微生物酶催化腈水解反应的研究进展

腈的酶法水解不仅反应条件温各,而且高效性、高选择性,在光学活性羧酸及其衍生物的合成中具有巨大的应用潜力。综述了催化腈水解的酶类、反应类型、反应特性、影响反应的因素及其在工业上的应用前景。     

微生物酶催化腈水解反应的研究进展*

腈的酶法水解不仅反应条件温和 ,而且高效性、高选择性 ,在光学活性羧酸及其衍生物的合成中具有巨大的应用潜力。综述了催化腈水解的酶类、反应类型、反应特性、影响反应的因素及其在工业上的应用前景。     

腈水解酶在羧酸合成中的研究进展

有机羧酸是有机合成中的重要中间体,用腈水解酶催化有机腈实现有机羧酸的合成不仅具有反应条件温和、污染少和易处理等优点,而且更重要的是能实现一般化学法所不能达到的高度化学、区域和立体选择性。综述了腈水解酶的来源、特性和作用机理,介绍了腈水解酶在有机合成中的研究进展以及该酶在工业上的应用前景。     

微生物法生产丙烯酰胺的研究(Ⅰ)——腈水合酶产生菌株的培养和高活力的表达

研究了丙烯酰胺生产菌株的培养条件。通过对培养过程pH值调控、培养基补料以及诱导剂加入量的研究 ,使发酵液的腈水合酶的活力达到了 6567u mL菌液。这一酶活是国内外所见报道中最高的。进一步进行了丙烯腈的酶催化水合实验 ,产物中并没有发现副产物丙烯酸 ,说明在提高腈水合酶的同时 ,酰胺酶的活力并没有明显体现这一试验结果为工厂化生产改造以及新工艺的研究打下了基础.     

原核微生物腈转化酶研究进展

腈类物质的生物转化符合绿色化工的要求,具有重要应用潜力。系统阐述了产腈转化酶微生物多样性和生物转化特点。从菌株的分离、产酶及诱导物的种类;酶作用的底物、获得的产物;基因表达、酶的耐受性以及其应用价值等方面进行了比较全面的综述。     

精细化学品绿色合成用工具酶酌定向开发

近年来随着人们环境保护意识的增强以及对可持续发展技术的追求,使得以绿色环保为特色的生物催化技术越来越受到人们的关注。生物催化剂作为生物催化技术的工具,发挥着至关重要的核心作用。本文综述了生物催化技术发展各个阶段生物催化剂定向开发的最新进展。     

工业生物催化技术在绿色精细化学品合成中的机遇

生物催化技术具有反应条件温和、专一性强、环境友好等特点,是传统化学方法无法比拟的。本文从生物催化技术的特点及其在精细化学品合成中的应用现状出发,从产品战略、技术策略等方面提出生物催化技术应用的方向与目标：生物催化与有机合成技术的集成、交叉将会是未来生物催化技术发展的重要方向;有机合成技术为生物催化技术的发展、应用指明并提供了更多技术、产品及产业机会。     

腈类物降解菌多样性和产腈水合酶研究进展

腈水合酶催化反应在有机合成领域已有广泛的应用。作为一类重要的催化剂,腈水合酶可以将腈类物质转化为相应的酰胺。由于这种酶具有固有的立体和区域选择性,在精细化工领域已成为绿色、温和、对同分异构体具有选择性的催化剂。同时腈水合酶在生物修复和环境保护中也起着重要作用。综述了目前国内外腈水合酶的研究进展,包括降解腈类的微生物多样性、腈水合酶的催化特性、产腈水合酶菌株的改造以及腈水合酶相关基因的克隆与研究。对固定化酶和腈水合酶的应用也进行了叙述。     

融合双亲短肽提高腈水合酶的热稳定性研究

腈水合酶(Nitirle hydratase, NHase)催化腈类物质转化为酰胺类物质,目前用于工业生产丙烯酰胺。但在催化过程中释放的热量易导致酶分子失活。研究通过蛋白质融合技术对腈水合酶进行分子改造,提高热稳定性。将2种双亲自组装肽（self-assembling peptides, SAPs）EAK16和ELK16分别融合至恶臭假单胞菌Pseudomonas putida NRRL-18668来源NHase非催化亚基β的N末端,构建出2种融合型NHase：EAK16-NHase和ELK16-NHase。经过表达、纯化后测定酶活力,发现EAK16-NHase和ELK16 NHase的酶活力分别为(426±14) U/mg和(372±12) U/mg,保留野生型酶活力的97%和85%。在50 ℃条件下孵育0~60 min,每5 min取样后测定残存酶活力,EAK16-NHase和ELK16-NHase酶活力半衰期（T₅₀）分别为35 min和40 min,野生型NHase为20 min。说明融合EAK16和ELK16均能提高NHase的热稳定性。研究表明融合SAPs能在不显著影响酶活力的条件下提高酶的热稳定性。     

