微生物脂肪酶活性构像的形成及激活过程中的影响因子

微生物脂肪酶是一类具有重要应用价值的生物催化剂。作为一种胞外酶,其活性构像的形成及激活是一个高度复杂而特异性的生理过程。综述了作为微生物脂肪酶的结构成分,参与脂肪酶折叠和激活过程中的各种因子及其作用机制,这些因子包括脂肪酶特异性的折叠酶、脂肪酶激活因子、前序列、钙离子和二硫键等。     

细菌脂肪酶分泌的研究进展

细菌脂肪酶是一类重要的工业用酶,其分泌系统有着严谨的机制。革兰阳性细菌利用Sec-转运系统使脂肪酶跨过质膜完成分泌;革兰氏阴性细菌的外泌蛋白通过Sec-转运系统、Tat-转运系统或其他机制跨越内膜后,还必须利用Ⅰ型、Ⅱ型、Ⅲ型、Ⅳ型与Ⅴ型分泌系统来完成跨外膜分泌。详细介绍细菌脂肪酶分泌主要依赖的Sec-或Tat-跨内膜的转运系统及革兰氏阴性细菌的Ⅰ型、Ⅱ型与Ⅴ型自分泌系统的3种不同分泌方式。细菌脂肪酶分泌的研究对人们认识其分泌机制,并利用基因工程的手段提高其外泌产量等具有重要的指导意义。     

前导肽介导的蛋白折叠机制及其对脂肪酶影响的研究进展

大多数蛋白质的形成过程主要由合成前体蛋白和合成功能蛋白两个步骤组成.在这个过程中,前导肽能够辅助蛋白质折叠或抑制它的活性.前导肽作为脂肪酶结构中重要的一段多肽链,通常作为分子内分子伴侣来辅助脂肪酶的折叠,同时该序列上包括糖基化位点在内的一些特殊位点,对酶的活性、极端环境稳定性、甲醇耐受性和底物特异性等性质具有重要影响....     

阿维链霉菌Streptomyces avermitilis碱性脂肪酶LpsA2的表达和酶学性质分析

【目的】本研究将推测的阿维链霉菌(Streptomyces avermitilis)脂肪酶基因lpsA2在大肠杆菌(Escherichia coli)中进行异源表达及系统的酶学性质分析。【方法】提取阿维链霉菌基因组,设计特异性引物,PCR扩增脂肪酶基因lpsA2,使其在大肠杆菌中异源表达,利用6个组氨酸标签纯化脂肪酶LpsA2,并进行酶学性质分析;对LpsA2进行序列比对和进化分析。【结果】氨基酸序列比对显示LpsA2具有脂肪酶典型的由Ser、His和Asp构成的活性部位,即Ser130-Asp221-His25,其中Ser位于保守的五肽结构(Gly128-His129-Ser130-Gln131-Gly132)中;分子系统学分析显示,LpsA2是脂肪酶第一家族亚家族成员(Subfamily I.7);实验测得纯化的重组脂肪酶LpsA2的最适反应pH为8.0,最适反应温度为50℃;最适底物为对硝基苯酚豆蔻酸酯;在10℃-50℃范围内该酶的激活自由能为6.3 kcal/mol;1 mmol/L Co2+、Hg2+、Zn2+可使酶活性提高至250%以上;15%的二甲基甲酰胺和二甲基亚砜使酶活分别提高至110.7%和138%;0.1%和1%的Span-20可使酶活性分别提高至352.7%和189.7%。【结论】本研究对推测的来源于S.avermitilis的脂肪酶基因lpsA2进行了异源表达和酶学功能鉴定,不仅为脂肪酶的研究积累了更多数据,也为具有优良性能的脂肪酶生物工程菌的筛选奠定了基础,更为其在食品加工、药物合成等工业生产中的应用提供了依据。     

