Convergent Cascade Catalyzed by Monooxygenase–Alcohol Dehydrogenase Fusion Applied in Organic Media

With the aim of applying redox-neutral cascade reactions in organic media, fusions of a type II flavin-containing monooxygenase (FMO-E) and horse liver alcohol dehydrogenase (HLADH) were designed. The enzyme orientation and expression vector were found to influence the overall fusion enzyme activity. The resulting bifunctional enzyme retained the catalytic properties of both individual enzymes. The lyophilized cell-free extract containing the bifunctional enzyme was applied for the convergent cascade reaction consisting of cyclobutanone and butane-1,4-diol in different microaqueous media with only 5 % (v/v) aqueous buffer without any addition of external cofactor. Methyl tert-butyl ether and cyclopentyl methyl ether were found to be the best organic media for the synthesis of γ-butyrolactone, resulting in about 27 % analytical yield.     

Discovery and Characterization of a Baeyer-Villiger Monooxygenase Using Sequence Similarity Network Analysis

Baeyer-Villiger monooxygenases (BVMOs) are important flavin-dependent enzymes which perform oxygen insertion reactions leading to valuable products. As reported in many studies, BVMOs are usually unstable during application, preventing a wider usage in biocatalysis. Here, we discovered a novel NADPH-dependent BVMO which originates from Halopolyspora algeriensis using sequence similarity networks (SSNs). The enzyme is stable at temperatures between 10 °C to 30 °C up to five days after the purification, and yields the normal ester product. In this study, the substrate scope was investigated for a broad range of aliphatic ketones and the enzyme was biochemically characterized to identify optimum reaction conditions. The best substrate (86 % conversion) was 2-dodecanone using purified enzyme. This novel BVMO could potentially be applied as part of an enzymatic cascade or in bioprocesses which utilize aliphatic alkanes as feedstock.     

新型层状交联酶聚集体的制备条件优化与性质研究

以假丝酵母脂肪酶（Candida sp.lipase）为研究对象,开发了一种新型层状交联酶聚集体。用蛋白或氨基酸对纳米氧化锌粒子进行修饰,继以交联剂交联后作为核芯,酶分子再交联在纳米核表面形成层状结构。实验结果表明牛血清白蛋白（BSA）是纳米氧化锌适宜的修饰剂。并且对纳米芯层状交联酶聚集体（BSA-N-LCLEAs）其他制备条件进行了优化,优化后BSA-N-LCLEAs制备条件为：沉淀剂硫酸铵饱和度为58%,交联剂戊二醛浓度为3.5%,交联温度和时间分别为0℃和2 h。BSA-N-LCLEAs酶活收率较传统CLEAs提高了196.5%。扫描电镜表征表明BSA-N-LCLEAs较传统CLEAs孔道大幅增加。纳米芯层状CLEAs的pH稳定性和热稳定性也都比传统CLEAs有所提高,并将该固定化酶用于催化维生素E琥珀酸酯的合成,反应五批次后反应产率还能达90%左右,说明该新型交联酶聚集体具有良好的催化活性和操作稳定性。     

Cross‐Linked Amorphous Nitrilase Aggregates for Enantioselective Nitrile Hydrolysis

A recombinant Escherichia coli strain was constructed which efficiently expressed the enantioselective nitrilase from Alcaligenes faecalis DSMZ 30030 as a hisitidine‐tagged enzyme variant under the control of a rhamnose inducible promoter. The enzyme was purified from cell extracts and used for the preparation of cross‐linked enzyme aggregates (CLEAs). The conditions for the preparation of the CLEAs were optimized using various organic solvents and cross‐linking agents and a procedure was developed which combined a precipitation with 85 % (v/v) isopropyl alcohol and a cross‐linking with 30 mM glutaraldehyde. Thus, about 80 % of the initial nitrilase activity could be incorporated into the CLEAs. The hydrolysis of racemic mandelonitrile to (R)‐mandelic acid was compared between the soluble nitrilase preparations and their CLEAs (nit‐CLEAs). The nitrilase activity in the CLEAs was at 30 °C and 60 °C about 5 times more stable than in the soluble preparations. The CLEAs could be reused 5 times with only about 10 % reduction in activity. The enantioselectivity of the nitrilase for the formation of (R)‐mandelic acid from racemic mandelonitrile decreased for both preparations with increasing temperatures (10 °C to 50 °C), but this effect was significantly less pronounced for the CLEAs. A detailed analysis of solvent effects on nitrilase enantioselectivity allowed thermodynamic insights into contributions from free energy component (activation enthalpy and entropy) to chiral preference of nitrilase in such non conventional media.     

