Biochemical and Structural Characterization of Two Site-Directed Mutants of <Emphasis Type="Italic">Staphylococcus xylosus</Emphasis> Lipase

Staphylococcus xylosus AF208229 lipase was expressed in E. coli containing an histidine-tag (WT-Val). In the present work, in order to check the importance of the residue 309 in the specific activity, the amino acid side chain residue valine 309 was substituted by aspartate or lysine through site-directed mutagenesis. Both mutant lipases (MUT-Lys and MUT-Asp) were expressed in E. coli and the recombinant histidine-tagged lipases were purified by immobilized metal ion affinity chromatography. The enzyme activity was determined using p-nitrophenyl butyrate as substrate and secondary structure content was evaluated by circular dichroism. MUT-Lys and MUT-Asp presented significant increase of lipase activity (P < 0.05) in comparison to WT-Val, although highest activities for the three enzymes were observed at the same pH and temperature (pH 9.0 and 42°C). The wild type and mutant lipases presented high thermal stability, after 30 min of incubation at 80°C all enzymes retained their initial activities.     

Characterization of two soil metagenome-derived lipases with high specificity for <Emphasis Type="Italic">p</Emphasis>-nitrophenyl palmitate

Two novel genes (pwtsB and pwtsC) encoding lipases were isolated by screening the soil metagenomic library. Sequence analysis revealed that pwtsB encodes a protein of 301 amino acids with a predicted molecular weight of 33 kDa, and pwtsC encodes a protein of 323 amino acids with a predicted molecular weight of 35 kDa. Furthermore, both genes were cloned and expressed in Escherichia coli BL21 (DE3) using pET expression system. The expressed recombinant enzymes were purified by Ni-nitrilotriacetic acid affinity chromatography and characterized by spectrophotometric with different p-nitrophenyl esters. The results showed that PWTSB displayed a high degree of activity and stability at 20°C with an optimal pH of around 8.0, and PWTSC at 40°C with an optimal pH of around 7.0. P-nitrophenyl palmitate (p-NPP) was identified as the best substrate of PWTSB and PWTSC. The specific activities of PWTSB and PWTSC were 150 and 166 U/mg, respectively toward p-NPP at 30°C, about 20-fold higher than that toward p-nitrophenyl butyrate (C4) and caprylate (C8). In conclusion, our results suggest that PWTSB is a cold adapt lipase and PWTSC is a thermostable lipase to long-chain p-nitrophenyl esters. P. Wei and L. Bai contributed equally to this work.     

The short form of the recombinant CAL-A-type lipase UM03410 from the smut fungus <Emphasis Type="Italic">Ustilago maydis</Emphasis> exhibits an inherent <Emphasis Type="Italic">trans</Emphasis>-fatty acid selectivity

The Ustilago maydis lipase UM03410 belongs to the mostly unexplored Candida antarctica lipase (CAL-A) subfamily. The two lipases with […] the highest identity are a lipase from Sporisorium reilianum and the prototypic CAL-A. In contrast to the other CAL-A-type lipases, this hypothetical U. maydis lipase is annotated to possess a prolonged N-terminus of unknown function. Here, we show for the first time the recombinant expression of two versions of lipase UM03410: the full-length form (lipUMf) and an N-terminally truncated form (lipUMs). For comparison to the prototype, the expression of recombinant CAL-A in E. coli was investigated. Although both forms of lipase UM03410 could be expressed functionally in E. coli, the N-terminally truncated form (lipUMs) demonstrated significantly higher activities towards p-nitrophenyl esters. The functional expression of the N-terminally truncated lipase was further optimized by the appropriate choice of the E. coli strain, lowering the cultivation temperature to 20 °C and enrichment of the cultivation medium with glucose. Primary characteristics of the recombinant lipase are its pH optimum in the range of 6.5–7.0 and its temperature optimum at 55 °C. As is typical for lipases, lipUM03410 shows preference for long chain fatty acid esters with myristic acid ester (C14:0 ester) being the most preferred one. More importantly, lipUMs exhibits an inherent preference for C18:1Δ9 trans and C18:1Δ11 trans-fatty acid esters similar to CAL-A. Therefore, the short form of this U. maydis lipase is the only other currently known lipase with a distinct trans-fatty acid selectivity.     

