Assembly of graphene oxide‐formate dehydrogenase composites by nickel‐coordination with enhanced stability and reusability

Featuring unique planar structure, large surface area and biocompatibility, graphene oxide (GO) has been widely taken as an ideal scaffold for the immobilization of various enzymes. In this regard, nickel‐coordinated graphene oxide composites (GO‐Ni) were prepared as novel supporters for the immobilization of formate dehydrogenase. The catalytic activity, stability and morphology were studied. Compared with GO, the enzyme loading capacity of GO‐Ni was enhanced by 5.2‐fold, besides the immobilized enzyme GO‐Ni‐FDH exhibited better thermostability, storage stability and reuse stability than GO‐FDH. GO‐Ni‐FDH retained 40.9% of its initial activity after 3 h at 60°C, and retained 31.4% of its initial relative activity after 20 days’ storage at 4°C. After eight times usages, GO‐Ni‐FDH maintained 63.8% of its initial activity. Mechanism insights of the multiple interactions of enzyme with the GO‐Ni were studied, considering coordination bonds, hydrogen bonds, electrostatic forces, coordination bonds, and etc. A practical and simple immobilization strategy by metal ions coordination for multimeric dehydrogenase was developed.     

Selective oxidation and reduction reactions with cofactor regeneration mediated by galactitol-, lactate-, and formate dehydrogenases immobilized on magnetic nanoparticles

Rapid immobilization with the one-pot purification of galactitol dehydrogenase (GatDH) and formate dehydrogenase (FDH) is achieved by using iminodiacetic acid (IDA) with chelated Co²⁺ modified magnetic nanoparticles as a carrier. Lactate dehydrogenase (LDH) from recombinant Escherichia coli and FDH commencing Candida methylica were used as an auxiliary enzyme for the regeneration of NADH/NAD⁺ with a representative synthesis of (S)-1,2-propanediol and l-tagatose starting from hydroxyacetone and galactitol. The affinity magnetic nanoparticles were characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR), while the purity of GatDH and FDH was assayed by SDS-PAGE analysis. The immobilized two-enzyme system, reflecting the pH dependence of its constituent enzymes, showed optimal activity at pH 7 and 8 for (S)-1,2-propanediol and l-tagatose production, respectively. The immobilized enzyme system retained up to 70% of its activity after one week of repeated use. The use of affinity magnetic nanoparticles offers the advantage of a one-pot purification of His(6)-tagged GatDH and FDH followed by the production of rare sugar and chiral diol.     

磁性纳米颗粒固定化乙醇脱氢酶的研究

本研究采用3-丙氨基三乙氧基硅烷(APTES)和戊二醛修饰包裹有SiO2磁性Fe3O4纳米颗粒表面,将其作为固定化载体固定化乙醇脱氢酶,研究固定化条件对固定化效率的影响,并对固定化酶性质进行分析。研究发现,当Fe3O4@SiO2纳米颗粒修饰上氨基和醛基后依然具有良好的水分散性和胶体稳定性,适合作为固定化载体。通过单因素优化,发现当最适给酶量为11. 3U/100 mg,搅拌转速为150 r/min,固定化p H和固定化温度分别控制在6. 5和5℃～15℃,固定化时长为45 min时,具有较好的固定化效果,固定化率可达到60. 2%。在此条件下制备得到的固定化酶与游离酶相比,固定化酶具有良好的耐高温和耐碱性。所得固定化乙醇脱氢酶在连续使用8次后,固定化率仍保留在57%左右,表明该固定化酶具有较好的操作稳定性,可为连续生产NADH提供技术依据。     

CO2生物转化关键酶固定体系构筑研究进展与挑战

酶催化CO₂还原制备高值化学品对缓解全球环境和能源危机具有重要意义,利用甲酸脱氢酶(formate dehydrogenase,FDH)或多酶级联还原CO₂制备甲酸/甲醇具有选择性高、条件温和的优势,但关键酶活性低、稳定性差和重复利用率低的问题限制了其规模化应用,酶的固定化为这些问题提供了有效解决方案。本文总结了近年来利用膜、无机材料、金属有机框架和共价有机框架等载体对酶进行固定化的研究进展,阐释了不同固定材料和固定方式的特点和优势;进一步总结了固定化酶与电催化或光催化耦联反应体系对CO₂还原的协同效果及应用,同时指出酶固定化技术和耦联反应体系目前存在的问题并对其发展前景进行了展望。     

Engineering bi‐functional enzyme complex of formate dehydrogenase and leucine dehydrogenase by peptide linker mediated fusion for accelerating cofactor regeneration