华根霉前导肽和成熟肽脂肪酶基因的克隆表达及性质研究

以华根霉(Rhizopus chinensis CCTCCM201021)为出发菌株,提取总DNA,PCR扩增出前导肽脂肪酶基因(proRCL)及成熟肽脂肪酶基因(mRCL),克隆到原核载体pET-28a,转化E-coli BL21(DE3),诱导表达并纯化出活性目的蛋白.经SDS-PAGE分析,重组蛋白ProRCL和MRCL的分子量分别约43 kD和33 kD.两重组脂肪酶主要酶学性质基本相似,最适反应温度均为35℃,40℃下较为稳定;最适反应pH相近,分别为8.0和8.5;以pNP脂肪酸酯为底物,均对短链脂肪酸酯底物特异性较高.MPCL上述性质与野生型RCL基本一致.研究结果表明,通过条件控制可以利用E.coli表达系统表达具有活性的华根霉前导肽脂肪酶ProRCL和成熟肽脂肪酶MRCL.与米根霉脂肪酶(ROL)不同,前导序列对华根霉脂肪酶的主要酶学特性没有显著影响.     

陆地棉脂肪酶基因克隆及氨基酸序列分析

根据陆地棉(Gossypium hirsutum L.)EST序列设计一对引物,采用RT-PCR方法从萌发的棉籽中获得了脂肪酶(triacylglycerol acylhydrolases,EC 3.1.1.3)基因,该基因编码483个氨基酸;比对结果显示,棉籽脂肪酶与拟南芥、水稻、蓖麻等脂肪酶相似性较低,具有由Ser-Asp-His组成的三联体催化活性中心,在亲核Ser残基周围有GXSXG保守序列.软件预测显示该脂肪酶为可溶性蛋白,分子量约为55.4 kD,等电点为9.07,不含N端信号肽,亚细胞定位可能是过氧化物酶体或胞质溶胶.     

利用非经典分泌蛋白质实现脂肪酶A的分泌表达

【目的】以枯草芽孢杆菌脂肪酶A(Lipase A)为报告蛋白,尝试利用4种非经典分泌蛋白质及其前50个氨基酸作为分泌信号以实现其分泌表达。【方法】我们扩增了脂肪酶A的编码基因和非经典分泌蛋白质的编码序列,构建了8种针对脂肪酶A的分泌表达载体,并转化至枯草芽孢杆菌WB800菌株,通过测定重组菌株的酶活、利用蛋白质电泳和免疫印迹等技术检测脂肪酶A的分泌情况【结果】以Pdh A的氨基酸序列和Sod A、Eno的前50氨基酸序列作为分泌信号的重组菌株较好的实现了脂肪酶A的分泌表达。【结论】部分非经分泌蛋白质的编码基因或其前50个氨基酸序列能够引导脂肪酶A分泌至细胞外。     

扩展青霉碱性脂肪酶基因在毕赤酵母中的高效表达

将编码扩展青霉碱性脂肪酶 (PEL)的cDNA克隆到酵母整合型质粒pPIC3.5K ,电转化His4缺陷型巴斯德毕赤酵母 (Pichiapastoris)GS115 ,通过橄榄油 MM平板及PCR方法筛选和鉴定重组子。重组子发酵液经SDS PAGE分析、橄榄油检验板鉴定 ,表明扩展青霉碱性脂肪酶基因在巴斯德毕赤酵母中获得了高效表达。表达蛋白分泌至培养基中 ,分子量约 2 8kD ,与扩展青霉碱性脂肪酶大小一致 ,占分泌蛋白的 95 %。橄榄油检验板检验表明该表达蛋白可分解橄榄油 ,通过优化该表达菌的发酵条件 ,以橄榄油为底物进行酶活测定 ,其发酵液酶活可达 2 6 0u mL。     