Cloning,Expression, Purification,and Characterization of a Novel Esterase from <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis>

Lactobacillus plantarum is an important lactic acid bacterium, usually found as natural inhabitant of food, such as fermented vegetables and meat products. However, little information about lactic acid bacteria, especially concerning L. plantarum, as a source of useful enzymes has been reported. The aim of this study was to clone, express in Escherichia coli, purify, and characterize an esterase from L. plantarum ATCC 8014. The esterase gene (1014 bp) was amplified and cloned in pET14b expression vector to express a His₆-tagged protein in E. coli. Recombinant L. plantarum esterase was purified by Ni-NTA resin, presenting an apparent molecular mass of about 38 kDa. It presented highest activity at pH 6.0 and 40°C. Also, it presented preference for p-nitrophenyl butyrate, but hydrolyzed more efficiently p-nitrophenyl acetate. Besides, this study shows, for the first time, CD data about secondary structure of an esterase from L. plantarum.     

Culture of Staphylococcus xylosus in fish processing by‐product‐based media for lipase production

Aims: The objective of this study was to demonstrate that fish‐processing by‐products could be used as sole raw material to sustain the growth of Staphylococcus xylosus for lipase production. Methods and Results: Bacterial growth was tested on supernatants generated by boiling (100°C for 20 min) of tuna, sardine, cuttlefish and shrimp by‐products from fish processing industries. Among all samples tested, only supernatants generated from shrimp and cuttlefish by‐products sustained the growth of S. xylosus. Shrimp‐based medium gave the highest growth (A₆₀₀ = 22) after 22 h of culture and exhibited the maximum lipase activity (28 U ml^?1). This effect may be explained by better availability of nutrients, especially, in shrimp by‐products. Standard medium (SM) amendments to sardine and tuna by‐product‐based media stimulated the growth of S. xylosus and the highest A₆₀₀ values were obtained with 75% SM. Lipase activity, however, remained below 4 U ml^?1 for both sardine and tuna by‐product‐based media. Conclusions: Fish by‐products could be used for the production of highly valuable enzymes. Significance and Impact of the Study: The use of fish by‐products in producing S. xylosus‐growth media can reduce environmental problems associated with waste disposal and, simultaneously, lower the cost of biomass and enzyme production.     

Alphitobius diaperinus Panzer (Insecta,Coleoptera) in a single house of a broiler production facility as a potential source of pathogenic bacteria for broilers and humans

Pest infestation in any stage can lead to a quality reduction in the finished products. This study aimed to detect Escherichia coli, Salmonella spp., Campylobacter spp., and Staphylococcus aureus in Alphitobius diaperinus adults, and in samples from broiler swabs, administered water and feed collected in a single house from a broiler production facility in central Italy. Three samplings were carried out, each collecting ninety adult beetles for microbial detection in the external, faecal and internal content; ten cloacal swab samples; and one sample of both administered feed and water. Microbiological cultures and biochemical identification were performed on suspected cultures and confirmed by species-specific PCRs. A. diaperinus was abundantly found near the windows, under the manger and in the corners of the facility. Salmonella enterica serovar Cholerasuis was found at the external surface of the beetles, while Staphylococcus xylosus and E. coli were in the faecal content. The latter micro-organism together with Staphylococcus lentus, S. xylosus and other staphylococcal species were detected in the internal microbiota. E. coli and Campylobacter spp. were observed in cloacal swabs, and S. xylosus in one feed sample. The study findings support evidence for Salmonella spp. and E. coli, and remark that adherence to sanitation rules and biosecurity procedures are required.     

Overexpression of Cold Shock Protein A of <Emphasis Type="Italic">Psychromonas arctica</Emphasis> KOPRI 22215 Confers Cold-Resistance

A polar bacterium was isolated from Arctic sea sediments and identified as Psychromonas artica, based on 16S rDNA sequence. Psychromonas artica KOPRI 22215 has an optimal growth temperature of 10 °C and a maximum growth temperature of 25 °C, suggesting this bacterium is a psychrophile. Cold shock proteins (Csps) are induced upon temperature downshift by more than 10 °C. Functional studies have researched mostly Csps of a mesophilic bacterium Escherichia coli, but not on those of psychrophilic bacteria. In an effort to understand the molecular mechanisms of psychrophilic bacteria that allow it withstand freezing environments, we cloned a gene encoding a cold shock protein from P. artica KOPRI 22215 (CspA_Pa) using the conserved sequences in csp genes. The 204 bp-long ORF encoded a protein of 68 amino acids, sharing 56% homology to previously reported E. coli CspA protein. When CspA_Pa was overexpressed in E. coli, it caused cell growth-retardation and morphological elongation. Interestingly, overexpression of CspA_Pa drastically increased the host’s cold-resistance by more than ten times, suggesting the protein aids survival in polar environments.     