This study reports the application of peptide linker in the construction of bi‐functional formate dehydrogenase (FDH) and leucine dehydrogenase (LeuDH) enzymatic complex for efficient cofactor regeneration and L‐tert leucine (L‐tle) biotransformation. Seven FDH‐LeuDH fusion enzymes with different peptide linker were successfully developed and displayed both parental enzyme activities. The incorporation order of FDH and LeuDH was investigated by predicting three‐dimensional structures of LeuDH‐FDH and FDH‐LeuDH models using the I‐TASSER server. The enzymatic characterization showed that insertion of rigid peptide linker obtained better activity and thermal stability in comparison with flexible peptide linker. The production rate of fusion enzymatic complex with suitable flexible peptide linker was increased by 1.2 times compared with free enzyme mixture. Moreover, structural analysis of FDH and LeuDH suggested the secondary structure of the N‐, C‐terminal domain and their relative positions to functional domains was also greatly relevant to the catalytic properties of the fusion enzymatic complex. The results show that rigid peptide linker could ensure the independent folding of moieties and stabilized enzyme structure, while the flexible peptide linker was likely to bring enzyme moieties in close proximity for superior cofactor channeling.     

纳米磁性壳聚糖微球固定化酵母醇脱氢酶的研究

建立了以纳米级磁性壳聚糖微球(magnetic chitosan microspheres , M-CS)为载体固定化酵母醇脱氢酶(yeast alcohol dehydrogenase,YADH)的方法,优化了YADH的固定化条件,考察了固定化酶的性质。结果表明,M-CS 呈规则的圆球形,粒径在30nm 左右,具有较好的磁响应性。酵母醇脱氢酶固定化适宜条件为:50 mg 磁性壳聚糖微球,加入20mL 0.25 mg/mL 酵母醇脱氢酶(蛋白质含量)磷酸盐缓冲液(0.05 mol/L ,pH 7.0) ,在4 ℃固定2h。M-CS 容易吸附酵母醇脱氢酶,但吸附的酶量受载体与酶的比例、溶液的离子浓度、溶液pH的影响明显,而温度对吸附的酶量的影响则相对较弱。相对于游离的酵母醇脱氢酶,固定化酶的最适温度略有升高,可明显改善其热稳定性、酸碱稳定性、操作稳定性和贮存稳定性。     

Improvement in thermal stability of l-fucose dehydrogenase by chemical modification and additives for use as an NADPH recycling reagent

Chemical modification and addition of stabilizers were evaluated to stabilize l-fucose dehydrogenase (FDH) in mild alkaline which is required for NADPH recycling used in a glutamate dehydrogenase (GDH)-catalyzed elimination of ammonia. FDH lost all its activity at pH 9 after 10 min at 50 °C, while FDH conjugated with dextran retained about 30% activity. EDTA or citric acid, at 50–100 mM, increased the thermal stability of FDH with retention of more than 70–80% activity after heat-treatment. FDH was then functional for NADPH regeneration.     

Immobilization Increases the Stability and Reusability of Pigeon Pea NADP<Superscript>+</Superscript> Linked Glucose-6-Phosphate Dehydrogenase

Immobilization of enzymes is valuably important as it improves the stability and hence increases the reusability of enzymes. The present investigation is an attempt for immobilization of purified glucose-6-phosphate dehydrogenase from pigeon pea on different matrix. Maximum immobilization was achieved when alginate was used as immobilization matrix. As compared to soluble enzyme the alginate immobilized enzyme exhibited enhanced optimum pH and temperature. The alginate immobilized enzyme displayed more than 80% activity up to 7 continuous reactions and more than 50% activity up to 11 continuous reactions.     

甲酸脱氢酶及其在手性生物制造中的应用

氧化还原生物合成体系在绿色生物制造手性化合物中具有重要应用价值.甲酸脱氢酶(formate dehydrogenase,FDH)能氧化甲酸盐生成二氧化碳,同时将NAD(P)+还原为NAD(P)H,是氧化还原生物合成中辅酶再生体系的关键酶.但天然的FDH催化效率低、稳定性差、辅酶利用率不高等缺点制约了其在工业生产中的应用...     

