Rhodococcus boritolerans FW815腈水合酶分离纯化及其酶学性质研究

Rhodococcus boritolerans FW815具有高效水合2,2-二甲基环丙甲腈活性,利用蛋白质柱层析技术从中分离得到R.boritolerans FW815腈水合酶。实验结果表明,该腈水合酶分子量为40 kDa,由大小分别为24 kDa、22 kDa两种亚基构成。R.boritolerans FW815腈水合酶最适作用温度为37℃,最适作用pH值7.0。Cu2+、Zn2+、Ni2+等金属离子及EDTA对该腈水合酶有较强的抑制作用。除2,2-二甲基环丙甲腈、2-氨基-2,3-二甲基丁腈之外,R.boritolerans FW815腈水合酶对2-氧代-1-吡咯烷基丁腈、丙烯腈、2-氨基-4-甲硫基丁腈也表现出腈水合酶活性。     

生物法生产丙烯酰胺过程中腈水合酶的抑制和失活

为改进生物法生产丙烯酰胺工艺，研究了腈水合酶催化丙烯腈水合生产丙烯酰胺过程中影响腈水合酶反应速率和酶失活的因素. 实验证明，水合过程中体系pH值的变化基本不影响酶反应速率；底物丙烯腈的浓度低于10 g/L时，酶反应速率与底物浓度成正比，大于75 g/L后，对酶有抑制作用；产物丙烯酰胺显著抑制腈水合酶的活性；菌体细胞内可能存在可以稳定腈水合酶的物质，胞内的腈水合酶在40℃下的半衰期可以达到59.9 h；丙烯酰胺与温度的协同作用是腈水合酶失活的重要原因.     

耦合末端盐桥与定点突变的重组腈水合酶催化动力学

双亚基腈水合酶是催化丙烯腈水合生产丙烯酰胺的重要工业酶。通过Discovery Studio 2.5软件对C-末端盐桥耦合定点突变改造的耐热型基因重组腈水合酶NHaseM-TH-SBM(S344K-S346K-L347E-N362S-435DT436(+))进行盐桥网络分析,发现其全局盐桥总数有所下降,但双亚基界面间盐桥数量提高到7个。在重组大肠杆菌中表达了原腈水合酶NHaseM-TH(TH)、盐桥突变酶NHaseM-TH-SB(SB)和盐桥耦合定点突变的重组酶NHaseM-TH-SBM(SBM),研究了3种酶的催化反应动力学和失活动力学。结果表明,SBM的腈水合酶活性为543.9 U·mg?1,比TH提高了31.0%;其米氏方程催化速率常数Kcat比TH提高了20%。SBM的表观失活常数KD在25~42℃范围内均明显低于对照TH,在42℃时为TH的68.0%,显示了良好的热稳定性。SBM是活性和稳定性同时提高的重组腈水合酶。     

游离细胞腈水合酶催化丙烯腈水合反应的双稳态反应动力学

腈水合酶是能够催化丙烯腈水合生成丙烯酰胺的一种重要的工业酶。本研究建立了游离细胞腈水合酶催化丙烯腈水合反应的双稳态反应动力学模型,关联了底物浓度、产物浓度和温度等主要因素对反应速率(表观酶活)的影响。在实验研究的基础上,通过麦夸特及全局最优化算法求解了动力学模型。结果表明,游离细胞腈水合酶催化的双稳态反应动力学模型是比较典型的产物抑制型,当产物浓度逐渐增大时,高浓度的产物将抑制腈水合酶的活性。当底物浓度10g·L-1时,由于底物加入反应体系时产生的局部瞬时高浓度,腈水合酶催化的丙烯腈水合反应的表观反应速率不随底物浓度变化。当底物浓度≥10g·L-1时,底物产物浓度对反应速率具有显著影响。温度对酶活的影响也十分显著,相同底物产物浓度下,28℃时的酶催化水合反应速率是15℃时的3.3倍。     

腈水合酶固定化方法和催化特性的研究

以一种能够产生腈水合酶诺卡氏菌为研究对象,针对原来的海藻酸盐包埋法存在的固定化细胞强度较小、通透性较差等问题,改进了对酶的固定化方法,并对固定化腈水合酶的催化特性:最适反应温度、pH值、底物丙烯腈浓度、和表面活性剂性质和丙烯酰胺累积浓度对酶活性的影响等五个方面进行了研究。其中固定化腈水合酶最适反应温度在15~25°C;pH 7.0左右;底物丙烯腈浓度为3%~4%;Triton X-100对酶活性基本无影响,而Tween 80和Tween 60对酶活力有抑制作用;固定化腈水合酶在丙烯酰胺累积浓度为15%~20%之间时,酶活性较好。     