The insertion of (LK) residues at the N-terminus of Staphylococcus xylosus lipase affects its catalytic properties and its enantioselectivity

Two residues (LK) were inserted at the N-terminus of Staphylococcus xylosus lipase (SXL). The S. xylosus lipase wild-type (WT) and its constructed mutant (LK-SXL) were expressed in Escherichia coli BL21 (DE3) and purified to homogeneity using Sephacryl S-200 gel filtration, anion exchange and cation exchange chromatography.A comparative study on the kinetics and interfacial properties of the SXL wild-type and its mutant was performed using emulsified and monolayer substrate. The first important observation was that the insertion of two residues (LK) resulted in an improvement in temperature activity, a better stability, a 3-fold specific activity and a 6-fold catalytic efficiency (k_cat/K_mapp), compared to the SXL wild-type.Using the monolayer technique, this study showed that the inserted residues (LK) at the N-terminus do not affect the adsorption step of the S. xylosus lipase into a monomolecular film of diC₁₂-phosphatidyl-choline.A kinetics study on the surface pressure dependency, stereoselectivity, and regioselectivity of the SXL wild-type and its mutant was also performed using the three dicaprin isomers spread as monomolecular films at the air-water interface. Furthermore, the insertion of two residues (LK) at the N-terminus of S. xylosus lipase was shown to be accompanied by the switch of its enantioselectivity.To confirm that the insertion of LK residues at the N-terminus of SXL affects both the hydrolysis properties and the synthesis, new experiments were performed and the production of butyl acetate by SXL before and after LK insertion was assessed. The insertion of these residues may have some valuable applications in industry.     

A novel cold-adapted lipase from <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. XMZ-26: gene cloning and characterisation

Acinetobacter sp. XMZ-26 (ACCC 05422) was isolated from soil samples obtained from glaciers in Xinjiang Province, China. The partial nucleotide sequence of a lipase gene was obtained by touchdown PCR using degenerate primers designed based on the conserved domains of cold-adapted lipases. Subsequently, a complete gene sequence encoding a 317 amino acid polypeptide was identified. Our novel lipase gene, lipA, was overexpressed in Escherichia coli. The recombinant protein (LipA) was purified by Ni-affinity chromatography, and then deeply characterised. The LipA resulted to hydrolyse pNP esters of fatty acids with acyl chain length from C2 to C16, and the preferred substrate was pNP octanoate showing a k _cat = 560.52 ± 28.32 s⁻¹, K _m = 0.075 ± 0.008 mM, and a k _cat/K _m = 7,377.29 ± 118.88 s⁻¹ mM⁻¹. Maximal LipA activity was observed at a temperature of 15°C and pH 10.0 using pNP decanoate as substrate. That LipA peaked at such a low temperature and remained most activity between 5°C and 35°C indicated that it was a cold-adapted enzyme. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents, including Ninol, Triton X-100, methanol, PEG-600, and DMSO. This cold-adapted lipase may find applications in the detergent industry and organic synthesis.     

Gene cloning,expression and characterization of a cold-adapted lipase from a psychrophilic deep-sea bacterium Psychrobacter sp. C18

A psychrophilic bacterium Psychrobacter sp. C18 previously isolated from the Southern Okinawa Trough deep-sea sediments showed extracellular lipolytic activity towards tributyrin. A genomic DNA library was constructed and screened to obtain the corresponding lipase gene. The sequenced DNA fragment contains an open reading frame of 945 bp, which was denoted as the lipX gene, from which a protein sequence LipX was deduced of 315 amino acid residues with a molecular mass of 35,028 Da. This protein contained the bacterial lipase GNSMG (GxSxG, x represents any amino acid residue) and HG consensus motifs. The recombinant pET28a(+)/lipX gene was overexpressed in heterologous host Escherichia coli BL21 (DE3) cells to overproduce the lipase protein LipX_His with a 6× histidine tag at its C-terminus. Nickel affinity chromatography was used for purification of the expressed recombinant lipase. The maximum lipolytic activity of the purified recombinant lipase was obtained at temperature of 30°C and pH 8.0 with p-nitrophenyl myristate (C14) as a substrate. Thermostability assay indicated that the recombinant LipX_His is a cold-adapted lipase, which was active in 10% methanol, ethanol, acetone and 30% glycol, and inhibited partially by Zn²⁺, Co²⁺, Mn²⁺, Fe³⁺ and EDTA. Most non-ionic detergents, such as DMSO, Triton X-100, Tween 60 and Tween 80 enhanced the lipase activity but 1% SDS completely inhibited the enzyme activity. Additionally, the highest lipolytic rate of the recombinant LipX_His lipase was achieved when p-nitrophenyl myristate was used as a substrate, among all the p-nitrophenyl esters tested.     

