The 110kDa glutathione transferase of Yarrowia lipolytica is encoded by a homologue of the TEF3 gene from Saccharomyces cerevisiae: cloning, expression, and homology modeling of the recombinant protein

The TEF4 gene of the non-saccharomyces yeast Yarrowia lipolytica encodes an EF1Bgamma protein with structural similarity to the glutathione transferases (GSTs). This 1203bp gene was cloned, over-expressed in Escherichia coli, and the recombinant protein characterized. DNA sequencing of the cloned gene agreed with the recently completed Y. lipolytica genome and showed 100% identity to a previously reported 30-residue N-terminal sequence for a 110kDa Y. lipolytica GST, except that it encoded two additional N-terminal residues (N-Met-Ser-). The recombinant protein (subunit M(r) 52kDa) was found not to possess GST activity with 1-chloro-2,4-dinitrobenzene. Partial tryptic digestion released two fragments of M(r) 22 and 18kDa, which we interpret as N- and C-terminal domains. Homology modeling confirmed that the N-terminal domain of Y. lipolytica TEF4 encodes a GST-like protein.     

Secretion of pro- and mature Rhizopus arrhizus lipases by Pichia pastoris and properties of the proteins

The lipases of Rhizopus spp. share a high 1,3-regiospecificity toward triacylglycerols, which makes them important enzymes in lipid modification. In the present study, the extracellularly active production of recombinant Rhizopus arrhizus lipase was carried out with genes encoding the mature region (mRAL) and the mRAL having the prosequence (ProRAL) in Pichia pastoris. Two transformed P. pastoris clones containing the multicopy of mRAL and ProRAL genes were separately selected for the production of recombinant enzymes. In a fed-batch cultivation, where methanol feeding was controlled by an on-line methanol analyzer, the supernatant contained 91 mg/L recombinant pro-form lipase (rProRAL) and 80 mg/L recombinant mature lipase (rRAL) after 92 h of cultivation. rProRAL and rRAL were purified by ultrafiltration, SP-Sepharose Rast Flow chromatography, and Butyl-Sepharose Fast Flow chromatography. Molecular weights of rProRAL and rRAL are 32 kDa and 29 kDa, respectively. The amino-terminal analysis showed that the 32-kDa protein was mRAL attached with 28 amino acids of the carboxy-terminal part of the prosequence (rPro28RAL). The specific lipase activities of mRAL attached with 28 amino acids of the carboxy-terminal part of the prosequence (rPro28RAL) and rRAL were 1543 U/mg and 2437 U/mg. The rPro28RAL was more stable than rRAL at pH 4.0–7.0, whereas rRAL was more stable at pH 7.0–10.0. The rPro28RAL had the highest lipase activity toward tributyrin (C₄), whereas rRAL had the highest lipase activity toward tricaprylin (C₈).     

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.     

Secretory expression and characterization of a highly Ca-activated thermostable L2 lipase

Thermostable lipases are important biocatalysts, showing many interesting properties with industrial applications. Previously, a thermophilic Bacillus sp. strain L2 that produces a thermostable lipase was isolated. In this study, the gene encoding for mature thermostable L2 lipase was cloned into a Pichia pastoris expression vector. Under the control of the methanol-inducible alcohol oxidase (AOX) promoter, the recombinant L2 lipase was secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. After optimization the maximum recombinant lipase activity achieved in shake flasks was 125 U/ml. The recombinant 44.5 kDa L2 lipase was purified 1.8-fold using affinity chromatography with 63.2% yield and a specific activity of 458.1 U/mg. Its activity was maximal at 70 °C and pH 8.0. Lipase activity increased 5-fold in the presence of Ca²⁺. L2 lipase showed a preference for medium to long chain triacylglycerols (C₁₀–C₁₆), corn oil, olive oil, soybean oil, and palm oil. Stabilization at high temperature and alkaline pH as well as its broad substrate specificity offer great potential for application in various industries that require high temperature operations.     

A novel organic solvent tolerant lipase from Bacillus sphaericus 205y: extracellular expression of a novel OST-lipase gene

An organic solvent tolerant (OST) lipase gene from Bacillus sphaericus 205y was successfully expressed extracellularly. The expressed lipase was purified using two steps purification; ultrafiltration and hydrophobic interaction chromatography (HIC) to 8-fold purity and 32% recovery. The purified 205y lipase revealed homogeneity on denaturing gel electrophoresis and the molecular mass was at approximately 30 kDa. The optimum pH for the purified 205y lipase was 7.0-8.0 and its stability showed a broad range of pH value between pH 5.0 to 13.0 at 37 degrees C. The purified 205y lipase exhibited an optimum temperature of 55 degrees C. The activity of the purified lipase was stimulated in the presence of Ca2+ and Mg2+. Ethylenediaminetetraacetic acid (EDTA) has no effect on its activity; however inhibition was observed with phenylmethane sulfonoyl fluoride (PMSF) a serine hydrolase inhibitor. Organic solvents such as dimethylsulfoxide (DMSO), methanol, p-xylene and n-decane enhanced the activity. Studies on the effect of oil showed that the lipase was most active in the presence of tricaprin (C10). The lipase exhibited 1,3 positional specificity. Keywords: Bacter     

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.     

