S5 Lipase: an organic solvent tolerant enzyme

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.     

Production of acidic lipase by a mutant of Aspergillus niger NCIM 1207 in submerged fermentation

Mutants of Aspergillus niger NCIM 1207, isolated by subjecting conidia to UV-irradiation, were tested for the production of lipase (glycerol ester hydrolase EC 3.1.1.3). Mutants UV-10 and ANCR-1 showed seven fold and five fold enhanced productivity of enzyme, respectively, over the wild strain in shake flask culture when grown in SOB medium containing 1% olive oil. Maximum lipase activity (41 IU/ml) was obtained in the culture broth when UV-10 was grown in medium supplemented with 0.5% Triton X-100. A higher concentration of oil in the medium did not help lipase production in the case of mutant UV-10. Similarly no increase in enzyme levels was observed when mutant UV-10 was grown in medium supplemented with glucose. However, the addition of glucose in the medium resulted in increased levels of lipase production by wild strain, Aspergillus niger NCIM 1207.     

Isolation of a novel lipase producing fungal isolate Aspergillus fumigatus and production optimization of enzyme

Abstract

Fungal lipases occupy a place of prominence among biocatalysts owing to their novel, multifold applications and resistance to high temperature and other operational conditions. In the present study, Aspergillus fumigatus isolated from oil-contaminated soil produced good amount of lipase activity with galactose (1%) as carbon source and peptone (0.1%) as nitrogen source after 72?h of incubation in the production medium at 45?°C and pH 10.0. The isolated enzyme was found to give its optimum reaction temperature at 40?°C and pH 9.0 with the substrate used as p-nitrophenyl benzoate. The activity of lipase was inhibited by the presence of metal ions. A 6.68-fold increase for lipase production was obtained by one variable at a time. Based on the findings of present study, lipase of A. fumigatus is a potential lipase and a candidate for industrial applications such as bioremediation, detergent, leather and pharmaceutical industries.     

Staphylococcus warneri BW 94--a new source of lipase

Staphylococcus isolated from a common Indian sweet viz. basundi was tested for its ability to produce lipase. The colorless zone of hydrolysis around the colony grown on Baird Parker agar containing egg yolk produced extracellular lipase. Colony morphology, coagulase production, haemolysis, acid production in carbohydrate medium and enzyme activity studies showed that the organism was Staphylococcus warneri. Growth of S. warneri was obtained after 11 hr at 37 degrees C, pH 7.5, while the maximum production of lipase was obtained at 30 degrees C at pH 6.5 after 9 hr of incubation. Agitation did not increase lipase production. A sudden fall in the activity of lipase was noted after 11 hr. Addition of sucrose which is a growth stimulant for Staphylococcus, did not stimulate production of lipase by these organisms. Also, addition of oleic acid, Tween 80 or ethanol did not stimulate formation of lipase.     

Overproduction of lipase by<Emphasis Type="Italic"> Yarrowia lipolytica</Emphasis> mutants

Non-genetically modified mutants with increased capacities of extracellular lipase production were obtained from Yarrowia lipolytica strain CBS6303 by chemical mutagenesis. Of the 400 mutants isolated, LgX64.81 had the highest potential for the development of an industrial lipase production process. This mutant exhibits lipase production uncoupled from catabolite repression by glucose, and a 10-fold increased productivity upon addition of oleic acid. Using a LIP2- LacZ reporter gene, we demonstrate that the mutant phenotype originates from a trans-acting mutation. The glucose uptake capacity of LgX64.81 is reduced 2.5-fold compared to the wild-type-strain, and it exhibits high lipase production on glucose medium. A trans-acting mutation in a gene involved in glucose transport could thus explain this mutant phenotype.     

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.     

产脂肪酶菌株C7828-5的筛选、鉴定以及产酶条件的优化

以花生油为唯一碳源,从海口市各地被油脂污染土样中分离筛选出1株中温碱性脂肪酶菌株C7828-5。形态学、生理生化特征和分子生物学鉴定结果表明,该菌株为铜绿假单胞菌(Pseudomonas aeruginosa)。该菌所产脂肪酶的最适温度为37℃,最适pH为8.0。优化了菌株的产酶条件,最适产酶培养基(g/L)为:蔗糖5、牛肉膏20、(NH_4)_2SO_41、MgSO_4·7H_2O 0.5、CaCl_20.5,聚乙烯醇花生油乳化液120 mL,发酵72 h,获得高达8.08 U/mL的脂肪酶表达量。     

Production and properties of an alkaline,thermophilic lipase from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> NS2W

