Properties and functions of the thiamin diphosphate dependent enzyme transketolase

This review highlights recent research on the properties and functions of the enzyme transketolase, which requires thiamin diphosphate and a divalent metal ion for its activity. The transketolase-catalysed reaction is part of the pentose phosphate pathway, where transketolase appears to control the non-oxidative branch of this pathway, although the overall flux of labelled substrates remains controversial. Yeast transketolase is one of several thiamin diphosphate dependent enzymes whose three-dimensional structures have been determined. Together with mutational analysis these structural data have led to detailed understanding of thiamin diphosphate catalysed reactions. In the homodimer transketolase the two catalytic sites, where dihydroxyethyl groups are transferred from ketose donors to aldose acceptors, are formed at the interface between the two subunits, where the thiazole and pyrimidine rings of thiamin diphosphate are bound. Transketolase is ubiquitous and more than 30 full-length sequences are known. The encoded protein sequences contain two motifs of high homology; one common to all thiamin diphosphate-dependent enzymes and the other a unique transketolase motif. All characterised transketolases have similar kinetic and physical properties, but the mammalian enzymes are more selective in substrate utilisation than the nonmammalian representatives. Since products of the transketolase-catalysed reaction serve as precursors for a number of synthetic compounds this enzyme has been exploited for industrial applications. Putative mutant forms of transketolase, once believed to predispose to disease, have not stood up to scrutiny. However, a modification of transketolase is a marker for Alzheimer’s disease, and transketolase activity in erythrocytes is a measure of thiamin nutrition. The cornea contains a particularly high transketolase concentration, consistent with the proposal that pentose phosphate pathway activity has a role in the removal of light-generated radicals.     

Comparison of soluble and immobilized trypsin kinetics: Implications for peptide synthesis

The protease trypsin was immobilized to porous glass in both the presence and absence of acetylated soybean trypsin inhibitor (STI) to determine whether immobilization could alter enzyme activity in favor of aminolysis over hydrolysis. Actiive-site titration with 4-methylumbelliferylguanidinobenzoate (MUGB) showed that only about 10% of immobilized trypsin had catalytic activity. Immobilization in the presence of STI produced a higher yield of active enzyme accessible to the inhibitor but did not increase the total yield of MUGB-active immobilized enzyme. Thus, enzyme inactivation upon immobilization could not be attributed to an inaccessible enzyme orientation, nor did STI prevent inactivation by stabilizing the active-site conformation. Kinetic parameters were determined for soluble and immobilized trypsin for two esters, N-tosyl-L-arginine methyl ester (TAME) and N-benzoyl-L-arginine ethyl ester (BAEE), and two amides, N-benzoyl-L-arginine p-nitroanilide (BAPNA) and N-t-boc-leucylglycylarginine p-nitroanilide (LGRNA). In all cases, immobilization caused a greater decrease in k(cat) for amidase activity than for esterase activity. The ratio [k(cat)/ K(m) (ester)]/[k(cat)/K(m) (amide)] increased slightly or stayed the same (for I.GRNA) or decreased sharply (for BAPNA). Including STI during immobilization had little effect on the active enzyme's intrinsic kinetics. A direct comparison of energy diagrams and free energies of activation for BAEE and BAPNA indicates that immobilization raises the free energy barriers for both amide and ester hydrolysis and lowers the energy barrier for aminolysis. In practice, these effects should lower the amidase activity and increase the aminolysis-hydrolysis ratio, rendering the immobilized enzyme a more efficient catalyst for peptide synthesis. (c) 1993 John Wiley & Sons, Inc.     

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.     

Structure and function of the transketolase from Mycobacterium tuberculosis and comparison with the human enzyme

The transketolase (TKT) enzyme in Mycobacterium tuberculosis represents a novel drug target for tuberculosis treatment and has low homology with the orthologous human enzyme. Here, we report on the structural and kinetic characterization of the transketolase from M. tuberculosis (TBTKT), a homodimer whose monomers each comprise 700 amino acids. We show that TBTKT catalyses the oxidation of donor sugars xylulose-5-phosphate and fructose-6-phosphate as well as the reduction of the acceptor sugar ribose-5-phosphate. An invariant residue of the TKT consensus sequence required for thiamine cofactor binding is mutated in TBTKT; yet its catalytic activities are unaffected, and the 2.5 Å resolution structure of full-length TBTKT provides an explanation for this. Key structural differences between the human and mycobacterial TKT enzymes that impact both substrate and cofactor recognition and binding were uncovered. These changes explain the kinetic differences between TBTKT and its human counterpart, and their differential inhibition by small molecules. The availability of a detailed structural model of TBTKT will enable differences between human and M. tuberculosis TKT structures to be exploited to design selective inhibitors with potential antitubercular activity.     

