假单胞菌属脂肪酶的分子生物学研究进展

微生物脂肪酶是商品化脂肪酶的主要来源,并广泛应用于诸多工业领域。与其他微生物脂肪酶相比,细菌脂肪酶催化反应的类型更多、活性更高、稳定性更好,其中又以假单胞菌属(Pseudomonas)脂肪酶的性能最为优越。作为性能最为优越、应用最为广泛的一类脂肪酶,假单胞菌属脂肪酶研究一直是脂肪酶领域的热点。就假单胞菌属脂肪酶的分子生物学研究进展进行归纳和述评,包括基因资源挖掘及克隆、基因表达调控及分泌机制、活性过表达策略、蛋白质结晶及3D结构解析、蛋白质工程,并对其未来研究方向做出展望,以期为后续研究提供有益参考。     

产碱假单胞菌脂肪酶的克隆表达及酶学性质研究

【目的】克隆产碱假单胞菌的脂肪酶基因,实现其在大肠杆菌中异源表达并进行酶学性质研究。【方法】通过基因文库构建和PCR,获得脂肪酶基因,并以pET30a(+)为表达载体、E.coli BL21(DE3)为宿主菌,在大肠杆菌中进行异源表达,表达产物经HisTrapTM亲和层析柱纯化后进行酶学性质研究。【结果】从产碱假单胞菌中克隆得到一个脂肪酶基因,大小为1 575 bp(GenBank登录号为JN674069)。该酶分子量为55 kD,最适底物为p-NPO,最适反应温度和pH分别为35°C、pH 9.0。重组酶经1 mmol/L的Cu2+处理30 min可使酶活提高至156%。在最适反应条件下重组酶的比活力为275 U/mg,Km和Vmax分别为80μmol/L和290 mmol/(min.g protein)。【结论】产碱假单胞菌脂肪酶基因的克隆与表达不仅积累了脂肪酶基因的资源,并为其在手性拆分中的应用奠定基础。     

立体选择性脂肪酶的筛选及其在薄荷醇拆分中的应用

采用双平板透明圈方法,筛选得到1株能够选择性水解L-薄荷醇丙酸酯的微生物。通过16sDNA测序,鉴定为产碱假单胞菌Pseudomonas alcaligenes。该菌所产脂肪酶可水解4对薄荷醇丙酸酯,在35℃时绝对转化率达到50％时,表现出较高的对映体过量值（e.e.p〉99％）与非对映体过量值（d．e．p〉90％）。这一结果表明,产碱假单胞菌脂肪酶是具有工业应用前景的生物催化剂。     

假单胞菌研究现状及应用前景

假单胞菌是自然界分布最广的微生物之一,广泛的分布必然面对多样的生境,多样的生境造就了假单胞菌丰富的遗传多样性,此遗传多样性不仅为假单胞菌的环境适应性奠定了物质基础,也为人类提供了宝贵的遗传资源,具有巨大的理论和应用价值。从环境的生物修复、生物防治、生物转化、铜绿假单胞菌的耐药机制等4个方面总结假单胞菌的国内外研究现状,并对假单胞菌的应用前景进行展望。     

荧光假单胞菌脂肪酶固定化及催化制备生物柴油的工艺研究

研究了不同因素对制备固定化荧光假单胞菌脂肪酶的影响及固定化酶的酶学性质,并初步探讨了利用该固定化酶制备生物柴油的工艺。以海藻酸钠明胶为复合载体,采用包埋法制备固定化荧光假单胞菌脂肪酶,考察了载酶量、颗粒直径等因子对固定化效果的影响,并用制备的固定化酶进行了酶促酯交换合成生物柴油的工艺研究,考察了反应条件如酶量、反应温度、甲醇流加方式、醇油比等因素对甲酯得率的影响。试验结果表明,制备固定化荧光假单胞菌脂肪酶的最优条件为:每克载体给酶量为300 IU,选用6号注射器针头(内径为0.5 mm);通过酯交换,催化大豆油合成生物柴油的最佳反应工艺参数为:固定化酶25%,醇油比4:1,含水量6%,反应温度40℃;此条件下反应35 h后,甲酯的最高得率可达82%。     