Eighteen bacterial strains were isolated from soil samples and screened for alkaline, thermophilic lipase production. Pseudomonas fluorescens NS2W was selected and its production of lipase was optimized in shake flasks using a statistical experimental design. Cell growth and lipase production were studied in shake flasks and in a 1-l fermenter in the optimized medium. Maximum lipase yields were 69.7 and 68.7 U ml⁻¹, respectively. The optimized medium resulted in about a five-fold increase in the enzyme production, compared to that obtained in the basal medium. The lipase had an optimal activity at pH 9.0 and was stable over a wide pH range of 3–11 with more than 70% activity retention. The lipase had an optimal activity at 55°C and was stable up to 60°C with more than 70% activity retention for at least 2 h. Journal of Industrial Microbiology & Biotechnology (2002) 28, 344–348 DOI: 10.1038/sj/jim/7000254 Received 06 September 2001/ Accepted in revised form 15 March 2002     

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.     

Influence of environmental factors on lipase production by Lactobacillus plantarum

A strain of Lactobacillus plantarum, DSMZ 12028 (Deutsch Sammlung von Mikroorganismen und Zellkulturen), isolated from a Portuguese dry fermented sausage, “chouriço”, was found to produce true lipase, producing free fatty acids from triolein (olive oil). This enzymatic activity was found in whole cells, but was negligible in comparison to lipolytic activity in culture supernatant. Therefore, only extracellular activity was studied. The effect of pH, temperature and glucose concentration on extracellular lipase production was studied in continuously stirred tank reactors, the first time this technology has been used to study the production of this enzyme in lactobacilli. Maximum lipase production was achieved at a pH of 5.5 and 30?°C and was kept at a significant level over a wide range of dilution rates (0.05–0.4 h^?1); the production of lipase was still significant for low pH values, temperature and glucose concentration, conditions that are close to the ones present during chouriço ripening. The effect of glucose concentration was also studied in a batch system. The control of lipase production was found to be related both to glucose concentration in the medium and to the growth rate/dilution rate. Glucose concentration was found to be important for fast lipase production, although it did not influence the maximum lipase activity reached in a batch culture.     

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.     

Studies on the enhanced production of extracellular lipase by Staphylococcus epidermidis

Staphylococcus epidermidis isolated from spoiled frozen marine fish samples exhibited optimum lipase activity of 8.1 U within 72 h in batch fermentation. Inducible effect of different sugars, nitrogen sources, salts and metal ions were studied on enzyme production. Trybutyrin induced the enzyme production by twofold. Addition of lactose in the production medium further improved lipase production. Sodium chloride increased lipase production whereas the presence of metals in the media had an inhibitory effect. Cells of immobilized S. epidermidis in agar beads (3%) increased lipase production compared with free cells. The optimum temperature and pH for enzyme activity was 20 degrees C and 7.0 respectively. Lipase retained its 85% stability at pH 6.0 and at 40 degrees C. Immobilized cells with high lipolytic activity and stability may provide commercial advantages over conventional methods of lipase production.     

Production of Tricarboxylic Acid Anhydrides from Fatty Acids by a Lipase-producing Strain of Pseudomonas cepacia

A lipase-producing strain of Pseudomonas cepacia isolated from a soil sample was found to produce five compounds when oleic acid was added to the culture medium as lipase inducer. The five compounds were isolated by solvent extraction, silicagel column chromatography and preparative HPLC, and their structural elucidation was performed by mass spectrometry, and infrared and nuclear magnetic resonance spectroscopies. The products were identified as dec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (product 1 ), undec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (product 2 ), dodec-3-ene-I,3,4-tricarboxylic acid 3,4-anhydride (product 3 ), dodec-3,8-diene-1,3,4-tricarboxylic acid 3,4-anhydride (product 4 ) and dodec-3,6-diene-I,3,4-tricarboxylic acid 3,4-anhydride (product 5 ). Accumulation of these compounds in the culture medium started after oleic acid consumption and followed a pattern similar to that found for cell growth and for lipase production. The five compounds were radioactively labeled when [U- 14 C]oleic acid was supplied to the culture medium, thus showing that they were produced by transformation of the acid. When isolated from cultures containing [1,2- 13 C]acetic acid and oleic acid as the sole sources of carbon, the compounds showed to contain the 13 C isotope only in the first five atoms of carbon of the molecule. Several long chain fatty acids also acted as precursors of these compounds, with maximal yields for chain lengths between 11 and 18 atoms of carbon. None of the five compounds acted as lipase inducer when added to the culture medium instead of oleic acid. The compounds showed moderate antibacterial and antifungal activities when tested in solid media bioassays.     

