medium. All the selected lipases were tested as biocatalysts in several organic reactions using unnatural substrates. Lipases from the fungi Fusarium. oxysporum and O. sulphureo-ochraceum gave the best yields and enantioselectivities in the esterification of carboxylic acids. F. oxysporum and Penicillium chrysogenum lipases were the most active ones for the acylation of alcohols without steric hindrance. A. murorum lipase is very useful for the esterification of menthol. F. oxysporum and Fusarium. solani lipases were very stereoselective in the synthesis of carbamates.

Novel microbial lipases: catalytic activity in reactions in organic media.

2,000 microbial strains were isolated from soil samples and tested to determine their lipolytic activity by employing screening techniques on solid and in liquid media. Culture broths were initially tested with 1,2-O-dilauryl-rac-glycero-3-glutaric acid-resorufinyl ester, a chromogenic substrate specific for lipases. Fourteen lipase-producing microorganisms were selected and their taxonomic identification was carried out. Hydrolysis of tributyrin or olive oil and the esterification of oleic acid with heptanol were selected to preliminary evaluate the catalytic activity of these lipases. All the selected lipases catalysed this esterification reaction with good yields. Resolution of (R,S)-2-(4-isobutylphenyl) propionic acid, (R,S)-1-phenylethanol, (R,S) 1-phenylethylamine and of (R) or (S) glycidol were performed to evaluate the stereoselectivity of these novel enzymes as biocatalysts in reactions in organic media. Lipases from the fungi Fusarium oxysporum and Ovadendron sulphureo-ochraceum gave the best yields and enantioselectivities in the resolution of racemic ibuprofen and 1-phenylethanol. Several lipases displayed a high stereoselectivity in the resolution of chiral amines by an alcoxycarbonylation reaction.

Three New Lipases from Actinomycetes and Their Use in Organic Reactions

Three novel lipase-producing microorganisms have been isolated from 526 actinomycete strains by employing screening techniques on solid media. Time-course and scale-up of enzyme production were analyzed. The lipases, produced by microorganisms belonging to the Streptomyces genus, were tested in several reactions in organic medium using unnatural substrates. The lyophilized crude lipases are stable at least for 1 month at 4°C (100% recovered activity). The lipase activity per milliliter of cell culture broth was higher than described in the literature for other lipases from actinomycetes. The three selected lipases displayed better activity than commercial lipase from Candida rugosa in the resolution of chiral secondary alcohols. The lipase from S. halstedii also displayed very good activity in the synthesis of carbamates.

Use of continuous culture to screen for lipase-producing microorganisms and interesterification of butter fat by lipase isolates

The continuous cultivation technique was used to investigate the screening for lipase-producing microorganisms from four commercial starters suitable for the degradation of domestic wastes. Using this technique, three strains of lipase-producing bacteria were isolated and identified: Pantoea agglomerans (BB96CC1, BB168CC2) and Pseudomonas fluorescens (BW96CC1). In addition, butter fat induced more lipase production when present in the growth medium. Interesterification of butter fat triacylglycerols by enzymatic extracts of the isolated strains of microorganisms resulted in an appreciable interesterification yield, implying that hydrolysis was suppressed and interesterification of butter fat triacylglycerols was maximized in a microemulsion free-cosurfactant system.

Screening and selection of a microbial lipase for the stereospecific hydrolysis of Verlukast

A search was implemented for a microbial lipase capable of bioconverting a diester (dimethyl 5-(3-(2-(7-chloroquinolin-2-yl)ethyl)phenyl)4,6-dithianon to its S-ester acid, an intermediate in the production of Verlukast (a leukotriene receptor antagonist). Required properties of the sought-after enzyme included a high enantiomeric selectivity (e.e. >98%), the formation of only trace amounts of diacid and a high bioconversion rate. This search yielded 57 lipase-producing microorganisms, 18 of which presented detectable bioconversion activity. Thirteen of these microbes were selected for further study based upon their lipase production level and enzyme stability at harvest. Despite their common enzymatic property, namely the hydrolysis of triglycerides, these lipase preparations presented diverse ester acid specific synthesis rates (from <0.01 μg/unit/h to 0.98 μg/unit/h) and diacid formation levels (from 0% to 35%). One of these microbes, identified asPseudomonas aeruginosa (strain MB 5001), was found to produce a lipase having all of the above-listed required properties. The initial fermentation process developed in shake flasks was rapidly and successfully scaled up in 23-liter labora bioreactors, achieving a maximum production of 35 units/ml of lipase after 48 h of cultivation.

Screening for lipase-producing microorganisms with a continuous cultivation system

The enrichment of lipase-producing bacteria, isolated from the environment, was evaluated using a continuous cultivation system. Continuous cultivations were performed using a synthetic medium where the carbon and energy source, triolein, was provided physically entrapped in an external loop. Using this system, pure cultivations of Pseudomonas fluorescens 378 showed an increase in lipase-producing ability, instead of declining as in more conventional systems. Enrichment cultures were obtained with environmental samples originating from a vegetable oil processing plant using the same system. Three types of lipase-producing bacteria were identified: P. alcaligenes, Enterobacter intermedium and a Gram-negative, oxidase-positive rod.

Isolation and Identification of Alkaline Lipase Producing Microorganisms, Cultural Conditions and Some Properties of Crude Enzymes

Two bacterial strains 26.IB (I) and 22.39B (II) were isolated from soil as alkaline lipase producing microorganisms. Strain 26. IB (I) was identified as Pseudomonas nitroreducens nov. var. thermotolerans, and strain 22.39B (II) was identified as Ps. fragi. When they were cultivated aerobically in a 20-liter jar fermentor in medium containing 2.0% soybean meal, they secreted a large amount of alkaline lipases. The enzymes were recovered efficiently as precipitates by adjusting the pH of the culture fluids to 4.0 with HC1. The enzymatic characteristics included: optimum pH, 9.5 (I,II); stable pH range, 5~11 (I,II); optimum temperatures, 50°C (I) and 75~80°C (II); and more than 95% of the enzyme activity remained after incubated at 70°C for 20 min (I,II). Lipase activities were inhibited remarkably in the presence of anionic surfactants (I,II) and bile salts (I,II).