Factors affecting growth and lipase production by meat lactobacilli strains and Brochothrix thermosphacta

Lipolytic activity of lactobacilli strains and Brochothrix thermosphacta was cell-related; no significant activity was found in the supernatant fluids. Most lipase was produced during the logarithmic phase of growth and was greatly affected by growth conditions. The optimal temperatures for growth and lipase production were respectively 24 degrees C for B. thermosphacta and 30 degrees C for lactobacilli. For all strains, an initial pH of around 7.0 for the medium and low glucose concentration stimulated lipase production. Tributyrin inhibited both growth and lipase production at a concentration of 0.1% for B. thermosphacta or 1% for lactobacilli. Butyric acid (0.1%) and anaerobic culture inhibited lipase production by B. thermosphacta while these two factors had no effect on enzyme production by lactobacilli.     

Growth of lipolytic psychrotrophic pseudomonads in raw and ultra-heat-treated milk

The lipolytic floras of 36 raw milk samples showing lipolytic defects were dominated by pseudomonads. Representative lipolytic isolates were selected and tested for growth, lipase activity and lipolysis in ultra-heat-treated milk at temperatures ranging from 5 degrees to 30 degrees C. Pseudomonas fluorescens was the most frequently encountered species but Ps. fragi was found to cause more severe lipolytic defects in both single and mixed strain milk cultures. A representative strain of Ps. fragi multiplied faster in cold-stored milk than did three representative strains of Ps. fluorescens. The lipases produced by Ps. fragi strains were more heat-stable than those produced by Ps. fluorescens strains.     

Lipolytic activity from Halobacteria: screening and hydrolase production

Strains of Halobacteria from an Algerian culture collection were screened for their lipolytic activity against p-nitrophenyl butyrate (PNPB) and p-nitrophenyl palmitate (PNPP). Most strains were active on both esters and 12% hydrolyzed olive oil. A strain identified as Natronococcus sp. was further studied. It grew optimally at 3.5 M NaCl, pH 8 and 40 degrees C. An increase in temperature shifted the optimum salt concentration range for growth from a wider range of 2-4 M, obtained at 25-30 degrees C, to a narrower range of 3.5-4 M, obtained at 35-40 degrees C. At 45 degrees C the optimum salt concentration was 2 M. These results show a clear correlation between salt and temperature requirement. The optimum conditions for the production of hydrolytic activity during growth were: 3.5 M NaCl and pH 8 for PNPB hydrolytic activity and 4 M NaCl and pH 7.5 for PNPP hydrolytic activity; both at 40 degrees C. The clear supernatant of cells grown at 4 M NaCl showed olive oil hydrolysis activity (in presence of 4 M NaCl) demonstrating the occurrence of a lipase activity in this strain. To our knowledge, this is the first report of a lipase activity at such high salt concentration.     

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.     

Low-temperature lipase from psychrotrophic Pseudomonas sp. strain KB700A

We have previously reported that a psychrotrophic bacterium, Pseudomonas sp. strain KB700A, which displays sigmoidal growth even at -5 degrees C, produced a lipase. A genomic DNA library of strain KB700A was introduced into Escherichia coli TG1, and screening on tributyrin-containing agar plates led to the isolation of the lipase gene. Sequence analysis revealed an open reading frame (KB-lip) consisting of 1,422 nucleotides that encoded a protein (KB-Lip) of 474 amino acids with a molecular mass of 49,924 Da. KB-Lip showed 90% identity with the lipase from Pseudomonas fluorescens and was found to be a member of Subfamily I.3 lipase. Gene expression and purification of the recombinant protein were performed. KB-Lip displayed high lipase activity in the presence of Ca2+. Addition of EDTA completely abolished lipase activity, indicating that KB-Lip was a Ca2+-dependent lipase. Addition of Mn2+ and Sr2+ also led to enhancement of lipase activity but to a much lower extent than that produced by Ca2+. The optimal pH of KB-Lip was 8 to 8.5. The addition of detergents enhanced the enzyme activity. When p-nitrophenyl esters and triglyceride substrates of various chain-lengths were examined, the lipase displayed highest activity towards C10 acyl groups. We also determined the positional specificity and found that the activity was 20-fold higher toward the 1(3) position than toward the 2 position. The optimal temperature for KB-Lip was 35 degrees C, lower than that for any previously reported Subfamily I.3 lipase. The enzyme was also thermolabile compared to these lipases. Furthermore, KB-Lip displayed higher levels of activity at low temperatures than did other enzymes from Subfamily I.3, indicating that KB-Lip has evolved to function in cold environments, in accordance with the temperature range for growth of its psychrotrophic host, strain KB700A.     

