“生物技术实践”教学研讨(2) “酶的应用”的教学组织

“酶的应用”包括“研究酶的存在和简单制作方法”、“尝试利用酶活力测定的一般原理和方法”、“探讨酶在食品制造中的应用”、“探讨酶在洗涤等方面的应用”、“尝试制备和应用固相化酶”等5项具体内容标准。下面谈谈对“酶的应用”专题的教学组织。     

融合酶构建技术在酶的改性以及多功能酶的构建方面的应用

融合酶技术是酶的改造技术之一。应用融合酶技术还可以创造出多功能的新酶,这些新酶有望应用于食品、化工等领域。目前研究表明,融合酶在低聚糖制备,生物燃料,生物材料,氨基酸发酵以及生物传感器等领域极具应用前景。融合酶的构建技术有理性设计和非理性设计,这两种技术各有利弊。整理了近年融合酶在以上领域中的研究成果,对融合酶的工业应用进行讨论。     

类过氧化物DNA酶在生物传感器领域的研究进展

随着对DNA酶研究的进展,DNA酶的很多优点已经超越了传统的蛋白质酶,具有过氧化物酶催化活性的DNA酶在电化学生物检测上拥有很大的潜力。我们简要阐述了DNA酶的特性和应用,描述了其基本性质,对DNA酶在生物分析领域的应用进行了展望。     

海藻酸盐裂解酶研究进展

海藻酸盐裂解酶是一类降解褐藻中海藻酸盐的酶。此酶已经在多种有机体中得到分离。对海藻酸盐裂解酶的生物特性、研究方法及其生物学功能进行了介绍。在酶学特性研究的基础上 ,通过酶解构建新型海藻酸盐多聚物 ,可增强和扩展海藻酸盐裂解酶在工业、农业、医药领域中的应用 ,使其在海藻多糖的高值化应用中发挥重要的作用。概述了海藻酸盐和海藻酸盐裂解酶过去和现在的研究状况 ,展望了海藻酸盐和海藻酸盐裂解酶将来的应用前景。     

微生物几丁质酶的特性、基因表达调控及应用

许多微生物均能产生几丁质酶。鉴于几丁质酶广泛的用途 ,尤其在植保上的应用 ,研究者对微生物几丁质酶的研究愈来愈深入。在此就微生物几丁质酶的特性、分子生物学进展及其应用作综述。     

酶化学修饰的应用潜力

本文评论了酶化学修饰在酶工程中的应用潜力,介绍了这方面的最新进展。事实证明,只要选择的化学修饰剂及修饰方法合适,有可能在较大范围内改变酶的性质,如稳定性和溶解度、酶催化活力和选择性等,从而创造天然酶所不具备的优良特性,扩大酶的应用范围。     

饲料用酶制剂的研究进展与趋势

饲料用酶目前已成为世界工业酶产业中增长速度最快、势头最强劲的一部分。畜牧业开发应用饲料用酶制剂有着重大意义:可缓解饲料资源短缺、人畜争粮的局面,有利于保障粮食安全;提供更为安全、优质的动物产品,有利于保障食品安全;减轻环境污染,保障养殖业的可持续发展。饲料用酶的应用效果已在世界范围内得到公认,但酶的使用量还较低,其主要原因在于饲料用酶的性质难以满足饲料工业的要求,以及饲料用酶表达量低,生产成本较高。因此,目前的研发趋势主要体现在:1)饲料用酶基因资源的高通量筛选技术,尤其是特殊环境微生物和未培养微生物中的基因资源;2)酶蛋白的分子改良技术,利用蛋白质工程技术定向改良,创造具有优良特性的酶蛋白质分子,进一步提高饲料用酶的应用性能;3)饲料用酶高效表达和生产技术;4)饲料用酶的应用效果快速评估技术和配套应用技术体系。     