Expression of recombinant hybrid peptide cecropinA(1-8)-magainin2(1-12) in Pichia pastoris: purification and characterization

Hybrid antibacterial peptide CA-MA (cecropinA(1-8)-magainin2(1-12)) is a linear cationic peptide that has potent antimicrobial properties without hemolytic activity. To explore a new approach of expression of hybrid peptide CA-MA in methylotrophic yeast, Pichia pastoris, the gene of CA-MA was obtained by recursive PCR (rPCR) and cloned into the vector pPICZalpha-A. The SalI-linearized plasmid pPICZalpha-CA-MA was transformed into P. pastoris SMD1168 by electroporation. The expression was induced for 96h with 1.0% methanol at 28 degrees C, pH 5.0. Recombinant CA-MA was purified by reversed-phase HPLC and 22 mg pure active CA-MA was obtained from 1L fermentation culture. Tricine-SDS-PAGE indicated that recombinant CA-MA protein molecular weight is 2.6 kDa. Mass spectrometry of purified CA-MA demonstrated a single large signal for the molecular ion [M+2H+](2+) at 1281.07 m/z, identical to that of the putative protein (2.56 kDa). Antimicrobial assays showed that CA-MA has a broad spectrum of antimicrobial property against fungi, as well as Gram-positive and Gram-negative bacteria. This is the first report on the heterologous expression of a hybrid antibacterial peptide with molecular weight below 3.0 kDa in P. pastoris. Our results demonstrate that functional CA-MA can be produced in sufficient quantities using P. pastoris for use in further studies on functionality and diagnostic applications.     

Gene Cloning,Expression, and Characterization of the <Emphasis Type="Italic">Geobacillus Thermoleovorans</Emphasis> CCR11 Thermoalkaliphilic Lipase

The gene for a Geobacillus thermoleovorans CCR11 thermostable lipase was recovered by PCR and cloned. Four genetic constructions were designed and successfully expressed in E. coli: (i) the lipase structural gene (lipCCR11) in the PinPoint Xa vector; (ii) the lipase structural gene (lipACCR11) in the pET-28a(+) vector; (iii) the lipase structural gene minus the signal peptide (lipMatCCR11) in the pET-3b vector; and (iv) the lipase structural gene plus its own promoter (lipProCCR11) in the pGEM-T cloning vector. The lipase gene sequence analysis showed an open reading frame of 1,212 nucleotides coding for a mature lipase of 382 residues (40 kDa) plus a 22 residues signal peptide. Expression under T7 and T7lac promoter resulted in a 40- and 36-fold increase in lipolytic activity with respect to the original strain lipase. All recombinant lipases showed an optimal activity at pH 9.0, but variations were found in the temperature for maximum activity and the substrate specificity among them and when compared with the parental strain lipase, especially in the recombinant lipases that contained fusion tags. Therefore, it is important to find the appropriate expression system able to attain a high concentration of the recombinant lipase without compromising the proper folding of the protein.     

带His-tag的解脂耶氏酵母脂肪酶Lip2在毕赤酵母中的表达及纯化

将去自身信号肽并且N-端带6×His标签的YlLip2基因克隆至表达载体pPIC9K中,电转化GS115获得高效表达脂肪酶His₆-YlLip2的基因工程菌。筛选到的阳性克隆子摇瓶发酵脂肪酶活力最高为400U/ml。对重组毕赤酵母在10 L发酵罐中表达His₆-YlLip2的分批补料发酵工艺进行了初步优化,探讨了培养基、pH、温度对生物量和重组蛋白表达量的影响。结果表明:采用FM22培养基,诱导温度为25℃,pH 5.0,甲醇诱导114 h后His₆-YlLip2的最高酶活力达到3160U/ml。SDS-PAGE分析表明,蛋白的分子量大约为38kDa。重组的His₆-YlLip2经镍柱一步纯化后的纯度达到95.43%,比酶活达到4250U/mg。     

长双歧杆菌α-唾液酸苷酶在大肠杆菌中的表达及酶学性质

[背景]唾液酸苷酶是一类水解唾液酸糖复合物末端唾液酸残基的糖苷水解酶,广泛存在于动物和微生物中,具有重要的生物学功能.[目的]克隆一个长双歧杆菌(Bifidobacterium longum)唾液酸苷酶基因(blsia42)并在大肠杆菌(Escherichia coli)中表达,探讨该重组酶的酶学性质.[方法]从长双歧...     

