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.     

Enhanced production of Penicillium expansum PED-03 lipase through control of culture conditions and application of the crude enzyme in kinetic resolution of racemic Allethrolone

Alkaline lipase production was performed in submerged fermentation by Penicillium expansum PED-03. It was found that the suitable carbon source and nitrogen source for lipase production were 0.5% starch and 4.0% soybean meal, respectively. The maximal lipase activity (850 U/mL) of production was achieved at initial pH 5.5-6.0, 26 degrees C, 72 h. Tween-80 was an effective enhancer for lipase production. Agitation speed of the fermentor played an important role, and the suitable agitation speed for lipase production was 500 r/min. The lipase was stable within the range of pH 7.0-10.0 and 20-40 degrees C, and the optimum conditions for the enzymatic reaction were 35 degrees C and pH 9.5. The enzymatic resolution of racemic allethrolone (4-hydroxy-3-methyl-2-(2-propenyl)-2- cyclopenten-1-one) was carried out by the lipase from P. expansum PED-03, and the conversion reached 48% with excellent enantioselectivity (E > 100), which showed a good application potential in the production of optically pure allethrolone.     

Improved catalytic performance of Bacillus megaterium epoxide hydrolase in a medium containing Tween-80

A new epoxide hydrolase with high enantioselectivity toward (R)-glycidyl phenyl ether (R-GPE) was partially purified from Bacillus megaterium strain ECU1001. The maximum activity of the isolated enzyme was observed at 30 degrees C and pH 6.5 in a buffer system with 5% (v/v) of DMSO as a cosolvent. The enzyme was quite stable at pH 7.5 and retained full activity after incubation at 40 degrees C for 6 h. Interestingly, when the cosolvent DMSO was replaced by an emulsifier (Tween-80, 0.5% w/v) as an alternative additive to help disperse the water-insoluble substrate, the apparent activity of the epoxide hydrolase significantly increased by about 1.8-fold, while the temperature optimum shifted from 30 to 40 degrees C and the half-life of the enzyme at 50 degrees C increased by 2.5 times. The enzymatic hydrolysis of rac-GPE was highly enantioselective, with an E-value (enantiomeric ratio) of 69.3 in the Tween-80 emulsion system, which is obviously higher than that (41.2) observed in the DMSO-containing system.     

Purification, crystallization and properties of triacylglycerol lipase from Pseudomonas fluorescens.

Triacylglycerol lipase of Pseudomonas fluorescens was purified from the crude enzyme by ammonium sulfate precipitation and chromatographies on Sephadex G-75 and DEAE-cellulose. The crystallization of the lipase was successfully carried out. The purified lipase was demonstrated to be homogenous on disc electrophoresis and its molecular weight was calculated to be 32 000 by gel filtration. The optimum pH for hydrolysis of sesame oil was 7.0. The enzyme was stable up to 40 degrees C under the condition of pH 7.0 for 30 min and had more than 80% of the remaining activity between pH 5.0--11.0 at 37 degrees C for 60 min. The lipase was strongly inhibited by iodine and partially inhibited by FeCl3 and N-bromosuccinimide, and showed the most activity on tricaproyglycerol, among the triacylglycerols used.     

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     

Heat stable lipase activity of thermotolerant bacteria from hot springs at Orissa, India

Thermotolerant bacteria (35 in toto) isolated from three hot springs (Atri, Taptapani and Deuljhari, Orissa), were screened for lipase activities. Of these, nine strains of Bacillus spp. and three strains of Pseudomonas spp. showed heat stable lipase activity at 60 degrees C. The hydrolytic activity of these bacteria was tested using Tween-20 and Tween-80 as substrates at different temperatures using plate assay and titration techniques. The hydrolytic activity at different pH values and salt concentrations was investigated.     

Expression and characterization of recombinant Rhizopus oryzae lipase for enzymatic biodiesel production

The Rhizopus oryzae lipase containing prosequence was expressed in Pichia pastoris. Recombinant lipase subunit showed a molecular mass of 32 kDa. The maximum activity of recombinant lipase obtained from Mut(s) recombinant was 90 IU/ml. The enzyme was stable in broad ranges of temperatures and pH, with the optimal temperature at 35 °C and pH 7.0. The crude recombinant R. oryzae lipase can be directly used for the transesterification of plant oils at high-water content of 60-100% (w/w) based on oil weight. The addition of 80% water to the transesterification systems resulted in the yield of methyl ester of 95%, 94% and 92% after 72 h using soybean oil, Jatropha curcas seed raw oil and Pistacia chinensis seed raw oil as raw material, respectively. These results indicate that the recombinant lipase is an effective biocatalyst for enzymatic biodiesel production.     