Enzymatic synthesis of β‐glucosylglycerol using a continuous‐flow microreactor containing thermostable β‐glycoside hydrolase CelB immobilized on coated microchannel walls

β‐Glucosylglycerol (βGG) has potential applications as a moisturizing agent in cosmetic products. A stereochemically selective method of its synthesis is kinetically controlled enzymatic transglucosylation from a suitable donor substrate to glycerol as acceptor. Here, the thermostable β‐glycosidase CelB from Pyrococcus furiosus was used to develop a microstructured immobilized enzyme reactor for production of βGG under conditions of continuous flow at 70°C. Using CelB covalently attached onto coated microchannel walls to give an effective enzyme activity of 30 U per total reactor working volume of 25 µL, substrate conversion and formation of transglucosylation product was monitored in dependence of glucosyl donor (2‐nitrophenyl‐β‐D ‐glucoside (oNPGlc), 3.0 or 15 mM; cellobiose, 250 mM), the concentration of glycerol (0.25–1.0 M), and the average residence time (0.2–90 s). Glycerol caused a concentration‐dependent decrease in the conversion of the glucosyl donor via hydrolysis and strongly suppressed participation of the substrate in the reaction as glucosyl acceptor. The yields of βGG were ≥80% and ≈60% based on oNPGlc and cellobiose converted, respectively, and maintained up to near exhaustion of substrate (≥80%), giving about 120 mM (30 g/L) of βGG from the reaction of cellobiose and 1 M glycerol. The structure of the transglucosylation products, 1‐O‐β‐D ‐glucopyranosyl‐rac‐glycerol (79%) and 2‐O‐β‐D ‐glucopyranosyl‐sn‐glycerol (21%), was derived from NMR analysis of the product mixture of cellobiose conversion. The microstructured reactor showed conversion characteristics similar to those for a batchwise operated stirred reactor employing soluble CelB. The advantage of miniaturization to the microfluidic format lies in the fast characterization of full reaction time courses for a range of process conditions using only a minimum amount of enzyme. Biotechnol. Bioeng. 2009;103: 865–872. © 2009 Wiley Periodicals, Inc.     

大肠杆菌转酮醇酶分子改造及催化酒石酸半醛合成

转酮醇酶(transketolase, TK, EC. 2.2.1.1)是一种焦磷酸硫胺素和二价阳离子依赖性酶,可催化二碳单位的转移,可逆形成C–C键,在多酶催化生产化学品、药物前体和不对称合成方面有广泛应用。文中以大肠杆菌(Escherichia coli) K12转酮醇酶TKTA为研究对象,通过定点饱和突变和组合突变提升对非磷酸化底物的反应活性,并探索突变酶TKTA＿M催化合成酒石酸半醛。结果表明：突变酶TKTA＿M (R358I/H461S/R520Q)最适反应温度为32℃,最适反应pH为7.0,以D-甘油醛为受体底物的比酶活为(6.57±0.14) U/mg,是野生型比酶活((0.71±0.02) U/mg)的9.25倍。在酶学性质研究的基础上,设计20 mL的反应体系,以50 mmol/L 5-酮基-D-葡萄糖酸和50 mmol/L非磷酸化乙醇醛为底物,TKTA＿M催化合成酒石酸半醛,最终酒石酸半醛的产量为3.71g,摩尔转化率为55.34%。研究结果为生物质制备L-(+)-酒石酸提供数据支撑,同时为转酮醇酶催化非磷酸化底物提供了借鉴。     

Application of immobilized enzyme technologies for the textile industry: a review

Abstract

A number of enzymes have applications in the textile sector, as several natural fibres (e.g. cotton, wool and flax) can be subjected to processing using such natural biocatalysts. In the latest two decades, demand for industrial enzymes has increased considerably; however, the textile industry requires highly stable enzymes, and good performance at extreme values of pH and temperature. New strategies are continuously emerging for the immobilization of enzymes with superior efficiency and usage. Enzymatic immobilization can stabilize enzymes and extend their useful life. Additionally, reduction in effluent treatment costs and improvement of efficiency is possible. This paper briefly reviews the most recent research efforts pertaining to immobilization of enzymes with potential application in the textile industry.     

