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.     

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。     

抑制剂对有机相酶促己酸乙酯合成中固定化脂肪酶影响

稳定的催化活性和选择性的调节与控制已成为研究有机相酶促反应机制和应用的重要内容。由于选择和优化反应条件的方法具有较大局限性．目前有机介质中酶催化选择性和稳定性的调节与控制的研究除了常用的固定化手段外,更关注通过改变酶分子自身的一些方法上。如蛋白质工程、酶的化学修饰和非共价修饰〔1.2〕。相比之下,其中非共价修饰具有方便、实用的特点。酶的修饰剂大多也是酶的抑制剂。抑制剂已用来研究脂肪酶的结构与代谢特征〔3〕。Russel,Guo等通过其改变酶的构象和界面特征来调节和控制酶稳定的特异性〔4.5〕。我们在对微生物脂肪酶正庚烷中合成短链芳香酯研究基础上〔6.7〕。本文报道用具有两亲特性的表面活性剂、胆汁盐和金属离子这些脂肪酶的抑制剂对庚烷中脂肪酶酶促己酸乙酯酯化反应的影响,以促进酶活性的调节和控制的研究和应用。     

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。     

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。     

微生物脂肪酶的共价键固定化

以N-羟基琥珀酰亚胺为活性官能团、丙烯酸为单体制备了一种含酯基的新型聚合物载体.利用微生物脂肪酶中的氨基易于氨解活化载体中的酯基生成酰胺的特点固定微生物脂肪酶,HPLC 和FT-IR 表征说明微生物脂肪酶与载体以共价键方式结合.探讨了微生物脂肪酶的最佳固定条件,固定脂肪酶酶活为游离酶活的75%,与游离酶相比,最适温度提高5℃(45℃),最适pH 值提高0.5(7.5);5 次使用后仍具有75.7% 的相对活力.     

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。     

基于空腔填充效应构建伯克霍尔德菌脂肪酶LipA的热稳定性突变体

【目的】中温伯克霍尔德菌胞外脂肪酶LipA在工业领域具有重要的应用价值。利用蛋白质工程技术来提高其热稳定性,对开发脂肪酶LipA酶制剂及提高其应用范围及应用效果,具有重要的意义。【方法】利用生物信息学软件Castp、Voronoia和Cave分析LipA分子中存在的空腔及其组成氨基酸残基;利用FoldX软件构建上述氨基酸残基的突变体电子文库,并基于空腔效应(体积变小)、自由能变化值(降低)和空间结构特点等对前述突变体电子文库进行筛选。从突变体电子文库中选择具有代表性的突变体,通过基因工程技术,引入突变。经诱导表达后,实验验证并筛选出热稳定性的突变体。【结果】构建了一个由58个突变体组成的电子文库;并对其中17个代表性的突变体进行了实验验证;筛选到2个热稳定性有明显提高的突变体LipA-His15Pro和LipA-Ala210Val;其叠加突变体LipA-His~(15)Pro/Ala~(210)Val的T50~(12)较野生型LipA提高了8°C,在55°C下的半衰期较野生型脂肪酶LipA提高了23.1倍。【结论】基于空腔填充技术构建热稳定性伯克霍尔德菌胞外脂肪酶LipA突变体,是一种行之有效的策略。     

Technical methods to improve yield,activity and stability in the development of microbial lipases

Lipases are ubiquitous biocatalysts that catalyze various reactions in organic solvents or in solvent-free systems and are increasingly applied in various industrial fields. In view of the excellent catalytic activities and the huge application potential, more than 20 microbial lipases have been realized in large-scale commercial production. The potential for commercial exploitation of a microbial lipase is determined by its yield, activity, stability and other characteristics. This review will survey the various technical methods that have been developed to enhance yield, activity and stability of microbial lipases from four aspects, including improvements in lipase-producing strains, modification of lipase genes, fermentation engineering of lipases and downstream processing technology of lipase products.     

Microbial lipases: at the interface of aqueous and non-aqueous media. A review

In recent times, biotechnological applications of microbial lipases in synthesis of many organic molecules have rapidly increased in non-aqueous media. Microbial lipases are the 'working horses' in biocatalysis and have been extensively studied when their exceptionally high stability in non-aqueous media has been discovered. Stability of lipases in organic solvents makes them commercially feasibile in the enzymatic esterification reactions. Their stability is affected by temperature, reaction medium, water concentration and by the biocatalyst's preparation. An optimization process for ester synthesis from pilot scale to industrial scale in the reaction medium is discussed. The water released during the esterification process can be controlled over a wide range and has a profound effect on the activity of the lipases. Approaches to lipase catalysis like protein engineering, directed evolution and metagenome approach were studied. This review reports the recent development in the field ofnon-aqueous microbial lipase catalysis and factors controlling the esterification/transesterification processes in organic media.     

Towards the development of systems for high-yield production of microbial lipases

Microbial lipases are a versatile and attractive class of biocatalysts for a wide variety of applications. Lipases can be produced by bacteria, yeasts or filamentous fungi. Nevertheless, they are often not optimal for direct use in industrial conditions due to low yields, low specific activities and a limited spectrum of activities. Improvements in the productivity of lipases have been made by genetic manipulation of the cell factory production hosts and by optimizing production media and conditions. Advances in protein engineering technology, ranging from directed evolution to rational design, have also been able to tailor lipases to particular applications. This review describes various approaches used to improve lipase production and applications.     

工业微生物：创新与突破专刊序言(2021)

工业微生物及其产品广泛用于工业、农业、医药等诸多领域,相关产业在国民经济中具有举足轻重的地位。高效的菌株是提高生产效率的核心,而先进发酵技术和仪器平台对充分开发菌株代谢潜能也很重要。近年来,工业微生物领域的研究取得了快速进展,人工智能、高效基因组编辑技术和合成生物学技术逐渐广泛使用,相关产业应用也在不断扩展。为进一步促进工业微生物在生物制造等领域的应用,《生物工程学报》特组织出版专刊,从微生物菌株的多样性和生理代谢、菌株改造技术、发酵过程优化和放大,高通量微液滴培养装备开发以及工业微生物应用等方面,分别阐述目前的研究进展,并展望未来的发展趋势,为促进工业微生物及生物制造等产业的发展奠定基础。     

Biocatalysts: application and engineering for industrial purposes

Enzymes are widely applied in various industrial applications and processes, including the food and beverage, animal feed, textile, detergent and medical industries. Enzymes screened from natural origins are often engineered before entering the market place because their native forms do not meet the requirements for industrial application. Protein engineering is concerned with the design and construction of novel enzymes with tailored functional properties, including stability, catalytic activity, reaction product inhibition and substrate specificity. Two broad approaches have been used for enzyme engineering, namely, rational design and directed evolution. The powerful and revolutionary techniques so far developed for protein engineering provide excellent opportunities for the design of industrial enzymes with specific properties and production of high-value products at lower production costs. The present review seeks to highlight the major fields of enzyme application and to provide an updated overview on previous protein engineering studies wherein natural enzymes were modified to meet the operational conditions required for industrial application.     

Recombinant microbial lipases for biotechnological applications

Lipases, mainly of microbial origin, represent the most widely used class of enzymes in biotechnological applications and organic chemistry. Modern methods of genetic engineering combined with an increasing knowledge of structure and function will allow further adaptation to industrial needs and exploration of novel applications. Production of such tailored lipases requires their functional overexpression in a suitable host. Hence, this article describes the functional heterologous production of commercially important microbial lipases. Based on the knowledge of different lipases' substrate binding sites, the most suitable lipase for a particular application may be selected.