Lipase-induced hydrolysis of castor oil: effect of various metals

The ability of an extracellular lipase from Pseudomonas aeruginosa KKA-5 to commence hydrolysis of castor oil in the presence of various metal chlorides, was investigated. Apart from CaCl₂ (commonly used for castor oil hydrolysis), AlCl₃ (group IIIB), CrCl₃ (group VIA) and MgCl₂ (group IIA) displayed enhanced hydrolysis capability. Specifically, our statistics show that with respect to time, when Cr³⁺ was used, hydrolysis of castor oil was four times faster than that of calcium, and 1.6 times faster with regards to Al³⁺. The chlorides of group VIII and alkali metals had no effect on hydrolysis. Group IV metal chlorides did not enhance lipase activity and inhibited castor oil hydrolysis. The effect of metal ions from other groups on lipase activity is also reported. Received 14 August 1998/ Accepted in revised form 22 October 1998     

Lipase from Pseudomonas aeruginosa LP602: biochemical properties and application for wastewater treatment

Lipase from Pseudomonas aeruginosa LP602, a bacterial strain isolated from a domestic wastewater sample, was preliminarily characterized. The enzyme exhibited maximum lipolytic activity at pH 8.0 where it was also stably maintained. At 55°C, the lipase had the highest activity but not stability. The enzyme was insensitive to EDTA and to many ions tested except Zn²⁺. It was sensitive to SDS but not to Tween-20, Tween-80 or Triton X-100. The enzyme was active towards a number of commercial food grade fats and oils. A suitable medium formula for lipase production was MMP containing 6.25% whey as a carbon source, 1% soybean oil as inducer and 0.5% yeast extract supplement. The culture was fed with glucose to a final concentration of 0.1% at the 15th hour of incubation. Lipase production under this condition was 3.5 U ml⁻¹. Both P. aeruginosa LP602 cells and the lipase were shown to be usable for lipid-rich wastewater treatment. Received 21 April 1998/ Accepted in revised form 6 August 1998     

PARTIAL PURIFICATION AND CHARACTERIZATION OF THE ORGANIC SOLVENT-TOLERANT LIPASE PRODUCED BY Pseudomonas fluorescens RB02-3 ISOLATED FROM MILK

Eighty-five putative Pseudomonas isolates were obtained from various raw milk and pasteurized milk samples using Pseudomonas CFC agar. Among them, 36 isolates were identified as Pseudomonas fluorescens, and one isolate was identified as Pseudomonas putida. Lipase activity of the strains was quantitatively measured by the spectrophotometric method using p-nitrophenyl palmitate (p-NPP) as substrate. Detected lipase activity of the strains was between 10.03 U/mL and 22.16 U/mL. Pseudomonas fluorescens RB02-3 possessed the highest lipase activity. The extracellular lipase of P. fluorescens RB02-3 strain was homogeneously purified using a combination of ammonium sulfate precipitation, dialysis, and gel filtration column chromatography. This purification procedure resulted in 2.97-fold purification with 20.3% recovery. The enzyme was characterized, and exhibited maximum activity at pH 7.0 and 50°C; after it was incubated for 1 h it was activated in the presence of hexane, ethyl acetate, isopropanol, and ethanol and remained stable after the incubation was extended for 2 hr. The lipase was slightly inhibited in the presence of Zn²⁺, Co²⁺, Cu²⁺, Ni²⁺ salts, and ethylenediamine tetraacetic acid (EDTA), whereas Cd²⁺, sodium dodecyl sulfate (SDS), and Tween-80 had no effect on its activity.     

Bioreactor operated production of lipase: castor oil hydrolysis using partially-purified lipase.

A highly stable lipase from Pseudomonas aeruginosa KKA-5 was produced by batch cultivation technique employing shake flask and 5 L-bioreactor. The bioreactor was run at different airflow rates. Low airflow rates (1 and 3 L/min), did not lead to effective growth and lipase production. Growth increased by about one order and lipase production increased by about 6 times, at an airflow rate of 5 L/min. Lipase production occurred during decelerated cell growth. A highly stable lipase was produced which retained its activity in the running bioreactor, even after a period of one month. This stable lipase was partially-purified using ammonium sulphate precipitation technique. Castor oil was hydrolyzed using 300U crude and partially-purified lipase, each. Approximately 21-fold, partially-purified lipase could hydrolyze 81% castor oil within a period of 96 hr, where as only 63% hydrolysis was obtained, in 216 hour, when crude lipase was used.     