Series-type enzyme deactivations: Influence of intermediate activity on deactivation kinetics

A series-type enzyme deactivation model involving an active enzyme precursor is proposed wherein the enzyme activity is a weighted function of the active enzyme states. The active enzyme precursor may be less active, as active or more active than the initial enzyme form. The proposed model is shown to fit the soluble and immobilized enzyme deactivation data presented reasonably well. Some enzymes exhibit a ‘compensation-like’ effect. In other enzymes, if the deactivation rate coefficient for the second step, k₂, is zero, then the activity may stabilize to a value that depends upon the relative activities of the two active enzyme states.     

褐藻降解菌群筛选及其复合酶固定化研究

以大连地区的褐藻为材料,筛选了褐藻降解菌群,其在30℃、pH7．5条件下培养74h时,酶活达1．883IU／mL。利用硅藻土吸附法经冷冻干燥制备了固定化复合酶,分析了复合酶系的酶学性质,其最适反应温度为45℃,并在40℃～55℃范围内具有良好的热稳定性;最适pH为7．5,并在pH7．0～8．5之间pH稳定性良好。利用固定化酶进行褐藻酸钠制备,提取率达48．3％,粘度为2．9Pa·s,与传统方法相比均有显著提高,为工程化生产褐藻酸钠提供一定基础。     

Immobilization and properties of carminomycin 4-O-methyltranferase, the enzyme which catalyzes the final step in the biosynthesis of daunorubicin in Streptomyces peucetius

The carminomycin 4-O-methyltransferase enzyme from Streptomyces peucetius was covalently immobilized on 3M Emphaze ABI-activated beads. Optimal conditions of time, temperature, pH, ionic strength, enzyme, substrate (carminomycin), and cosubstrate (S-adenosyl-L-methionine) concentrations were defined for the immobilization reaction. Protein immobilization yield ranged from 52% to 60%. Including carminomycin during immobilization had a positive effect on the activity of the immobilized enzyme but a strongly negative effect on the coupling efficiency. The immobilized enzyme retained at least 57% of its maximum activity after storage at 4 degrees C for more than 4 months. The properties of the free and immobilized enzyme were compared to determine whether immobilization could alter enzyme activity. Both soluble and bound enzyme exhibited the same pH profile with an optimum near 8.0. Immobilization caused an approximately 50% decrease in the apparent K(m) (K'(m)) for carminomycin while the K'(m) for S-adenosyl-L-methionine was approximately doubled. A 57% decrease in the V(max) value occurred upon immobilization. These changes are discussed in terms of active site modifications as a consequence of the enzyme immobilization. This system has a potential use in bioreactors for improving the conversion of carminomycin to daunorubicin. (c) 1995 John Wiley & Sons, Inc.     

Biosilicification of dual-fusion enzyme immobilized on magnetic nanoparticle

Rapid recovery, immobilization, and silica encapsulation of a dual-fusion enzyme was achieved by using iminodiacetic acid (IDA) modified magnetic nanoparticle as a carrier. D-amino acid oxidase (DAAO) of Rhodosporidium toruloides was used as a model enzyme in which a silica-precipitating peptide R5 and a metal ion complexing peptide (His)(6) were fused to its N- and C-terminal, respectively. After charging the magnetic particle with Cu(2+), the dual-fusion DAAO of 0.43 g could be directly recovered from the recombinant E. coli crude extract and immobilized on 1 g of the magnetic particle. Once in contact with hydrolyzed tetramethoxysilane (TMOS), the homogeneously dispersed immobilized dual-fusion DAAO was biosilicificated to form aggregates with size about 50 microm. The silica-encapsulated immobilized DAAO demonstrated a pyruvic acid production rate comparable with that of the naked immobilized DAAO in five repeated batch reactions when D-alanine was used as substrate. Furthermore, 85% of its activity remained after incubation at 60 degrees C for 1 h while the naked immobilized DAAO lost all its activity. This process provides the advantages that recombinant fusion enzyme can be directly recovered from crude extract, silica encapsulation protects the enzyme from leakage and denaturation, and the enzyme activity can be easily retrieved by applying a magnetic field.     