人类新基因netrin-G 2的初步分析

Netrins是与层粘连蛋白相关的、高度保守的小分子分泌蛋白家族成员,在细胞迁移和轴突导向活动中具有重要的作用,其同源物在多种模式动物中均已发现.Netrins分为2个亚家族:netrins和netrin-Gs;其中的netrin-G亚家族各成员之间具有高度的相似性.目前,在人类中得到确证的只有netrin-G₁,推测在人类中应该还有其它的netrin-G亚家族成员以及编码它的基因.从20周龄人胚中提取脑组织的总RNA,用PCR cDNA文库试剂盒制备脑组织cDNA文库,再用特异性引物进行PCR扩增,得到1条人netrin-G的全长cDNA,命名为人netrin-G₂,用Northern杂交研究其表达,用进化树分析其与netrin家族各成员间的关系;证实人netrin-G₂确实为netrin-G亚家族的1个新成员,该基因位于染色体的9q34,大小为2 428 bp,含有1个1 593 bp的假定开放阅读框,起始密码子为86位的甲硫氨酸,终止密码子为1 678位的TGA,可编码1个大小为530个氨基酸的蛋白质;Northern杂交显示,人netrin-G₂在脑组织中特异性表达,而在其它组织中却很少发现,推测人netrin-G₂可能在中枢神经系统的发育过程中具有重要的作用,可能与刺激性神经冲动的传递以及神经调节有关.     

扩展青霉PF898碱性脂肪酶cDNA的克隆及序列分析

扩展青霉 (Penicilliumexpansum)PF898可产生一种具有工业价值的碱性脂肪酶 (PEL) .在测定了其N端 12个氨基酸残基序列的基础上 ,通过RT PCR、5′RACE、基因克隆及序列测定 ,获得了PEL完整的cDNA序列 (GenBank登录号为AF2 84 0 6 4 ) .cDNA全长 10 5 0bp ,包括PEL编码区、3′非翻译区和部分 5′非翻译区基因的序列 .编码区cDNA由 85 5个碱基组成 ,编码 1个由 2 85个氨基酸残基组成的酶蛋白 ,其信号肽及前肽部分由 2 7个氨基酸残基组成 ,成熟肽部分由 2 5 8个氨基酸残基组成 .根据氨基酸组成推导该脂肪酶蛋白的分子量为 2 7 3kD .该脂肪酶的氨基酸序列 130～ 134位上有各类脂肪酶中普遍存在的G X S X G保守序列     

Cloning and sequence analysis of the lipase and lipase chaperone-encoding genes from Acinetobacter calcoaceticus RAG-1, and redefinition of a proteobacterial lipase family and an analogous lipase chaperone family

The genes encoding the lipase (LipA) and lipase chaperone (LipB) from Acinetobacter calcoaceticus RAG-1 were cloned and sequenced. The genes were isolated from a genomic DNA library by complementation of a lipase-deficient transposon mutant of the same strain. Transposon insertion in this mutant and three others was mapped to a single site in the chaperone gene. The deduced amino acid (aa) sequences for the lipase and its chaperone were found to encode mature proteins of 313 aa (32.5kDa) and 347 aa (38.6kDa), respectively. The lipase contained a putative leader sequence, as well as the conserved Ser, His, and Asp residues which are known to function as the catalytic triad in other lipases. A possible trans-membrane hydrophobic helix was identified in the N-terminal region of the chaperone. Phylogenetic comparisons showed that LipA, together with the lipases of A. calcoaceticus BD413, Vibrio cholerae El Tor, and Proteus vulgaris K80, were members of a previously described family of Pseudomonas and Burkholderia lipases. This new family, which we redefine as the Group I Proteobacterial lipases, was subdivided into four subfamilies on the basis of overall sequence homology and conservation of residues which are unique to the subfamilies. LipB, moreover, was found to be a member of an analogous family of lipase chaperones. We propose that the lipases produced by P. fluorescens and Serratia marcescens, which comprise a second sequence family, be referred to as the Group II Proteobacterial lipases. Evidence is provided to support the hypothesis that both the Group I and Group II families have evolved from a combination of common descent and lateral gene transfer.     

