微生物脂肪酶蛋白质工程*

微生物脂肪酶催化的化学反应具有严格的立体选择性、位点选择性等专一性,催化活性高而副反应少,催化反应不需要辅助因子等特点,因此广泛应用于工农业生产中的诸多领域。利用蛋白质工程技术,提高微生物脂肪酶的特异性、活性和稳定性,对提高微生物脂肪酶制剂产品的市场竞争能力,扩大微生物脂肪酶的应用领域,具有重要的意义。综述了蛋白质工程技术在微生物脂肪酶改性方面的应用现状、存在问题及前景。     

微生物脂肪酶的重组表达

微生物脂肪酶在传统和新型工业催化领域中的应用越来越广泛与深入。作为脂肪酶规模化制备主要途径的高效重组表达,为脂肪酶催化剂的最终形成及工业催化奠定了坚实的技术基础。概述并讨论了微生物脂肪酶重组表达的最新策略和发展趋势,阐述密码子优化、融合共表达、杂合启动子、同源高效表达、细胞表面展示和表达文库的高通量筛选等技术特点及其表达应用现状,指出细胞表面展示表达和表达文库高通量筛选体系为脂肪酶重组表达注入强劲活力;在此基础上对几种代表性微生物脂肪酶的重组表达进展作一综述,为微生物脂肪酶的重组表达研究及工业生产提供借鉴。     

基于核糖体基因序列快速鉴定产脂肪酶微生物

利用含罗丹明B的橄榄油检测平板从中国各省市油污土壤中分离、筛选产脂肪酶微生物菌株,扩增细菌的核糖体基因16S rDNA序列和真菌的ITS2序列,分析核糖体基因簇DNA,并结合形态学特征从而对产脂肪酶菌株进行分子生物学鉴定.核糖体基因16S rDNA序列分析及系统发育分析表明,分离得到的产脂肪酶细菌分别属于枯草芽孢杆菌(Bacillus subtilis)、产碱假单胞菌(Pseudomonas alcaligenes)、洋葱伯克霍尔德氏(Burkholderia cepacia)、琼氏不动杆菌(Acinetobater jurii)、嗜麦芽窄食单孢菌(Stenotrophomonas maltophilia)和荧光假单胞菌(Pseudomonas sp.);真菌核糖体基因转录间隔区(ITS2)序列及同源性分析表明产脂肪酶真菌分别属于黑曲酶(Aspergillus niger)、白地酶(Galactomyces geotrichum)、解脂耶氏酵母(Yarrowia lipolytica)、丝孢酵母(Trichosporon guehoae)和假丝酵母(Candida sp.).研究结果表明,核糖体基因簇的DNA分析技术为从自然界分离、鉴定产脂肪酶菌种提供了一种快速有效的手段,为产脂肪酶微生物资源开发利用奠定了技术基础.     

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。     

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…     

产酯酶微生物菌种的筛选研究

研究了产酯酶微生物的筛选,包括筛选模型、酶活力检测方法及菌株的分布。对30多份土样以及实验室保存的菌种进行了大量的筛选,以添加三醋酸甘油酯、乳酸乙酯酯类物质对土样等样品富集,采用添加显色剂溴甲酚紫的快速简便平板显色法,观察水解变色圈直径和菌落直径的大小进行初筛。获得两者直径之比相对大的菌株174株,采用平板打孔检测法和摇瓶发酵比色法测酶活力相结合进行复筛,最终得到酯酶活力较高的24株菌株。就初筛和复筛方法及结果加以比较分析,复筛菌株做不同底物的酶活力检测,建立了一个有效、简便及快速的微生物酯酶的筛选模型。并对酯酶产生菌的立体选择专一性进行了初步考察。     

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。     

提高微生物脂肪酶产量、活性和稳定性的方法研究

微生物脂肪酶是一类广泛应用于诸多工业领域的生物催化剂。提高微生物脂肪酶的产量、活性和稳定性,增强产品的市场竞争力,一直是微生物脂肪酶研究的重点和热点。本文从产脂肪酶菌株的改造、脂肪酶基因的改良、脂肪酶发酵工程和脂肪酶后期处理等四个方面概述了提高微生物脂肪酶产量、活性和稳定性的方法,以期为微生物脂肪酶的规模化工业生产提供方法性指导。     