Production of Tricarboxylic Acid Anhydrides from Fatty Acids by a Lipase-producing Strain of Pseudomonas cepacia

A lipase-producing strain of Pseudomonas cepacia isolated from a soil sample was found to produce five compounds when oleic acid was added to the culture medium as lipase inducer. The five compounds were isolated by solvent extraction, silicagel column chromatography and preparative HPLC, and their structural elucidation was performed by mass spectrometry, and infrared and nuclear magnetic resonance spectroscopies. The products were identified as dec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (product 1 ), undec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (product 2 ), dodec-3-ene-I,3,4-tricarboxylic acid 3,4-anhydride (product 3 ), dodec-3,8-diene-1,3,4-tricarboxylic acid 3,4-anhydride (product 4 ) and dodec-3,6-diene-I,3,4-tricarboxylic acid 3,4-anhydride (product 5 ). Accumulation of these compounds in the culture medium started after oleic acid consumption and followed a pattern similar to that found for cell growth and for lipase production. The five compounds were radioactively labeled when [U- 14 C]oleic acid was supplied to the culture medium, thus showing that they were produced by transformation of the acid. When isolated from cultures containing [1,2- 13 C]acetic acid and oleic acid as the sole sources of carbon, the compounds showed to contain the 13 C isotope only in the first five atoms of carbon of the molecule. Several long chain fatty acids also acted as precursors of these compounds, with maximal yields for chain lengths between 11 and 18 atoms of carbon. None of the five compounds acted as lipase inducer when added to the culture medium instead of oleic acid. The compounds showed moderate antibacterial and antifungal activities when tested in solid media bioassays.     

Aggregation properties of cold-active lipase produced by a psychrotolerant strain of Pseudomonas palleroniana (GBPI_508)

Abstract

The present study aims to exploit microbial potential from colder region to produce lipase enzyme stable at low temperatures. A newly isolated bacterium GBPI_508 from Himalayan environment, was investigated for the production of cold-active lipase emphasizing on its aggregation properties. Plate based assays followed by quantitative production of enzyme was estimated under different culture conditions. Further characterization of partially purified enzyme was done for molecular weight determination and activity and stability under varying conditions of pH, temperature, and in presence of organic solvents, inhibitors, and metal ions. The psychrotolerant bacterium was identified as Pseudomonas palleroniana following 16S rRNA gene sequencing. Maximum lipase production by GBPI_508 was recorded in 7?days at 25?°C utilizing yeast extract as nitrogen source and olive oil as substrate in the lipase production medium. Triton X-100 (1%) in the medium as emulsifier significantly enhanced the lipase production. Lipase produced by bacterium showed aggregation which was confirmed by dynamic light scattering and native PAGE. SDS-PAGE followed by zymogram analysis of partially purified enzyme showed two active bands of ～50?kDa and ～54?kDa. Optimum activity of partially purified enzymatic preparation was recorded at 40?°C while the activity remained nearly consistent from pH 7.0 to 12.0, whereas, maximum stability was recorded at pH values 7.0 and 11.0 at 25?°C. Interestingly, lipase in the partially purified fraction retained 60% enzyme activity at 10?°C. Medium chain pNP ester (C10) was the most preferred substrate for the lipase of GBPI_508. The lipase possessed >50% residual activity when incubated with different organic solvents (25% v/v) except toluene and dichloromethane which inhibited the activity below 50%. Partially purified enzyme was also stable in the presence of metal ions and inhibitors. The study suggests applicability of GBPI_508 lipase in low temperature conditions such as cold-active detergent formulations and cold bioremediation.     

Lipase production of Aspergillus oryzae

Aspergillus oryzae produced a small amount of lipase (0.05–0.8 U/wet-g of solid medium) in solid cultures, in contrast to the larger amount (0.46 U/ml) in a shake-flask culture in a modified GYP medium containing 2% glucose, 1% yeast extract and 2% Polypepton. Optimum conditions of lipase production in the submerged culture of A. oryzae were determined in terms of pH, composition of medium, and temperature. In a shake-flask culture at 28°C, the maximum amount of lipase increased to 0.78 U/ml upon the addition of 3% soybean oil to the modified GYP medium. In a jar fermentor culture, 30 U/ml lipase activity was obtained after 72 h at 28°C under appropriate conditions. Lipase production was greatly influenced by the culture temperature, and the optimum temperature for lipase production was about 24°C with a narrow temperature range, which was 10 degrees lower than that for the growth. In the submerged cultures, two kinds of lipase at least exhibiting different substrate specificities were also suggested.     