Immunological evidence for two types of PQQ-dependent d-glucose dehydrogenase in bacterial membranes and the location of the enzyme in Escherichia coli

Abstract Using an antibody raised against d -glucose dehydrogenase (EC 1.1.99.17) purified from Pseudomonas fluorescens , immuno-cross-reactivity with the enzymes from several bacterial strains and localization of the enzyme in Escherichia coli were examined. The antibody cross-reacted with glucose dehydrogenases from various Gram-negative bacteria examined. As a result, it became apparent that the enzymes from Gluconobacter, Acetobacter, Pseudomonas and Acinetobacter , which existed as holoenzymes in the membranes, had lower molecular weights than those from E. coli and Klebsiella , which were apoenzymes.
Treatment with trypsin of right-side out and inside-out membrane vesicles from E. coli clearly demonstrated that d -glucose dehydrogenase was located on the outer surface of the cytoplasmic membrane of E. coli , as had been suggested for Pseudomonas .     

Immobilization of glucose oxidase and lactate dehydrogenase onto magnetic nanoparticles for bioprocess monitoring system

Glucose oxidase (GOD) and lactate dehydrogenase (LDH) were immobilized onto magnetic nanoparticles, viz. Fe₃O₄, via carbodiimide and glutaraldehyde. The immobilization efficiency was largely dependent upon the immobilization time and concentration of glutaraldehyde. The magnetic nanoparticles had a mean diameter of 9.3 nm and were superparamagnetic. The immobilization of GOD and LDH on the nanoparticles slightly decreased their saturation magnetization. However, the FT-IR spectra showed that GOD and LDH were immobilized onto the nanoparticles by different binding mechanisms, the reason for which was not well explained. The optimum pH values of the immobilized GOD and LDH were changed to 8 and 10, respectively. The free and immobilized enzyme kinetic parameters (K_m and V_max) were determined by Michaelis-Menten enzyme kinetics. The Km values for free and immobilized GOD were 0.168 and 0.324 mM, respectively, while those for free and immobilized LDH were 0.19 and 0.163 mM for NAD, and 2.976 and 4.785 mM for lactate, respectively. High operational stability was observed, with more than 80% of the initial enzyme activity being retained for the immobilized GOD up to 12 h and for the immobilized LDH up to 24 h. The immobilized GOD was applied to a sequential injection analysis system for the application of bioprocess monitoring.     

甲醛氧化途径关键酶重组蛋白的生化特性及固定化酶吸收甲醛效果研究

甲醛脱氢酶(formaldehyde dehydrogenase,ADH)与甲酸脱氢酶(formate dehydrogenase,FDH)是甲醛氧化途径的两个关键酶.恶臭假单胞菌(Pseudomonas putida)的PADH是一种不依赖谷胱甘肽可以把游离甲醛直接氧化为甲酸的脱氢酶,博伊丁假丝酵母菌(Candida boidinii)的FDH在有NAD+存在时可以把甲酸氧化为二氧化碳.以基因组DNA为模板用PCR方法,从P.putida中扩增出PADH基因的编码区(padh),从C.boidinii中扩增出FDH的编码区(fdh),然后亚克隆到pET-28a(+)中分别构建这两个基因的原核表达载体pET-28a-padh和pET-28a-fdh,转化大肠杆菌,利用IPTG诱导重组蛋白PADH和FDH的表达.通过优化条件使重组蛋白的表达量占菌体总蛋白的70%以上,通过亲和层析法纯化出可溶性PADH和FDH重组蛋白.对重组蛋白的生化特性分析结果表明:PADH在最适反应温度50℃的活性为1.95 U/mg;FDH在最适反应温度40℃的活性为0.376 U/mg.所表达的重组蛋白与之前报道过的相比,具有更好的热稳定性和更广的温度适应范围.将PADH、FDH两个重组蛋白及辅因子NAD+固定到聚丙烯酰胺载体基质上,对固定化酶甲醛吸收效果的初步分析结果显示固定化酶对空气中的甲醛有一定的吸收效果,说明这两种酶被固定后具有开发成治理甲醛污染环保产品的潜力.     

Efficient immobilization technique for enhancement of cellobiose dehydrogenase activity on silica gel

In this study, cellobiose dehydrogenase (CDH) of Phanerochaete chrysosporium ATCC 32629 was immobilized on silica gel for the further application of CDH in the saccharification process of biomass. To prevent the loss of enzyme activity during enzyme immobilization, the pretreatment of CDH was performed by various pretreatment materials before immobilization. When pretreated enzymes were used in immobilization, the activities of immobilized CDH were higher than non-pretreated CDH even in same amounts of immobilized protein. The specific activity of pretreated immobilized CDH with lactose was about two times higher than that of non-pretreated immobilized CDH. Moreover, the pretreated immobilized CDH showed better reusability than non-pretreated immobilized CDH, with 67.3% of its original activity being retained after 9 reuses.     