牦牛瘤胃中产脂肪酶微生物的分离与鉴定

【背景】脂肪酶是一类特殊的酯键水解酶,广泛应用于工业化生产中,微生物是工业脂肪酶的主要来源。瘤胃中微生物种类繁多、数量庞大,已有关于瘤胃微生物产纤维素酶的报道,尚无产脂肪酶瘤胃微生物的分离筛选报道。【目的】从牦牛瘤胃中分离筛选出能够产脂肪酶的微生物,并进行菌株鉴定及其酶学性质的研究。【方法】以橄榄油为唯一碳源,通过中性红油脂平板进行初步筛选后,用改进铜皂-分光光度法测定酶活力进行复筛;再经形态学观察、生理生化实验和16S rRNA基因序列分析进行菌种鉴定;研究3种脂肪酶的最适作用温度、pH值及金属离子、有机溶剂和表面活性剂对酶活力的影响。【结果】筛选出6株酶活力较高的菌株,其中3株为液化沙雷氏菌,2株为白地霉,1株为卷枝毛霉。脂肪酶的酶学性质研究表明:液化沙雷氏菌、白地霉和卷枝毛霉所产脂肪酶的最适作用温度为45、35和40°C;最适pH为8.0、7.0和7.0;Ca2+和Mg2+对3种脂肪酶均有激活作用;Zn2+对3种脂肪酶有不同程度的抑制作用,EDTA、SDS可使3种脂肪酶失活;3种脂肪酶对丙三醇的耐受力较高,卷枝毛霉脂肪酶对甲醇、乙醇、丙酮的耐受力较高。【结论】从牦牛瘤胃中分离出3种产脂肪酶的微生物,且证实瘤胃微生物在脂肪酶研究方面具有较高的价值。     

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.     

Kinetic resolution of (±)-diethyl- and dibenzyl hydroxy(phenyl)methanephosphonates and their acyl derivatives with lipases

Abstract

A wide variety of commercially available lipases and microbial whole cells were tested for biotransformations of (±)-diethyl and dibenzyl hydroxyl(phenyl)methanephosphonates. Biocatalytic hydrolysis of acylated hydroxyphosphonates by whole cells of Bacillus subtilis gave optically active compounds with 95%ee _S. Enantioselectivities obtained when using commercially available enzymatic preparations were less satisfactory, leading to both compounds with an enantiomeric excess in the range 15 35%. Screening lipases for their ability to acylate these phosphonates or to hydrolyze their acylated derivatives enabled selection of enzymes and organisms suitable for use in both processes.     

Influence of precursors and additives on microbial lipases stabilized by sol-gel entrapment

Sol-gel entrapment of microbial lipases from Candida cylindracea (Cc lipase), Pseudomonas fluorescens (Lipase AK), and Pseudomonas cepacia (Lipase PS), using as precursors tetraethoxysilane (TEOS) and silanes of type R-Si(OEt)₃ with alkyl or aryl R groups, has been investigated. Three different methods using these precursors were tried exhibiting protein immobilization yields in the range of 20-50%. Hydrolysis of emulsified olive oil, esterification of lauric acid with 1-octanol and enantioselective acylation of 2-pentanol have been used as model reactions for testing the properties of the encapsulated lipases. The recovery yields of the enzyme activity in the esterification reaction were between 20-68%, the best performance being achieved with phenyltriethoxysilane and tetraethoxysilane precursors at 3:1 molar ratio. When testing the entrapped Lipase AK in the enantioselective acylation reaction of 2-pentanol, activity recovery yields up to 32% related to the free enzyme were obtained and the immobilization increased the enantioselectivity of the enzyme.     

Improvement of lipase obtaining system by orange waste-based solid-state fermentation: production,characterization and application

Abstract

Lipases are an economic important group of biocatalysts that can be produced by some fungal under solid-state fermentation. Orange wastes are source of lipases and potential substrates for lipases production. This work assessed 19 fugal strains cultivated in Citrus sinensis cv. Hamlin orange wastes (peel, frit and core) for production of lipases in order to generate compounds with antioxidant, antimicrobial and cytotoxic properties. Fifteen of those fungi grew and produced lipases, mainly the Aspergillus brasiliensis [National Institute of Quality Control (INCQS) 40036]/frit system, which showed 99.58?U/g total lipase. The substrate with the highest production of lipase was frit with 26.67 and 78.91?U/g of total lipases produced on average by the 15 microorganisms. Aspergillus niger 01/frit (33.53?U/g) and Aspergillus niger (INCQS 40015)/frit (34.76?U/g) systems showed the highest specificity values in all the herein tested synthetic substrates with 4, 12 and 16 carbons. Analysis of the fatty acid profile of hydrolysis products obtained in the most prominent systems applied to corn and sunflower oils showed: palmitic acid, linoleic acid, oleic acid, and stearic acid. These acids showed antioxidant capacity of up to 58% DPPH (2,2-diphenyl-1-pierylhydrazyl) radical reduction and antibacterial activity against Escherichia coli, Listeria monocytogenes, Pseudomonas aureginosa, Salmonella Enteritidis and Staphylococcus aureus, as well as cytotoxicity to SCC9 cells (squamous cancer cells).     