The sucked-flow analyser: A simple apparatus for automatic determination of steady-state enzyme kinetics

The sucked-flow analyser is a modified stopped-flow apparatus for automatic measurements of initial rates of enzyme reactions at varying concentrations of substrate or inhibitor. The flow through the system is driven by a water suction pump and regulated by magnetic valves. Sample and reagent solution are aspired through tubes whose resistances to laminar flow determine a precise ratio of mixing in the observation cell. A minicomputer controls all operations, collects the data and calculates the results. Measurements on teh β-galactosidase and the cytochome P-450 reactions are presented.     

Enzymatic synthesis of chiral amino‐alcohols by coupling transketolase and transaminase‐catalyzed reactions in a cascading continuous‐flow microreactor system

Rapid biocatalytic process development and intensification continues to be challenging with currently available methods. Chiral amino‐alcohols are of particular interest as they represent key industrial synthons for the production of complex molecules and optically pure pharmaceuticals. (2S,3R)‐2‐amino‐1,3,4‐butanetriol (ABT), a building block for the synthesis of protease inhibitors and detoxifying agents, can be synthesized from simple, non‐chiral starting materials, by coupling a transketolase‐ and a transaminase‐catalyzed reaction. However, until today, full conversion has not been shown and, typically, long reaction times are reported, making process modifications and improvement challenging. In this contribution, we present a novel microreactor‐based approach based on free enzymes, and we report for the first time full conversion of ABT in a coupled enzyme cascade for both batch and continuous‐flow systems. Using the compartmentalization of the reactions afforded by the microreactor cascade, we overcame inhibitory effects, increased the activity per unit volume, and optimized individual reaction conditions. The transketolase‐catalyzed reaction was completed in under 10 min with a volumetric activity of 3.25 U ml^?1. Following optimization of the transaminase‐catalyzed reaction, a volumetric activity of 10.8 U ml^?1 was attained which led to full conversion of the coupled reaction in 2 hr. The presented approach illustrates how continuous‐flow microreactors can be applied for the design and optimization of biocatalytic processes.

     

One-pot synthesis of amino-alcohols using a de-novo transketolase and beta-alanine: pyruvate transaminase pathway in Escherichia coli

Biocatalysis continues to emerge as a powerful technique for the efficient synthesis of optically pure pharmaceuticals that are difficult to access via conventional chemistry. The power of biocatalysis can be enhanced if two or more reactions can be achieved by a single whole cell biocatalyst containing a pathway designed de-novo to facilitate a required synthetic sequence. The enzymes transketolase (TK) and transaminase (TAm) respectively catalyze asymmetric carbon--carbon bond formation and amine group addition to suitable substrate molecules. The ability of a transaminase to accept the product of the transketolase reaction can allow the two catalysts to be employed in series to create chiral amino-alcohols from achiral substrates. As proof of principle, the beta-alanine: pyruvate aminotransferase (beta-A:P TAm) from Pseudomonas aeruginosa has been cloned, to create plasmid pQR428, for overexpression in E.coli strain BL21gold(DE3). Production of the beta-A:P TAm alongside the native transketolase (overexpressed from plasmid pQR411), in a single E.coli host, has created a novel biocatalyst capable of the synthesis of chiral amino alcohols via a synthetic two-step pathway. The feasibility of using the biocatalyst has been demonstrated by the formation of a single diastereoisomer of 2-amino-1,3,4-butanetriol (ABT) product, in up to 21% mol/mol yield, by the beta-A:P TAm, via transamination of L-erythrulose synthesized by TK, from achiral substrates glycolaldehyde (GA) and beta-hydroxypyruvate (beta-HPA). ABT synthesis was achieved in a one-pot process, using either whole cells of the dual plasmid strain or cell lysate, while the dual alcohol-amine functionality of ABT makes it an excellent synthon for many pharmaceutical syntheses.     

Evaluation of simple enzyme kinetics by response surface modelling

The activity of a horse liver alcohol dehydrogenase catalysed reduction of cyclohexanone was investigated by using a central composite circumscribed design in which two parameters (pH and cyclohexanone concentration) were varied. By log transformation of the substrate concentration an adequate model could be obtained from which reliable kinetic constants and pH profiles were determined.     