Lipase and esterase formation by psychrophilic and mesophilic Acinetobacter species.

Acinetobacter O16, a psychrophilic species, produced extracellular lipase (measured by hydrolysis of olive oil, tributyrin, or beta-naphthyl laurate) when grown on a complex medium (peptone plus yeast extract). Most lipase was produced during the logarithmic phase of growth. Very little cell-bound lipase was formed. These cells also produced an esterase (measured by the hydrolysis of beta-naphthyl acetate). At first, all esterase was cell bound; significant amounts appeared in the external medium late in growth. Breaking the cells did not increase cell-bound lipase activity. After breaking of the cells, most of the cell-bound lipase and esterase activity was solubilized, even after very high speed centrifugation. No appreciable amounts of these enzymes were released by osmotic shock. Lipase formation was greatly affected by nutrient conditions. Lowering either the yeast extract of the peptone content of the normal complex medium lowered or abolished lipase formation. Esterase activity was lowered to a lesser extent. Cells growing in synthetic amino acid plus vitamin medium or in acid-hydrolyzed casein produced substantial amounts of esterase but no cell-free or cell-bound lipase. However, if sodium taurocholate was added to these media, lipase was produced. Greatest production occurred if a mixture of di- and poly-peptides was also present. Taurocholate also stimulated lipase production in the normal complex medium. Adding Tween 80 or ethanol to the normal complex medium inhibited lipase production. Sodium acetate, oleic acid, olive oil, or Tween 20 added to synthetic media did not affect lipase production. The psychrophile grew more quickly at 30 degrees C than at 15 or 20 degrees C but produced more lipase at the lower temperatures. Esterase production was about the same at 20 and 30 degrees C. A mesophilic Acinetobacter species produced the same amount of lipase and esterase at 20 and 30 degrees C. The best production of lipase by the psychrophile occurred in standing cultures.     

Study of the experimental conditions for the lipase production by a newly isolated strain of Pseudomonas aeruginosa for the enantioselective hydrolysis of (±)-methyl trans-3(4-methoxyphenyl) glycidate

A lipase producing bacterium has been isolated from the soil to enantiospecifically hydrolyze the (±)-methyl trans-3(4-methoxyphenyl) glycidate (MPGM), an intermediate in the synthesis of cardiovascular drug, diltiazem. This hydrolysis provided the desired (−)-MPGM in 44% yield with 99% enantiomeric excess. The organism was identified and confirmed as Pseudomonas aeruginosa by 16S rRNA sequencing. The various physiochemical parameters have been optimized for the maximum production of lipase in shake flask. Beef extract was found to be the best nitrogen source for lipase production. The optimized cultivation conditions were 30°C with an initial medium pH 8 in shake flask. Both inoculum age and inoculum concentration have positive effect on the lipase production and (±)-MPGM (3 mM) was found to be the optimal inducer.An erratum to this article can be found at     

Partial characterization of a crude cold-active lipase from Rhodococcus cercidiphylli BZ22

Cold-active lipase production by the psychrophilic strain Rhodococcus cercidiphylli BZ22 isolated from hydrocarbon-contaminated alpine soil was investigated. Depending on the medium composition, high cell densities were observed at a temperature range of 1–10 °C in Luria–Bertani (LB) broth or 1–30 °C in Reasoner’s 2A (R2A). Maximum enzyme production was achieved at a cultivation temperature of 1–10 °C in LB medium. About 70–80 % of the secreted enzyme was bound to the cell and was highly active as a cell-immobilized lipase which exhibited good reusability; more than 60 % of the initial lipase activity was retained after five-fold reuse. The properties of the lipase produced by the investigated strain were compared with those of a mesophilic porcine pancreatic lipase (PPL). The thermal stability of the cell-immobilized bacterial lipase was higher than that of the extracellular enzyme. Highest activity was detected at 30 °C for the cell-immobilized enzyme and for PPL, while the extracellular enzyme displayed highest activity at 10–20 °C. The bacterial lipase hydrolyzed p-nitrophenyl (p-NP) esters with different acyl chain lengths (C₂–C₁₈). The highest hydrolytic activity was obtained with p-NP-butyrate (C₄) as substrate, while the highest substrate affinity was obtained with p-NP-dodecanoate (C₁₂) as substrate, indicating a clear preference of the enzyme for medium acyl chain lengths.