酶的分子定向进化及其应用

酶的分子定向进化是20世纪90年代初兴起的一种蛋白质工程的新策略,是一种在生物体外模拟自然进化过程的、具有一定目的性的快速改造蛋A质的方法.该方法引起了生物催化技术领域的又一次革命.目前分子定向进化技术已被广泛应用于工业、农业及制药业等的相关领域.本文详细综述了酶的分子定向进化的概念、过程、基本策略及其核心技术,并着重介绍了酶的分子定向进化技术在提高酶的活力、稳定性、底物特异性和对映体选择性等几方面的应用及取得的相关成果.     

工业生物催化技术

以蛋白质酶的工程应用为核心的工业生物催化技术,被认为是生物技术继生物医药和转基因植物之后的第三次浪潮。它的发展与应用将对人类的工业化学过程带来根本的变革。工业生物催化的兴起与以下的两个关键技术因素有密切的关系：(1)蛋白质定向进化技术的出现,(2)基因组学和蛋白质组学的发展。探讨了工业生物催化技术的现状和发展趋势,并对我国如何发展该领域的基础和应用研究提出一些见解。     

多酶共固定化反应体系的研究进展

近年来,生物催化为化学、生物学和生物工程学等领域提供了一种绿色研究工具,其中多酶体系在这些领域中的应用越来越受到关注,其克服了以往单个酶不能满足催化需求的局限性,同时多酶共固定化在级联反应过程中,可增加酶周围的反应物浓度,并将不同酶的催化特性结合起来,能排除干扰因素,从而提高酶的整体催化效率。对多酶共固定化反应体系的研究进展进行了综述,包括多酶反应体系的类别、共固定化技术的特点以及相关应用,并对共固定化多酶反应体系进行了展望。     

工业酶结构与功能的构效关系

工业酶是绿色生物制造的"芯片",支撑着下游数十倍甚至百倍的产业。解析工业酶结构与功能关系是对其设计改造并应用于工业生产的基础。近年来随着蛋白结构解析技术和计算模拟技术的发展,酶结构与功能的构效关系得到更加深刻的认识,使得酶理性设计,甚至是从头设计成为可能。文中围绕酶结构的可塑性及其催化功能的多样性,综述工业酶结构与功能构效关系的研究进展及应用,并展望该领域的未来发展前景。     

合成生物制造2022

本文对2022年《生物工程学报》发表的与合成生物制造相关的综述和研究论文进行了评述,重点讨论了DNA测序、DNA合成、DNA编辑、基因表达调控和数学细胞模型等底层技术,酶的设计、改造和应用技术,化学品生物催化、氨基酸及其衍生物、有机酸、天然化合物、抗生素与活性肽、功能多糖、功能蛋白质等重要产品的生物制造技术,一碳化合物和生物质原料利用技术以及合成微生物组技术,以帮助读者从一个侧面了解合成生物制造相关技术和产业的发展情况。     

角质酶的研究进展

角质酶(EC3.1.1.74)是一种可以降解角质并产生大量脂肪酸单体的水解酶。角质酶是一种多功能酶,可水解可溶性酯、不溶性甘油三酯和各种聚酯,同时还能催化酸与醇的酯化、脂肪酸盐与醇的转酯化反应,因此在食品工业、化工工业等诸多领域都具有广泛的应用。近年来研究发现,角质酶可实现棉纤维的生物精练和合成纤维的生物改性,是推动纺织工业清洁生产的关键酶制剂。     

蛋白质去糖基化酶及其应用

果糖氨氧化酶和果糖氨激酶能催化去糖基化反应而释放出自由氨基。本文主要探讨阿马多里酶的生物化学特征。如结构和功能的关系,动力学机制和底物的特异性,并探讨这些酶的生物作用和在蛋白中去糖基化中的应用,希望为糖基化相关疾病的治疗提供很好的治疗手段。     

作为人造极端酶的交联酶晶体

作为人造极端酶的交联酶晶体林影卢荣德郭勇（华南理工大学生物工程系,广州５１０６４０关键词交联酶晶体极端酶酶作为生物催化剂,催化效率高,立体选择性和底物专一性强,是普通化学催化剂所不可比拟的。然而,在酶的应用过程中还有很多令人不满意之处,如在环境中常常...     

基于纳米酶的比色生物传感器在生物医学检测中的应用

比色生物传感技术由于具有灵敏度高、方法简单并且容易操作等优点，已广泛应用于生物环境中污染物检测、生物体内重要标志物的检测以及癌症筛查等多个领域。基于纳米酶的比色生物传感器主要是借助纳米酶自身所具有的催化能力，模拟类过氧化物酶活性，将显色剂氧化生成有色溶液，从而实现可视化检测，并通过对有色溶液吸光度的检测得到相关物质的含量。与无纳米酶的比色生物传感器相比，基于纳米酶的比色生物传感器具有选择性更高、检测更快以及灵敏度更高等优点。纳米酶在具有天然酶活性的同时还具有成本低、稳定性好的、易于合成等优点，其相关研究越来越广泛。目前，基于纳米酶的比色生物传感器已成为辅助相关医学检测的重要方法，同时也广泛应用于便携和实时性相关检测当中，为医学检测提供了重要的支持和保障。为了提高比色生物传感器的灵敏度以及应用范围，研究人员也在致力于增加可检测物质的种类以及纳米酶种类的多样化等。本文主要介绍基于纳米酶的比色生物传感器的检测原理、几类典型的纳米酶，以及基于纳米酶的比色生物传感器在生物医学检测领域中的应用情况和研究进展。     

