脂肪酶生物印迹研究进展

脂肪酶是一种广泛应用的水解酶。生物印迹技术是一项新兴技术,它使脂肪酶在有机溶剂中发挥更好的催化能力,如更高的活性和稳定性,从而扩大了脂肪酶作为生物催化剂在工业上的应用。本文介绍了脂肪酶蛋白质的结构特点,脂肪酶生物印迹的过程和原理,对生物印迹过程中印迹模板的种类和选择原则、保护剂的作用等主要影响因素进行了讨论,并对不同种类脂肪酶的生物印迹条件和适用的反应及生物印迹效果作了归纳总结,指出了目前脂肪酶生物印迹技术存在的问题和发展前景。     

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。     

微生物脂肪酶特性及工业应用

脂肪酶(Lipase, EC 3.1.1.3,即甘油三酯水解酶),不仅具有催化甘油三酯水解生成甘油及脂肪酸,而且具有催化酯交换反应、转酯反应等活性。由于脂肪酶的作用温度、作用pH值范围较广,且耐有机溶剂、催化反应不需要辅酶、催化效率比较高及对环境友好等性质,使得其在食品、洗涤工业、生物传感器、医药、化妆品、生物柴油及环境治理等方面具有广泛应用。本综述着重对脂肪酶的性质及其在工业中的应用两方面进行分析。为脂肪酶相关研究提供科学依据。同时,对脂肪酶的未来发展前景进行展望。     

脂肪酶及其在化学品合成中的应用

脂肪酶催化过程具有高效和高选择性、条件温和以及环境友好等特点。目前可再生能源和绿色化工领域对新型酶催化转化技术的迫切需求使得越来越多的脂肪酶被应用到生物柴油、精细化学品和医药中间体合成的领域。本文主要介绍了脂肪酶的催化技术及其在化学品合成中的应用。     

脂肪酶TLL在毕赤酵母中表达及催化制备生物柴油初探

[目的]拟实现疏棉状嗜热丝孢菌(Thermomyces lanuginosus)脂肪酶TLL在毕赤酵母菌中的高效表达;初步探索该脂肪酶催化生物柴油的可行性及催化条件,为大规模工业生产生物柴油提供一种可行方案。[方法]比较分析α-信号肽和脂肪酶tll基因自身的信号肽对其分泌表达量的影响;构建脂肪酶tll基因多拷贝表达框,提高该基因在宿主基因组中的剂量,从而实现高效表达;直接以液体脂肪酶TLL为催化剂,采用单因子方法初步探索了其制备生物柴油的条件。[结果]α-信号肽融合和多拷贝均能提升TLL脂肪酶的表达水平。在14 L发酵罐中培养144 h后,发酵上清液的酶活可达到769 U/m L;液体脂肪酶TLL可成功地催化制备生物柴油,且获得88.3%的转酯率。[结论]该研究显著地提高了脂肪酶TLL的表达水平,初步确定了液体脂肪酶TLL可以直接制备生物柴油。     

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.     

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。     

非水介质中脂肪酶催化的手性拆分研究进展

脂肪酶催化的外消旋体动力学拆分是不对称催化领域中极具吸引力且发展非常迅速的重要技术。对这一领域的最新研究进展进行了调研,并将注意力集中于脂肪酶在非水溶剂中的应用。引用最近五年中的一些文献案例,说明了脂肪酶作为立体选择性生物催化剂在外消旋体拆分中的多样化功能和应用。     

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。     

固态发酵生产微生物脂肪酶的研究进展

脂肪酶在水相和非水相中都具有催化活性,在众多工业领域应用前景十分广阔。但脂肪酶的生产成本仍然过高,限制了其在某些工业领域的大规模使用。固体发酵因具有设备比较简单、能耗低、成本低、对环境危害小、易于推广等诸多优点,已逐渐成为微生物脂肪酶生产的一个重要方式。由于能源成本的抬高和人们环保意识的加强,自上世纪90年代以来,原来一直认为技术含量较低的固态发酵技术重新受到重视并得到了快速的发展。综述了固态发酵在脂肪酶生产中的应用研究,重点介绍了固态发酵生产脂肪酶的特点、脂肪酶固态发酵的影响因素及其生物反应器。     

Hydrolysis of oils by using immobilized lipase enzyme: A review

This review focuses on the use of immobilized lipase technology for the hydrolysis of oils. The importance of lipase catalyzed fat splitting process, the various immobilization procedures, kinetics, deactivation kinetics, New immobilized lipases for chiral resolution, reactor configurations, and process considerations are all reviewed and discussed.     