Cloning and sequence analysis of the lipase and lipase chaperone-encoding genes from Acinetobacter calcoaceticus RAG-1, and redefinition of a Proteobacterial lipase family and an analogous lipase chaperone family

The genes encoding the lipase (LipA) and lipase chaperone (LipB) from Acinetobacter calcoaceticus RAG-1 were cloned and sequenced. The genes were isolated from a genomic DNA library by complementation of a lipase-deficient transposon mutant of the same strain. Transposon insertion in this mutant and three others was mapped to a single site in the chaperone gene. The deduced amino acid (aa) sequences for the lipase and its chaperone were found to encode mature proteins of 313 aa (32.5 kDa) and 347 aa (38.6 kDa), respectively. The lipase contained a putative leader sequence, as well as the conserved Ser, His, and Asp residues which are known to function as the catalytic triad in other lipases. A possible trans-membrane hydrophobic helix was identified in the N-terminal region of the chaperone. Phylogenetic comparisons showed that LipA, together with the lipases of A. calcoaceticus BD413, Vibrio cholerae El Tor, and Proteus vulgaris K80, were members of a previously described family of Pseudomonas and Burkholderia lipases. This new family, which we redefine as the Group I Proteobacterial lipases, was subdivided into four subfamilies on the basis of overall sequence homology and conservation of residues which are unique to the subfamilies. LipB, moreover, was found to be a member of an analogous family of lipase chaperones. We propose that the lipases produced by P. fluorescens and Serratia marcescens, which comprise a second sequence family, be referred to as the Group II Proteobacterial lipases. Evidence is provided to support the hypothesis that both the Group I and Group II families have evolved from a combination of common descent and lateral gene transfer.     

A novel fluorogenic substrate for the measurement of endothelial lipase activity

Endothelial lipase (EL) is a phospholipase A1 (PLA1) enzyme that hydrolyzes phospholipids at the sn-1 position to produce lysophospholipids and free fatty acids. Measurement of the PLA1 activity of EL is usually accomplished by the use of substrates that are also hydrolyzed by lipases in other subfamilies such as PLA2 enzymes. In order to distinguish PLA1 activity of EL from PLA2 enzymatic activity in cell-based assays, cell supernatants, and other nonhomogeneous systems, a novel fluorogenic substrate with selectivity toward PLA1 hydrolysis was conceived and characterized. This substrate was preferred by PLA1 enzymes, such as EL and hepatic lipase, and was cleaved with much lower efficiency by lipases that exhibit primarily triglyceride lipase activity, such as LPL or a lipase with PLA2 activity. The phospholipase activity detected by the PLA1 substrate could be inhibited with the small molecule esterase inhibitor ebelactone B. Furthermore, the PLA1 substrate was able to detect EL activity in human umbilical vein endothelial cells in a cell-based assay. This substrate is a useful reagent for identifying modulators of PLA1 enzymes, such as EL, and aiding in characterizing their mechanisms of action.     

Differential behaviour of Pseudomonas sp. 42A2 LipC,a lipase showing greater versatility than its counterpart LipA

Growth of Pseudomonas sp. 42A2 on oleic acid releases polymerized hydroxy-fatty acids as a result of several enzymatic conversions that could involve one or more lipases. To test this hypothesis, the lipolytic system of strain Pseudomonas sp. 42A2 was analyzed, revealing the presence of at least an intracellular carboxylesterase and a secreted lipase. Consensus primers derived from a conserved region of bacterial lipase subfamilies I.1 and I.2 allowed isolation of two secreted lipase genes, lipA and lipC, highly homologous to those of Pseudomonas aeruginosa PAO1. Homologous cloning of the isolated lipA and lipC genes was performed in Pseudomonas sp. 42A2 for LipA and LipC over-expression. The overproduced lipases were further purified and characterized, both showing preference for medium fatty acid chain-length substrates. However, significant differences could be detected between LipA and LipC in terms of enzyme kinetics and behaviour pattern. Accordingly, LipA showed maximum activity at moderate temperatures, and displayed a typical Michaelis–Menten kinetics. On the contrary, LipC was more active at low temperatures and displayed partial interfacial activation, showing a shift in substrate specificity when assayed at different temperatures, and displaying increased activity in the presence of certain heavy metal ions. The versatile properties shown by LipC suggest that this lipase could be expressed in response to variable environmental conditions.     