脂肪酶产生菌的筛选、鉴定及其产酶条件优化

目的:寻找合适的产酶菌。方法:从富油土壤中分离到一株脂肪酶产生菌,并通过16S rRNA部分序列分析和系统发育分析将其鉴定为假单胞菌属,定名为:Pseudomonas sp.26-2。本研究进一步通过正交试验设计对该菌株的产脂肪酶条件进行了优化。结果:在摇瓶培养条件下,其最适产酶条件为:淀粉1.5%,酵母提取物3%,硫酸镁0.05%,K2HPO40.2%,橄榄油0.2%;反应起始pH值为7.0,发酵温度为30℃。在此条件下,发酵脂肪酶活力可达15.5U/ml。结论:所获得的假单胞菌26-2具有一定的脂肪酶生产能力,并为该菌株的菌种改良以及脂肪酶的高效基因工程菌的构建奠定了基础。     

铜绿假单胞菌铁摄取调节子Fur诱导表达突变株构建及表型分析

铁摄取调节子 (Ferric uptake regulator,Fur) 是细菌控制细胞内铁平衡的一类重要的调节子。铜绿假单胞菌Pseudomonas aeruginosa的fur为必需基因,不能直接敲除。文中通过构建诱导型缺失突变株Δfur/attB::PBAD-fur,来研究该基因对铜绿假单胞菌的生长、生物被膜形成、运动能力和抗氧应激能力等方面的影响。结果表明,当Fur低表达时,铜绿假单胞菌在高铁和低铁环境中出现了生长阻滞的现象;低表达Fur的铜绿假单胞菌抵抗H2O2的能力降低,形成生物被膜的能力减弱,游动、颤动 (Twitching) 和丛集 (Swarming) 运动能力也出现了减弱的现象。Fur的表达直接影响铜绿假单胞菌荧光嗜铁素的产量。在铜绿假单胞菌体内表达来自格瑞菲斯瓦尔德磁螺菌Fur超级家族中的蛋白,可以部分恢复铜绿假单胞菌荧光嗜铁素的产量。由此说明,Fur对铜绿假单胞菌的生长、生物被膜形成、抗氧应激能力和运动能力方面都起着至关重要的作用。本研究为铜绿假单胞菌的防治提供理论指导。     

乳杆菌对致病假单胞菌的抑制作用研究进展*

假单胞菌污染事件在临床就医和日常饮食中频发,屡次产生致病、致死等恶劣后果,有效抑制致病假单胞菌并降低其耐药性作为解决该问题的关键手段,是目前的研究重点。相关研究表明益生菌等天然活性成分对假单胞菌产生多方面影响,以应用范围最广的益生菌——乳杆菌为例,综合国内外最新研究进展,论述了乳杆菌对假单胞菌的生物膜结构、生长活性、生物毒性、黏附细胞表面能力及被假单胞菌感染后的小鼠等产生的影响。深入挖掘乳杆菌等益生菌及其代谢产物成分的作用机制,是防治假单胞菌等微生物污染和感染的关键。     

顺反式肉桂醛对肉源隆德假单胞菌生物被膜和致腐性的抑制作用

【目的】为比较反式和顺式肉桂醛对肉源假单胞菌生物被膜和致腐性的影响。【方法】通过平板计数测定两种肉桂醛对隆德假单胞菌的最小抑菌浓度(MIC),结晶紫法、珠涡流法、激光共聚焦显微镜观察、福林法等检测亚抑菌浓度肉桂醛处理下隆德假单胞菌生物被膜形成、运动性和胞外酶活性变化。荧光定量RT-PCR检测肉桂醛对隆德假单胞菌粘附lapA、鞭毛fliC、蛋白酶aprX和脂肪酶lip基因表达量的影响。【结果】反式和顺式肉桂醛对隆德假单胞菌的MIC分别为200μg/mL和225μg/mL,1/8 MIC、1/4MIC、1/2MIC亚抑菌浓度肉桂醛显著降低隆德假单胞菌生物被膜结晶紫和粘附性,其中1/2MIC反式和顺式肉桂醛处理下被膜分别减少60.27%和52.05%,菌体粘附降低56.35%和61.10%。亚抑菌浓度肉桂醛显著减少被膜厚度,反式肉桂醛还能显著杀灭被膜菌。且肉桂醛能显著抑制菌体的泳动性,反式肉桂醛对生物被膜和泳动性的抑制效果更强。肉桂醛还能抑制隆德假单胞菌蛋白酶和脂肪酶活性,其中1/2MIC反式和顺式肉桂醛处理下菌体蛋白酶分别减少61.90%和76.19%,脂肪酶降低40.17%和47.01%。且发现肉桂醛显著降低lapA、fliC、aprX和lip表达量,其中1/2MIC反式和顺式肉桂醛分别降低4个基因表达量至对照组的0.05–0.16和0.02–0.12倍。【结论】两种亚抑菌浓度肉桂醛异构体显著抑制隆德假单胞菌生物被膜和致腐性,其中反式肉桂醛对生物被膜抑制较强,而顺式肉桂醛更有效地降低致腐酶活性,其与肉桂醛下调相应基因表达密切相关。     