Influence of precursors and additives on microbial lipases stabilized by sol-gel entrapment

Sol-gel entrapment of microbial lipases from Candida cylindracea (Cc lipase),Pseudomonas fluorescens (Lipase AK), and Pseudomonas cepacia (Lipase PS), using as precursors tetraethoxysilane (TEOS) and silanes of type R-Si(OEt)₃ with alkyl or aryl R groups, has been investigated. Three different methods using these precursors were tried exhibiting protein immobilization yields in the range of 20–50%. Hydrolysis of emulsified olive oil, esterification of lauric acid with 1-octanol and enantioselective acylation of 2-pentanol have been used as model reactions for testing the properties of the encapsulated lipases. The recovery yields of the enzyme activity in the esterification reaction were between 20–68%, the best performance being achieved with phenyltriethoxysilane and tetraethoxysilane precursors at 3:1 molar ratio. When testing the entrapped Lipase AK in the enantioselective acylation reaction of 2-pentanol, activity recovery yields up to 32% related to the free enzyme were obtained and the immobilization increased the enantioselectivity of the enzyme.     

酵母脂肪酶的制备及应用新进展

脂肪酶是工业领域应用非常广泛的一类绿色生物催化剂,由于脂肪酶可催化酯水解、酯化、转酯化、醇解和氨解等多种反应,在食品加工,有机合成,制备生物柴油等方面均得到了较为广泛的应用,是目前的研究热点.微生物是脂肪酶的重要来源之一,其中酵母脂肪酶被认为是非常安全的一类脂肪酶,也是应用最为广泛的一类脂肪酶.该文介绍了酵母脂肪酶的制备和应用研究概况,重点综述了其在多个应用领域中的最新研究进展.挖掘更多的新型高活性脂肪酶,降低脂肪酶的生产成本,提高酶的重复使用率是今后脂肪酶应用研究亟待解决的问题.     

Improved catalytic properties of immobilized lipases by the presence of very low concentrations of detergents in the reaction medium

The addition of a very small concentration of a detergent (in many instances under the critical micellar concentration (cmc)) has been found to greatly increase the activity of immobilized lipases, using those from Pseudomonas fluorescens (PFL) and Candida antarctica (isoform B) as model enzymes. However, the detergents may also have a negative effect on enzyme activity; in fact, for all enzyme preparations and substrates the activity/detergent concentration curve reached a maximum value and started to decrease, in many instances even under the initial value. The concentration and nature of the detergent (SDS, CTAB, Triton X-100, or X-45) that permitted the maximum hyperactivation was different depending on the substrate. The best hyperactivation values promoted by the presence of detergent were over a 20-fold factor. The presence of detergents permitted the inhibition of lipases by irreversible covalent inhibitors (e.g., 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride) (AEBSF) while the enzyme, in the absence of detergent, is not inhibited by these irreversible inhibitors. This suggested that the main effect of the detergents is to shift the conformational equilibrium of lipases toward the open form. Moreover, the presence of detergents also permitted to improve the enantioselectivity exhibited by the immobilized lipases in some cases. For example, the enantioselectivity of PFL-glyoxyl agarose increased from 40 to more than 100 in the hydrolysis of (+/-)-2-hydroxy-4-phenylbutyric acid ethyl ester by using 0.1% CTAB.     

脂肪酶活力测定研究进展

脂肪酶催化作用发生在油-水界面上,是一种典型的界面酶,因此其活力测定有别于其他的水相酶。如何准确测定酶活力的大小,对于研究该酶的特性及应用具有重要的意义。本文对脂肪酶检测的常用方法进行了综述与评价。     

低温脂肪酶的研究现状与应用前景

低温脂肪酶在低温下仍保持高酶活,因此在应用中有着中温脂肪酶无法取代的优越性,而具有高活性的低温脂肪酶因其具有理论和应用上的双重意义成为了近年来的研究热点。本文从描述产低温脂肪酶的低温微生物特征入手,系统阐述了低温脂肪酶的来源、分类、特征、研究方法及最新进展,并简述了低温脂肪酶在食品、洗涤、制药以及低温环境修复等工业上的应用前景。     

根霉脂肪酶的研究进展

根霉属微生物是脂肪酶的一个重要来源,多种根霉脂肪酶已被分离并鉴定。本文将对根霉脂肪酶的菌种筛选、发酵、纯化、结构、分子生物学研究及应用进行概述。