Characterization of thermostable lipase from thermophilic Geobacillus sp. TW1

A novel lipase-producing thermophilic strain TW1, assigned to Geobacillus sp. TW1 based on 16S rRNA sequence, was isolated from a hot spring in China. Based on this strain, a lipase gene encoding 417 amino acids was cloned. Subsequently, the lipase gene was expressed in Escherichia coli and purified as a fusion protein with glutathione S-transferase. The results showed that the recombinant lipase had an activity optimum at 40 degrees C and pH at 7.0-8.0. It was active up to 90 degrees C at pH 7.5, and stable over a wide pH ranging from 6.0 to 9.0. The recombinant lipase was stable in 1 mM enzyme inhibitors (EDTA, 2-ME, SDS, PMSF or DTT), as well as in 0.1% detergents (Tween 20, Chaps or Triton X-100). Its catalytic function was enhanced in the presence of Ca(2+), Mg(2+), Zn(2+), Fe(2+) or Fe(3+), but inhibited by Cu(2+), Mn(2+), and Li(+). By comparison with the crude lipase, the recombinant lipase had similar properties and was characteristic of thermostable enzymes. Our study presented a rapid overexpression and purification of the lipase gene from thermophile, aimed at improving the enzyme yield for industrial applications.     

Isolation and properties of oxaloacetate keto-enol-tautomerases from bovine heart mitochondria

Two highly purified proteins with quite different properties capable of oxaloacetate keto-enol-tautomerase activity (oxaloacetate keto-enol-isomerase, EC 5.3.2.2) were isolated from the bovine heart mitochondrial matrix. The first protein has an apparent molecular mass of 37 kDa as determined by SDS-gel electrophoresis and Sephacryl SF-200 gel filtration. It is quite stable upon storage at 40 degrees C and reaches the maximal catalytic activity at pH 8.5 with a half-maximal activity at pH 7.0. The enzyme is specifically inhibited by oxalate and diethyloxaloacetate. When assayed in the enol----ketone direction at 25 degrees C (pH 9.0), the enzyme obeys a simple substrate saturation kinetics with Km and Vmax values of 45 microM and 74 units per mg of protein, respectively; the latter value corresponds to the turnover number of 2700 min-1. The second protein has an apparent molecular mass of 80 kDa as determined by SDS-gel electrophoresis and Sephacryl SF-300 gel filtration. The enzyme is rapidly inactivated at 40 degrees C and shows a sharp pH optimum of activity at pH 9.0. The enzyme can be completely protected from thermal inactivation by oxaloacetate and dithiothreitol. The kinetic parameters of the enzyme as assayed in the enol----ketone direction at 25 degrees C (pH 9.0) are: Km = 220 microM and Vmax = 20 units per mg of protein; the latter corresponds to the turnover number of 1600 min-1. The enzyme activity is specifically inhibited by maleate and pyrophosphate. About 30% of the total oxaloacetate tautomerase activity in crude mitochondrial matrix is represented by the 37 kDa enzyme and about 70% by the 80 kDa protein.     

Characterization of a thermostable endo-beta-1,4-D-galactanase from the hyperthermophile Thermotoga maritima

A putative endo-beta-1,4-D-galactanase gene of Thermotoga maritima was cloned and overexpressed in Escherichia coli. The recombinant enzyme hydrolyzed pectic galactans and produced D-galactose, beta-1,4-D-galactobiose, beta-1,4-D-galactotriose, and beta-1,4-D-galactotetraose. The enzyme displayed optimum activity at 90 degrees C and pH 7.0. It was slowly inactivated above pH 8.0 and below pH 5.0 and stable at temperatures up to 80 degrees C.     