冷链食品中铜绿假单胞菌的检测方法研究及进化树构建

铜绿假单胞菌是条件致病菌,对于患者具有较大的感染风险与危害,因此建立冷链食品中铜绿假单胞菌的检测方法并完成其溯源分子进化树构建工作,对于保障食品安全至关重要。以冷链食品中的铜绿假单胞菌为研究对象,应用实时荧光PCR检测技术建立快速检测方法。通过冷链食品抽样调查检测出铜绿假单胞菌后,将样品中检测出的铜绿假单胞菌菌株进行测序分析并建立铜绿假单胞菌生物进化树完成溯源分析。结果利用该方法成功从随机采集的271份冷链食品中检出10株病原菌,病原菌总体检出率为3.69%(10/271),并通过分子进化树的构建成功溯源铜绿假单胞菌的污染来源并完成菌种种属定位。研究成果可以为冷链食品中铜绿假单胞菌等相关病原菌的检测溯源分析提供思路与方法。     

Gene synthesis, expression in E. coli, and in vitro refolding of Pseudomonas sp. KWI 56 and Chromobacterium viscosum lipases and their chaperones

Pseudomonas lipases are industrially used as detergent additives, in the food industry, and in organic synthesis. Currently, these lipases are either isolated from wild-type strains or overexpressed in recombinant Pseudomonas host strains which may be subject to special safety regulations and thus be unsuitable for enzyme engineering via directed evolution. Here we describe the heterologous expression of two Pseudomonas lipases in Escherichia coli. The lipase genes of Pseudomonas sp. KWI 56 (recently reclassified as Burkholderia cepacia) and Chromobacterium viscosum and the genes of their specific chaperones, which are required for correct folding, were synthesized with an optimized nucleotide sequence and overexpressed (up to 50%) in E. coli. However, both lipases were inactively expressed inside inclusion bodies. Quantitative in vitro refolding of the lipases in the presence of their specific chaperones yielded 310,000 U/g (Pseudomonas sp. KWI 56) and 190,000 U/g (C. viscosum) wet cells. In addition, these lipases could be demonstrated to refold efficiently in the presence of chaperones of related lipases.     

Purification, characterization, and molecular cloning of lactonizing lipase from Pseudomonas species

An extracellular lipase catalyzing the synthesis of macrocyclic lactones in anhydrous organic solvents was purified to homogeneity from Pseudomonas nov. sp. 109, and characterized. The lipase showed a pI of 5.3 on isoelectric focusing and a Mr of 29,000 +/- 1,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With respect to substrate specificity, optimum chain length for acyl moiety varied depending on the type of reaction catalyzed: C18 in monomer lactone formation, C11 or shorter in dimer lactone formation, and C8 in ester hydrolysis. The amino-terminal 19 amino acid residues of the purified lipase were determined as Ser-Thr-Tyr-Thr-Gln-Thr-Lys-Tyr-Pro-Ile-Val-Leu-Ala-His-Gly-Met-Leu-Gly- Phe, and the gene encoding the lipase was identified by hybridization to a synthetic 20-nucleotide probe, cloned, and sequenced. Nucleotide sequence analysis predicted a 311-amino acid open reading frame, a putative ribosome-binding site, and a 26-amino acid sequence at the amino terminus of the sequence that is not found in the mature protein. This 26-amino acid sequence has many of the characteristics common to known signal peptides. The lipase gene encoded a sequence of Val-Asn-Leu-Ile-Gly-His-Ser-His-Gly-Gly which is very well conserved among lipases, and showed 38-40% overall homology to the amino acid sequences of lipases from Pseudomonas fragie and Pseudomonas cepacia, but showed little homology to those of other lipases, suggesting that some structural features are required for catalyzing macrocyclic lactone synthesis in organic solvents and are restricted to lipases of the Pseudomonas origin.     