耦合末端盐桥与定点突变的重组腈水合酶催化动力学

双亚基腈水合酶是催化丙烯腈水合生产丙烯酰胺的重要工业酶。通过Discovery Studio 2.5软件对C-末端盐桥耦合定点突变改造的耐热型基因重组腈水合酶NHase^M-TH-SB^M（S344K-S346K-L347E-N362S-435DT436（+））进行盐桥网络分析,发现其全局盐桥总数有所下降,但双亚基界面间盐桥数量提高到7个。在重组大肠杆菌中表达了原腈水合酶NHase^M-TH（TH）、盐桥突变酶NHase^M-TH-SB（SB）和盐桥耦合定点突变的重组酶NHase^M-TH-SB^M（SB^M）,研究了3种酶的催化反应动力学和失活动力学。结果表明,SB^M的腈水合酶活性为543.9 U·mg^-1,比TH提高了31.0%;其米氏方程催化速率常数K_cat比TH提高了20%。SB^M的表观失活常数K_D在25～42℃范围内均明显低于对照TH,在42℃时为TH的68.0%,显示了良好的热稳定性。SB^M是活性和稳定性同时提高的重组腈水合酶。     

Nocardia sp.腈水合酶的纯化过程研究

对Nocardiasp.高活力腈水合酶进行了纯化研究。在细胞破碎中,超声时间对腈水合酶比酶活存在一个最优值,超声时间为20.00min时得到的比酶活最高。在离子交换层析过程中,采用DEAE-Sepharose作为层析介质,分别对平衡缓冲液pH、离子强度和线性梯度洗脱体积进行了优化。结果表明,采用pH7.20、50mmolL-1的Na2HPO4-NaH2PO4溶液作为平衡缓冲液,0.00~1.00molL-1的NaCl线性梯度洗脱,洗脱体积为20~25倍柱体积,此条件下腈水合酶的纯化倍数和酶活收率较佳。以Phenyl-SepharoseFF为层析介质研究了疏水层析精制腈水合酶的工艺过程,采用两步层析优化方法纯化出的Nocardiasp.腈水合酶比酶活达到2648.0U穖g-1,酶活收率为40.84%,用SDS-PAGE检测其纯度为99.00%以上。Nocardiasp.腈水合酶的两个亚基分子量分别为22.90kDa和27.38kDa。     

腈水合酶区域选择性与对映选择性的研究进展

介绍了生物法制备丙烯酰胺用酶——腈水合酶的研究现状,系统阐述了腈水合酶在除丙烯酰胺以外的其他酰胺类产品生产中的应用潜力,综述了腈水合酶区域选择性及对映选择性的研究近况。     

Nocardia sp.RS合成腈水合酶过程及其高酶活的表达工艺

对Nocardia sp．RS合成腈水合酶的过程进行了分析，研究了菌体生长和酶活表达的相互关系、pH调控和葡萄糖消耗对产酶速率的影响．根据腈水合酶在发酵过程中的生成特性，设计开发了葡萄糖—Co^2 耦合补加工艺优化产酶过程，通过对产酶过程的优化，大大提高了腈水合酶的发酵水平，酶活达到了10195U／ml，比优化前提高了43．2倍．     

腈水合酶/酰胺酶体系制备蛋氨酸羟基类似物

西洼湖戈登氏菌CGMCC 4.218 4含有一个高效的腈水合酶/酰胺酶体系。本文利用该菌株全细胞中的腈水合酶(Nitrile hydratas)催化2-羟基-4-甲硫基丁腈(HMTBN)水合为2-羟基-4-甲硫基丁酰胺(HMTBAm),利用酰胺酶(Amidase)以原位串联的方式将2-羟基-4-甲硫基丁酰胺水解为2-羟基-4-甲硫基丁酸(HMTBA,蛋氨酸羟基类似物)。考察了底物浓度、细胞质量浓度、温度、pH、反应时间对腈水合酶和酰胺酶活性的影响,对腈水合酶、酰胺酶的热稳定性和pH稳定性进行了测定。结果表明,最优的反应条件为:50 mmol/L HMTBN,3 g/L干重细胞,温度30℃,pH=8.0,反应时间30 min。在最优反应条件下,采用分批补料策略,细胞可使用23批次,HMTBA的累积量在44 h内达到164 g/L,其产率为95%。     

游离静止细胞中腈水合酶的失活动力学

Nitrile hydratase (NHase) is an important industrial enzyme used for acrylamide production from acrylonitrile. The deactivation kinetics of NHases in free resting cells of Rhodococcus sp. was presented based on a bi-steady state assumption. Effects of hydration temperature, product concentration and substrate concentration on NHase deactivation were investigated experimentally and correlated with a first order deactivation kinetics. The results showed that the hydration temperature and product concentration were major factors governing the deactivation of NHases under substrate-feeding conditions. When acrylamide concentration was higher than 250 g•L－1, the deactivation of NHases became serious and the bi-steady state assumption was not applicable. When the hydration temperature was controlled at a relatively higher level such as 28°C, the total deactivation rate constant was about 2.8-fold of that at 20°C.     