改进酶稳定性的定向进化技术

酶作为一种生物催化剂,以其独特的优良特性,在绿色化学和清洁生产中得到了广泛的应用。随着酶定向进化技术的建立和发展,通过定向进化改进酶稳定性的研究越来越多。详细综述了各种定向进化方法的特点及在提高酶稳定性方面的应用,并从结构和功能的角度进一步解释了相关机理。     

Engineering biocatalytic systems in organic media with low water content

The use of organic media in biocatalysis stems from the fact that in many cases biocatalytic processes can hardly be conducted (if at all) in aqueous solutions because of extremely low solubilities of substrates and/or unfavorable shift of the reaction equilibrium in water. The growing interest in this biotechnological area that has sprung up over the past few years has resulted in various approaches to enzyme stabilization against organic solvents. Thus, the main goal of the present review is to formulate a comprehensive classification of numerous successful nonaqueous biocatalytic systems based on a few fundamental principles. Typical examples are considered, along with the advantages and drawbacks inherent in each of the approaches discussed.     

A novel framework for the cell-free enzymatic production of glucaric acid

Glucaric acid (GlucA) is a valuable glucose-derived chemical with promising applications as a biodegradable and biocompatible chemical in the manufacturing of plastics, detergents and drugs. Recently, there has been a significant focus on producing GlucA microbially (in vivo) from renewable materials such as glucose, sucrose and myo-inositol. However, these in vivo GlucA production processes generally lack efficiency due to toxicity problems, metabolite competition and suboptimal enzyme ratios. Synthetic biology and accompanying cell-free biocatalysis have been proposed as a viable approach to overcome many of these limitations. However, cell-free biocatalysis faces its own limitations for industrial applications due to high enzyme costs and cofactor consumption. We have constructed a cell-free GlucA pathway and demonstrated a novel framework to overcome limitations of cell-free biocatalysis by i) the combination of both thermostable and mesophilic enzymes, ii) incorporation of a cofactor regeneration system and iii) immobilisation and recycling of the pathway enzymes. The cell-free production of GlucA was achieved from glucose-1-phosphate with a titre of 14.1 ± 0.9 mM (3.0 ± 0.2 g l⁻¹) and a molar yield of 35.2 ± 2.3% using non-immobilised enzymes, and a titre of 8.1 ± 0.2 mM (1.70 ± 0.04 g l⁻¹) and a molar yield of 20.2 ± 0.5% using immobilised enzymes with a total reaction time of 10 h. The resulting productivities (0.30 ± 0.02 g/h/l for free enzymes and 0.170 ± 0.004 g/h/l for immobilised enzymes) are the highest productivities so far reported for glucaric acid production using a synthetic enzyme pathway.     

Enzyme stabilization: state of the art

Enzyme stabilization is one of the most important fields in basic and applied enzymology. In basic enzymology, it is of particular relevance to understand enzyme stabilization principles first elucidating how and why the enzymes lose their biological activity and then deriving structure-stability relationships existing in enzymatic molecules. In applied enzymology, the most significant goal is to achieve useful compounds by biocatalysis. Enzymes are good catalysts in terms of high catalytic and specific activity with ability to function under mild conditions. However, they are not always ideal catalysts for practical applications because they are generally unstable and they inactivate rapidly through several mechanisms. In order to enhance enzyme stability, many strategies have been pursued in recent years. The present article is an attempt to provide detailed information about these strategies.     

Homogeneous Biocatalysis in Organic Solvents and Water-Organic Mixtures

Biocatalysis in non-aqueous media has undergone tremendous development during the last decade, and numerous reactions have been introduced and optimized for synthetic applications. In contrast to aqueous enzymology, biotransformations in organic solvents offer unique industrially attractive advantages, such as: drastic changes in the enantioselectivity of the reaction, the reversal of the thermodynamic equilibrium of hydrolysis reactions, suppression of water-dependent side reactions, and resistance to bacterial contamination. Currently, the field is dominated by heterogeneous biocatalysis based primarily on lyophilized enzyme powders, cross-linked crystals, and enzymes immobilized on inert supports that are mainly applied in enantioselective synthesis. However, low reaction rates are an inherent problem of the heterogeneous biocatalysis, while the homogeneous systems have the advantage that the elimination of diffusional barriers of substrates and products between organic and water phases results in an increase in the reaction rate. Here the discussion is focused on the correlation between activity and structure of the intact enzymes dissolved in neat organic solvents, as well as modifications of natural enzymes, which make them soluble and catalytically active in non-aqueous environment. Factors that influence conformation and stability of the enzymes are also discussed. Current developments in non-aqueous biocatalysts that combine advantages of protein modification and immobilization, i.e., HIP plastics, enzyme chips, ionic liquids, are introduced. Finally, engineering enzymes for biotransformations in non-conventional media by directed evolution is summarized.     

