新型耐热腈水合酶的异源表达及其催化工艺研究

腈水合酶(nitrile hydratase,NHase,EC 4. 2. 1. 84)是一种金属酶,能将腈类物质催化水合生成酰胺类物质,在工业上主要应用于烟酰胺和丙烯酰胺的生产。腈水合酶在应用过程中,普遍存在热稳定性较差的缺陷,而腈类的水合催化属于放热反应,在工业催化过程中,过高的反应温度使得腈水合酶的催化效率、重复利用率均较低,故寻找一种耐热型腈水合酶对工业应用以及理论研究具有重要意义。该研究以提高腈水合酶热稳定性为出发点,从美国国立生物技术信息中心通过BLAST筛选到一种新型耐热腈水合酶,该腈水合酶来源为温泉热碱芽孢杆菌(Caldalkalibacillus thermarum TA2. A1),该菌株最适生长温度为65～70℃。将来源于Cal. thermarum TA2. A1的腈水合酶基因序列进行密码子优化,基因合成后在大肠杆菌BL21(DE3)宿主中异源表达,通过在该腈水合酶β亚基的C端添加strep标签成功纯化该酶,测定其在65℃的半衰期为3 h,在30℃下催化烟腈的比酶活为395 U/mg,其全细胞催化反应速率比已报道数据将近快1倍,烟酰胺的最终产量提高了25%,更有...     

基于底物通道工程提升嗜热腈水合酶对烟腈的催化性能

腈水合酶(nitrile hydratase, NHase, EC 4.2.1.84)是一类将腈类化合物经水合反应生产酰胺类化合物的金属酶,工业上用该酶进行生物法生产烟酰胺。由于水合过程放热,工业用酶对其稳定性提出了更高的要求。实验室前期通过基因挖掘获得了一种来源于嗜热菌温泉热碱芽胞杆菌(Caldalkalibacillus thermarum) TA2.A1的腈水合酶基因,但其催化活性不足,难以满足应用需求。该研究基于底物通道工程,将该酶β亚基的第42位甘氨酸突变为赖氨酸后,其催化3-氰基吡啶的比酶活力提高为野生酶的2.5倍,且仍保留较好的稳定性,经5 L发酵罐培养,细胞酶活力达到5 539.0 U/mL,具有巨大的应用潜力。     

协同两类腈水合酶高效催化烟酰胺及丙烯酰胺生物合成

腈水合酶(nitrile hydratase, NHase)可以将腈类物质通过水合作用转化生成更有利用价值的酰胺类化合物,在工业上被用于生产烟酰胺、丙烯酰胺等重要化学品。然而,尚无兼具对底物和产物耐受性高以及催化活性高的腈水合酶,导致底物不能被彻底利用、酰胺产物的产量受限。该研究协同表达了高催化活性的低分子质量腈水合酶(L-NHase)和高耐受性的高分子质量腈水合酶(H-NHase),并优化两类酶在质粒上的排列位置。其中BAE-BAG菌株对底物、产物的耐受性与H-NHase菌株相仿;在以烟腈或者丙烯腈为底物进行全细胞催化时,该菌株的催化速率与L-NHase菌株相仿,最终烟酰胺产量可达到516 g/L,是目前报道的最高产量;丙烯酰胺产量比原始BAE或BAG菌株提高19.1%以上。该文成功协同两类腈水合酶,同时提高了合成烟酰胺和丙烯酰胺的催化反应速率及产量,对工业应用具有一定的指导意义。     

β-乳球蛋白与丙烯酰胺的相互作用研究

为了研究β-乳球蛋白对丙烯酰胺的抑制情况,文章采用液质联用和分子对接手段探究两者相互作用的可能性及其结合位点。研究结果表明,β-乳球蛋白会一定程度上抑制丙烯酰胺的含量,且浓度越大抑制率越高;对接结果表明二者存在相互作用的可能性,其复合物最佳的吉布斯自由能为-3.6 kcal/mol,此时丙烯酰胺结合在β-乳球蛋白内部由氨基酸残基Leu46、Leu54、Ile56、Val92、Val94、Leu103、Phe105和Leu122共同形成的柱状疏水空腔内。上述8个氨基酸残基都对丙烯酰胺均具有疏水作用,进一步实验表明Leu、Val、Ile、Phe都对丙烯酰胺有抑制作用,实验为探究β-乳球蛋白与丙烯酰胺同时被摄入时降低丙烯酰胺对人体负面影响提供了理论基础。     

高分子量腈水合酶工程菌生产烟酰胺的工艺建立

为提高表达玫瑰色红球菌(Rhodococcus rhodochrous J1)来源的高分子量腈水合酶大肠杆菌Escherichia coli BL21(DE3)/pET-24a(+)-nhh Brbs Arbs G(BAG)的菌体酶活,建立了合适的烟腈全细胞催化工艺以及重组工程菌的高密度发酵工艺。采用正交试验L_9(3~4)对摇瓶培养基中各组分进行优化,比较烟腈底物流加和底物分批补料工艺,对重组菌进行分批补料高密度发酵培养。培养基优化后其菌体生物量提高到4.42 gDCW/L,细胞比酶活提高到30.91 U/mgDCW;与烟腈底物流加相比,烟腈分批补料工艺更适合烟酰胺的生产,通过此工艺的反应,摇瓶发酵后的BAG能够生产390 g/L烟酰胺;对BAG进行5 L发酵罐分批补料扩大培养,菌体生物量最高达到73.97 gDCW/L(OD600=200),细胞比酶活最高为42.70 U/mgDCW,单位发酵液酶活最高为2 813 U/mL,用较高密度的发酵罐培养后重组菌进行烟腈催化反应,产物质量浓度能达到537 g/L,是已报道的大肠杆菌重组表达腈水合酶产烟酰胺所得终质量浓度的最高水平。     