粘质沙雷氏菌几丁质酶基因的研究进展

几丁质酶具有降解几丁质的作用,其降解产物氨基寡糖和几丁低聚糖在农业、食品、医药等领域具有重要的应用价值和广泛的应用前景。自然界中多种微生物可以产生几丁质酶,而其在细菌体内的合成受精密调控。粘质沙雷氏菌作为一种高产几丁质酶的菌种,在农业生防领域具有很大的应用潜力。本综述从粘质沙雷氏菌几丁质酶基因的分类和结构特征、几丁质酶基因克隆、表达和调控以及几丁质酶的应用等方面论述了粘质沙雷氏菌几丁质酶基因的研究进展,为粘质沙雷氏菌几丁质酶基因和功能蛋白的利用提供理论基础。     

生物催化中酰胺酶立体选择性的影响因素

立体选择性酰胺酶是一种重要的手性合成工具酶,在制备手性羧酸及其衍生物方面具有广阔的应用前景,日益受到重视。在酰胺酶的应用中,其立体选择性影响巨大。从底物、反应温度、pH、添加共溶剂和微生物来源5个方面综述了其对酰胺酶立体选择性的影响,对提高酰胺酶的立体选择性,扩大其在制备光学活性化合物领域的应用具有重要的意义。     

固定化酶:从策略到材料设计

酶是一种高效、高选择性、催化条件温和的绿色催化剂,在生物催化、生物传感、生物分离等领域具有广泛的应用价值。然而,游离酶的操作稳定性差、回收和再利用困难等缺点限制了其进一步应用。固定化酶技术应运而生,它的出现和发展为解决酶的工业化应用提供了优良的解决方案。本文中,笔者主要从酶的固定化方法、固定化酶的载体和固定化酶的应用这三方面详细介绍近几年固定化酶的研究现状,结合笔者所在课题组和国内外同行近年来的最新研究进展,重点总结了具有结构可调、孔隙率高、结晶度良好的金属-有机框架材料(MOFs)和共价有机框架材料(COFs)作为新型载体在固定化酶方面的研究进展。     

Cold-adapted enzymes: from fundamentals to biotechnology

Psychrophilic enzymes produced by cold-adapted microorganisms display a high catalytic efficiency and are most often, if not always, associated with high thermosensitivity. Using X-ray crystallography, these properties are beginning to become understood, and the rules governing their adaptation to cold appear to be relatively diverse. The application of these enzymes offers considerable potential to the biotechnology industry, for example, in the detergent and food industries, for the production of fine chemicals and in bioremediation processes.     

Cold active microbial lipases: some hot issues and recent developments

Lipases are glycerol ester hydrolases that catalyze the hydrolysis of triglycerides to free fatty acids and glycerol. Lipases catalyze esterification, interesterification, acidolysis, alcoholysis and aminolysis in addition to the hydrolytic activity on triglycerides. The temperature stability of lipases has regarded as the most important characteristic for use in industry. Psychrophilic lipases have lately attracted attention because of their increasing use in the organic synthesis of chiral intermediates due to their low optimum temperature and high activity at very low temperatures, which are favorable properties for the production of relatively frail compounds. In addition, these enzymes have an advantage under low water conditions due to their inherent greater flexibility, wherein the activity of mesophilic and thermophilic enzymes are severely impaired by an excess of rigidity. Cold-adapted microorganisms are potential source of cold-active lipases and they have been isolated from cold regions and studied. Compared to other lipases, relatively smaller numbers of cold active bacterial lipases were well studied. Lipases isolated from different sources have a wide range of properties depending on their sources with respect to positional specificity, fatty acid specificity, thermostability, pH optimum, etc. Use of industrial enzymes allows the technologist to develop processes that closely approach the gentle, efficient processes in nature. Some of these processes using cold active lipase from C. antarctica have been patented by pharmaceutical, chemical and food industries. Cold active lipases cover a broad spectrum of biotechnological applications like additives in detergents, additives in food industries, environmental bioremediations, biotransformation, molecular biology applications and heterologous gene expression in psychrophilic hosts to prevent formation of inclusion bodies. Cold active enzymes from psychrotrophic microorganisms showing high catalytic activity at low temperatures can be highly expressed in such recombinant strains. Thus, cold active lipases are today the enzymes of choice for organic chemists, pharmacists, biophysicists, biochemical and process engineers, biotechnologists, microbiologists and biochemists.     