Cloning,Expression and Purification of <Emphasis Type="Italic">Microcystis viridis</Emphasis> Lectin in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Microcystis viridis lectin (MVL), a sugar-binding protein originally isolated from freshwater blue-green algae Microcystis viridis, has been reported to have potent anti-HIV activity. In this paper, we described the expression and purification of recombinant-MVL (R-MVL) gene in E. coli. The results demonstrated that the R-MVL in shake flask cultures was primarily expressed either in the form of inclusion bodies at 37°C or in the soluble fraction at 23 °C. Secondly, a one-step purification based on nickel-affinity chromatography was employed and 15 mg of highly purified (>95%) R-MVL from 1 l of cell cultures was yielded. The purified R-MVL was then subjected to MALDI-TOF–MS analysis for protein identification. In conclusion, for the first time, the R-MVL was successfully cloned and expressed in E. coli, which is useful for further study and large-scale cost-effective production of MVL protein.     

Enzymatic propyl gallate synthesis in solvent-free system: Optimization by response surface methodology

The ability of a non-commercial immobilized Staphylococcus xylosus lipase to catalyze the esterification of propanol with gallic acid was investigated and the antioxidant as well as the antimicrobial activities of the ester formed were evaluated. The response surface methodology, based on a three variables Box–Behnken design (reaction temperature, enzyme amount and 1-propanol/gallic acid molar ratio), was used to optimize the experimental conditions of propylgallate synthesis. The maximum conversion yield (90% ±3.5) was obtained by using 400 IU of immobilized lipase and a propanol/gallic acid at a molar ratio of 160 at 52 °C. The obtained ester was characterized by spectroscopic methods, NMR and FTIR. The antioxidant activity of propyl gallate was evaluated and compared to the synthetic classical antioxidants, BHA and ascorbic acid, taken as references. In addition, the antimicrobial activity of the propyl gallate was tested against S. xylosus, Escherchia coli and Staphylococcus aureus using disc diffusion and macrodilution methods. Our results show that the synthesized propyl gallate ester presents a higher antioxidant and antimicrobial power than the parent gallic acid as well as the synthetic classical antioxidants.     

Screening Escherichia coli, Enterococcus faecalis, and Clostridium perfringens as Indicator Organisms in Evaluating Pathogen-Reducing Capacity in Biogas Plants

This study was conducted to identify an indicator organism(s) in evaluating the pathogen-reducing capacity of biogas plants. Fresh cow manure containing 10⁴ to 10⁵ colony forming unit (CFU) per milliliter of Escherichia coli and Enterococcus faecalis along with an inoculated Clostridium perfringens strain were exposed to 37°C for 15 days, 55°C for 48 h, and 70°C for 24 h. C. perfringens was the most heat-resistant organism followed by E. faecalis, while E. coli was the most heat-sensitive organism. E. coli was reduced below detection limit at all temperatures with log₁₀ reductions of 4.94 (10 s), 4.37 (40 min), and 2.6 (5 days) at 70°C, 55°C, and 37°C, respectively. Maximum log₁₀ reductions for E. faecalis were 1.77 at 70°C (1 day), 1.7 at 55°C (2 days) and 3.13 at 37°C (15 days). For C. perfringens, maximum log₁₀ reduction at 37°C was 1.35 log₁₀ units (15 days) compared to less than 1 unit at 55 and 70°C. Modeling results showed that E. faecalis and C. perfringens had higher amount of heat-resistant fraction than E. coli. Thus, E. faecalis and C. perfringens can be used as indicator organisms to evaluate pathogen-reducing capacity in biogas plants at high temperatures of 55°C and 70°C while at 37°C E. coli could also be included as indicator organism.     