Affinity and stability modifications of immobilized alcohol dehydrogenase through multipoint copolymerization

Yeast alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1), a potentially useful enzyme for cofactor regeneration processes, was covalently immobilized in a multipoint fashion by activation with acryloyl chloride and subsequent copolymerization in a polyacrylamide gel. Several properties such as the activity and stability were systematically studied for the free enzyme, the acryloate-enzyme and the immobilized enzyme. The activation energy was significantly lowered upon immobilization. The thermal stability of the immobilized enzyme was, however, greatly increased. But its maximum activity was observed at a lower temperature. These results suggest an important effect of the diffusional restrictions and of the mode of activation and immobilization on the activity and the stability of the enzyme.     

Kinetics and stability of immobilized chicken liver xanthine dehydrogenase.

Xanthine dehydrogenase (EC 1.2.1.37) was isolated from chicken livers and immobilized by adsorption to a Sepharose derivative, prepared by reaction of n-octylamine with CNBr-activated Sepharose 4B. Using a crude preparation of enzyme for immobilization it was observed that relatively more activity was adsorbed than protein, but the yield of immobilized activity increased as a purer enzyme preparation was used. As more activity and protein were bound, relatively less immobilized activity was recovered. This effect was probably due to blocking of active xanthine dehydrogenase by protein impurities. The kinetics of free and immobilized xanthine dehydrogenase were studied in the pH range 7.5-9.1. The Km and V values estimated for free xanthine dehydrogenase increase as the pH increase; the K'm and V values for the immobilized enzyme go through a minimum at pH 8.1. By varying the amount of enzyme activity bound per unit volume of gel, it was shown that K'm is larger than Km are result of substrate diffusion limitation in the pores of the support material. Both free and immobilized xanthine dehydrogenase showed substrate activation at low concentrations (up to 2 microM xanthine). Immobilized xanthine dehydrogenase was more stable than the free enzyme during storage in the temperature range of 4-50 degrees C. The operational stability of immobilized xanthine dehydrogenase at 30 degrees C was two orders of magnitude smaller than the storage stability, t 1/2 was 9 and 800 hr, respectively. The operational stability was, however, better than than of immobilized milk xanthine oxidase (t 1/2 = 1 hr). In addition, the amount of product formed per unit initial activity in one half-life, was higher for immobilized xanthine dehydrogenase than for immobilized xanthine oxidase. Unless immobilized milk xanthine oxidase can be considerable stabilized, immobilized chicken liver xanthine dehydrogenase is more promising for application in organic synthesis.     

Aspartate 142 is involved in both hydrolase and dehydrogenase catalytic centers of 10-formyltetrahydrofolate dehydrogenase

The enzyme 10-formyltetrahydrofolate dehydrogenase (FDH) catalyzes conversion of 10-formyltetrahydrofolate to tetrahydrofolate in either a dehydrogenase or hydrolase reaction. The hydrolase reaction occurs in a 310-residue amino-terminal domain of FDH (N(t)-FDH), whereas the dehydrogenase reaction requires the full-length enzyme. N(t)-FDH shares some sequence identity with several 10-formyltetrahydrofolate-utilizing enzymes. All these enzymes have a strictly conserved aspartate, which is Asp(142) in the case of N(t)-FDH. Replacement of the aspartate with alanine, asparagine, glutamate, or glutamine in N(t)-FDH resulted in complete loss of hydrolase activity. All the mutants, however, were able to bind folate, although with lower affinity than wild-type N(t)-FDH. Six other aspartate residues located near the conserved Asp(142) were substituted with an alanine, and these substitutions did not result in any significant changes in the hydrolase activity. The expressed D142A mutant of the full-length enzyme completely lost both hydrolase and dehydrogenase activities. This study shows that Asp(142) is an essential residue in the enzyme mechanism for both the hydrolase and dehydrogenase reactions of FDH, suggesting that either the two catalytic centers of FDH are overlapped or the dehydrogenase reaction occurs within the hydrolase catalytic center.     

Increased activity of glucose dehydrogenase co-immobilized with a redox mediator in a bioreactor with electrochemical NAD+ regeneration

An electrochemical bioreactor with glucose dehydrogenase immobilized on to the electrode surface produced gluconic acid from glucose with concomitant recycling of the NAD⁺ coenzyme at 0.7 V. Since the enzyme is deactivated during operation at this redox potential, co-immobilization of 3,4-dihydroxybenzaldehyde as mediator allowed the system to operate at 0.2 V and increased both the activity (2.4-times) and the stability of the immobilized enzyme (2.2-times). The different effective electrochemical surfaces resulting from the different mediator immobilization modes are important in determining these three properties.     