Structural insights into cold inactivation of tryptophanase and cold adaptation of subtilisin S41

A wide variety of enzymes can undergo a reversible loss of activity at low temperature, a process that is termed cold inactivation. This phenomenon is found in oligomeric enzymes such as tryptophanase (Trpase) and other pyridoxal phosphate dependent enzymes. On the other hand, cold-adapted, or psychrophilic enzymes, isolated from organisms able to thrive in permanently cold environments, have optimal activity at low temperature, which is associated with low thermal stability. Since cold inactivation may be considered "contradictory" to cold adaptation, we have looked into the amino acid sequences and the crystal structures of two families of enzymes, subtilisin and tryptophanase. Two cold adapted subtilisins, S41 and subtilisin-like protease from Vibrio, were compared to a mesophilic and a thermophilic subtilisins, as well as to four PLP-dependent enzymes in order to understand the specific surface residues, specific interactions, or any other molecular features that may be responsible for the differences in their tolerance to cold temperatures. The comparison between the psychrophilic and the mesophilic subtilisins revealed that the cold adapted subtilisins have a high content of acidic residues mainly found on their surface, making it charged. The analysis of the Trpases showed that they have a high content of hydrophobic residues on their surface. Thus, we suggest that the negatively charged residues on the surface of the subtilisins may be responsible for their cold adaptation, whereas the hydrophobic residues on the surface of monomeric Trpase molecules are responsible for the tetrameric assembly, and may account for their cold inactivation and dissociation.     

Comparative studies on lipogenic enzyme activities in brown adipose tissue and liver of the rat during starvation-refeeding transition and cold exposure

1. The effect of starvation-refeeding transition and cold exposure on the activity of lipogenic enzymes in brown adipose tissue (BAT) and liver from rats was compared. 2. Starvation caused a decrease of lipogenic enzyme activities in BAT and liver. 3. Refeeding of the animals with a high carbohydrate diet caused an increase of lipogenic enzymes in these tissues. 4. Cold exposure (4 degrees C for 30 days) led to the increase of BAT enzyme activities to the values observed in rats fed a high carbohydrate diet. 5. Under the same conditions the activity of hepatic lipogenic enzymes also increased but never reached the values observed in the liver of rats fed with a high carbohydrate diet. 6. Therefore BAT and liver lipogenic enzymes showed, in general, a similar pattern of variation under identical nutritional conditions, but substantial differences between these two organs occurred as far as the response to cold exposure was concerned. 7. The experiments also revealed that in the control animals BAT displayed a higher lipogenic potential than the liver.     

低温微生物及其酶类的研究概况

广泛分布在地球寒冷生境 ,如南北两极、高山、深海以及冰川中的低温微生物 ,不但为研究低温生态系统、生命起源与进化以及生物适冷机制提供了丰富的材料 ,同时在生物工程方面也具有潜在的巨大开发价值。国内外越来越多的科研人员对低温微生物及其产物的研究表现出了浓厚的兴趣。关于细胞膜和低温酶的研究 ,是目前微生物适冷机制研究中的 2个热点。就低温微生物的研究现状和适冷机制以及低温酶类的研究进行了综述。     

Function and biotechnology of extremophilic enzymes in low water activity

ABSTRACT: Enzymes from extremophilic microorganisms usually catalyze chemical reactions in non-standard conditions. Such conditions promote aggregation, precipitation, and denaturation, reducing the activity of most non-extremophilic enzymes, frequently due to the absence of sufficient hydration. Some extremophilic enzymes maintain a tight hydration shell and remain active in solution even when liquid water is limiting, e.g. in the presence of high ionic concentrations, or at cold temperature when water is close to the freezing point. Extremophilic enzymes are able to compete for hydration via alterations especially to their surface through greater surface charges and increased molecular motion. These properties have enabled some extremophilic enzymes to function in the presence of non-aqueous organic solvents, with potential for design of useful catalysts. In this review, we summarize the current state of knowledge of extremophilic enzymes functioning in high salinity and cold temperatures, focusing on their strategy for function at low water activity. We discuss how the understanding of extremophilic enzyme function is leading to the design of a new generation of enzyme catalysts and their applications to biotechnology.     

