Optimization of Culture Conditions of Brevibacterium SP. A4 for the Production of Nitrile Hydratase

Optimum culture conditions of Brevibacterium sp. A4 for production of nitrile hydratase were determined by two mathematical methods: the Hadamard method and graphic analysis of response areas. A minimal medium was optimized and the basic roles of Fe²⁺ and Mg²⁺ were clearly shown. The influence of physico-chemical factors (pH, temperature and light conditions) on the culture and on nitrile hydratase were also studied. Various results permit the production of Brevibacterium sp. A4 cells with low protease and high nitrile hydratase contents.     

耐底物腈水合酶融合子的产酶条件优化

采用正交设计法对耐底物腈水合酶融合子的发酵条件进行优化,以发酵液起始pH,发酵周期,接种量,装料系数作为考察因素,最终确定最佳发酵条件为:起始pH8.0、发酵周期54h、接种量12％、装液系数12％.在此优化条件下融合子腈水合酶的活力达到1100万U/ml,较优化前提高了83.3％.通过响应面法对发酵培养基配方进行优化研究,采用Plackett-Burman法对8个因素进行了筛选,结果表明,葡萄糖、尿素、磷酸氢二钾、磷酸二氢钾是影响发酵液腈水合酶产量的主效应因子.用最陡爬坡试验及Central composite design设计进一步优化,利用Design-Expert软件进行二次回归分析,得到各因素的最佳浓度为:葡萄糖22.62g/L、尿素9.76g/L、K2HP04 1.22g/L、KH2PO41.268g/L.在此培养基优化配方下融合子腈水合酶的活力达到1280万U/ml,较原配方的酶活提高了16.4％.     

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。     

一株产丙烯腈水合酶菌株的研究

从山东省泰山地区土壤中分离到一株能产生丙烯腈水合酶的微生物菌株８６－１６３,经分类学特征鉴定可归属于诺卡氏菌属,其培养特征、生理生化特征及产酶活性等方面与已报导的诺卡氏菌有差别,暂定名为Ｎｏｃａｒｄｉａｓｐ．８６－１６３。     

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。     

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.     

产腈水合酶重组大肠杆菌的质粒稳定性研究

成功构建了腈水合酶(nitrile hydratase,NHase)高表达的重组大肠杆菌E.coliBL21(DE3)/pETNH^M(Kan^r),研究了重组质粒pETNH^M在重组菌株中的质粒稳定性。结果表明,pETNH^M具有较好的结构稳定性,连续传代60代后质粒的基因序列没有明显缺失,且能够正常表达腈水合酶。pETNH^M具有分离不稳定性,在无抗生素选择压力下,连续传代48代后质粒丢失的无质粒细胞开始出现。琼脂糖凝胶电泳定量分析表明,2/3的质粒pETNH^M以二聚体形式存在,导致质粒拷贝数的下降。进一步研究表明,重组细胞的连续高速分裂及腈水合酶的高表达也会造成质粒拷贝数的下降,从而降低其分离稳定性。反之,重组菌株相对于宿主菌株的较高比生长速率有利于保持含质粒细胞的生长优势,卡那霉素的选择压力则能够保证质粒的稳定遗传。     

Rhodococcus ruber CGMCC3090腈水合酶纯化、酶学性质及结晶研究

Rhodococcus ruber CGMCC309菌株为酰胺酶及腈水解酶双重缺陷菌株,研究表明该菌能产宽泛底物特异性的腈水合酶。对该菌株产生的新型腈水合酶(NHase-3090)进行纯化和结晶,并研究了其酶学性质。采用疏水、离子交换及凝胶过滤3种层析方法,使该酶纯化倍数达到17.14,得率高达26.2%。电泳分析表明,全酶分子量为105 kDa,由α(24.3 k Da)和β(28.0k Da)2个亚基组成,并构成α2β2四聚体。酶的最适p H和温度分别为7.5和30℃。该酶明显受不同金属离子影响。动力学研究表明,Km为178.8 m M;Vmax为209.1μmol/L·min·mg。研究发现3种金属离子Zn~(2+),CO~(2+)和Cd~(2+)有利于酶蛋白结晶。结晶最佳条件是:采用112-34#试剂(0.05mol/L水合硫酸镉、0.1mol/L HEPS和1.0mol/L三水醋酸钠),蛋白质浓度为15 mg/ml,结晶温度为16℃,p H为7.5,结晶时间为30 d。腈水合酶蛋白单晶经X射线衍射,分辨率达到了3.7。该腈水合酶的纯化和结晶为进一步深入研究其结构和功能奠定了基础。     

Identification of an Active Site-bound Nitrile Hydratase Intermediate through Single Turnover Stopped-flow Spectroscopy