DBF (Disulfide Bond Forming) Enzyme from the Hyperthermophilic Archaebacterium Sulfolobus Solfataricus Behaves Like a Molecular Chaperone

DBF enzyme from the hyperthermophilic archaebacterium Sulfolobus solfataricus greatly enhances the refolding at 30°C of denatured and reduced bovine pancreatic ribonuclease (Guagliardi et al., 1992). Here we show that DBF behaves like a molecular chaperone: it affects in an ATP-dependent manner the in vitro refolding at 50°C of two thermostable dehydrogenases, an alcohol dehydrogenase and a glutamate dehydrogenase from S. solfataricus. This paper also reports the complete amino acid sequence of DBF. The role of molecular chaperones from thermophilic microorganisms in applied biocatalysis is discussed.     

DBF (Disulfide Bond Forming) Enzyme from the Hyperthermophilic Archaebacterium Sulfolobus Solfataricus Behaves Like a Molecular Chaperone

DBF enzyme from the hyperthermophilic archaebacterium Sulfolobus solfataricus greatly enhances the refolding at 30°C of denatured and reduced bovine pancreatic ribonuclease (Guagliardi et al., 1992). Here we show that DBF behaves like a molecular chaperone: it affects in an ATP-dependent manner the in vitro refolding at 50°C of two thermostable dehydrogenases, an alcohol dehydrogenase and a glutamate dehydrogenase from S. solfataricus. This paper also reports the complete amino acid sequence of DBF. The role of molecular chaperones from thermophilic microorganisms in applied biocatalysis is discussed.     

Development of Ionic Liquid‐Water‐Based Thermomorphic Solvent (TMS)‐Systems for Biocatalytic Reactions

The applicability of ionic liquid‐water‐based thermomorphic solvent (TMS)‐systems with an upper critical solution temperature for homogeneous biocatalysis is investigated. Cholinium‐ and imidazolium‐based ionic liquids are used to facilitate a temperature‐dependent phase change, which can be easily fine‐tuned by adding salts or polar organic solvents. Within the TMS‐system, a high enzymatic activity and subsequent full conversion is achieved in the intermittent monophasic reaction system of the TMS‐system. Therefore, the biocatalyst can be easily recycled after separating the phases at lower temperatures.     

Cross-linking and immobilisation of different proteins with recombinant Verrucomicrobium spinosum tyrosinase

This paper reports on the cross-linking and immobilisation of various proteins by the recombinant tyrosinase from Verrucomicrobium spinosum (Vs-tyrosinase). In general it is found that Vs-tyrosinase can readily cross-link proteins with a low degree of complexity, such as casein, but that the enzyme cannot readily cross-link well folded protein substrates such as lysozyme, myoglobin, cytochrome c or Candida antarctica lipase B (CALB). However, the inclusion of phenolic compounds (phenol or caffeic acid) to reaction mixtures of these proteins can greatly enhance the levels of cross-linking. For example it is possible to prepare cross-linked aggregates of industrially applicable enzymes such as CALB by simply incubating it with Vs-tyrosinase and phenol. The resulting aggregates can be collected by centrifugation and retain high levels of activity and may find applications in biocatalysis.     

Aspects Of Biocatalyst Stability In Organic Solvents

The stability of biocatalysis in systems containing organic solvents is reviewed. Among the examples presented are homogeneous mixtures of water and water-miscible organic solvents, aqueous/organic two-phase systems, solid biocatalysts suspended in organic solvents, enzymes in reverse micelles and modified enzymes soluble in water immiscible solvents. The stability of biocatalysts in organic solvents depends very much on the conditions. The hydrophobicity or the polarity of the solvent is clearly of great importance. More hydrophobic solvents (higher log P values) are less harmful to enzymes than less hydrophobic solvents. The water content of the system is a very important parameter. Some water is essential for enzymatic activity; however, the stability of enzymes decreases with increasing water content. Mechanisms of enzyme inactivation are discussed.     

A window into biocatalysis and biotransformations

Eight papers were presented in this year's symposium "Advances in Biocatalysis" at the 232nd ACS National Meeting, accentuating the most recent development in biocatalysis. Researchers from both industry and academia are addressing several fundamental problems in biocatalysis, including the limited number of commercially available enzymes that can be provided in bulk quantities, the limited enzyme stability and activity in nonaqueous environments, and the permeability issue and cell localization problems in whole-cell systems. A trend that can be discerned from these eight talks is the infusion of new tools and technologies in addressing various challenges facing biocatalysis. Nanotechnology, bioinformatics, cellular membrane engineering and metabolic engineering (for engineering whole-cell catalysts), and protein engineering (to improve enzymes and create novel enzymes) are becoming more routinely used in research laboratories and are providing satisfactory solutions to the problems in biocatalysis. Significant progress in various aspects of biocatalysis from discovery to industrial applications was highlighted in this symposium.