Immobilization and characterization of lipase from an indigenous Bacillus aryabhattai SE3-PB isolated from lipid-rich wastewater

Abstract

Extracellular lipase from an indigenous Bacillus aryabhattai SE3-PB was immobilized in alginate beads by entrapment method. After optimization of immobilization conditions, maximum immobilization efficiencies of 77%?±?1.53% and 75.99%?±?3.49% were recorded at optimum concentrations of 2% (w/v) sodium alginate and 0.2?M calcium chloride, respectively, for the entrapped enzyme. Biochemical properties of both free and immobilized lipase revealed no change in the optimum temperature and pH of both enzyme preparations, with maximum activity attained at 60?°C and 9.5, respectively. In comparison to free lipase, the immobilized enzyme exhibited improved stability over the studied pH range (8.5–9.5) and temperature (55–65?°C) when incubated for 3?h. Furthermore, the immobilized lipase showed enhanced enzyme-substrate affinity and higher catalytic efficiency when compared to soluble enzyme. The entrapped enzyme was also found to be more stable, retaining 61.51% and 49.44% of its original activity after being stored for 30 days at 4?°C and 25?°C, respectively. In addition, the insolubilized enzyme exhibited good reusability with 18.46% relative activity after being repeatedly used for six times. These findings suggest the efficient and sustainable use of the developed immobilized lipase for various biotechnological applications.     

The inhibitory kinetics and mechanism of glycolic acid on lipase

Abstract

Obesity is prone to cause a variety of chronic metabolic diseases, and it has aroused people’s attention that the rapid increase in the global population of obese people in the past years. As a kind of weight-loss drug acting in the intestine, lipase inhibitor does not enter the bloodstream without producing central nervous side effects. Because they do not affect the metabolism system, lipase inhibitors and obesity have become one of the hot spots in recent years. Glycolic acid is a new substrate analog inhibitor with the value of the semi-inhibitory concentration of lipase is estimated to be 17.29?±?0.14?mM. Using the plots of Lineweaver-Burk, the inhibition mechanism of lipase by glycolic acid was reversible and the inhibition type belongs to competitive inhibition with a K_I value of 19.61?±?0.26?mM. The inhibitory kinetics assay showed that the microscopic velocity constant k₊₀ of inhibition kinetics is 1.79?×?10^?3?mM^?1s^?1, and k_?0 is 0.73?×?10^?3 s^?1. The results of UV full-wavelength scanning on product cumulative, fluorescence quenching and molecular simulation also indicated that glycolic acid and substrate competitive with lipase by binding to Lys137. Thereby glycolic acid inhibiting the oxidation-catalyzed reaction and reducing the product of the enzyme and substrate. This adds a new direction for the search for lipase inhibitors and provides new ideas about the development of anti-obesity drugs.

Communicated by Ramaswamy H. Sarma     

国内脂肪酶研究状况分析

基于中国期刊全数据库(CJFD)1995～2001年中脂肪酶检索数据,从基础研究和应用研究两个方面对其进行了统计分析,客观地概述和评价了国内该领域的研究状况。着重分析了国内脂肪酶研究的现状、发展趋势。结果表明,中国脂肪酶研究多集中在基础研究领域,基础研究各方面发展不平衡,国内9个城市是脂肪酶研究的核心区域,高等学校是脂肪酶研究的主要机构。     

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.     

Residue Tyr224 is critical for the thermostability of Geobacillus sp. RD-2 lipase

A thermophilic lipase (lipGRD) from Geobacillus sp. RD-2, isolated from a hot spring in Yunnan, China, was cloned and over-expressed in Escherichia coli. The function of the conserved residue, Tyr224, near the presumed temperature switch site was analyzed by site-directed saturation mutagenesis. The activity of the wild type lipGRD was optimal at 55??C and pH?7.5, but that from mutant Y224C was optimally active at 35??C, whereas Y224P lipase was optimally active at 65??C. Furthermore, the latter lipase retained 60% of its activity after incubation at 65??C for 5?h. The conserved residue Tyr224, which is close to the lid helix, is the key amino acid residue determining the thermostability of the thermostable lipase.     