Comparison of two experimental methods for the determination of Michaelis-Menten kinetics of an immobilized enzyme

For the application of immobilized enzymes, the influence of immobilization on the activity of the enzyme should be Known. This influence can be obtained by determining the intrinsic kinetic parameters of the immobilized enzyme, and by comparing them with the kinetic parameters of the suspended enzyme. This article deals with the determination of the intrinsic kinetic parameters of an agarose-gel bead immobilized oxygen-consuming enzyme: L-lactate 2-monooxygenase. The reaction rate of the enzyme can be described by Michaelis-Menten kinetics. Batch conversion experiments using a biological oxygen monitor, as well as steady-state profile measurements within the biocatalyst particles using an oxygen microsensor, were performed. Two different mathematical methods were used for the batch conversion experiments, both assuming a pseudosteady-state situation with respect to the shape of the profile inside the bead. One of the methods used an approximate relation for the effectiveness factor for Michaelis-Menten kinetics which interpolates between the analytical solutions for zero- and first-order kinetics. The other mathematical method was based on a numerical solution and combined a mass balance over the reactor with a mass balance over the bead. The main difference in the application of the two methods is the computer calculation time; the completely numerical calculation procedure was about 20 times slower than the other calculation procedure.The intrinsic kinetic parameters resulting from both experimental methods were compared to check the reliability of the methods. There was no significant difference in the intrinsic kinetic parameters obtained from the two experimental methods. By comparison of the kinetic parameters for the suspended enzyme with the intrinsic kinetic parameters for the immobilized enzyme, it appeared that immobilization caused a decrease in the value of V(m) by a factor of 2, but there was no significant difference in the values obtained for K(m).     

Probing altered enzyme activity in the biochemical characterization of cancer

Enzymes have evolved to catalyze their precise reactions at the necessary rates, locations, and time to facilitate our development, to respond to a variety of insults and challenges, and to maintain a healthy, balanced state. Enzymes achieve this extraordinary feat through their unique kinetic parameters, myriad regulatory strategies, and their sensitivity to their surroundings, including substrate concentration and pH. The Cancer Genome Atlas (TCGA) highlights the extraordinary number of ways in which the finely tuned activities of enzymes can be disrupted, contributing to cancer development and progression often due to somatic and/or inherited genetic alterations. Rather than being limited to the domain of enzymologists, kinetic constants such as k_cat, K_m, and k_cat/K_m are highly informative parameters that can impact a cancer patient in tangible ways—these parameters can be used to sort tumor driver mutations from passenger mutations, to establish the pathways that cancer cells rely on to drive patients’ tumors, to evaluate the selectivity and efficacy of anti-cancer drugs, to identify mechanisms of resistance to treatment, and more. In this review, we will discuss how changes in enzyme activity, primarily through somatic mutation, can lead to altered kinetic parameters, new activities, or changes in conformation and oligomerization. We will also address how changes in the tumor microenvironment can affect enzymatic activity, and briefly describe how enzymology, when combined with additional powerful tools, and can provide us with tremendous insight into the chemical and molecular mechanisms of cancer.     

A spin label study of immobilized enzyme spectral subpopulations

Electron spin resonance (ESR) spin label studies have been carried out to examine the active site conformation of alpha-chymotrypsin before and after immobilization on two types of organic polymer supports: Amberlite XAD-8 and XAD-2. alpha-Chymotryspin was first chemically modified by reaction with methyl-4-phenylbutyrimidate and then inhibited by the active site spin label 4-(2,2,6,6-tetramethyl-piperdine-1-oxyl)-m-flurosulfonylbenzamide. In general, the ESR spectra of the active site lable revealed no significant changes in conformation for most of the enzyme before or after derivatization. On the other hand, two spectral subpopulations (A and B) of spin-labeled enzyme were characterized on the basis of their ESR spectra after immobilization on Amberlite XAD-8. Spectral subpopulation A (distinguished by a highly restrained spectrum) appeared to retain its active site structure and conformation and represented a large majority of the labeled chymotrypsin on the beads. Its presence correlated with the high activity and stability of phenylbutyramidinated chymotryspin on the Amberlite XAD-8 beads. Spectral subpopulation B (distinguished by a very weakly constrained spectrum) appeared to reflect loosely bound or denatured enzyme which was removable upon washing with 40% (v/v) ethylene glycol. Two methods for examining solvent accessibility to the active site lable of the kinetics of ascorbate reduction suggested that both spectral subpopulations had identical accessibilities to the bulk solvent. Paramagnetic broadening of the signal by K(3)Fe(CN)(6) revealed differences in the spin-spin broadening of the A and B components but is deemed and inappropriate indicator of solvent accessibility.