Enzyme Fusions in Biocatalysis: Coupling Reactions by Pairing Enzymes

One approach to bringing enzymes together for multienzyme biocatalysis is genetic fusion. This enables the production of multifunctional enzymes that can be used for whole-cell biotransformations or for in vitro (cascade) reactions. In some cases and in some aspects, such as expression and conversions, the fused enzymes outperform a combination of the individual enzymes. In contrast, some enzyme fusions are greatly compromised in activity and/or expression. In this Minireview, we give an overview of studies on fusions between two or more enzymes that were used for biocatalytic applications, with a focus on oxidative enzymes. Typically, the enzymes are paired to facilitate cofactor recycling or cosubstrate supply. In addition, different linker designs are briefly discussed. Although enzyme fusion is a promising tool for some biocatalytic applications, future studies could benefit from integrating the findings of previous studies in order to improve reliability and effectiveness.     

Investigation of a New Type I Baeyer–Villiger Monooxygenase from Amycolatopsis thermoflava Revealed High Thermodynamic but Limited Kinetic Stability

Baeyer–Villiger monooxygenases (BVMOs) are remarkable biocatalysts, but, due to their low stability, their application in industry is hampered. Thus, there is a high demand to expand on the diversity and increase the stability of this class of enzyme. Starting from a known thermostable BVMO sequence from Thermocrispum municipale (TmCHMO), a novel BVMO from Amycolaptosis thermoflava (BVMO_Flava), which was successfully expressed in Escherichia coli BL21(DE3), was identified. The activity and stability of the purified enzyme was investigated and the substrate profile for structurally different cyclohexanones and cyclobutanones was assigned. The enzyme showed a lower activity than that of cyclohexanone monooxygenase (CHMO_Acineto) from Acinetobacter sp., as the prototype BVMO, but indicated higher kinetic stability by showing a twofold longer half-life at 30 °C. The thermodynamic stability, as represented by the melting temperature, resulted in a T_m value of 53.1 °C for BVMO_Flava, which was comparable to the T_m of TmCHMO (ΔT_m=1 °C) and significantly higher than the T_m value for CHMO_Acineto ((ΔT_m=14.6 °C)). A strong deviation between the thermodynamic and kinetic stabilities of BVMO_Flava was observed; this might have a major impact on future enzyme discovery for BVMOs and their synthetic applications.     

One-dimensional crosslinked enzyme aggregates in SBA-15: Superior catalytic behavior to conventional enzyme immobilization

α-Chymotrypsin (CT) and lipase (LP) were immobilized in SBA-15 mesoporous silica by crosslinking adsorbed enzymes. This simple approach resulted in one-dimensional crosslinked enzyme aggregates (CLEAs) in the linear pore channels of SBA-15, which was very effective in preventing the enzyme leaching and consequently improving the enzyme stability. Both CLEAs of CT and LP showed negligible activity decrease under harsh shaking condition for one week while the conventional approaches including adsorption and covalent attachment resulted in more than 50–90% enzyme inactivation under the same condition. This effective stabilization results from the bent pore structure of SBA-15 with a high aspect ratio, which prevents the leaching of one-dimensional CLEAs and thereby achieves the higher enzyme loading capacity. Along with the higher specific activity than that of adsorbed enzymes, this CLEA approach is much simpler than that of covalent attachment by obviating the tedious processes for silica functionalization and enzyme attachment.     

Switch in Cofactor Specificity of a Baeyer–Villiger Monooxygenase

Baeyer–Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to esters or lactones by using molecular oxygen and a cofactor. Type I BVMOs display a strong preference for NADPH. However, for industrial purposes NADH is the preferred cofactor, as it is ten times cheaper and more stable. Thus, we created a variant of the cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMO_Acineto); this used NADH 4200‐fold better than NADPH. By combining structure analysis, sequence alignment, and literature data, 21 residues in proximity of the cofactor were identified and targeted for mutagenesis. Two combinatorial variants bearing three or four mutations showed higher conversions of cyclohexanone with NADH (79 %) compared to NADPH (58 %) as well as specificity. The structural reasons for this switch in cofactor specificity of a type I BVMO are especially a hydrogen‐bond network coordinating the two hydroxy groups of NADH through direct interactions and bridging water molecules.     