A thermoalkaliphilic lipase of <Emphasis Type="Italic">Geobacillus</Emphasis> sp. T1

A thermoalkaliphilic T1 lipase gene of Geobacillus sp. strain T1 was overexpressed in pGEX vector in the prokaryotic system. Removal of the signal peptide improved protein solubility and promoted the binding of GST moiety to the glutathione-Sepharose column. High-yield purification of T1 lipase was achieved through two-step affinity chromatography with a final specific activity and yield of 958.2 U/mg and 51.5%, respectively. The molecular mass of T1 lipase was determined to be approximately 43 kDa by gel filtration chromatography. T1 lipase had an optimum temperature and pH of 70°C and pH 9, respectively. It was stable up to 65°C with a half-life of 5 h 15 min at pH 9. It was stable in the presence of 1 mM metal ions Na⁺, Ca²⁺, Mn²⁺, K⁺ and Mg²⁺ , but inhibited by Cu²⁺, Fe³⁺ and Zn²⁺. Tween 80 significantly enhanced T1 lipase activity. T1 lipase was active towards medium to long chain triacylglycerols (C10–C14) and various natural oils with a marked preference for trilaurin (C12) (triacylglycerol) and sunflower oil (natural oil). Serine and aspartate residues were involved in catalysis, as its activity was strongly inhibited by 5 mM PMSF and 1 mM Pepstatin. The T _m for T1 lipase was around 72.2°C, as revealed by denatured protein analysis of CD spectra.     

Molecular and enzymatic characterization of a subfamily I.4 lipase from an edible oil-degrader <Emphasis Type="Italic">Bacillus</Emphasis> sp. HH-01

An edible-oil degrading bacterial strain HH-01 was isolated from oil plant gummy matter and was classified as a member of the genus Bacillus on the basis of the nucleotide sequence of the 16S rRNA gene. A putative lipase gene and its flanking regions were cloned from the strain based on its similarity to lipase genes from other Bacillus spp. The deduced product was composed of 214 amino acids and the putative mature protein, consisting of 182 amino acids, exhibited 82% amino acid sequence identity with the subfamily I.4 lipase LipA of Bacillus subtilis 168. The recombinant product was successfully overproduced as a soluble form in Escherichia coli and showed lipase activity. The gene was, therefore, designated as lipA of HH-01. HH-01 LipA was stable at pH 4–11 and up to 30°C, and its optimum pH and temperature were 8–9 and 30°C, respectively. The enzyme showed preferential hydrolysis of the 1(3)-position ester bond in trilinolein. The activity was, interestingly, enhanced by supplementing with 1 mM CoCl₂, in contrast to other Bacillus lipases. The lipA gene seemed to be constitutively transcribed during the exponential growth phase, regardless of the presence of edible oil.     

Enantioselective Nitrilase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis>: Cloning,Heterologous Expression,and Bioreactor Studies

Nitrilases have attracted tremendous attention for the preparation of optically pure carboxylic acids. This article aims to address the production and utilization of a highly enantioselective nitrilase from Pseudomonas putida MTCC 5110 for the hydrolysis of racemic mandelonitrile to (R)-mandelic acid. The nitrilase gene from P. putida was cloned in pET 21b(+) and over-expressed as histidine-tagged protein in Escherichia coli. The histidine-tagged enzyme was purified from crude cell extracts of IPTG-induced cells of E. coli BL21 (DE3). Inducer replacement studies led to the identification of lactose as a suitable and cheap alternative to the costly IPTG. Effects of medium components, various physico-chemical, and process parameters (pH, temperature, aeration, and agitation) for the production of nitrilase by engineered E. coli were optimized and scaled up to a laboratory scale bioreactor (6.6 l). Finally, the recombinant E. coli whole-cells were utilized for the production of (R)-(−)-mandelic acid.     

Characterization of a thermostable lipase showing loss of secondary structure at ambient temperature

A gene encoding extracellular lipase was cloned and characterized from metagenomic DNA extracted from hot spring soil. The recombinant gene was expressed in E. coli and expressed protein was purified to homogeneity using hydrophobic interactions chromatography. The mature polypeptide consists of 388 amino acids with apparent molecular weight of 43 kDa. The enzyme displayed maximum activity at 50°C and pH 9.0. It showed thermal stability up to 40°C without any loss of enzyme activity. Nearly 80% enzyme activity was retained at 50°C even after incubation for 75 min. However above 50°C the enzyme displayed thermal instability. The half life of the enzyme was determined to be 5 min at 60°C. Interestingly the CD spectroscopic study carried out in the temperature range of 25–95°C revealed distortion in solution structure above 35°C. However the intrinsic tryptophan fluorescence spectroscopic study revealed that even with the loss of secondary structure at 35°C and above the tertiary structure was retained. With p-nitrophenyl laurate as a substrate, the enzyme exhibited a K _m , V _max and K _cat of 0.73 ± 0.18 μM, 239 ± 16 μmol/ml/min and 569 s⁻¹ respectively. Enzyme activity was strongly inhibited by CuCl₂, HgCl₂ and DEPC but not by PMSF, eserine and SDS. The protein retained significant activity (~70%) with Triton X-100. The enzyme displayed 100% activity in presence of 30% n-Hexane and acetone.     