Purification and properties of the extracellular lipase, LipA, of Acinetobacter sp. RAG-1.

An extracellular lipase, LipA, extracted from Acinetobacter sp. RAG-1 grown on hexadecane was purified and properties of the enzyme investigated. The enzyme is released into the growth medium during the transition to stationary phase. The lipase was harvested from cells grown to stationary phase, and purified with 22% yield and > 10-fold purification. The protein demonstrates little affinity for anion exchange resins, with contaminating proteins removed by passing crude supernatants over a Mono Q column. The lipase was bound to a butyl Sepharose column and eluted in a Triton X-100 gradient. The molecular mass (33 kDa) was determined employing SDS/PAGE. LipA was found to be stable at pH 5.8-9.0, with optimal activity at 9.0. The lipase remained active at temperatures up to 70 degrees C, with maximal activity observed at 55 degrees C. LipA is active against a wide range of fatty acid esters of p-nitrophenyl, but preferentially attacks medium length acyl chains (C6, C8). The enzyme demonstrates hydrolytic activity in emulsions of both medium and long chain triglycerides, as demonstrated by zymogram analysis. RAG-1 lipase is stabilized by Ca2+, with no loss in activity observed in preparations containing the cation, compared to a 70% loss over 30 h without Ca2+. The lipase is strongly inhibited by EDTA, Hg2+, and Cu2+, but shows no loss in activity after incubation with other metals or inhibitors examined in this study. The protein retains more than 75% of its initial activity after exposure to organic solvents, but is rapidly deactivated by pyridine. RAG-1 lipase offers potential for use as a biocatalyst.     

Isolation and characterization of a thermophilic lipase from Bacillus thermoleovorans ID-1

A thermophilic microorganism, Bacillus thermoleovorans ID-1, isolated from hot springs in Indonesia, showed extracellular lipase activity and high growth rates on lipid substrates at elevated temperatures. On olive oil (1.5%, w/v) as the sole carbon source, the isolate ID-1 grew very rapidly at 65 degrees C with its specific growth rate (2.50 h(-1)) and its lipase activity reached the maximum value of 520 U l(-1) during the late exponential phase and then decreased. In addition to this, isolate ID-1 could grow on a variety of lipid substrates such as oils (olive oil, soybean oil and mineral oil), triglycerides (triolein, tributyrin) and emulsifiers (Tween 20, 40). The excreted lipase of ID-1 was purified 223-fold to homogeneity by ammonium sulfate precipitation, DEAE-Sephacel ion-exchange chromatography and Sephacryl S-200 gel filtration chromatography. As a result, the relative molecular mass of the lipase was determined to be 34 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme showed optimal activity at 70-75 degrees C and pH 7.5 and exhibited 50% of its original activity after 1 h incubation at 60 degrees C and 30 min at 70 degrees C and its catalytic function was activated in the presence of Ca(2+) or Zn(2+).     