Coupled reactions on bioparticles: Stereoselective reduction with cofactor regeneration on PhaC inclusion bodies

Chiral alcohols are important building blocks for specialty chemicals and pharmaceuticals. The production of chiral alcohols from ketones can be carried out stereo selectively with alcohol dehydrogenases (ADHs). To establish a process for cost‐effective enzyme immobilization on solid phase for application in ketone reduction, we used an established enzyme pair consisting of ADH from Rhodococcus erythropolis and formate dehydrogenase (FDH) from Candida boidinii for NADH cofactor regeneration and co‐immobilized them on modified poly‐p‐hydroxybutyrate synthase (PhaC)‐inclusion bodies that were recombinantly produced in Escherichia coli cells. After separate production of genetically engineered and recombinantly produced enzymes and particles, cell lysates were combined and enzymes endowed with a Kcoil were captured on the surface of the Ecoil presenting particles due to coiled‐coil interaction. Enzyme‐loaded particles could be easily purified by centrifugation. Total conversion of 4'‐chloroacetophenone to (S)‐4‐chloro‐α‐methylbenzyl alcohol could be accomplished using enzyme‐loaded particles, catalytic amounts of NAD⁺ and formate as substrates for FDH. Chiral GC‐MS analysis revealed that immobilized ADH retained enantioselectivity with 99 % enantiomeric excess. In conclusion, this strategy may become a cost‐effective alternative to coupled reactions using purified enzymes.     

On the role of conserved histidine 106 in 10-formyltetrahydrofolate dehydrogenase catalysis: connection between hydrolase and dehydrogenase mechanisms

The enzyme, 10-formyltetrahydrofolate dehydrogenase (FDH), converts 10-formyltetrahydrofolate (10-formyl-THF) to tetrahydrofolate in an NADP(+)-dependent dehydrogenase reaction or an NADP(+)-independent hydrolase reaction. The hydrolase reaction occurs in a 310-amino acid long amino-terminal domain of FDH (N(t)-FDH), whereas the dehydrogenase reaction requires the full-length enzyme. The amino-terminal domain of FDH shares some sequence identity with several other enzymes utilizing 10-formyl-THF as a substrate. These enzymes have two strictly conserved residues, aspartate and histidine, in the putative catalytic center. We have shown recently that the conserved aspartate is involved in FDH catalysis. In the present work we studied the role of the conserved histidine, His(106), in FDH function. Site-directed mutagenesis experiments showed that replacement of the histidine with alanine, asparagine, aspartate, glutamate, glutamine, or arginine in N(t)-FDH resulted in expression of insoluble proteins. Replacement of the histidine with another positively charged residue, lysine, produced a soluble mutant with no hydrolase activity. The insoluble mutants refolded from inclusion bodies adopted a conformation inherent to the wild-type N(t)-FDH, but they did not exhibit any hydrolase activity. Substitution of alanine for three non-conserved histidines located close to the conserved one did not reveal any significant changes in the hydrolase activity of N(t)-FDH. Expressed full-length FDH with the substitution of lysine for the His(106) completely lost both the hydrolase and dehydrogenase activities. Thus, our study showed that His(106), besides being an important structural residue, is also directly involved in both the hydrolase and dehydrogenase mechanisms of FDH. Modeling of the putative hydrolase catalytic center/folate-binding site suggested that the catalytic residues, aspartate and histidine, are unlikely to be adjacent to the catalytic cysteine in the aldehyde dehydrogenase catalytic center. We hypothesize that 10-formyl-THF dehydrogenase reaction is not an independent reaction but is a combination of hydrolase and aldehyde dehydrogenase reactions.     

Immobilization of formate dehydrogenase on polyethylenimine‐grafted graphene oxide with kinetics and stability study

Graphene oxide‐based nanomaterials are promising for enzyme immobilization due to the possibilities of functionalizing surface. Polyethylenimine‐grafted graphene oxide was constructed as a novel scaffold for immobilization of formate dehydrogenase. Compared with free formate dehydrogenase and graphene oxide adsorbed formate dehydrogenase, thermostability, storage stability, and reusability of polyethylenimine‐grafted graphene oxide‐formate dehydrogenase were enhanced. Typically, polyethylenimine‐grafted graphene oxide‐formate dehydrogenase remained 47.4% activity after eight times’ repeat reaction. The immobilized capacity of the polyethylenimine‐grafted graphene oxide was 2.4‐folds of that of graphene oxide. Morphological and functional analysis of polyethylenimine‐grafted graphene oxide‐formate dehydrogenase was performed and the assembling mechanism based on multi‐level interactions was studied. Consequently, this practical and facile strategy will likely find applications in biosynthesis, biosensing, and biomedical engineering.