黑曲霉F044脂肪酶的分离纯化及酶学性质研究

黑曲霉F044脂肪酶发酵上清液经硫酸铵沉淀、透析、DEAESepharoseFastFlow阴离子交换层析和SephadexG-75凝胶过滤层析得到电泳纯的脂肪酶,纯化倍数为73·71倍,活性回收率为34%。对纯化脂肪酶性质研究表明:该脂肪酶分子量约为35~40kD,水解橄榄油的最适温度和最适pH分别为45℃和7·0,在60℃以下和pH2·0~9·0之间有很好的稳定性。该脂肪酶的水解活性对Ca2 表现明显的依赖性,而Mn2 、Fe2 和Zn2 对脂肪酶则有显著的抑制作用。在最适条件下水解pNPP的Km和Vmax分别为7·37mmol/L和25·91μmol/(min·mg)。其N-端的15个氨基酸序列为Ser(Glu/His)-Val-Ser-Thr-Ser-Thr-Leu-Asp-Glu-Leu-Gln-Leu-Phe-Ala-Gln。     

Purification and Characterization of a Cold-Adapted Lipase from Oceanobacillus Strain PT-11

We isolated a moderately halophilic lipase-producing bacterium from the saline soil. Based on the morphological, physiological, chemotaxonomic and phylogenetic analysis, the isolate PT-11 was postulated to be a novel species identified as Oceanobacillus rekensis PT-11. The lipase was purified 2.50-fold by Q-Sepharose FF and SP-Sepharose FF chromatography and its molecular mass was estimated to be 23.5 kDa by SDS-PAGE. It was highly active over the broad temperature ranging from 10 to 35°C and showed up to 80% of the maximum activity at 10°C indicating the lipase to be a typical cold-adapted enzyme. The enzyme activity was slightly enhanced by Na⁺, Li⁺ and K⁺. Incubation with detergents, such as Tween-20 and Tween-80, slightly inhibited the enzyme activity; while Triton X-100decreased the enzyme activity. The enzyme was fairly stable in the presence of long-chain alcohols but was highly denatured in hydrophilic solvents such as acetone or short-chain alcohols (C1–C3).     

Over-expression and properties of a purified recombinant Bacillus licheniformis lipase: a comparative report on Bacillus lipases

The gene coding for an extracellular lipase of Bacillus licheniformis was cloned using PCR techniques. The sequence corresponding to the mature lipase was subcloned into the pET 20b(+) expression vector to construct a recombinant lipase protein containing 6 histidine residues at the C-terminal. High-level expression of the lipase by Escherichia coli cells harbouring the lipase gene-containing expression vector was observed upon induction with IPTG at 30 degrees C. A one step purification of the recombinant lipase was achieved with Ni-NTA resin. The specific activity of the purified enzyme was 130 units/mg with p-nitrophenyl-palmitate as substrate. The enzyme showed maximum activity at pH 10-11.5 and was remarkably stable at alkaline pH values up to 12. The enzyme was active toward p-nitrophenyl esters of short to long chains fatty acids but with a marked preference for esters with C(6) and C(8) acyl groups. The amino acid sequence of the lipase shows striking similarities to lipases from Bacillus subtilis and Bacillus pumilus. Based on the amino acid identity and biochemical characteristics, we propose that Bacillus lipases be classified into two distinct subfamilies of their own.     

Characterization of turkey pancreatic lipase

Turkey pancreatic lipase (TPL) was purified from delipidated pancreases. Pure TPL (glycerol ester hydrolase, EC 3.1.1.3) was obtained after ammonium sulfate fractionation, Sephacryl S-200 gel filtration, anion exchange chromatography (DEAE-Sepharose) and size exclusion column using high performance liquid chromatography system (HPLC). The pure lipase, which is not a glycoprotein, was presented as a monomer having a molecular mass of about 45 kDa. The lipase activity was maximal at pH 8.5 and 37 degrees C. TPL hydrolyses the long chains triacylglycerols more efficiently than the short ones. A specific activity of 4300 U/mg was measured on triolein as substrate at 37 degrees C and at pH 8.5 in the presence of colipase and 4 mM NaTDC. This enzyme presents the interfacial activation when using tripropionin as substrate. TPL was inactivated when the enzyme was incubated at 65 degrees C or at pH less than 5. Natural detergent (NaTDC), synthetic detergent (Tween-20) or amphipatic protein (beta-lactoglobulin A) act as potent inhibitors of TPL activity. To restore the lipase activity inhibited by NaTDC, colipase should be added to the hydrolysis system. When lipase is inhibited by synthetic detergent or protein, simultaneous addition of colipase and NaTDC was required to restore the TPL activity. The first 22 N-terminal amino acid residues were sequenced. This sequence was similar to those of mammal's pancreatic lipases. The biochemical properties of pancreatic lipase isolated from bird are similar to those of mammals.     