Factors affecting growth and lipase production by meat lactobacilli strains and Brochothrix thermosphacta

Lipolytic activity of lactobacilli strains and Brochothrix thermosphacta was cellrelated; no significant activity was found in the supernatant fluids. Most lipase was produced during the logarithmic phase of growth and was greatly affected by growth conditions. The optimal temperatures for growth and lipase production were respectively 24°C for B. thermosphacta and 30°C for lactobacilli. For all strains, an initial pH of around 7-0 for the medium and low glucose concentration stimulated lipase production. Tributyrin inhibited both growth and lipase production at a concentration of 0-1% for B. thermosphacta or 1% for lactobacilli. Butyric acid (0-1%) and anaerobic culture inhibited lipase production by B. thermosphacta while these two factors had no effect on enzyme production by lactobacilli.     

Continuous hydrolysis of oil by immobilized lipase in a countercurrent reactor

Lipase from Pseudomonas fluorescens biotype I was immobilized by adsorption of anion exchange resin using glutaraldehyde to enhance the adsorption. The activity yield of the immobilized lipase was very low (below 1%) when lipase activity was measured using emulsion substrate. The activity yield was 10-70% when lipase activity was measured using non-emulsion substrate. Countercurrent reactors for hydrolysis of oil using non-emulsion substrate were studied. A fluidized bed reactor was found to be superior to a fixed bed one since in a fixed bed reactor the separation rate of the two layers was slow and the flow rate of the reactor had to be slower than the separation rate. A fluidized bed reactor system equipped with settling compartments and stirring compartments was devised. Continuous lipolysis at 60 degrees C and continuous separation of oily product and water soluble product were performed. After continuous operation for more than 3 months, 70% of the initial activity of the immobilized lipase was observed at the end of the reaction.     

Nucleotide sequence of the lipase gene lip2 from the antarctic psychrotroph Moraxella TA144 and site-specific mutagenesis of the conserved serine and histidine residues

The lip2 gene from the antarctic psychotroph Moraxella TA144 was sequenced. The primary structure of the Lip2 preprotein deduced from the nucleotide sequence is composed of 433 amino acids with a predicted Mr of 47,222. This enzyme contains a Ser-centered consensus sequence and a conserved His-Gly dipeptide found in most lipase amino-terminal domains. These sequences are involved in the lipase active site conformation since substitution of the conserved Ser or His residues by Ala and Gln, respectively, results in the loss of both lipase and esterase activities. Structural factors that would allow proper enzyme flexibility at low temperatures are discussed. It is suggested that only subtle changes in the primary structure of these psychrotrophic enzymes can account for their ability to catalyze lipolysis at temperatures close to 0 degrees C.     

Purification and characterization of lipase from psychrophilic Acinetobacter calcoaceticus LP009

A lipase-producing bacterium, Acinetobacter calcoacetius LP009, was isolated from raw milk. The optimum conditions for growth and lipase production by A. calcoaceticus LP009 were 15 degrees C with shaking at 200 rpm in LB supplemented with 1.0% (v/v) Tween 80. The crude lipase was purified to homogeneous state by ultrafiltration and gel filtration chromatography on Sephadex G-100. Its molecular weight determined by SDS-PAGE was 23 kDa and it exhibited maximum activity at pH 7.0 and 50 degrees C. It was stable over the pH range of 4.0 to 8.0 and at temperatures lower than 45 degrees C. It was a metalloenzyme that is positionally non-specific and had the ability to improve fat hydrolysis in soybean meal and in premixed animals feed.     

Production, properties and application to nonaqueous enzymatic catalysis of lipase from a newly isolated Pseudomonas strain

A potent bacterium for lipase production was isolated from soil and identified as Pseudomonas species. It produced lipase constitutively. A mutant of this strain with a lipase productivity 3.25-fold higher was obtained by treatment with ultraviolet (UV) and nitrosoguanidine (NTG). Its fermentation condition was optimized to a lipase yield of 87.5 U/ml. The lipase had maximum activity at pH 9.0 and 45 degrees C. It was stable at pHs from 7.0 to 11.0 and below 60 degrees C. The effects of metal ions, surfactants and bile salts were also studied. The lipase was 1,3-specific. In organic solvents, the thermal stability of the lipase was significantly enhanced. Its optimum temperature was also slightly increased. The optimum water activity was found between 0.5 and 0.6. The lipase was successfully applied in organic phase to catalyze the glycerolysis of palm oil for monoglyceride (MG) production, and the enantioselective esterification of (R,S)-2-octanol. The enantioselectivity of the lipase could be enhanced substantially by treatment with an amphipathic.     

