脂肪酶的表面展示及其应用

脂肪酶是一种广泛应用的水解酶类。脂肪酶的表面展示技术不仅是脂肪酶蛋白质工程中一种有效的高通量筛选方法,而且展示的脂肪酶与自由酶相比具备更高的温度稳定性、有机溶剂稳定性等优点,其作为全细胞催化剂与传统的固定化脂肪酶相比也具备诸多优点。脂肪酶表面展示的宿主包括噬菌体、细菌以及酵母等,本文将分别介绍这三种宿主中脂肪酶表面展示的概况以及其作为高通量筛选和全细胞等方面的应用。     

脂肪酶表面展示技术

脂肪酶是一种广泛应用的水解酶类.脂肪酶的表面展示技术不仅是脂肪酶蛋白质工程中一种有效的高通量筛选方法,而且展示的脂肪酶与自由酶相比具备更高的温度稳定性、有机溶剂稳定性等优点,其作为全细胞催化剂与传统的固定化脂肪酶相比也具备诸多优点.脂肪酶表面展示的宿主包括噬菌体、细菌以及酵母等,将分别介绍这三种宿主中脂肪酶表面展示的概况及其作为高通量筛选和全细胞等方面的应用.     

细菌脂肪酶在大肠杆菌细胞表面的功能性展示

细胞表面展示技术已广泛应用于突变文库的高通量筛选,有力地促进了蛋白质工程的发展。以来自于铜绿假单胞菌的自转运蛋白Est A的羧基端结构域作为锚定区,构建脂肪酶LipA与EstA羧基端结构域的融合基因,并将融合基因插入到改造后的pACYC-Duet表达载体中,获得表面展示载体pBCCB-X1。将载体pBCCB-X1分别导入到大肠杆菌JK321和大肠杆菌UT5600菌株中,以IPTG诱导融合基因的表达。分别用三丁酸甘油酯定性检测和pNPO定量检测诱导表达后的全细胞脂肪酶的水解活性。试验结果表明,脂肪酶LipA在大肠杆菌JK321和大肠杆菌UT5600细胞表面均得到功能性展示,水解活性分别为(2.8±0.1)U/OD和(2.6±0.06)U/OD。脂肪酶LipA在大肠杆菌细胞表面的功能性展示,为后续高通量筛选LipA突变基因文库,奠定了坚实的基础。     

微生物脂肪酶资源挖掘及其催化性能改良策略

脂肪酶催化在食品、医药、化工、能源等领域发挥重要作用.开发新型微生物脂肪酶资源,对脂肪酶进行修饰改良,是脂肪酶催化领域的重要研发内容.极端微生物和不可培养微生物脂肪酶的发掘是获取新型工业催化剂的热点;体外定向进化、杂合酶、表面展示等蛋白质工程等分子生物学技术手段为开发特定性质"新酶"提供了有力工具;生物印迹、pH记忆、定向固定化、交联酶晶体、脂质体包埋等高效物理化学修饰方法拓宽了脂肪酶原有的催化性质.微生物脂肪酶资源挖掘及其改良将推动脂肪酶的生物催化产业快速发展.     

毕赤酵母组成型表达脂肪酶及其高通量筛选方法

【目的】获得组成型表达脂肪酶毕赤酵母,建立利用橄榄油罗丹明B平板高通量筛选组成型表达华根霉脂肪酶基因的有效方法。【方法】运用PCR技术从pGAPZαA表达载体上扩增得到GAP启动子片段,插入到表达载体pPIC9K-proRCL中,构建组成型表达载体pGAPK-proRCL。在保留含有同源双交换重组序列的诱导型启动子AOX1序列的基础上,电转化后华根霉Rhizopus chinensis CCTCC M201021脂肪酶基因proRCL表达盒在毕赤酵母基因组上发生双交换整合事件,从而组成型表达单拷贝的华根霉脂肪酶基因。【结果】重组菌发酵144 h后,脂肪酶最高酶活为130 U/mL。利用橄榄油罗丹明B平板高通量筛选组成型表达华根霉脂肪酶基因。【结论】该方法将初筛时间从12 d缩短为3 d,排除了多拷贝突变株的干扰,为后续脂肪酶的定向进化及筛选奠定了基础。     

核糖体展示技术

组合化学在分子生物学中的应用,大大提高了所构建分子文库的容量,而高通量筛选技术对于大容量文库的筛选是至关重要的。核糖体展示技术克服了传统筛选技术库容量小和筛选效率低等缺陷。该技术完全在体外进行,其文库容量不受细胞转染效率的影响,库容量和筛选效率得到很大提高,可与其他技术相组合,在体外分子高效表达和定向进化方面具有广泛的应用前景。该文介绍核糖体展示技术的基本原理、技术特点以及最新研究进展。     