Stopped-flow kinetic data were obtained for the iron-type nitrile hydratase from Rhodococcus equi TG328-2 (ReNHase) using methacrylonitrile as the substrate. Multiple turnover experiments suggest a three-step kinetic model that allows for the reversible binding of substrate, the presence of an intermediate, and the formation of product. Microscopic rate constants determined from these data are in good agreement with steady state data confirming that the stopped-flow method used was appropriate for the reaction. Single turnover stopped-flow experiments were used to identify catalytic intermediates. These data were globally fit confirming a three-step kinetic model. Independent absorption spectra acquired between 0.005 and 0.5 s of the reaction reveal a significant increase in absorbance at 375, 460, and 550 nm along with the hypsochromic shift of an Fe³⁺←S ligand-to-metal charge transfer band from 700 to 650 nm. The observed UV-visible absorption bands for the Fe³⁺-nitrile intermediate species are similar to low spin Fe³⁺-enzyme and model complexes bound by NO or N₃⁻. These data provide spectroscopic evidence for the direct coordination of the nitrile substrate to the nitrile hydratase active site low spin Fe³⁺ center.     

腈水合酶基因克隆与调控表达的研究进展

微生物腈水合酶作为新型生物催化剂得到日益广泛的应用 ,但野生菌株本身存在的酶稳定性差等问题制约了这一绿色工艺的发展 ,基因工程菌为解决这个难题开辟了新的思路。总结了各种菌株中腈水合酶的序列研究进展 ,虽然基因序列和蛋白序列同源性不高 ,但它们都以基因簇的形式存在 ,并具有相同的活性中心序列。归纳了克隆并表达腈水合酶基因的基本步骤和方式 ,并提出几种有效增强重组腈水合酶活性表达的方法。     

DA-F127水凝胶包埋固定化含腈水合酶细胞

腈水合酶是一类可催化腈类化合物转化生成相应酰胺类物质的酶。含腈水合酶的游离细胞催化水合反应存在酶容易失活、细胞无法重复利用、分离纯化困难等缺陷,细胞固定化技术可有效解决这些问题。为探索合适的固定化方法,以含腈水合酶的重组E.coli细胞为研究对象,以固定化酶活回收率和批次反应情况为评价指标,筛选比较了几种常用的包埋固定化方法。结果表明,DA-F127水凝胶包埋固定化细胞不仅具有较高的酶活回收率,而且稳定性也很好。对该方法进行了固定化条件和操作稳定性优化,当DA-F127浓度为15%、UV光源距离为20cm、光照时间为6min、菌体含量为20mg/g 固定化细胞时,酶活回收率为89.74%,并且可以催化9批次150g/L的3-氰基吡啶完成转化,第九批次转化率可达98.26%。与游离细胞催化过程相比,单位质量游离细胞的烟酰胺产量提高了12倍,具有良好的工业应用前景。     

营养及环境因素对黄色短杆菌发酵生产L-亮氨酸的影响

目的:研究葡萄糖、玉米浆、蛋氨酸等主要营养物质以及环境因素对发酵生产L-亮氨酸的影响。方法:应用单因素实验确定发酵条件,采用高效液相色谱法测定产量。结果:在最优发酵条件下,以种龄36h、接种量为10%,摇瓶产L-亮氨酸的量可达19.4g/L,采用10L罐分批补料发酵64h可积累29.47g/L的亮氨酸。结论:营养及环境因素对发酵生产L-亮氨酸具有重要影响。     

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。     

Operational stability of Brevibacterium imperialis CBS 489-74 nitrile hydratase

Brevibacterium imperialis CBS 489-74 was grown in broths prepared with yeast and malt extract, bacteriological peptone and 2% glucose or differently modified with the addition of Na-phosphate buffer, FeSO₄, MgSO₄ and CoCl₂. The peak production of nitrile hydratase (NHase) did not change significantly. At the stationary growth phase, the units per milliliter of broth (60 units ml⁻¹) were more important than those at the exponential growth phase.

The NHase operational stability of whole resting cells was monitored following the bioconversion of acrylonitrile to acrylamide in continuous and stirred UF-membrane reactors. The rate of inactivation was independent on buffer molarity from 25 to 75 mM and on pH from 5.8 to 7.4. Enzyme stability and activity remained unchanged in distilled water. The initial reaction rate increased from 12.8 to 23.8 g acrylamide/g dry cell/h, but NHase half-life dropped from 33 to roughly 7 h when temperature was varied from 4°C to 10°C. The addition of butyric acid up to 20 mM did not improve enzyme operational stability, and largely reduced (94%) enzyme activity. Acrylonitrile caused an irreversible damage to NHase activity. High acrylonitrile conversion (86%) was attained using 0.23 mg cells/ml in a continuously operating reactor.     