Three in One: Temperature,Solvent and Catalytic Stability by Engineering the Cofactor‐Binding Element of Amine Transaminase

Amine transaminase (ATA) catalyse enantioselectively the direct amination of ketones, but insufficient stability during catalysis limits their industrial applicability. Recently, we revealed that ATAs suffer from substrate‐induced inactivation mechanism involving dissociation of the enzyme–cofactor intermediate. Here, we report on engineering the cofactor‐ring‐binding element, which also shapes the active‐site entrance. Only two point mutations in this motif improved temperature and catalytic stability in both biphasic media and organic solvent. Thermodynamic analysis revealed a higher melting point for the enzyme–cofactor intermediate. The high cofactor affinity eliminates the need for pyridoxal 5′‐phosphate supply, thus making large‐scale reactions more cost effective. This is the first report on stabilising a tetrameric ATA by mutating a single structural element. As this structural “hotspot” is a common feature of other transaminases it could serve as a general engineering target.     

Kinetic and transport effects on enzymatic biocatalysis resulting from the PEGylation of cofactors

The utilization of cofactor‐dependent redox enzymes in bioprocess technologies requires low cost cofactor regeneration methods. PEGylated NAD(H) (PEG‐NAD(H)) has been utilized in enzyme membrane reactors as a means to recover the cofactor; however, there is a lack of understanding of the effect of PEGylation on enzymatic activity, especially on the relationship between biocatalysis and transport phenomena. To explore this further, two redox enzymes (formate dehydrogenase (FDH) from Saccharomyces cerevisiae and NAD(H)‐dependent d ‐lactate dehydrogenase (nLDH) from Escherichia coli) have been chosen and the kinetic effects caused by cofactor modifications (with PEG of three different chain lengths) have been investigated. The PEGylation did not impact the cofactor dissociation constants and mass transfer was not the rate‐limiting step in biocatalysis for either enzyme. However, the PEG chain length had different impacts on the formation of enzyme/cofactor and/or enzyme/cofactor/substrate ternary complexes for the enzymes. © 2017 American Institute of Chemical Engineers AIChE J, 63: 12–17, 2018     

手性化合物酶法制备中辅酶再生体系的构建与应用进展

辅酶的再生与反复利用对氧化还原型生物催化剂在手性化合物中的生产具有重要作用。辅酶的酶催化法再生具有高效、专一的特点,根据其催化环境可分为单细胞内辅酶再生体系、双细胞耦合体系及胞外酶耦合体系。本文详细阐述了酶法再生中各种辅酶再生体系的构建方法,特别指出了辅酶再生体系使用中可能遇到的问题并给出了解决方法,如利用离子液体解决有机底物低水溶性的问题,利用共价固定方法解决小分子辅酶的留存问题,无细胞抽提液中内源性辅酶再生酶的利用等。除此之外,对每种辅酶再生方法与体系在工业催化中应用的优势与局限性作了评述,特别关注了辅酶再生体系反复多批次利用的问题。     

Potential of Different Enzyme Immobilization Strategies to Improve Enzyme Performance

Enzyme biocatalysis plays a very relevant role in the development of many chemical industries, e.g., energy, food or fine chemistry. To achieve this goal, enzyme immobilization is a usual pre‐requisite as a solution to get reusable biocatalysts and thus decrease the price of this relatively expensive compound. However, a proper immobilization technique may permit far more than to get a reusable enzyme; it may be used to improve enzyme performance by improving some enzyme limitations: enzyme purity, stability (including the possibility of enzyme reactivation), activity, specificity, selectivity, or inhibitions. Among the diverse immobilization techniques, the use of pre‐existing supports to immobilize enzymes (via covalent or physical coupling) and the immobilization without supports [enzyme crosslinked aggregates (CLEAs) or crystals (CLECs)] are the most used or promising ones. This paper intends to give the advantages and disadvantages of the different existing immobilization strategies to solve the different aforementioned enzyme limitations. Moreover, the use of nanoparticles as immobilization supports is achieving an increasing importance, as the nanoparticles versatility increases and becomes more accessible to the researchers. We will also discuss here some of the advantages and drawbacks of these non porous supports compared to conventional porous supports. Although there are no universal optimal solutions for all cases, we will try to give some advice to select the optimal strategy for each particular enzyme and process, considering the enzyme properties, nature of the process and of the substrate. In some occasions the selection will be compulsory, for example due to the nature of the substrate. In other cases the optimal biocatalyst may depend on the company requirements (e.g., volumetric activity, enzyme stability, etc).     