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。     

微生物表面展示技术及其在环境污染治理中的应用

微生物表面展示技术是通过基因工程手段,将短的外源肽或蛋白质表达在微生物细胞表面,该技术可以应用于开发活的细菌疫苗、筛选抗体库、生产生物细胞吸附剂以及制备整细胞生物催化剂。通过金属高效结合肽的肽库筛选和微生物展示技术,将金属结合肽直接展示在微生物的表面,用于处理环境中的重金属污染,为环境中重金属污染的防治提供了一条崭新的途径。利用微生物表面展示技术制备整细胞催化剂,用于有毒有机污染物的处理,可以极大地加快污染物的降解速率。简要介绍了微生物表面展示技术及其在重金属污染治理和毒性有机污染物的脱毒等环境生物修复方面的最新研究进展。     

酵母细胞表面展示技术及其在非水相酶催化合成中的应用

酵母展示技术是将外源蛋白与酵母细胞壁蛋白融合,并将外源蛋白表达在酵母细胞表面。酵母展示技术已广泛应用于各种功能蛋白的表达及筛选。以下重点介绍酵母展示技术在脂肪酶展示体系构建及其在脂肪酸甲酯、短链芳香酯及糖酯生物合成中的应用。     

细胞表面展示技术在环境修复方面的研究进展*

人类社会工业化导致各种有毒物质被排放到环境中造成严重的污染。除了自然降解外,传统的处理方法包括化学转化、物理吸附、离子交换和电化学方法等,但存在二次污染、能源需求高、投资成本高、再生效率低、低浓度废水处理效率低等缺点。细胞表面展示技术是一种通过表面锚定蛋白在细胞表面连接功能肽的新型、高效的生物技术。与细胞内和分泌物表达系统相比,微生物表面展示的蛋白质对有机溶剂、蛋白酶、温度和pH的变化表现出更强的稳定性。通过细胞培养就可以获得固定在细胞表面的蛋白酶,避免了蛋白质纯化、浓缩等繁琐的程序。此外,细胞表面展示技术是良好的单细胞水平突变体文库高通量筛选平台。综述细胞表面展示技术在环境生物修复方面的研究进展,重点介绍该技术的应用和未来发展前景。     

利用冰核蛋白N末端结构域在大肠杆菌表面展示粘质沙雷氏菌脂肪酶

【目的】利用细胞表面工程技术将活性脂肪酶展示于大肠杆菌细胞表面并对展示脂肪酶的酶学性质进行研究。【方法】将丁香假单胞菌冰核蛋白N末端结构域序列与粘质沙雷氏菌脂肪酶编码基因融合,构建成脂肪酶表面展示载体,并转化大肠杆菌BL21(DE3)。【结果】重组菌以终浓度0.05 mmol/L异丙基硫代-D-半乳糖苷(IPTG)、25°C条件下诱导培养,16 h后表面展示脂肪酶活力达到最大值1 852 U/g细胞干重。表面展示酶的最适pH为9.0,最适反应温度为40°C,表面展示酶热稳定性较游离酶有较大提高,在40°C孵育1 h后仍能保持90%以上的酶活力。【结论】以上结果表明细菌表面展示技术为脂肪酶固定提供了一个很有前景的替代方法。     

High-throughput screening of B factor saturation mutated Rhizomucor miehei lipase thermostability based on synthetic reaction

Conventional lipase screening methods are mostly based on hydrolytic activity, which may not always be the best method to assess the enzyme activity, especially for evaluating synthetic activity. Here we developed a high throughput and visual method to screen clones with high synthetic activity and used it to assess lipases thermostability. All mutants' lipase synthetic activity were identified through esterification of caprylic acid and ethanol with methyl red as the pH indicator adding in the substrates on according to the color change halo around the colony on culture plates since synthetic reaction was often accompanied with a rise in pH. After two rounds operation with the pH indicator screening method, we obtained a double mutant Asn120Lys/Lys131Phe from the Rhizomucor miehei lipase saturation mutated library based on amino acid residue B factors. The mutant's initial synthetic activity was a little higher than wild type and its thermostability in synthetic reaction was enhanced, which remained 63.1% residual activity after being heated at 70°C for 5h comparing to 51.0% of wild type. The double mutant with the two residue replacements balanced well between stability and activity. Yeast surface display technology and the pH indicator method, combined with colony screening were shown to facilitate high-throughput screening for lipase synthetic activity.     