Novel zinc-binding center and a temperature switch in the Bacillus stearothermophilus L1 lipase

The bacterial thermoalkalophilic lipases optimally hydrolyze saturated fatty acids at elevated temperatures. They also have significant sequence homology with staphylococcal lipases, and both the thermoalkalophilic and staphylococcal lipases are grouped as the lipase family I.5. We report here the first crystal structure of the lipase family I.5, the structure of a thermoalkalophilic lipase from Bacillus stearothermophilus L1 (L1 lipase) determined at 2.0-A resolution. The structure is in a closed conformation, and the active site is buried under a long lid helix. Unexpectedly, the structure exhibits a zinc-binding site in an extra domain that accounts for the larger molecular size of the family I.5 enzymes in comparison to other microbial lipases. The zinc-coordinated extra domain makes tight interactions with the loop extended from the C terminus of the lid helix, suggesting that the activation of the family I.5 lipases may be regulated by the strength of the interactions. The unusually long lid helix makes strong hydrophobic interactions with its neighbors. The structural information together with previous biochemical observations indicate that the temperature-mediated lid opening is triggered by the thermal dissociation of the hydrophobic interactions.     

Crystal Structure of Proteus mirabilis Lipase,a Novel Lipase from the Proteus/Psychrophilic Subfamily of Lipase Family I.1

Bacterial lipases from family I.1 and I.2 catalyze the hydrolysis of triacylglycerol between 25–45°C and are used extensively as biocatalysts. The lipase from Proteus mirabilis belongs to the Proteus/psychrophilic subfamily of lipase family I.1 and is a promising catalyst for biodiesel production because it can tolerate high amounts of water in the reaction. Here we present the crystal structure of the Proteus mirabilis lipase, a member of the Proteus/psychrophilic subfamily of I.1lipases. The structure of the Proteus mirabilis lipase was solved in the absence and presence of a bound phosphonate inhibitor. Unexpectedly, both the apo and inhibitor bound forms of P. mirabilis lipase were found to be in a closed conformation. The structure reveals a unique oxyanion hole and a wide active site that is solvent accessible even in the closed conformation. A distinct mechanism for Ca²⁺ coordination may explain how these lipases can fold without specific chaperones.     

Purification and properties of the lipases from Rhodotorula pilimanae Hedrick and Burke

Summary The Rhodotorula pilimanae CBS 5804 strain secretes into the culture medium two lipases: their pH optima are 4 and 7. The two lipases were purified by precipitation with acetone followed by chromatography on SP-Sephadex C50 and Sephadex G200. The purification factors achieved in comparison with the supernatant culture were x74 for lipase I and x90 for lipase II. The molecular weights were estimated at 172,800 and 21,400 for lipase I and lipase II, respectively. Their activities are optimal between 45°C and 55°C. The activation energies were 5.9 kcal·mole^-1 for lipase I and 12.4 kcal·mole^-1 for lipase II. The inactivation energies were about 21.9 and 17.7 kcal·mole^-1 for lipase I and lipase II, respectively. The enzymes are slightly inhibited by Cu²⁺, Co²⁺, Hg²⁺, Mn²⁺, N-acetylacetone, acetic acid and sodium lauryl sulphate. EDTA did not affect their enzymatic activity. These two lipases are secreted in the culture media in the absence of inducer; their biosynthesis is not inhibited by glucose. These lipases hydrolyse primarily the 1-(or 3-)position of all triglycerides tested.     