酶的寡聚结构与催化稳定性

酶的结构与催化稳定性是生物催化与转化过程中的研究热点之一。与单体酶相比,寡聚酶在进化过程中亚基之间的聚合使其在结构和功能上具有一定的优越性,然而寡聚酶独特的四级结构导致其在制备和应用中存在诸多问题,如制备效率低、催化位点利用率低、催化稳定性差等,其中亚基解离导致的催化稳定性问题在很大程度上限制了其工业化应用。目前,介质工程、多亚基固定化、亚基界面工程和融合蛋白策略被应用于寡聚酶的催化稳定性改造,而寡聚酶至单体酶的改造策略则试图从根本上解决寡聚酶的制备和应用问题,具有较好的应用前景。本文介绍了酶的寡聚结构演替所产生的新功能,总结了寡聚酶在制备和应用中存在的问题,重点阐述了提高寡聚酶制备效率和催化稳定性的策略。     

Harnessing P450 Enzyme for Biotechnology and Synthetic Biology

Cytochrome P450 enzymes (P450s, CYPs) catalyze the oxidative transformation of a wide range of organic substrates. Their functions are crucial to xenobiotic metabolism and steroid transformation in humans and other organisms. The enzymes are promising for synthetic biology applications but limited by several drawbacks including low turnover rates, poor stability, the dependance of expensive cofactors and redox partners, and the narrow substrate scope. To conquer these obstacles, emerging strategies including substrate engineering, usage of decoy and decoy-based small molecules auxiliaries, designing of artificial enzyme cascades and the incorporation of materials have been explored based on the unique properties of P450s. These strategies can be applied to a wide range of P450s and can be combined with protein engineering to improve the enzymatic activities. This minireview will focus on some recent developments of these strategies which have been used to leverage P450 catalysis. Remaining challenges and future opportunities will also be discussed.     

Methods for stabilizing and activating enzymes in ionic liquids—a review

Ionic liquids (ILs) have evolved as a new type of non‐aqueous solvents for biocatalysis, mainly due to their unique and tunable physical properties. A number of recent review papers have described a variety of enzymatic reactions conducted in IL solutions; on the other hand, it is important to systematically analyze methods that have been developed for stabilizing and activating enzymes in ILs. This review discusses the biocatalysis in ILs from two unique aspects (1) factors that impact the enzyme's activity and stability, (2) methods that have been adopted or developed to activate and/or stabilize enzymes in ionic media. Factors that may influence the catalytic performance of enzymes include IL polarity, hydrogen‐bond basicity/anion nucleophilicity, IL network, ion kosmotropicity, viscosity, hydrophobicity, the enzyme dissolution, and surfactant effect. To improve the enzyme's activity and stability in ILs, major methods being explored include the enzyme immobilization (on solid support, sol–gel, or CLEA), physical or covalent attachment to PEG, rinsing with n‐propanol methods (PREP and EPRP), water‐in‐IL microemulsions, IL coating, and the design of enzyme‐compatible ionic solvents. It is exciting to notice that new ILs are being synthesized to be more compatible with enzymes. To utilize the full potential of ILs, it is necessary to further improve these methods for better enzyme compatibility. This is what has been accomplished in the field of biocatalysis in conventional organic solvents. Copyright © 2010 Society of Chemical Industry     

A facile technique to prepare cross-linked enzyme aggregates using <Emphasis Type="Italic">p</Emphasis>-benzoquinone as cross-linking agent

To obtain robust and thermo-stable enzyme aggregates, p-benzoquinone was used as cross-linker and bovine serum albumin (BSA) as crowding macromolecules to prepare cross-linked enzyme aggregates (CLEAs) of lipase. Effects of cross-linking time and cross-linker content on the activity, thermal stability and characteristics of enzyme aggregates were examined carefully. It was observed that when the content of p-benzoquinone was 5 mM and amount of BSA was 125% of that of lipase (w/w), the specific activity of cross-linked co-aggregates of lipase and BSA was 79.8 U mg⁻¹, 2.44-fold of that of cross-linked enzyme aggregates of lipase without BSA. Moreover, after heat treatment for 96 h at 50 °C, the CLEAs prepared with this facile routine kept 75.18% of their initial activity, 5.01-fold more than that of the just CLEAs using glutaraldehyde. Furthermore, BSA macromolecules in lipase CLEAs enhanced the catalytic efficiency of free and just lipase CLEAs without BSA by 1.45 and 2.83 times, respectively. The proposed crosslinking technique would rank among the potential strategies for efficiently preparing robust and thermo-stable enzyme aggregates.     