Thermostable lipase of Bacillus Stearothermophilus: high-level production, purification, and calcium-dependent thermostability

An efficient expression system was developed for the production of the thermostable lipase from Bacillus stearothermophilus L1 in an Escherichia coli system. A structural gene corresponding to mature lipase was subcloned in the pET-22b(+) expression vector and its expression was induced by IPTG at 30 degrees C in E. coli cells. The lipase activity in a cell-free extract was as high as 448,000 units/g protein, which corresponds to as much as 26% of the total cellular protein and is 77 times higher than that of E. coli RR1/pLIP1. Based on its pI (7.4) and pH stability data reported previously, the L1 lipase was efficiently purified to homogeneity with CM (at pH 6.0) and DEAE (at pH 8.8) column chromatographies with a recovery yield of 62%. The specific activity of the purified enzyme was 1700 units/mg protein when olive oil emulsion was used as a substrate. Its optimum temperature for the hydrolysis of olive oil was 68 degrees C and it was stable up to 55 degrees C for 30 min-incubation. The thermostability increased by about 8-10 degrees in the presence of calcium ions. This calcium-dependent thermostability was confirmed by the tryptophan fluorescence emission kinetics showing that the enzyme starts to unfold at 66 degrees C in the presence of calcium ions but at 58 degrees C in the absence of calcium ions, implying that the calcium ions bind to the thermostable enzyme and stabilize the protein tertiary structure even at such high temperatures.     

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.     

Production, purification, and characterization of lipase from thermophilic and alkaliphilic Bacillus coagulans BTS-3

A thermophilic isolate Bacillus coagulans BTS-3 produced an extracellular alkaline lipase, the production of which was substantially enhanced when the type of carbon source, nitrogen source, and the initial pH of culture medium were consecutively optimized. Lipase activity 1.16 U/ml of culture medium was obtained in 48 h at 55 degrees C and pH 8.5 with refined mustard oil as carbon source and a combination of peptone and yeast extract (1:1) as nitrogen sources. The enzyme was purified 40-fold to homogeneity by ammonium sulfate precipitation and DEAE-Sepharose column chromatography. Its molecular weight was 31 kDa on SDS-PAGE. The enzyme showed maximum activity at 55 degrees C and pH 8.5, and was stable between pH 8.0 and 10.5 and at temperatures up to 70 degrees C. The enzyme was found to be inhibited by Al3+, Co2+, Mn2+, and Zn2+ ions while K+, Fe3+, Hg2+, and Mg2+ ions enhanced the enzyme activity; Na+ ions have no effect on enzyme activity. The purified lipase showed a variable specificity/hydrolytic activity towards various 4-nitrophenyl esters.     

Extracellular solvent stable cold-active lipase from psychrotrophic Bacillus sphaericus MTCC 7526: partial purification and characterization

Psychrotropic Bacillus sphaericus producing solvent stable cold-active lipase upon growth at low temperature was isolated from Gangotri glacier. Optimal parameters for lipase production were investigated and the strain was able to produce lipase even at 15 °C. An incubation period of 48 h and pH 8 was found to be conducive for cold-active lipase production. The addition of trybutyrin as substrate and lactose as additional carbon source increased lipase production. The enzyme was purified up to 17.74-fold by ammonium sulphate precipitation followed by DEAE cellulose column chromatography. The optimum temperature and pH for lipase activity were found to be 15 °C and 8.0, respectively. The lipase was found to be stable in the temperature range 20–30 °C and the pH range 6.0–9.0. The protein retained more than 83 % of its initial activity after exposure to organic solvents. The lipase exhibited significant stability in presence of acetone and DMSO retaining >90 % activity. The enzyme activity was inhibited by 10 mM CuSO₄ and EDTA but showed no loss in activity after incubation with other metals or inhibitors examined in this study.     