Defying the activity–stability trade-off in enzymes: taking advantage of entropy to enhance activity and thermostability

The biotechnological applications of enzymes are limited due to the activity–stability trade-off, which implies that an increase in activity is accompanied by a concomitant decrease in protein stability. This premise is based on thermally adapted homologous enzymes where cold-adapted enzymes show high intrinsic activity linked to enhanced thermolability. In contrast, thermophilic enzymes show low activity around ambient temperatures. Nevertheless, genetically and chemically modified enzymes are beginning to show that the activity–stability trade-off can be overcome. In this review, the origin of the activity–stability trade-off, the thermodynamic basis for enhanced activity and stability, and various approaches for escaping the activity–stability trade-off are discussed. The role of entropy in enhancing both the activity and the stability of enzymes is highlighted with a special emphasis placed on the involvement of solvent water molecules. This review is concluded with suggestions for further research, which underscores the implications of these findings in the context of productivity curves, the Daniel–Danson equilibrium model, catalytic antibodies, and life on cold planets.     

Some like it cold: biocatalysis at low temperatures

In the last few years, increased attention has been focused on a class of organisms called psychrophiles. These organisms, hosts of permanently cold habitats, often display metabolic fluxes more or less comparable to those exhibited by mesophilic organisms at moderate temperatures. Psychrophiles have evolved by producing, among other peculiarities, "cold-adapted" enzymes which have the properties to cope with the reduction of chemical reaction rates induced by low temperatures. Thermal compensation in these enzymes is reached, in most cases, through a high catalytic efficiency associated, however, with a low thermal stability. Thanks to recent advances provided by X-ray crystallography, structure modelling, protein engineering and biophysical studies, the adaptation strategies are beginning to be understood. The emerging picture suggests that psychrophilic enzymes are characterized by an improved flexibility of the structural components involved in the catalytic cycle, whereas other protein regions, if not implicated in catalysis, may be even more rigid than their mesophilic counterparts. Due to their attractive properties, i.e., a high specific activity and a low thermal stability, these enzymes constitute a tremendous potential for fundamental research and biotechnological applications.     

Comparative void-volume analysis of psychrophilic and mesophilic enzymes: Structural bioinformatics of psychrophilic enzymes reveals sources of core flexibility

Background

Psychrophiles, cold-adapted organisms, have adapted to live at low temperatures by using a variety of mechanisms. Their enzymes are active at cold temperatures by being structurally more flexible than mesophilic enzymes. Even though, there are some indications of the possible structural mechanisms by which psychrophilic enzymes are catalytic active at cold temperatures, there is not a generalized structural property common to all psychrophilic enzymes.

Results

We examine twenty homologous enzyme pairs from psychrophiles and mesophiles to investigate flexibility as a key characteristic for cold adaptation. B-factors in protein X-ray structures are one way to measure flexibility. Comparing psychrophilic to mesophilic protein B-factors reveals that psychrophilic enzymes are more flexible in 5-turn and strand secondary structures. Enzyme cavities, identified using CASTp at various probe sizes, indicate that psychrophilic enzymes have larger average cavity sizes at probe radii of 1.4-1.5 Å, sufficient for water molecules. Furthermore, amino acid side chains lining these cavities show an increased frequency of acidic groups in psychrophilic enzymes.

Conclusions

These findings suggest that embedded water molecules may play a significant role in cavity flexibility, and therefore, overall protein flexibility. Thus, our results point to the important role enzyme flexibility plays in adaptation to cold environments.