Fe(III) and Co(III) centers with carboxamido nitrogen and modified sulfur coordination: lessons learned from nitrile hydratase

Nitrile hydratase (NHase) is a non-heme Fe(III) or non-corrinoid Co(III) metalloenzyme with an unprecedented coordination sphere comprising deprotonated carboxamido nitrogens and modified Cys-S (-SO(-) and -SO(2)(-)) sulfurs. We have synthesized model complexes derived from designed ligands that contain these donor groups. The model complexes mimic almost all the intrinsic properties of the unique M(III) (M = Fe, Co) active site of NHase. Even a functional Co(III) model has been synthesized that hydrolyzes nitriles catalytically at pH close to the optimum pH of the enzyme. Our studies have provided insight into how the unusual donor atoms dictate the overall properties of the biological M(III) sites.     

Cold-active citrate synthase: mutagenesis of active-site residues

A comparison of the crystal structure of the dimeric enzymecitrate synthase from the psychrophilic Arthrobacter strainDS2-3R with that of the structurally homologous enzyme fromthe hyperthermophilic Pyrococcus furiosus reveals a significantdifference in the accessibility of their active sites to substrates.In this work, we investigated the possible role in cold activityof the greater accessibility of the Arthrobacter citrate synthase.By site-directed mutagenesis, we replaced two alanine residuesat the entrance to the active site with an arginine and glutamateresidue, respectively, as found in the equivalent positionsof the Pyrococcus enzyme Also, we introduced a loop into theactive site of the psychrophilic citrate synthase, again mimickingthe situation in the hyperthermophilic enzyme. Analysis of thethermoactivity and thermostability of the mutant enzymes revealsthat cold activity is not significantly compromised by the mutations,but rather the affinity for one of the substrates, acetyl-CoA,is dramatically increased. Moreover, one mutant (Loop insertion/K313L/A361R)has an increased thermostability but a reduced temperature optimumfor catalytic activity. This unexpected relationship betweenstability and activity is discussed with respect to the natureof the dependence of catalytic activity on temperature.     

High-Throughput Screening of Signal Peptide Library with Novel Fluorescent Probe

Nitrile hydratase (NHase) is an excellent biocatalyst for the synthesis of amide compounds and is composed of two heterologous subunits. However, the secretory expression of NHase has been difficult to achieve because of its complex expression mechanism. In this work, a novel fluorescent probe Rho-IDA-CoII was synthesized by a one-pot method. Rho-IDA-CoII could specifically label His-tagged proteins in vitro, such as for staining in-gel, Western blot, and ELISA analysis. Furthermore, Rho-IDA-CoII combined with dot blots could quantitatively detect His-tagged proteins at between 1–10 pmol and perform high-throughput screening for the NHase signal peptide library. Recombinant Bacillus subtilis WB800/phoB-HBA with the extracellular expression of NHase was screened (ca. 6500 clones). After optimization of fermentation conditions, the NHase activity in the culture supernatant reached 17.34±0.16 U/mL. This is the first time that secretory NHase has been expressed in B. subtilis successfully.     

理性设计提高β-葡萄糖醛酸苷酶的热稳定性

采用同源序列比对策略和脯氨酸效应的设计策略,以同源建模的三维结构为基础,结合定点突变技术,对重组产紫青霉β-葡萄糖醛酸苷酶(PGUS-E)进行理性设计,获得了2个热稳定性明显提高的突变体PGUS-E I130V和PGUS-E G280P,再将突变位点进行组合获得突变体PGUS-E I130V+G280P。相比PGUS-E,PGUS-E I130V、PGUS-E G280P和PGUS-E I130V+G280P在60℃下的半衰期T1/2分别比原始酶的23 min提高3.5倍,5倍和5.5倍,达到82 min,117 min和128min。突变体的动力学参数Kcat/Km值分别为1.534×107 mol-1·L·min-1,1.368×107mol-1·L·min-1和1.283×107 mol-1·L·min-1,与原始酶(1.316×107 mol-1·L·min-1)接近,对底物的亲和力基本不变。结果表明在蛋白质构象不稳定的区段中引入脯氨酸,以及在相应位置引入嗜热菌的氨基酸,均可提高蛋白质热稳定性。