腈水合酶NHaseK在大肠杆菌中的功能表达

腈水合酶由α亚基和β亚基组成,活化元件对其功能表达至关重要,研究腈水合酶基因簇中各元件的表达比例对酶重组表达的影响具有重要意义。以来源于Klebsiella oxytoca KCTC 1686的腈水合酶（NHaseK）为研究对象,构建了多种表达策略,以期实现α亚基、β亚基和活化元件17k差异表达。利用pETDuet-1质粒具有双T7启动子的特点,将上述基因以八种不同的组合方式分别插入于两个启动子之后。当将三段基因同时插入于第一个启动子之后时,亚基表达量均衡,比活力为0.78 U/mg蛋白,是亚基表达量比例为5：3时的124%。在此基础上,在第二个启动子之后插入活化元件基因,活化元件表达水平提升2倍,比活提升5%,为0.82 U/mg蛋白。当将α亚基和β亚基插入于不同启动子之后时,酶活仅为对照组的10%,说明NHaseK的亚基必须同时转录才可形成成熟蛋白。进一步考察质粒拷贝数对大肠杆菌表达NHaseK的影响,确定15~20的质粒拷贝数足够实现NHaseK的功能表达。结果表明,亚基的均衡表达以及活化元件的充分表达对NHaseK的重组表达具有积极作用。     

Enzymatic hydrolysis of nitriles by an engineered nitrile hydratase (papain Gln19Glu) in aqueous-organic media

A mutant of the cysteine protease papain, displaying nitrile hydratase and amidase activities, was expressed in Pichia pastoris and used for the hydrolysis of peptide nitriles in aqueous-organic media. The rate of hydrolysis of these nitriles is lowered by increasing acetone concentration. This is caused by an increase of the Michaelis constant, and a variation of Vmax proportional to the amount of water in the mixture. The hydrolysis of the amide is less affected by the increase in co-solvent, which results in lower accumulation of this intermediate product. With the peptide nitrile tested, high nitrile concentrations could be used to promote the production of the amide and prevent its hydrolysis to the acid by diminishing the relative rate of amide hydrolysis. A number of non-peptidyl nitriles were also tested as potential substrates but activity was detected for only one compound with structural resemblance to peptide nitriles.     

Nitrile Hydratase and Its Application to Industrial Production of Acrylamide

Nitrile hydratase (NHase) was discovered in our laboratory. This enzyme was purified and characterized from various microorganisms. NHases are roughly classified into two groups according to the metal involved: Fe-type and Co-type. NHases are expected to have great potential as catalysts in organic chemical processing because they can convert nitriles to the corresponding higher-value amides under mild conditions. We have used microbial enzymes for the production of useful compounds; NHase has been used for the industrial production (production capacity: 30,000 tons/year) of acrylamide from acrylonitrile. This is the first successful example of a biotransformation process for the manufacture of a commodity chemical. This review summarizes the history of NHase studied not only from a basic standpoint but also from an applied point of view.     

Enzymatic hydrolysis of cyanohydrins with recombinant nitrile hydratase and amidase from hodococcus erythropolis

Nitrile hydratase and amidase from Rhodococcus erythropolis CIMB11540 were both cloned and expressed in Escherichia coli.Crude cell free extracts were used for the hydrolysis of different aromatic cyanohydrins. Nitrile hydratase expression was increased up to 5-fold by redesign of the expression cassette. The recombinant enzymes were successfully used for the conversion of several cyanohydrins to the corresponding α-hydroxy amides and acids while retaining enantiopurity.     

Nitrile-converting enzymes as a tool to improve biocatalysis in organic synthesis: recent insights and promises

Nitrile-converting enzymes, including nitrilase and nitrile hydratase (NHase), have received increasing attention from researchers of industrial biocatalysis because of their critical role as a tool in organic synthesis of carboxylic acids and amides from nitriles. To date, these bioconversion approaches are considered as one of the most potential industrial processes using resting cells or purified enzymes as catalysts for production of food additives, pharmaceutical, and agrochemical precursors. This review focuses on the distribution and catalytic mechanism research of nitrile-converting enzymes in recent years. Molecular biology aspects to improve the biocatalytic performance of microbial nitrilase and NHase are demonstrated. The process developments of microbial nitrilase and NHase for organic synthesis are also discussed.     