Nitrile hydrolysing activities of deep-sea and terrestrial mycolate actinomycetes

Nitrile metabolising actinomycetes previously recovered from deep-sea sediments and terrestrial soils were investigated for their nitrile transforming properties. Metabolic profiling and activity assays confirmed that all strains catalysed the hydrolysis of nitriles by a nitrile hydratase/amidase system. Acetonitrile and benzonitrile, when used as growth substrates for enzyme induction experiments, had a significant influence on the biotransformation activities towards various nitriles and amides. The specific activities of selected deep-sea and terrestrial acetonitrile-grown bacteria against a suite of nitriles and amides were higher than those of the only other reported marine nitrile-hydrolysing R. erythropolis, isolated from a shallow sediment. The increase of nitrile chain length appeared to have negative influence on the nitrile hydratase activity of acetonitrile-grown bacteria, but the same was not true for benzonitrile-grown bacteria. The nitrile hydratases and amidases were constitutive in 10 of the 16 deep-sea and terrestrial actinomycetes studied, and one strain showed an inducible hydratase and a constitutive amidase. Most of the deep-sea strains had constitutive activities and showed some of the highest activities and broadest substrate specificities of organisms included in this study. This revised version was published online in August 2006 with corrections to the Cover Date.     

产生L-异亮氨酸的黄色短杆菌的代谢途径分析

目的:代谢工程要解决的主要问题是改变某些途径中的碳架物质流量或改变碳架物质流在不同途径中的流量分布,其目标就是修饰初级代谢,将碳架物质流导入目的产物的载流途径,以获得产物的最大转化率。方法:利用途径分析方法对黄色短杆菌生产L-异亮氨酸的途径进行了分析。结果:建立了9种基础模型,确定L-异亮氨酸理论最高摩尔产率是1;确定了黄色短杆菌生产L-异亮氨酸的最佳途径的通量分布,并以此为依据进行发酵溶氧控制优化,溶氧分阶段控制发酵生产L-异亮氨酸比溶氧恒定控制方式发酵的产率提高了8.2%。结论:根据途径分析结果,通过改变发酵过程有关参数,可使目的产物产率得到提高。     

短杆菌变异株FMP92814产苯丙氨酸的研究

经过多次的人工诱变筛选,获得一株来自乳糖发酵短杆菌ＡＴＣＣ１３８６９的变异株ＦＭＰ９２８１４。它对多种苯丙氨酸结构类似物具有抗性,并兼有酪氨酸营养缺陷型特性。我们对ＦＭＰ９２８１４菌株的发酵生产苯丙氨酸条件进行了研究。在５Ｌ台式小罐的发酵试验中,该菌株的苯丙氨酸产量可达２５．２ｇ／Ｌ,糖酸转化率１６．８％．。     

Production of Proteases from Brevibacterium Linens

Protease formation in submerged cultivations of Brevibacterium linens was studied. The effect of several proteinaceous materials on the production of proteolytic enzymes was investigated in mineral media containing 0.2% malt extract for bacterial growth. The addition (0.5%) of yeast extract or enzymatically hydrolyzed casein considerably increased the amount of protease formed, whereas ammonium salts supplied additionally in most cases had a repressive effect on enzyme formation. Furthermore, the kinetic of protease formation was determined in a highly instrumented fermenter system. Respiration activity indicated several phases of bacterial growth. Most of the proteolytic activity was synthesized during active growth; there was only a small increase in the stationary phase. A total proteolytic activity of 36 U/ml was formed in 24 hr. Concentration of α-amino nitrogen decreased steadily and ammonium ions accumulated during bacterial growth. Electrophoretic analysis revealed the occurrence of one leucine aminopeptidase (26 kDa monomer) and several proteases. There is a broad spectrum of proteolytic active proteins in the range of 11-66 kD which may be caused by some auto-degrading effects.     

腺苷蛋氨酸发酵条件及发酵培养基的优化

腺苷蛋氨酸具有转甲基、转硫和转氨丙基等重要生理作用 ,已成为治疗疾病的重要药物。研究了腺苷蛋氨酸发酵条件及发酵培养基的优化。结果表明 :发酵培养基的最适初始pH值为5.0 ;最佳发酵温度为 30℃ ;最适底物浓度为 1 % ;8%蔗糖为最佳碳源 ;0.5 %的NH4NO3 为最佳氮源 ;无机离子的最佳组合为 0.01 %MgSO4·7H2 O、0.03%CaCl2 、0.04%FeSO4·7H2 O和 0.01%LiCl。     

Purification and characterization of the enantioselective nitrile hydratase from Rhodococcus sp. AJ270

Nitrile hydratase (NHase, EC 4.2.1.84) from Rhodococcus sp. AJ270 was purified with 23.96% yield after sonication, ammonium sulfate fractionation, ion exchange, hydrophobic and gel-filtration column chromatography. The enzyme showed intriguing characteristics: it hydrated not only aliphatic and heterocyclic nitriles but also aromatic ones. Some substrates were also hydrated enantioselectively to the corresponding amides. The enantiomeric excess (ee) value of the enzyme hydrating trans-2,2-dimethyl-3-phenylcyclopropanecarbonitrile was 84.7. The enzyme is composed of two subunits: an alpha subunit and beta subunit of 22 975 Da and 23 493 Da, respectively. The optimal temperature and pH for the catalytic reaction of the enzyme was 25 degrees C and pH 7.6. The enzyme activity of the purified NHase was strongly inhibited by some oxidizing agents and heavy metals.