乳糖酶交联酶聚体的制备及性能

以双醛淀粉(DAS)为交联剂,分别制备了乳酸克鲁维酵母和米曲霉来源的乳糖酶交联酶聚体(CLEAs),同时添加牛血清白蛋白(BSA)作为保护剂以提高CLEAs活性。当DAS质量分数为10%、氧化度为80%、BSA与酶蛋白质量比为1∶8时得到的酵母和曲霉乳糖酶CLEAs的活力保留分别为53.84%和55.25%。CLEAs的最适pH值较游离酶有所降低。曲霉乳糖酶CLEAs在60℃下具有较好的热稳定性,并且在37℃下重复使用5次(20 h)后活力可保留52%。     

酶/MOFs复合材料催化性能调控及应用

金属有机框架（MOFs）具有比表面积大、设计性强和生物相容性好等优良特性，可以作为固定化酶的理想载体，从而提高游离酶的稳定性和催化性能，许多酶/MOFs复合材料也显示出比游离酶更好的催化性能。因此，酶/MOFs复合材料已应用于生物传感、检测、催化等领域，已成为传统催化剂的环保替代品。综述了酶在MOFs上的3种固定化方法（表面固定、孔封装和原位包埋法），重点介绍了4种影响酶/MOFs复合材料催化性能的因素及调控方法，对酶/MOFs复合材料在催化方面的应用也进行了总结，并对酶固定化的未来进行了展望。     

Improvement of chymotrypsin enzyme stability as single enzyme nanoparticles

Individual enzyme molecules were coated with a nanometer-scale polymer network in order to increase their stability, providing longer lifetime of enzymes for biochemical processes. This polymer nanolayer is thin and porous enough [Kim, J., Grate, J.W., 2003. Single-enzyme nanoparticles armored by a nanometer-scale organic/inorganic network. Nano Letters 3, 1219-1222] to allow practically unhindered diffusion of the substrate from the solution to the active site of the enzyme, and unhindered transfer of the product from the active site to the solution. During the three-step preparation of the individual enzyme nanoparticles, the chymotrypsin enzyme investigated can lose 30-50% of its original activity. The main cause for a decrease in the enzyme's activity is the UV-light irradiation used during the polymerization step of the pretreatment. The UV-light can destroy the tertiary structure of the enzyme, and this can lead to reduced activity. The enzyme modification and solubilization steps of the treatment methodology do not essentially lower enzyme activity. The morphology and size of enzyme nanoparticles prepared was examined by transmission electron microscopy and demonstrated in this paper. The activity's change of the free and the covered enzyme was investigated at different temperatures, shaking frequencies and pH values. It has been proved that the preparation of enzyme nanoparticles can essentially stabilize the enzyme. Its activity changes much slower than that of native enzyme. The pretreated enzyme can have relatively high residual activity under extreme pH values and temperature, as well. The essence of the results presented is that stability of the enzyme can be increased significantly by covering individual enzymes with a thin, porous polymer layer.     

金属有机骨架材料载体用于酶固定化的研究进展

现有的固定化酶技术存在低稳定性、低回收利用率等缺点,而金属有机骨架材料（MOFs）凭借自身独特的性质如特定的主-客体相互作用及限域效应,极大提高了酶的负载率以及限制酶分子的流失,甚至极端环境下仍能维持酶的活性,近年来成为固定化酶载体的研究热点。本文从酶-MOF复合物的合成策略出发,介绍了利用MOFs载体完成酶固定化包括表面吸附、共价结合、孔道包埋和原位合成这4种方法的优劣势,重点概述了酶-MOF复合物在微反应器构建、级联反应等应用领域的巨大潜力。研究进展表明,有巨大优势的新型MOFs载体将有力促进酶-MOF复合物作为优异的催化剂、生物传感器等在多学科领域的发展。