Characterization of Betaine Aldehyde Dehydrogenase from Cylindrocarpon didymum M-1

To obtain a lipase which effectively hydrolyzes castor oil, bacteria were isolated from 500 soil samples. The best strain was examined; its microbiological characteristics suggested that it belongs to the genus Pseudomonas. A lipase from this strain was purified by ammonium sulfate fractionation and chromatographies on DEAE-cellulose and DEAE-Toyopearl 650 M. The enzyme was purified about 400-fold with a yield of 13%. The purified enzyme was electrophoretically homogeneous and its molecular weight was 30,000. The optimum pH and temperature for the hydrolysis of olive oil emulsion were 7.0 and 60°C. The enzyme was stable up to 35°C at pH 7.0 for 30min and also stable from pH 9.0 to 10.0 at 4°C for 22 hr. The activity was inhibited by Fe³⁺ , Hg²⁺ , pCMB, and anionic surfactants, and enhanced by nonionic surfactants and bile salts. The enzyme efficiently hydrolyzed castor oil.     

Single-step purification of lipase from Burkholderia multivorans using polypropylene matrix

Lipase from Burkholderia multivorans was purified with high yields directly from fermentation broth by a single-step purification protocol involving adsorption and desorption. The crude enzyme (lyophilized powder) from B. multivorans was loaded on Accurel (Membrana, Germany), a polypropylene matrix, using butanol as the solvent in a buffer at pH 9.0 and ambient temperature for a period of 12 h. The enzyme adsorbed onto the matrix with high specific activity (33 units mg^–1 protein). This was followed by desorption of the enzyme from the matrix using Triton X-100 as the eluent. The enzyme was finally recovered by precipitation with acetone (50%, v/v). Thus, an overall enzyme yield of 66% with a 3.0-fold purification was obtained. The purity of the enzyme was ascertained by SDS-PAGE. The phenomenon of adsorption and desorption on Accurel was studied for three more lipases, viz. Mucor meihei lipase (Sigma–Aldrich Co.), Lipolase (Novo Nordisk, Denmark) and Pseudomonas aeruginosa lipase (laboratory isolate).     

A Nonhemolytic Phospholipase C from Burkholderia cepacia

Burkholderia cepacia is an opportunistic pathogen that causes serious pulmonary infections in cystic fibrosis patients. Although several potential virulence factors—a protease, lipase, and two phospholipases C (one hemolytic and one nonhemolytic)—have been identified, only two, the protease and the lipase, have been described in detail. The goal of this study was to purify and characterize a nonhemolytic phospholipase C secreted by B. cepacia strain Pc224c. The enzyme was concentrated from culture supernatants and purified by polyacrylamide gel electrophoresis. The 54-kDa protein was stable in the presence of sodium dodecyl sulfate (up to 10%) and at 4°, 22°, and 37°C; it was, however, inactivated at 100°C. The enzyme bound to glass, chromatography matrices, and polyvinylidene difluoride and cellulose membranes, suggesting that it is hydrophobic.  In a genetic approach, primers based on conserved sequences of a B. cepacia Pc69 hemolytic phospholipase C and both the Pseudomonas aeruginosa hemolytic and nonhemolytic proteins were designed to identify the Pc224c nonhemolytic phospholipase C gene. One polymerase chain reaction product was identified; it was sequenced and the sequence compared with sequences in the BLAST database. The best match was the Pseudomonas aeruginosa hemolytic phospholipase C. Ten additional B. cepacia strains were screened for the gene by Southern hybridization; five had the 4-kb band, suggesting that these strains have a similar form of the PLC gene. Nine of the ten strains reacted with the probe, suggesting that similar sequences were present, but in another form. Received: 13 October 1998 / Accepted: 6 November 1998     

Partial purification and characterization of the organic solvent-tolerant lipase produced by Pseudomonas fluorescens RB02-3 isolated from milk

Eighty-five putative Pseudomonas isolates were obtained from various raw milk and pasteurized milk samples using Pseudomonas CFC agar. Among them, 36 isolates were identified as Pseudomonas fluorescens, and one isolate was identified as Pseudomonas putida. Lipase activity of the strains was quantitatively measured by the spectrophotometric method using p-nitrophenyl palmitate (p-NPP) as substrate. Detected lipase activity of the strains was between 10.03 U/mL and 22.16 U/mL. Pseudomonas fluorescens RB02-3 possessed the highest lipase activity. The extracellular lipase of P. fluorescens RB02-3 strain was homogeneously purified using a combination of ammonium sulfate precipitation, dialysis, and gel filtration column chromatography. This purification procedure resulted in 2.97-fold purification with 20.3% recovery. The enzyme was characterized, and exhibited maximum activity at pH 7.0 and 50 °C; after it was incubated for 1 h it was activated in the presence of hexane, ethyl acetate, isopropanol, and ethanol and remained stable after the incubation was extended for 2 hr. The lipase was slightly inhibited in the presence of Zn2+, Co2+, Cu2+, Ni2+ salts, and ethylenediamine tetraacetic acid (EDTA), whereas Cd2+, sodium dodecyl sulfate (SDS), and Tween-80 had no effect on its activity.     