Enantioselective recognition mechanism of secondary alcohol by surfactant-coated lipases in nonaqueous media.

The enantioselective recognition mechanism of secondary alcohol by lipases originated from Candida rugosa and Pseudomonas cepacia was elucidated on the basis of the kinetic study of the esterification of alcohol with lauric acid in isooctane. To obtain inherent kinetic parameters, we utilized a surfactant-coated lipase whose conformation is considered to be an "open" form in a homogeneous organic solvent. Based on the experimental results, the enantioselectivity of lipases was found to be derived from the difference in the V(max) values between the two enantiomers. The same result was observed when lipases of different origin and substrates with different molecular structures were applied. © 1999 John Wiley & Sons, Inc.     

脂肪酶的特异性折叠酶

以Pseudomonas aeruginosa为代表的Ⅰ.1亚家族脂肪醇和以Burkholderia cepacia为代表的Ⅰ.2亚家族脂肪酶具有优良的手性化合物拆分性能,在绿色化工领域具有良好的开发潜力及其应用前景.Ⅰ.1亚家族脂肪酶和Ⅰ.2亚家族脂肪醇均采用Ⅱ型分泌机制进行分泌,其分泌依赖于一种分子伴侣--脂肪酶的特异性折叠酶(lipase specific foldase,Lif)的协助.从Lif蛋白的分类、结构特点、介导对应脂肪酶折叠的特异性及机制、lif基因表达调控机制及尚待探讨的问题等方面系统介绍Lif蛋白的研究进展.     

The cold-active lipase of Pseudomonas fragi. Heterologous expression, biochemical characterization and molecular modeling.

A recombinant lipase cloned from Pseudomonas fragi strain IFO 3458 (PFL) was found to retain significant activity at low temperature. In an attempt to elucidate the structural basis of this behaviour, a model of its three-dimensional structure was built by homology and compared with homologous mesophilic lipases, i.e. the Pseudomonas aeruginosa lipase (45% sequence identity) and Burkholderia cepacia lipase (38%). In this model, features common to all known lipases have been identified, such as the catalytic triad (S83, D238 and H260) and the oxyanion hole (L17, Q84). Structural modifications recurrent in cold-adaptation, i.e. a large amount of charged residues exposed at the protein surface, have been detected. Noteworthy is the lack of a disulphide bridge conserved in homologous Pseudomonas lipases that may contribute to increased conformational flexibility of the cold-active enzyme.     

Production of optically active esters and alcohols from racemic alcohols by lipase-catalyzed stereoselective transesterification in non-aqueous reaction system

Microbial lipase-catalyzed transesterification between vinyl acetate and (RS)-2-octanol or (RS)-1-phenylethanol was investigated in a reaction system without addition of aqueous or organic solvents. From a screening test with various lipases, it was found that the enzymes from Pseudomonas species could efficiently catalyze the reaction, and R-enantiomers of the racemic alcohols were preferentially esterified by them. Enantiomeric purities of the optically active alcohols (S) and esters (R) obtained from (RS)-1-phenylethanol by the stereoselective transesterification of these lipases were all more than 95%.     

Substrate specificities of bacterial polyhydroxyalkanoate depolymerases and lipases: bacterial lipases hydrolyze poly(omega-hydroxyalkanoates).