High-level production of extracellular lipase by Yarrowia lipolytica mutants from methyl oleate

The yeast Yarrowia lipolytica degrades efficiently low-cost hydrophobic substrates for the production of various added-value products such as lipases. To obtain yeast strains producing high levels of extracellular lipase, Y. lipolytica DSM3286 was subjected to mutation using ethyl methanesulfonate (EMS) and ultraviolet (UV) light. Twenty mutants were selected out of 1600 mutants of Y. lipolytica treated with EMS and UV based on lipase production ability on selective medium. A new industrial medium containing methyl oleate was optimized for lipase production. In the 20 L bioreactor containing new industrial medium, one UV mutant (U6) produced 356 U/mL of lipase after 24h, which is about 10.5-fold higher than that produced by the wild type strain. The properties of the mutant lipase were the same as those of the wild type: molecular weight 38 kDa, optimum temperature 37°C and optimum pH 7. Furthermore, the nucleotide sequences of extracellular lipase gene (LIP2) in wild type and mutant strains were determined. Only two silent substitutions at 362 and 385 positions were observed in the ORF region of LIP2. Two single substitutions and two duplications of the T nucleotide were also detected in the promoter region. LIP2 sequence comparison of the Y. lipolytica DSM3286 and U6 strains shows good targets to effective DNA recombinant for extracellular lipase of Y. lipolytica.     

Optimization of a Thermostable Lipase from Bacillus stearothermophilus P1: Overexpression, Purification, and Characterization

An expression library was generated from a partial NcoI and HindIII digest of genomic DNA from the thermophilic bacterium, Bacillus stearothermophilus P1. The DNA fragments were cloned into the expression vector pQE-60 and transformed into Escherichia coli M15[EP4]. Sequence analysis of a lipase gene showed an open reading frame of 1254 nucleotides coding a 29-amino-acid signal sequence and a mature sequence of 388 amino acids. The expressed lipase was isolated and purified to homogeneity in a single chromatographic step. The molecular mass of the lipase was determined to be approximately 43 kDa by SDS-PAGE and mass spectrometry. The purified lipase had an optimum pH of 8.5 and showed maximal activity at 55°C. It was highly stable in the temperature range of 30–65°C. The highest activity was found with p-nitrophenyl ester-caprate as the synthetic substrate and tricaprylin as the triacylglycerol. Its activity was strongly inhibited by 10 mM phenylmethanesulfonyl fluoride and 1-hexadecanesulfonyl chloride, indicating that it contains a serine residue which plays a key role in the catalytic mechanism. In addition, it was stable for 1 h at 37°C in 0.1% Chaps and Triton X-100.     

High-level expression and characterization of Galactomyces geotrichum (BT107) lipase I in Pichia pastoris

The mature lipI gene, encoding the lipase I from Galactomyces geotrichum BT107, was obtained by PCR from genomic DNA, sequenced and cloned into a Pichia pastoris expression vector. Clones containing multiple copies of lipI integrated in their genome were analyzed to achieve high-level expression of the recombinant lipase I. One strain with four or more copies of the expression cassette was able to produce more than 200mg/L of extracellular heterologous protein. The lipase I was partially purified using anion exchange chromatography and its activity on monounsaturated (triolein) and polyunsaturated (triEPA) triglycerides was analyzed by a novel HPLC-MS assay.     

基因拷贝数和甲醇浓度对重组毕赤酵母产华根霉脂肪酶的影响

将华根霉脂肪酶基因克隆到甲基营养型毕赤酵母中表达,以甲醇利用快型菌株为宿主,在7 L发酵罐水平对脂肪酶基因拷贝数分别为3、5、6的3株基因重组菌——XY RCL-3、XY RCL-5、XY RCL-6进行高密度发酵调控,同时研究了甲醇浓度对表达华根霉脂肪酶的影响。结果表明,XY RCL-5在相同条件下发酵产酶能力高于XY RCL-6和XY RCL-3,最适甲醇诱导浓度控制在0.1%±0.02%时,酶活可达到12 500 U/mL,菌体干重达到204 g/L,蛋白浓度也能达到8.02 g/L。     

Production of pediocin PA-1 in the methylotrophic yeast Pichia pastoris reveals unexpected inhibition of its biological activity due to the presence of collagen-like material