High-level expression of Candida parapsilosis lipase/acyltransferase in Pichia pastoris

Candida parapsilosis has been previously shown to produce a lipase/acyltransferase (EC 3.1.1.3) that preferentially catalyses transfer reactions such as alcoholysis over hydrolysis in the presence of suitable nucleophiles other than water, even in aqueous media (aw > 0.9 ). This enzyme has been shown to belong to a new family of lipases. The present work describes the cloning of the gene coding for this lipase/acyltransferase in the yeast Pichia pastoris and the heterologous high-level expression of the recombinant enzyme. The lipase/acyltransferase gene, in which the sequence encoding the signal peptide was replaced by that of the alpha-factor of Saccharomyces cerevisiae, was placed under the control of the methanol inducible promoter of the alcohol oxidase 1 gene (AOX1). A transformed P. pastoris clone, containing five copies of the lipase/acyltransferase gene, was selected for the production of recombinant enzyme. The fed-batch culture supernatant contained 5.8 gl(-1) (weighted) of almost pure recombinant lipase/acyltransferase displaying the same catalytic behavior as the original enzyme.     

Display of lipase on the cell surface of Escherichia coli using OprF as an anchor and its application to enantioselective resolution in organic solvent

We have developed a new cell surface display system using a major outer membrane protein of Pseudomonas aeruginosa OprF as an anchoring motif. Pseudomonas fluorescens SIK W1 lipase gene was fused to the truncated oprF gene by C-terminal deletion fusion strategy. The truncated OprF-lipase fusion protein was successfully displayed on the surface of Escherichia coli. Localization of the truncated OprF-lipase fusion protein was confirmed by western blot analysis, immunofluorescence microscopy, and whole-cell lipase activity. To examine the enzymatic characteristics of the cell surface displayed lipase, the whole-cell enzyme activity and stability were determined under various conditions. Cell surface displayed lipase showed the highest activity at 37 degrees C and pH 8.0. It retained over 80% of initial activity after incubation for a week in both aqueous solution and organic solvent. When the E. coli cells displaying lipases were used for enantioselective resolution of racemic 1-phenylethanol in hexane, (R)-phenyl ethyl acetate was successfully obtained with the enantiomeric excess of greater than 96% in 36 h of reaction. These results suggest that E. coli cells displaying lipases using OprF as an anchoring motif can be employed for various biotechnological applications both in aqueous and nonaqueous phases.     

Towards novel biocatalysts via protein design: the case of lipases

Abstract: During the past 3 years, the tertiary structures of several lipases have been solved by X-ray analysis. The structures revealed unique features such as hydrophobic 'patches' on the surface, presumably involved in lipid supersubstrate binding, and a lid structure which covers the active site in the absence of substrate. Only very recently the first X-ray structure of a bacterial lipase has been solved, and further structural features different from lipases of eukaryotic origin became apparent. Many lipase genes have been cloned and sequenced recently, and expression systems for the preparation of recombinant enzymes in good yields are available. As an example, the lipase from Rhizopus oryzae has been successfully expressed by us in Escherichia coli , and the resulting inclusion bodies were renatured in high yields. Consequently, the mechanism of action of lipases is now being studied via site-directed mutagenesis, and the rational design of lipases for the selective transformation of substrates is presently addressed in several laboratories.     

Enhanced reactivity of Rhizopus oryzae lipase displayed on yeast cell surfaces in organic solvents: potential as a whole-cell biocatalyst in organic solvents

Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized Rhizopus oryzae lipase displayed on the cell surface of Saccharomyces cerevisiae in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with alpha-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 x 10(4)-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate and 3.8 x 10(4)-fold higher in an esterification reaction with palmitic acid and n-pentanol (0.2% H2O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three- to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.     

Characterization of two novel lipase genes isolated directly from environmental sample

Two novel lipase genes (lipJ02, lipJ03) were isolated directly from environmental DNA via genome-walking method. Lipase gene lipJ02 contained an open reading frame (ORF) of 1,425 bp and encoded a 474-amino acids lipase protein, while lipase gene lipJ03 contained an ORF of 1,413 bp and encoded a 470-amino acids lipase protein. The lipase genes were cloned into expression vector pPIC9K and successfully integrated into a heterologous fungal host, Pichia pastoris KM71, and the recombinant P. pastoris were screened via a high-throughput method. The recombinants were induced by methanol to secrete active lipases into cultural medium. The recombinant lipases were also purified and characterized. The optimum temperature for the purified lipase LipJ02 and LipJ03 was 30 and 35°C, respectively, at pH 8.0. They exhibited similar thermostability, but LipJ02 exhibited better pH stability than LipJ03.