The substrate specificities of extracellular lipases purified from Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas fluorescens, and Burkholderia cepacia (former Pseudomonas cepacia) and of extracellular polyhydroxyalkanoate (PHA) depolymerases purified from Comamonas sp., Pseudomonas lemoignei, and P. fluorescens GK13, as well as that of an esterase purified from P. fluorescens GK 13, to various polyesters and to lipase substrates were analyzed. All lipases and the esterase of P. fluorescens GK13 but none of the PHA depolymerases tested hydrolyzed triolein, thereby confirming a functional difference between lipases and PHA depolymerases. However, most lipases were able to hydrolyze polyesters consisting of an omega-hydroxyalkanoic acid such as poly(6-hydroxyhedxanoate) or poly(4-hydroxybutyrate). The dimeric ester of hydroxyhexanoate was the main product of enzymatic hydrolysis of polycaprolactone by P. aeruginosa lipase. Polyesters containing side chains in the polymer backbone such as poly (3-hydroxybutyrate) and other poly(3-hydroxyalkanoates) were not or were only slightly hydrolyzed by the lipases tested.     

Enhancement of the organic solvent‐stability of the LST‐03 lipase by directed evolution

LST‐03 lipase from an organic solvent‐tolerant Pseudomonas aeruginosa LST‐03 has high stability and activity in the presence of various organic solvents. In this research, enhancement of organic solvent‐stability of LST‐03 lipase was attempted by directed evolution. The structural gene of the LST‐03 lipase was amplified by the error prone‐PCR method. Organic solvent‐stability of the mutated lipases was assayed by formation of a clear zone of agar which contained dimethyl sulfoxide (DMSO) and tri‐n‐butyrin and which overlaid a plate medium. And the organic solvent‐stability was also confirmed by measuring the half‐life of activity in the presence of DMSO. Four mutated enzymes were selected on the basis of their high organic solvent‐stability in the presence of DMSO. The organic solvent‐stabilities of mutated LST‐03 lipase in the presence of various organic solvents were measured and their mutated amino acid residues were identified. The half‐lives of the LST‐03‐R65 lipase in the presence of cyclohexane and n‐decane were about 9 to 11‐fold longer than those of the wild‐type lipase, respectively. Some substituted amino acid residues of mutated LST‐03 lipases have been located at the surface of the enzyme molecules, while some other amino acid residues have been changed from neutral to basic residues. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Cold active microbial lipases: some hot issues and recent developments

Lipases are glycerol ester hydrolases that catalyze the hydrolysis of triglycerides to free fatty acids and glycerol. Lipases catalyze esterification, interesterification, acidolysis, alcoholysis and aminolysis in addition to the hydrolytic activity on triglycerides. The temperature stability of lipases has regarded as the most important characteristic for use in industry. Psychrophilic lipases have lately attracted attention because of their increasing use in the organic synthesis of chiral intermediates due to their low optimum temperature and high activity at very low temperatures, which are favorable properties for the production of relatively frail compounds. In addition, these enzymes have an advantage under low water conditions due to their inherent greater flexibility, wherein the activity of mesophilic and thermophilic enzymes are severely impaired by an excess of rigidity. Cold-adapted microorganisms are potential source of cold-active lipases and they have been isolated from cold regions and studied. Compared to other lipases, relatively smaller numbers of cold active bacterial lipases were well studied. Lipases isolated from different sources have a wide range of properties depending on their sources with respect to positional specificity, fatty acid specificity, thermostability, pH optimum, etc. Use of industrial enzymes allows the technologist to develop processes that closely approach the gentle, efficient processes in nature. Some of these processes using cold active lipase from C. antarctica have been patented by pharmaceutical, chemical and food industries. Cold active lipases cover a broad spectrum of biotechnological applications like additives in detergents, additives in food industries, environmental bioremediations, biotransformation, molecular biology applications and heterologous gene expression in psychrophilic hosts to prevent formation of inclusion bodies. Cold active enzymes from psychrotrophic microorganisms showing high catalytic activity at low temperatures can be highly expressed in such recombinant strains. Thus, cold active lipases are today the enzymes of choice for organic chemists, pharmacists, biophysicists, biochemical and process engineers, biotechnologists, microbiologists and biochemists.     

Blue native electrophoresis study on lipases

We have developed a modified blue native polyacrylamide gel electrophoresis (PAGE) protocol that can overcome aggregation of lipases seen in native PAGE. We have shown that two lipases, Pseudomonas aeruginosa lipase and Candida rugosa lipase, which aggregate in the native gel, can be resolved using our protocol. Activity staining was done to test for the functionality of the two lipases.