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.     

温和气单孢菌YH311硫酸软骨素裂解酶的分离纯化与固定化

通过硫酸铵沉淀、QAESephadex-A50柱层析及Sephadex-G150凝胶过滤等纯化步骤,对源自温和气单孢菌YH311的ChSase进行了分离纯化。结果表明,ChSase经上述纯化步骤后被纯化了55倍,其最终纯度可达95%以上,比活为31.86u/mg。经SDSPAGE及IFE测定可知该酶的分子量约为80kD,等电点为4.3～4.8。将纯化后的ChSase用海藻酸钠或纤维素固定化后,ChSase的热稳定性及贮存稳定性均可得到大幅度的提高：固定化酶用80℃水浴处理120min或于4℃冰箱放置30d后仍可保留50%以上的相对活力;但固定化酶的收率较低,仅为18.56%和18.86%。     

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.     

Integration of purification with immobilization of Candida rugosa lipase for kinetic resolution of racemic ketoprofen

The two processes for the partial purification and for the immobilization of a crude lipase preparation (Candida rugosa Lipase OF) have been successfully integrated into one by simple adsorption of the enzyme onto a cation ion exchanger resin (SP-Sephadex C-50) at pH 3.5. Due to selective removal of the unfavorable lipase isoenzyme (L1), the enzyme components (mainly L2 and L3) that are tightly fixed on the resin displayed a significantly improved enantioselectivity (E value: 50 versus 13 with addition of Tween-80) in the biocatalytic hydrolysis of 2-chloroethyl ester of rac-ketoprofen. The activity yields of the immobilized lipase were 48 and 70%, respectively when emulsified and non-emulsified substrates were employed for enzyme assay. Moreover, the concentration of Tween-80 was found to be a factor affecting the lipase enantioselectivity. By using such an immobilized enzyme as biocatalyst, the process for preparing enantiopure (S)-ketoprofen becomes simpler and more practical as compared with the previously reported procedures and the product was obtained with >94% ee at 22.3% conversion in the presence of an optimal concentration (0.5 mg/ml) of Tween-80 at pH 3.5. Furthermore, the operational stability of the immobilized biocatalyst was examined in different types of reactors. In an air-bubbled column reactor, the productivity was much higher than that in a packed-bed column reactor, in spite of a slightly lower stability. Under optimal conditions, the air-bubbled column reactor could be operated smoothly for at least 350 h, remaining nearly 50% activity.     

Optimized production of a thermostable lipase by recombinantPseudomonas putida 1641

A thermostable lipase gene fromPseudomonas fluorescence was cloned into a multicopy plasmid and electroporated intoP. putida. The recombinantP. putida produced soluble lipase into the broth. Using these recombinant cells, the optimum conditions for lipase production were investigated. Olive oil was the most effective carbon source, producing upto 60 unit/ml. Supplementation of the medium with a surfactant, such as Tween-80, increased the lipase productivity significantly. The optimum temperature and pH for lipase production were 26°C and 7.0, respectively.     

Purification and characterization of a lipase from Pseudomonas aeruginosa KKA-5 and its role in castor oil hydrolysis

An extracellular lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) from Pseudomonas aeruginosa KKA-5 hydrolyzed castor oil by 90%. Purification of this castor oil-hydrolyzing lipase included ammonium sulfate precipitation and successive hydroxylapatite column chromatography. The enzyme was purified 518-fold. It was homogeneous electrophoretically and its molecular weight was estimated to be 30 kDa. The enzyme was stable up to 45°C and retained its activity in the alkaline pH range. Lipase was highly stable in the presence of aqueous organic solvents like methanol and ethanol. It was weakly inhibited in the presence of acetone. The anionic surfactant, sodium dodecyl sulfate, was inhibitory while the cationic surfactants, Triton X-100 and Tween-80 appreciably enhanced activity. Lipase was stabilized significantly by Ca²⁺. Inactivation of the enzyme by EDTA was overcome by sequential CaCl₂ treatment. This finding suggests the existence of a calcium-binding site in Pseudomonas aeruginosa KKA-5 lipase. Received 22 January 1998/ Accepted in revised form 27 April 1998