Purification of extracellular lipases from Trichosporon fermentans WU-C12

Two types of extracellular lipases (I and II) from Trichosporon fermentans WU-C12 were purified by acetone precipitation and successive chromatographies on Butyl-Toyopearl 650 M, Toyopearl HW-55F and Q-Sepharose FF. The molecular weight of lipase I was 53 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and 160 kDa by gel filtration, while that of lipase II was 55 kDa by SDS-PAGE and 60 kDa by gel filtration. For the hydrolysis of olive oil, the optimum pH and temperature of both the lipases were 5.5 and 35°C, respectively. The lipases showed stable activities after incubation at 30°C for 24 h in a pH range from 4.0 to 8.0. The thermostability of lipase I for 30 min at a reaction pH of 5.5 was up to 40°C, while that of lipase II under the same conditions was up to 50°C. Both lipases could hydrolyze the 1-, 2-, and 3-positions of triolein, and cleave all three ester bonds, regardless of the position in the triglyceride.     

Analysis of Comparative Sequence and Genomic Data to Verify Phylogenetic Relationship and Explore a New Subfamily of Bacterial Lipases

Thermostable and organic solvent-tolerant enzymes have significant potential in a wide range of synthetic reactions in industry due to their inherent stability at high temperatures and their ability to endure harsh organic solvents. In this study, a novel gene encoding a true lipase was isolated by construction of a genomic DNA library of thermophilic Aneurinibacillus thermoaerophilus strain HZ into Escherichia coli plasmid vector. Sequence analysis revealed that HZ lipase had 62% identity to putative lipase from Bacillus pseudomycoides. The closely characterized lipases to the HZ lipase gene are from thermostable Bacillus and Geobacillus lipases belonging to the subfamily I.5 with ≤ 57% identity. The amino acid sequence analysis of HZ lipase determined a conserved pentapeptide containing the active serine, GHSMG and a Ca²⁺-binding motif, GCYGSD in the enzyme. Protein structure modeling showed that HZ lipase consisted of an α/β hydrolase fold and a lid domain. Protein sequence alignment, conserved regions analysis, clustal distance matrix and amino acid composition illustrated differences between HZ lipase and other thermostable lipases. Phylogenetic analysis revealed that this lipase represented a new subfamily of family I of bacterial true lipases, classified as family I.9. The HZ lipase was expressed under promoter P_lac using IPTG and was characterized. The recombinant enzyme showed optimal activity at 65°C and retained ≥ 97% activity after incubation at 50°C for 1h. The HZ lipase was stable in various polar and non-polar organic solvents.     

Cloning,expression and characterization of an organic solvent tolerant lipase from Pseudomonas fluorescens JCM5963

A novel lipase gene from an organic solvent degradable strain Pseudomonas fluorescens JCM5963 was cloned, sequenced, and overexpressed as an N-terminus His-tag fusion protein in E. coli. The alignment of amino acid sequences revealed that the protein contained a lipase motif and shared a medium or high similarity with lipases from other Pseudomonas strains. It could be defined as a member of subfamily I.1 lipase. Most of the recombinant proteins expressed as enzymatically active aggregates soluble in 20 mM Tris–HCl buffer (pH 8.0) containing sodium deoxycholate are remarkably different from most subfamily I.1 and I.2 members of Pseudomonas lipases expressed as inactive inclusion body formerly described in E. coli. The recombinant lipase (rPFL) was purified to homogeneity by Ni-NTA affinity chromatography and Sephacryl S-200 gel filtration chromatography. The purified lipase was stable in broad ranges of temperatures and pH values, with the optimal temperature and pH value being 55 °C and 9.0, respectively. Its activity was found to increase in the presence of metal ions such as Ca²⁺, Sn²⁺ and some non-ionic surfactants. In addition, rPFL was activated by and remained stable in a series of water-miscible organic solvents solutions and highly tolerant to some water-immiscible organic solvents. These features render this novel lipase attraction for biotechnological applications in the field of organic synthesis and detergent additives.