微生物脂肪酶稳定性研究进展

脂肪酶广泛应用于食品、药物、生物燃料、诊断、生物修复、化学品、化妆品、清洁剂、饲料、皮革和生物传感器等工业领域,微生物脂肪酶是商品化脂肪酶的重要来源。高温、酸性、碱性和有机溶剂等恶劣的工业生产环境使得脂肪酶的进一步工业应用受到限制,获取稳定性好的脂肪酶成为打破这一限制的关键环节。本文重点对提高微生物脂肪酶稳定性的策略进行了综述:挖掘极端微生物脂肪酶资源;利用定向进化、理性设计和半理性设计等蛋白质工程策略改造脂肪酶;利用物理吸附、封装、共价结合和交联等酶的固定化技术提高脂肪酶的稳定性;利用物理/化学修饰、表面展示以及多种改良策略相结合提高脂肪酶的稳定性。结合作者前期对酶工程的研究发现,新型酶催化剂的获得应该基于明确的设计思路,结合多种改造方法,基于定向进化-理性设计、定向进化-半理性设计、蛋白质工程-酶的固定化、蛋白质工程-物理/化学修饰、酶的固定化-物理/化学修饰等组合改造,比单一的改造方法具有更高的效率。     

微生物脂肪酶分子改造研究进展

有关脂肪酶分子改造的蛋白质工程研究 ,越来越引起研究人员的兴趣[1 - 4] 。在过去的短短几年中 ,关于如何定向改造蛋白质特定性状的研究有了一些明显的进展。1　脂肪酶简介脂肪酶被广泛用于催化三脂酰甘油酯及其他一些水不溶性酯类的水解、醇解、酯化、转酯化以及酯类逆向合成反应[5] 。它们是一类具有多种催化能力的酶 ,经常还能表现出磷脂酶、溶血磷脂酶、胆固醇酯酶、酰胺水解酶等其他一些酶的活性。目前 ,已有越来越多的各种微生物来源的脂肪酶被应用于洗涤剂、油脂与食品加工、有机合成、表面清洗、皮革与造纸工业等生产实践。虽然各…     

新型微生物脂肪酶资源开发

微生物脂肪酶是一类重要的工业生物催化剂, 广泛应用于工农业生产的诸多领域。筛选、挖掘和开发出具有新型催化活性或高稳定性的微生物脂肪酶一直是脂肪酶研究的重点。本文从极端微生物、宏基因组技术、基因组数据库挖掘、定向进化技术、固定化技术和化学修饰技术等方面介绍了当前新型脂肪酶开发的途径和方法。     

饲用植酸酶蛋白质工程研究进展*

植酸酶作为一种单胃动物的饲料添加剂,它的推广和应用受到酶性质的局限。本在植酸酶改性策略,以及提高饲用植酸酶的表达量,改善热稳定性,提高催化效率,改良最适pH等方面介绍了植酸酶蛋白质工程方面的研究进展。     

微生物脂肪酶及其应用

自然界中有许多微生物能利用天然油脂和脂肪作为自身生长的碳源,这是由于脂肪酶作用使其分解成能被直接利用的小分子物质,脂肪酶（Ec．3．1．1．3）[1]分解脂肪,催化分解三酸甘油脂,产生脂肪酸、甘油脂和甘油。它们的催化反应如图1。脂肪酸在生物体起有相当重要的生理诈用,脂肪酶的分解产物除供给生物能量外,还是合成磷脂等物质的材料,脂肪酸在动植物组织及多种微生物中普遍存在,其性质多年前就被确定了。早在六十年代我国就开展微生物脂肪酸研究。1969年研制成解脂假丝酵母脂肪酸制剂。但同淀粉酶、蛋白酶相比脂肪酸的工业应用还…     

微生物脂肪酶的研究进展

详细综述了脂态酶微生物产生菌的选育、诱变育种及发酵生产技术,对微生物脂肪酶的性质、分离、纯化、固定化技术及应用进行了简要概述     

蛋白质工程

<正> 随着科学技术的迅猛发展,人类对不同层次(分子、亚细胞、细胞、组织、器官乃至生命体本身)的生命过程的控制和利用已成为可能。作为生物功能的基本物质的蛋白质,由于其本身的多样性而为其多种应用提供了巨大潜能,人们一直在期待着对蛋白质功能的某种添加与除去的最终自主控制,并希望制造出比天然蛋白质性能更为优越的新     

华根霉胞内脂肪酶同功酶酶学性质研究

华根霉(Rhizopus chinensis CCTCCM201021)细胞破碎液经硫酸铵分级盐析、Phenyl-Sepharose FF疏水层析、DEAE-Sepharose FF离子交换层析和Sephadex G100凝胶层析得到两种脂肪酶同功酶:Lip1和Lip2。SDS-PAGE显示其亚基分子量分别为59.2kD和39.4kD。Lip1和Lip2的最适反应pH和最适反应温度相近,分别为8.0、8.5和40℃、35℃。但底物专一性差异明显:Lip1对pNP脂肪酸酯的长链脂肪酸有较高的专一性,Lip2对pNP脂肪酸酯的短链脂肪酸专一性较好;Lip1对三油酸甘油酯表现1,3-位置特异性,而Lip2没有位置选择性。1mmol/L的Ca2 、Mg2 对Lip1、Lip2有较好的激活作用;SDS强烈抑制酶活力。Lip1、Lip2在环己烷、正己烷、正庚烷和异辛烷(30%V/V)中稳定性良好。     

Lipases: Useful biocatalysts for the preparation of pharmaceuticals

Biocatalysis offers a clean and ecological way to perform chemical processes, in mild reaction conditions and with high degree of selectivity. The use of enzymes, specially lipases, in organic solvents proves an excellent methodology for the preparation of single-isomer chiral drugs. This review covers some general aspects and representative examples of the use of lipases for the enantioselective or regioselective preparation of alcohol and amine intermediates in the synthesis of pharmaceuticals.     