Influence of cultivation conditions on the production of a thermostable extracellular lipase from Amycolatopsis mediterranei DSM 43304

Among several lipase-producing actinomycete strains screened, Amycolatopsis mediterranei DSM 43304 was found to produce a thermostable, extracellular lipase. Culture conditions and nutrient source modification studies involving carbon sources, nitrogen sources, incubation temperature and medium pH were carried out. Lipase activity of 1.37 ± 0.103 IU/ml of culture medium was obtained in 96 h at 28°C and pH 7.5 using linseed oil and fructose as carbon sources and a combination of phytone peptone and yeast extract (5:1) as nitrogen sources. Under optimal culture conditions, the lipase activity was enhanced 12-fold with a twofold increase in lipase specific activity. The lipase showed maximum activity at 60°C and pH 8.0. The enzyme was stable between pH 5.0 and 9.0 and temperatures up to 60°C. Lipase activity was significantly enhanced by Fe³⁺ and strongly inhibited by Hg²⁺. Li⁺, Mg²⁺ and PMSF significantly reduced lipase activity, whereas other metal ions and effectors had no significant effect at 0.01 M concentration. A. mediterranei DSM 43304 lipase exhibited remarkable stability in the presence of a wide range of organic solvents at 25% (v/v) concentration for 24 h. These features render this novel lipase attractive for potential biotechnological applications in organic synthesis reactions.     

Purification and characterization of an organic solvent-tolerant lipase from Pseudomonas aeruginosa LX1 and its application for biodiesel production

An organic solvent-tolerant lipase from newly isolated Pseudomonas aeruginosa LX1 has been purified by ammonium sulfate precipitation and ion-exchange chromatography leading to 4.3-fold purification and 41.1% recovery. The purified lipase from P. aeruginosa LX1 was homogeneous as determined by SDS-PAGE, and the molecular mass was estimated to be 56 kDa. The optimum pH and temperature for lipase activity were found to be 7.0 and 40 °C, respectively. The lipase was stable in the pH range 4.5–12.0 and at temperatures below 50 °C. Its hydrolytic activity was found to be highest towards p-nitrophenyl palmitate (C16) among the various p-nitrophenol esters investigated. The lipase displayed higher stability in the presence of various organic solvents, such as n-hexadecane, isooctane, n-hexane, DMSO, and DMF, than in the absence of an organic solvent. The immobilized lipase was more stable in the presence of n-hexadecane, tert-butanol, and acetonitrile. The transesterification activity of the lipase from P. aeruginosa LX1 indicated that it is a potential biocatalyst for biodiesel production.     

Influence of precursors and additives on microbial lipases stabilized by sol-gel entrapment

Sol-gel entrapment of microbial lipases from Candida cylindracea (Cc lipase),Pseudomonas fluorescens (Lipase AK), and Pseudomonas cepacia (Lipase PS), using as precursors tetraethoxysilane (TEOS) and silanes of type R-Si(OEt)₃ with alkyl or aryl R groups, has been investigated. Three different methods using these precursors were tried exhibiting protein immobilization yields in the range of 20–50%. Hydrolysis of emulsified olive oil, esterification of lauric acid with 1-octanol and enantioselective acylation of 2-pentanol have been used as model reactions for testing the properties of the encapsulated lipases. The recovery yields of the enzyme activity in the esterification reaction were between 20–68%, the best performance being achieved with phenyltriethoxysilane and tetraethoxysilane precursors at 3:1 molar ratio. When testing the entrapped Lipase AK in the enantioselective acylation reaction of 2-pentanol, activity recovery yields up to 32% related to the free enzyme were obtained and the immobilization increased the enantioselectivity of the enzyme.     