Transformation of the herbicide 2,6-dichlorobenzonitrile to the persistent metabolite 2,6-dichlorobenzamide (BAM) by soil bacteria known to harbour nitrile hydratase or nitrilase

In soil the herbicide 2,6-dichlorobenzonitrile (dichlobenil) is degraded to the persistent metabolite 2,6-dichlorobenzamide (BAM) which has been detected in 19% of samples taken from Danish groundwater. We tested if common soil bacteria harbouring nitrile-degrading enzymes, nitrile hydratases or nitrilases, were able to degrade dichlobenil in vitro. We showed that several strains degraded dichlobenil stoichiometrically to BAM in 1.5–6.0 days; formation of the amide intermediate thus showed nitrile hydratase rather than nitrilase activity, which would result in formation of 2,6-dichlorobenzoic acid. The non-halogenated␣analogue benzonitrile was also degraded, but here the benzamide intermediate accumulated only transiently showing nitrile hydratase followed by amidase activity. We conclude that a potential for dichlobenil degradation to BAM is found commonly in soil bacteria, whereas further degradation of the BAM intermediate could not be demonstrated.     

对羟基苯乙腈水解酶产生菌的筛选及产酶条件研究

以对羟基苯乙腈为唯一氮源,从土壤中筛选到5株腈水解酶产生菌。在初筛的过程中,用薄板层析（TLC）对细胞转化液定性检测,高效液相色谱法（HPLC）定量分析。考察了培养时间、不同碳源对细胞酶产量的影响及在反应过程中金属离子对细胞酶产量的影响,实验结果发现,培养时间在70h左右时细胞酶产量最高,以葡萄糖为碳源质量,质量浓度在12．5～15g／L附近酶产量最高。Cu^2＋对细胞酶活力具有非常强烈的抑制作用,Co^2＋、Hg^2＋、Ni^2＋等对细胞酶活力具有明显的抑制,而Ba^2＋、Mg^2＋具有较明显的促进作用,但在高浓度下促进作用减弱。     

Comparative characterisation of two Rhodococcus species as potential biocatalysts for ammonium acrylate production

Two Rhodococcal isolates, one possessing a nitrile hydratase and an amidase enzyme, the other an aliphatic nitrilase enzyme have been isolated. The kinetic constants for the enzymes in each isolate have been determined. This data coupled with stability tests indicate that Rhodococcus ruber NCIMB 40757, the nitrilase containing organism, should be an excellent biocatalyst for the commercial production of ammonium acrylate. This is confirmed by a fed-batch bioconversion to produce 5.7 M ammonium acrylate.     

微生物法生产丙烯酰胺的研究（Ⅰ）—腈水合酶产生菌株的培养和高活力的表达

研究了丙烯酰胺生产菌株的培养条件。通过对培养过程pH值调控、培养基补料以及诱导剂加入量的研究,使发酵液的腈水合酶的活力达到了6567u/mL菌液。这一酶活是国内外所见报道中最高的。进一步进行了丙烯腈的酶催化水合实验,产物中并没有发现副产物丙烯酸,说明在提高腈水合酶的同时,酰胺酶的活力并没有明显体现这一试验结果为工厂化生产改造以及新工艺的研究打下了基础。     

新型工业酶制剂的进步对生物化学品工业生产过程的影响

工业酶制剂对化工和生物化工产品生产的影响有两方面,一是直接催化生产,另一方面是辅助发酵菌进行生产。以下简单回顾了酶制剂在工业领域的应用现状,注重讨论酶制剂在淀粉糖等方面的直接催化应用和在酒精发酵生产等方面的辅助作用,分析新型酶制剂对生产过程的影响及带来的益处,促进行业的可持续发展。     

Sizing of theRhodococcus sp. R312 genome by pulsed-field gel electrophoresis. Localization of genes involved in nitrile degradation

The two restriction enzymesAsnI andDraI were found to produce DNA fragment sizes that could be used for mapping theRhodococcus sp. R312 (formerlyBrevibacterium sp. R312) genome by pulsed-field gel electrophoresis.AsnI produced 24 fragments (4 to 727 kb) andDraI yielded 15 fragments (8.5 to 2400 kb). The fragment lengths in each digest were summed, indicating that the size of the chromosome ranged from 6.31 to 6.56 Mb, with a mean of 6.44 Mb. In addition, the wide-spectrum amidase gene (amiE) and the operon containing the enantiomer-selective amidase gene (amdA) and the nitrile hydratase structural gene (nthA, nthB) were localized on theAsnI andDraI fragments.