蛋白质与酶工程精品课程的建设

阐述在"蛋白质与酶工程"课程建设中,对教学内容进行结构性调整,新旧教学方法并行,进行考核评价手段与实践教学的创新,赋予学生自主选择教学内容、教学方法、考试方法、参与评价的权利,使学生得到全面发展,在教学改革方面积累经验。     

Protein Thermostability: Mechanism and Control Through Protein Engineering

Chemical modification and protein engineering especially are now the useful tools for thermo-stabilizing proteins, and also for elucidating the mechanism of protein stability. The information on the mechanism so far accumulated indicate that a single or few amino acid replacement(s) in a protein is/are sufficient to enhance protein thermostability. Salt bridges inside protein molecule or decrease of internal or external hydrophobicity, respectively, may contribute to increased thermostability. However, generalized molecular reasons for protein thermostability and generalized methods for protein stabilization have not yet been proposed. Some of typical examples of the application of protein engineering to stabilize proteins are presented. They are based on information concerning the tertiary structure of the proteins or their related proteins. Even if such structural information is unavailable, one can replace amino acid(s) in a protein by mutagenesis of the gene coding for the protein via the application of chemicals to the gene (or the plasmid harbouring the gene) or organism. A promising strategy involving transfer of the identified gene into a thermophile and subsequent growth at higher temperatures (thermal adaptation) is described.     

点饱和突变技术及其在蛋白质工程中的应用

点饱和突变技术是蛋白质工程中的一门新兴技术,它通过对目的蛋白的编码基因进行改造,短时间内获取靶位点氨基酸分别被其它19种氨基酸替代的突变子。此技术不仅是蛋白质定向改造的强有力工具,而且是蛋白质结构－功能关系研究的重要手段。本文概述了几种常用点饱和突变技术,介绍了其在蛋白质工程中的应用状况,讨论了其在应用中的问题,展望了其应用前景。     

Engineering a Thermostable Protein with Two DNA-binding Domains Using the Hyperthermophile Protein Sac7d

Abstract

The acid- and thermostable Sac7d is a small, non-specific DNA-binding protein of the hyperthermophile archaea Sulfolobus acidocaldarius. In this study, Sac7d was employed as a structural unit in the design of a thermostable protein containing two putative DNA-binding domains. By linking two Sac7d proteins together and comparing the DNA interaction of dimer to that of monomer, this study may provide structural insights into other dimeric DNA-binding proteins. The engineered protein, Sac7dK66C, was over-expressed and purified. Dimeric Sac7d was obtained by cross-linking two mutant Sac7d molecules through the C-terminal disulfide bond. Thermal stability and DNA-binding ability of dimeric Sac7d were assessed and compared to those of wild type Sac7d by gel retardation assay, circular dichroism spectroscopy, and crystallization experiments. Dimeric Sac7d was shown to be equally thermostable as wild type, and its ability to stabilize DNA duplex is the same as wild type. However, the interaction of dimeric Sac7d with DNA diverged from that of wild type, suggesting different DNA-binding modes for dimeric Sac7d. In addition, a large difference in extinction coefficient was observed in all dimer/DNA CD spectra, which was reminiscent of the spectrum of ψ-DNA. Conjugation of various chemical groups to mutant Sac7d is possible through the C-terminal thiol group. This offers a possible approach in the design of a thermostable biomolecule with novel functions.     

用蛋白质工程方法改变葡萄糖异构酶最适pH和最适温度

用寡核苷酸诱导的定点突变方法构建了葡萄糖异构酶基因的突变体（Ｎ１８４Ｄ和Ａ１９８Ｃ）。含突变体的重组质粒ｐＴＫＤ－ＧＩ１（Ｎ１８４Ｄ）和ｐＴＫＤ－ＧＩ２（Ａ１９８ｃ）在Ｅ．ｃｏｌｉＫ３８菌株中表达,用ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ ＦＦ和Ｓｅｐｈａｃｒｙｌ Ｓ－３００ＨＲ柱层析分离纯化突变酶。与野生型葡萄糖异构酶比较实验表明：（１）突变酶Ｎ１８４Ｄ的最适ｐＨ值下降了１个单位;等电点下降了０．６个单位     

Three New Lipases from Actinomycetes and Their Use in Organic Reactions

Three novel lipase-producing microorganisms have been isolated from 526 actinomycete strains by employing screening techniques on solid media. Time-course and scale-up of enzyme production were analyzed. The lipases, produced by microorganisms belonging to the Streptomyces genus, were tested in several reactions in organic medium using unnatural substrates. The lyophilized crude lipases are stable at least for 1 month at 4°C (100% recovered activity). The lipase activity per milliliter of cell culture broth was higher than described in the literature for other lipases from actinomycetes. The three selected lipases displayed better activity than commercial lipase from Candida rugosa in the resolution of chiral secondary alcohols. The lipase from S. halstedii also displayed very good activity in the synthesis of carbamates.     

蛋白质工程与生物制药

在论述蛋白工程基本概念和定义的基础之上,介绍了蛋白质工程的主要内容,着重介绍了基因重组和分子生物学等贩新技术在蛋白工程的应用,最后还介绍了蛋白质工程在生物制药方面的应用情况。