Extracellular cold-active lipase of Microbacterium luteolum isolated from Gangotri glacier,western Himalaya: Isolation,partial purification and characterization

A psychrophilic bacterium producing cold-active lipase upon growth at low temperature was isolated from the soil samples of Gangotri glacier and identified as Microbacterium luteolum. The bacterial strain produced maximum lipase at 15 °C, at a pH of 8.0. Beef extract served as the best organic nitrogen source and ammonium nitrate as inorganic for maximum lipase production. Castor oil served as an inducer and glucose served as an additional carbon source for production of cold-active lipase. Ferric chloride as additional mineral salt in the medium, highly influenced the lipase production with an activity of 8.01 U ml^?1. The cold-active lipase was purified to 35.64-fold by DEAE-cellulose column chromatography. It showed maximum activity at 5 °C and thermostability up to 35 °C. The purified lipase was stable between pH 5 and 9 and the optimal pH for enzymatic hydrolysis was 8.0. Lipase activity was stimulated in presence of all the solvents (5%) tested except with acetonitrile. Lipase activity was inhibited in presence of Mn²⁺, Cu²⁺, and Hg²⁺; whereas Fe⁺, Na⁺ did not have any inhibitory effect on the enzyme activity. The purified lipase was stable in the presence of SDS; however, EDTA and dithiothreitol inhibited enzyme activity. Presence of Ca²⁺ along with inhibitors stabilized lipase activity. The cold active lipase thus exhibiting activity and stability at a low temperature and alkaline pH appears to be practically useful in industrial applications especially in detergent formulations.     

Studies on the Lipoprotein Lipases of Microorganisms

Mucor javanicus IAM 6108 was cultivated aerobically at large scale in the medium containing corn steep liquor 3.0%, soluble starch 1.0%, soybean yuto 1.0% and inorganic salts, and the lipoprotein lipase produced was recovered by addition of ammonium sulfate (0.7 saturation). From this crude preparation, the enzyme was purified about 13 times, through ammonium sulfate fractionation (0~0.4 saturation), precipitation at pH 4.0, ethanol precipitation (80%) and Sephadex G-200 gel filtration. The purified lipoprotein lipase was sedimented as single peak in ultracentrifugal analysis in the presence of 1.0% sodium dodecylsulfate. The enzymatic properties of the purified enzyme was as follows; optimum pH was 7.0, stable pH range was from 5.0 to 7.0, optimum temperature was 40°C, inactivated rapidly above 50°C. The lipoprotein lipase activity was inhibited by 75% and 88% by 10^?2 m taurocholate and 1.0 m NaCl, respectively. ZnCl₂, CuCl₂, Pb(NO₃)₂, and SnCl₂ at 10^?3 m showed complete inhibition. The ratio of lipoprotein lipase to lipase activity was 10 : 1. Lipoprotein lipase activity was dependent on the concentration of blood plasma which could be substituted by bovine serum albumin or egg albumin to a certain degree. The results suggesting the preferential α-fatty acid hydrolysis was obtained.     

Purification,characterization and thermostability of lipase from a thermophilic Bacillus sp. J33

A thermostable lipase produced by a thermophilic Bacillus sp. J33 was purified to 175-fold with 15.6% recovery by ammonium sulphate and Phenyl Sepharose column chromatography. The enzyme is a monomeric protein having molecular weight of 45 kDa. It hydrolyzes triolein at all positions. The fatty acid specificity of lipase is broad with little preference for C12 and C4. The K_m and V_max for lipase with pNP-laurate as substrate was calculated to be 2.5 mM and 0.4 M min^-1 ml^-1 respectively. The immobilized enzyme was stable for 12 h at 60°C. Polyhydric alcohols such as ethylene glycol (2.5 M), sorbitol (2.5 M) and glycerol (2.5 M) were used as thermostabilizers. Lipase acquired a remarkable stability, since no deactivation occurred at 70°C for 150 min in the presence of additives.     

Purification and Properties of a Chromobacterium Lipase with a High Molecular Weight

A lipase with a high molecular weight was purified from Chromobacterium viscosum by chromatography using the Amberlite CG–50 and Sephadex G–75. The purified lipase (Lipase A) was found to be homogeneous by disc electrophoresis.

Lipase A had an optimum pH around 7 for lipolysis of olive oil and the enzyme was stable at the range of pH 4 to 9 and below 50°C. Zn²⁺, Cu²⁺, Fe³⁺ and high concentrations of l-cysteine, iodoacetic acid and NBS had remarkable inhibitory effects. Bile salts were activator. Lipase A was more active on water insoluble esters than water soluble esters. The isoelectric point of the enzyme was pH 4.7.     