Expression of the pedA gene from Pediococcus acidilactici, coding for mature bacteriocin Pediocin PA-1, was investigated using the yeast Pichia pastoris to obtain larger quantities of pediocin to support additional studies, including structure-function research. Following various cloning strategies, a KM71H (Mut(s)) strain was selected. A significant concentration (74 microg/ml) of extracellular recombinant pediocin was obtained but the pediocin showed no biological activity. Supernatant fluids from P. pastoris cultures, harboring or not pedA, inhibited the biological activity of natural pediocin PA-1. The recombinant pediocin appeared as a mixture of three main fractions (7-8, 11, 20 kDa vs. 4.6 kDa for natural pediocin PA-1). The recombinant pediocin was also less hydrophobic and behaved differently when subjected to isoelectric focusing. Strong evidence indicated that some "collagen-like" material was tightly associated, most probably via covalent binding, to the recombinant pediocin. The "collagen-like" material was most probably responsible for the lack of biological activity of the recombinant pediocin and for the differences observed regarding some of the physico-chemical properties. Both the recombinant pediocin and natural pediocin were sensitive to collagenase, suggesting that pediocin PA-1 may possess a somewhat "collagen-like" nature. Interestingly, recombinant pediocin preparations showed the ability to assemble into fibrils.     

基于基因的重新设计与高通量筛选策略选育解脂耶氏酵母脂肪酶高产菌株

脂肪酶（EC 3.1.1.3）是应用广泛的工业用酶。高效的脂肪酶产生菌是脂肪酶工业生产和应用的前提。通过基因的重新设计与合成技术优化了解脂耶氏酵母（Yarrowia lipolytica）脂肪酶YLL的密码子,并实现了其在毕赤酵母（Pichia pastoris）中的高效表达;通过高通量筛选策略获得了更高效的脂肪酶基因工程菌菌株SILVER。在14 L发酵罐条件下,菌株SILVER酶活达40 500 U/ml、蛋白质含量达2.52 g/L发酵液,为该类脂肪酶的产业化奠定了基础。     

少根根霉(Rhizopus arrhizus)脂肪酶基因在毕赤酵母中的分泌表达

利用PCR技术从少根根霉中扩增出脂肪酶基因(包括前导序列和成熟肽),并将其连接到酵母分泌表达载体pPIC9K中,转化毕赤酵母GS115。利用抗生素G418从重组阳性克隆中筛选得到高拷贝的转化子。在5 L的发酵罐中,当碳源耗尽后开始流加甲醇诱导脂肪酶的表达,经过96 h培养后发酵液上清液中重组脂肪酶(rRAL)的表达量约为90 mg/L。rRAL经过超滤,SP-Sepharose离子交换层析和Butyl-Sepharose疏水层析纯化。纯化后的蛋白在SDS-PAGE上为单一条带,表观分子量为32 kDa,比酶活为1 543 U/mg。N-端序列分析表明rRAL是经过加工后的产物。同时没有发现全长的Rhizopus arrhizus脂肪酶(RAL)被分泌表达。     

Expression in Pichia pastoris X33 of His-tagged lipase from a novel strain of Rhizopus oryzae and its mutant Asn 134 His: purification and characterization

The sequence corresponding to the mature lipase of Rhizopus oryzae WPG (ROLw) was subcloned in the pPIC9K expression vector, with a strong AOX₁ promoter, to construct a recombinant lipase protein containing six histidine residues at the N-terminal. The His-tagged lipase was expressed in Pichia Pastoris X₃₃ and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity chromatography (Ni-NTA resin). High level expression of the lipase by Pichia Pastoris X₃₃ cells harbouring the lipase gene containing expression vector was observed upon induction with 2.5 g/l methanol at 28°C; the specific activity of the purified His₆-ROLw was 1,500 or 760 U/mg using olive oil emulsion or tributyrin as substrates, respectively. To check the importance of Asn 134 His substitution in the affinity and substrate selectivity of ROLw, the mutant His₆-ROLw-N134H was overexpressed in Pichia Pastoris X33 and purified with the same nickel metal affinity column. The specific activity of the purified His-tagged ROLw-N134H was 5,900 and 35 U/mg using olive oil emulsion or tributyrin as substrate. A comparative study of the wild type (His₆-ROLw) and the mutant (His₆-ROLw-N134H) proteins was carried out. A 3D structure model of ROLw was built using the RNL structure as template. We have concluded that a slight increase in the exposed hydrophilic residues on the surface of ROLw as compared to RNL (ROLwN134H) could be responsible for a higher selectivity of ROlw for long and short chain triacylglycerols at the lipid/water interface and then explaining the importance of Asn 134 for the chain length specificity of ROLw. This property is quite rare among Rhizopus lipases and gives this new lipase great potential for use in the field of biocatalysis.