Characterization of oat lipase for lipolysis of rice bran oil

Summary Lipases from plant and microbial sources were screened for lipolysis of rice bran oil (RBO). Oat lipase was found to give reproducibly 60% lipolysis. Factors affecting the efficiency of lipolysis such as oil:emulsifier ratio, duration of homogenization for the substrate preparation, substrate:enzyme ratio, substrate pH, kinetics of lipolysis and efficiency of lipase as a function of pH, oat particle size, its quality and reuseability were studied to assess the feasibility of using of oat lipase for splitting RBO commercially. The efficiency of lipolysis was optimal at (a) RBO:emulsifier (0.5:1) ratio, homogenized for 2.5 min, (b) pH 7.5 and (c) substrate:enzyme ratio of 5 ml: 2 g.     

Sunflower seed lipase: extraction, purification, and characterization

A simple procedure for the extraction of the lipolytic activity from sunflower seed has been developed. Various conditions of extraction have been optimized in order to obtain maximum yield of lipase. A new lipase enzyme was purified by the fractional salt precipitation from the supernatant, dialysis on a cellulose membrane, and gel column chromatography on Sephadex G-75. The lipase was monomeric, with an apparent Mr of 22 kDa and a pI of 8, with the electrophoretic analysis. Kinetics of the enzyme activity versus substrate concentration showed typical lipase behavior, with Km and Vmax, values of 1.33 mM and 555 U/mg. All triglycerides were efficiently hydrolyzed by the enzyme, but this showed a preference towards triglycerides of natural mono unsaturated fatty acids. The optimum temperature, pH, and incubation time for lipolytic activity were 50 degrees C, 7.5, and 5 min, respectively. The stability of the sunflower lipase was investigated under operational and storage conditions. It was found that this enzyme preserved its lipolytic activity at temperatures between at 35-50 degrees C, alkaline pH, and for a period of about four months.     

Selection of pH buffers for use in conductimetric microbiological assays

The lipolytic floras of 36 raw milk samples showing lipolytic defects were dominated by pseudomonads. Representative lipolytic isolates were selected and tested for growth, lipase activity and lipolysis in ultra-heat-treated milk at temperatures ranging from 5° to 30°C. Pseudomonas fluorescens was the most frequently encountered species but Ps. fragi was found to cause more severe lipolytic defects in both single and mixed strain milk cultures. A representative strain of Ps. fragi multiplied faster in cold-stored milk than did three representative strains of Ps. fluorescens. The lipases produced by Ps. fragi strains were more heat-stable than those produced by Ps. fluorescens strains.     

Alkaline lipase production by Citrobacter freundii IIT-BT L139

Around 150 lipase producing bacterial isolates were screened from the local soils enriched with oil. Citrobacrer freundii IIT-BT L139, an isolated microbial strain, produced lipase that had high activity (8.8 U/ml) at pH 9.0 and 40 degrees C. The 16S rDNA phylogenetic studies showed that Citrobacter freundii belongs to the family Enterobacteriaceae and later confirmed by the microbial identification. Suitable C and N sources for lipase production were deduced to be starch and peptone-urea, respectively. In a controlled fermenter (1 L), the lipase activity was found to increase by 36% (12 Uml(-1)). The variation of lipase activity, pH and dissolved oxygen (DO) during growth of the organism in the controlled batch fermenter were monitored. The rheological characteristics of the fermentation broth indicated that it behaved like a Newtonian fluid throughout the fermentation. The fermentation time was comparatively short (60 h). The lipase was also found to be substantially resistant to common detergents. This lipase was, thus, characterized as alkaline, thermostable and solvent stable, which was essentially desirable in pharmaceutical, detergent and other industrial applications or production.