Production and characterization of a cold-active and n-hexane activated lipase from a newly isolated Serratia grimesii RB06-22

Abstract

A lipase-producing bacterium isolated from raw milk was identified as Serratia grimesii based on 16S rRNA sequence analysis. The extracellular lipase was partially purified by ammonium sulfate precipitation and ultrafiltration. Maximal activity was observed at 10°C, the optimum pH was 8.0 and the enzyme was stable at 5–30°C for 1 h. The K_m and V_max values were 1.7 mM and 0.3 mM/min respectively. It was found that the lipase had the highest hydrolytic activity towards sunflower oil and soybean oil. CaCl₂ had a stimulatory effect on lipase activity, while EDTA and iodoacetic acid slightly inhibited the lipase activity and the enzyme was strongly inhibited by PMSF. The enzyme was compatible with various non-ionic surfactants as well as sodium cholate and saponin. In addition, the enzyme was relatively stable towards oxidizing agents. This lipase exhibited maximum activity in 35% n-hexane retaining about 2191% activity for 1 h.     

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.     

Identification of the Pseudornonas aeruginosa acid phosphatase as a phosphorylcholine phosphatase activity

Summary Choline, betaine and N,N-dimethylglycine as the sole carbon and nitrogen source induced a periplasmic acid phosphatase activity in Pseudomonas aeruginosa. This enzyme produced the highest rates of hydrolysis in phosphorylcholine and phosphorylethanolamine among the various phosphoric esters tested. At saturating concentrations of Mg²⁺, the K_m values were 0.2 and 0.7 mM for phosphorylcholine and phosphorylethanolamine respectively. At high concentrations both compounds were inhibitors of the enzyme activity. The K _inf1 ^sups values for phosphorylcholine and phosphorylethanolamine were 1.0 and 3.0 mM respectively. The higher catalytic efficiency was that of phosphorylcholine. Considering these results it is possible to suggest that the Pseudomonas aeruginosa acid phosphatase is a phosphorylcholine phosphatase. The existence of this activity which is induced jointly with phospholipase C by different choline metabolites, in a high phosphate medium, suggests that the attack of Pseudomonas aeruginosa on the cell host may also be produced under conditions of high phosphate concentrations, when the alkaline phosphatase is absent.     

Co-expression of the lipase and foldase of Pseudomonas aeruginosa to a functional lipase in Escherichia coli

The lipA gene, a structural gene encoding for protein of molecular mass 48 kDa, and lipB gene, encoding for a lipase-specific chaperone with molecular mass of 35 kDa, of Pseudomonas aeruginosa B2264 were co-expressed in heterologous host Escherichia coli BL21 (DE3) to obtain in vivo expression of functional lipase. The recombinant lipase was expressed with histidine tag at its N terminus and was purified to homogeneity using nickel affinity chromatography. The amino acid sequence of LipA and LipB of P. aeruginosa B2264 was 99–100% identical with the corresponding sequence of LipA and LipB of P. aeruginosa LST-03 and P. aeruginosa PA01, but it has less identity with Pseudomonas cepacia (Burkholderia cepacia) as it showed only 37.6% and 23.3% identity with the B. cepacia LipA and LipB sequence, respectively. The molecular mass of the recombinant lipase was found to be 48 kDa. The recombinant lipase exhibited optimal activity at pH 8.0 and 37°C, though it was active between pH 5.0 and pH 9.0 and up to 45°C. K _m and V _max values for recombinant P. aeruginosa lipase were found to be 151.5 ± 29 μM and 217 ± 22.5 μmol min⁻¹ mg⁻¹ protein, respectively.     

Optimization of extracellular lipase production from the biocontrol fungus Nomuraea rileyi

Lipases are important cuticle-degrading enzymes that hydrolyze the ester bonds of waxes, fats and lipoproteins during the infection of insects by the fungus Nomuraea rileyi. Lipase production by the N. rileyi strain MJ was optimized by varying environmental and nutritional conditions in culture medium containing different vegetable oils at various concentrations with shaking at 150 rpm for 8 days at 25°C. The maximum lipase production was obtained using castor oil (30.5±0.6 U mL^?1), followed in order by coconut oil (20.8±0.4 U mL^?1), olive oil (20.8±0.4 U mL^?1) and cottonseed oil (20.6±0.4 U mL^?1). The highest lipase activity (37.7±0.4 U mL^?1) was obtained when castor oil was used at a concentration of 4% (v/v) of basal medium. When the surfactant Tween 80 was added at the fourth day rather than at the beginning of incubation, a maximum lipase activity of 44.9±3.5 U mL^?1 was obtained. The optimal temperature and pH for lipase production were 25°C and pH 8.0, respectively. This is the first report on lipase production by the biocontrol fungus N. rileyi.