Engineering of <Emphasis Type="Italic">Bacillus</Emphasis> lipase by directed evolution for enhanced thermal stability: effect of isoleucine to threonine mutation at protein surface

A lip gene from a Bacillus isolate was cloned and expressed in E. coli. By thermal denaturation analysis, T_1/2 of lipase was observed to be 7 min at 50°C with less than 10% activity after 1 h incubation at 50°C. To expand the functionality of cloned lipase, attempts have been made to create thermostable variants of lip gene. A lipase variant with an isoleucine to threonine amino acid substitution at the protein surface was isolated that demonstrated higher thermostability than its wild type predecessor. To explore the structure–function relationship, the lip gene product of wild type (WT) and mutant was characterized in detail. The mutation enhanced the specific activity of enzyme by 2-folds when compared with WT. The mutant enzyme showed enhanced T_1/2 of 21 min at 50°C. The kinetic parameters of the mutant enzyme were significantly altered. The mutant enzyme displayed higher affinity for substrate (decreased K _m ) in comparison to the wild type. The k _cat and catalytic efficiency (k _cat/K _m ) of mutant were also enhanced by two and five times, respectively, as compared with the WT. The mutation resides on the part of helix which is exposed to the solvent and away from the catalytic triad. The replacement of a solvent exposed hydrophobic residue (Ile) in WT with a hydrophilic residue (Thr) in mutant might impart thermostability to the protein structure.     

Expression and functional analysis of Euglena Gracilis chloroplast initiation factor 3

A portion of a cDNA predicted to encode the mature form of Euglena gracilis chloroplast translational initiation factor 3 (IF-3_chlM, molecular mass, 46402) and the portion of this factor homologous to bacterial IF-3 (IF-3_chlH, molecular mass 22829) have been cloned and expressed in Escherichia coli as histidine-tagged proteins. The homology domain can be expressed in reasonable levels in E. coli. However, IF-3_chlM is quite toxic and can only be produced in small amounts. Both forms of the chloroplast factor are associated with E. coli ribosomes. Purification procedures have been developed for both IF-3_chlM and IF-3_chlH using Ni-NTA affinity chromatography followed by ion exchange chromatography. IF-3_chlM and IF-3_chlH are active in promoting ribosome dissociation and in promoting the binding of fMet-tRNA to E. coli ribosomes. However, IF-3_chlH has at least 5-fold more activity than either native IF-3_chl or IF-3_chlM in promoting initiation complex formation on chloroplast 30S ribosomal subunits in the presence of a mRNA carrying a natural translational initiation signal. This observation suggests that regions of IF-3_chl lying outside of the homology domain may down-regulate the activity of this factor.This work was supported in part by National Institutes of Health Grant GM24963.     

Cloning,characterization and expression of the extracellular lipase gene from <Emphasis Type="Italic">Aureobasidium </Emphasis><Emphasis Type="Italic">pullulans</Emphasis> HN2-3 isolated from sea saltern

The extracellular lipase structural gene was isolated from cDNA of Aureobasidium pullulans HN2-3 by using SMART^TM RACE cDNA amplification kit. The gene had an open reading frame of 1245 bp long encoding a lipase. The coding region of the gene was interrupted by only one intron (55 bp). It encodes 414 amino acid residues of a protein with a putative signal peptide of 26 amino acids. The protein sequence deduced from the extracellular lipase structural gene contained the lipase consensus sequence (G-X-S-X-G) and three conserved putative N-glycosylation sites. According to the phylogenetic tree of the lipases, the lipase from A. pullulans was closely related to that from Aspergillus fumigatus (XP_750543) and Neosartorya fischeri (XP_001257768) and the identities were 50% and 52%, respectively. The mature peptide encoding cDNA was subcloned into pET-24a (+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21(DE3). The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 47 kDa was found. Enzyme activity assay verified the recombinant protein as a lipase. A maximum activity of 0.96 U/mg was obtained from cellular extract of E. coli BL21(DE3) harboring pET-24a(+)LIP1. Optimal pH and temperature of the crude recombinant lipase were 8.0 and 35 °C, respectively and the crude recombinant lipase had the highest hydrolytic activity towards peanut oil.