Biosensors for the evaluation of lipase activity

In the review a comprehensive analysis is given of instrumental analytical approaches to control the activity of lipolytic enzymes and their substrates. A special attention is attached to the methods based on the biosensor technology. A number of electrochemical, optical and mechanical biosensors that were developed in several laboratories are analyzed in detail. In addition, perspectives are evaluated for the development of these biosensors and their practical application. It was concluded that biosensors may provide all practical demands which are in enzymology field at the express determination of lipase activity and triacylglycerol levels in biological objects.     

宏基因组学技术在痤疮研究中的应用进展

高通量测序技术的发展提高了人们对微生物组的认识。宏基因组学技术因其全面和深入的分析功能被广泛应用于各种环境微生物组的研究中,尤其在阐明各种疾病与人体微生物组的关系中,宏基因组学技术具有重要作用。痤疮作为一种常见的皮肤疾病,严重影响人们皮肤美观度和心理健康。利用宏基因组学技术挖掘皮肤微生物与痤疮的关系,将有助于痤疮发病机理的研究和临床治疗方法的改进。通过介绍宏基因组学技术的发展背景、概述及其应用研究进展,探讨皮肤微生物与痤疮的关系,综述宏基因组学技术在痤疮研究中的应用现状,并总结目前宏基因组学技术在皮肤疾病研究中存在的问题,旨在为痤疮的宏基因组研究提供参考。     

Improvement of lipase activity by synergistic immobilization on polyurethane and its application for large-scale synthesizing vitamin A palmitate

Abstract

We have developed an improved and effective method to immobilize lipase on hydrophobic polyurethane foam (PUF) with different modifications. PUF was treated with hydrochloric acid to increase the active sites and then the active carboxyl groups and amino groups were exposed. Enzyme activity of lipase immobilized on PUF-HCL (8000?U/g) was 50% higher than that of lipase immobilized on PUF (5300?U/g). There is an increase in the activity of the immobilized lipase on AA/PEI-modified support (115,000?U/g), a 2.17-fold increase compared to lipase immobilized on the native support was observed. The activity of immobilized lipases was dependent on the PEI molecular weight, with best results from enzyme immobilized on PUF-HCL-AA/PEI (MW 70,000?Da, 12,800?U/g)), which was 2.41 times higher compared to that of the same enzyme immobilized on PUF. These results suggest that the activity of immobilized lipase is influenced by the support surface properties, and a moderate support surface micro-environment is crucial for improving enzyme activity. Finally, the immobilized lipase was used for the production of vitamin A palmitate. The immobilized lipase can be reused for up to 18 times with a conversion rate above 90% for 12?h in a 3?L bioreactor.

• Research highlights

• An efficient immobilization protocol on polyurethane foam was developed

• Polyethyleneimine and acetic acid were used to regulate the micro-environment concurrently

• The activity of lipase immobilized on PUF-HCL-AA/PEI was improved by 2.41 times

• Immobilized lipase exhibited excellent operational stability for vitamin A palmitate synthesis

     

Influence of environmental factors on lipase production by Lactobacillus plantarum

A strain of Lactobacillus plantarum, DSMZ 12028 (Deutsch Sammlung von Mikroorganismen und Zellkulturen), isolated from a Portuguese dry fermented sausage, “chouriço”, was found to produce true lipase, producing free fatty acids from triolein (olive oil). This enzymatic activity was found in whole cells, but was negligible in comparison to lipolytic activity in culture supernatant. Therefore, only extracellular activity was studied. The effect of pH, temperature and glucose concentration on extracellular lipase production was studied in continuously stirred tank reactors, the first time this technology has been used to study the production of this enzyme in lactobacilli. Maximum lipase production was achieved at a pH of 5.5 and 30?°C and was kept at a significant level over a wide range of dilution rates (0.05–0.4 h^?1); the production of lipase was still significant for low pH values, temperature and glucose concentration, conditions that are close to the ones present during chouriço ripening. The effect of glucose concentration was also studied in a batch system. The control of lipase production was found to be related both to glucose concentration in the medium and to the growth rate/dilution rate. Glucose concentration was found to be important for fast lipase production, although it did not influence the maximum lipase activity reached in a batch culture.