腈水合酶制备关键技术研究

<正>腈水合酶是一类可以催化腈类物质转化相应的酰胺类化合物的金属酶,工业上利用其催化丙烯腈转化为丙烯酰胺。该生物法制备丙烯酰胺工艺在1985年被开发,是生物法替代化学法的典型案例。随着丙烯酰胺的需求不断扩大,国内外研究机构及学者对腈水合酶产生菌的分离选育、菌株特性研究、酶的形成条件、酶的结构和性质、生物合成机制、翻译后的成熟机制等进行了深入研究。如中国专利CN201710456875.6公开了一种改造腈水合酶及其应用,该发明改造腈水合酶的抗逆性、耐热性和产物耐受     

Rhodococcus sp.SHZ-1腈水合酶的高酶活发酵工艺

通过研究发酵过程中溶氧、生物量、腈水合酶活力以及残糖的变化规律和搅拌速度、通气量、接种量及诱导剂对产腈水合酶的影响,确立了5L发酵罐中Rhodococcus sp.SHZ-1腈水合酶的高酶活发酵工艺参数。结果表明,发酵过程中溶氧控制在30%以上有利于菌体快速生长和腈水合酶的合成。在pH7.2,温度30℃的发酵条件下,适宜的腈水合酶合成工艺条件为:接种量10%,通气量1.0vvm,搅拌速度采用由初始的200r/min调至500r/min的变速调控方式,同时于48h补加产酶诱导剂,发酵液腈水合酶的最高酶活力达到了9500U/mL,是摇瓶培养时最高酶活力8208U/mL的1.2倍,且比未进行工艺优化时最大产酶期缩短了20～24h,其最佳产酶期为52～60h。     

产腈水合酶菌株的驯化选育及其产酶条件的优化

以Rhodococcussp YL 1为出发菌株 ,通过逐渐增加培养基中丙烯腈的浓度重复继代培养 ,得到 1株酶活为 79 8U/mg的腈水合酶高活力菌株YL 2 ,酶活比出发菌株YL 1提高了 87%。对菌株YL 2的产酶条件进行了优化。结果表明 ,葡萄糖、诱导剂脲、Co2 + 及pH是影响腈水合酶高效表达的主要因素。在葡萄糖浓度为 2 0g/L ,诱导剂脲的添加量为 0 0 6g/L ,钴离子加入量为 0 3g/L ;培养温度为 30℃ ,培养基初始 pH为 7 0的条件下培养 4 0h后 ,酶活可达 134 5U/mg ,比优化前提高了 6 9%。     

基于氨基酸热点突变对腈水合酶底物亲和力的改造

目的:以Rhodococcus erythropolis CCM2595来源的腈水合酶为研究对象,利用半理性设计以及定点突变以获取其对烟腈底物亲和力更高的突变体.方法:通过序列比对找到同源性很高的1AHJ蛋白并采用Swiss-Model和iTASSER等软件进行评估.利用Discovery Studio 2016(DS...     

微生物腈水合酶的研究现状

本文对微生物腈水合酶的结构、催化机理、产生菌的选育、酶的形成条件、分子生物学、酶学特性进行了阐述。     

Aldoxime dehydratase co-existing with nitrile hydratase and amidase in the iron-type nitrile hydratase-producer Rhodococcus sp. N-771

We identified an aldoxime dehydratase (Oxd) gene in the 5'-flanking region of the nitrile hydratase-amidase gene cluster in the photoreactive iron-type nitrile hydratase-producer, Rhodococcus sp. N-771. The enzyme showed 96.3%, 77.6%, and 30.4% identities with the Oxds of Rhodococcus globerulus A-4, Pseudomonas chlororaphis B23, and Bacillus sp. OxB-1, respectively. The enzyme was expressed in Escherichia coli under the control of the lac- or T7 promoters in its intact and His6-tagged forms, purified, and characterized. The enzyme had heme b as a prosthetic group, catalyzed a stoichiometric dehydration of aldoxime into nitrile, and exhibited the highest activity at neutral pH and at around 30 degrees C similar to the known Oxd from Bacillus sp. OxB-1. The activity was enhanced by reducing agents, such as Na2S, Na2S2(O4), 2-mercaptoethanol, and L-cysteine and supplementary additions of electron acceptors such as flavins, sulfite ion, and vitamin K3. The effect of various chemicals on the enzyme activity was different in the presence and absence of the reducing reagent, Na2S. The enzyme preferentially acts on aliphatic-type substrates and the substrate specificity of the enzyme coincides with that reported for nitrile hydratase produced by the strain.     

丙烯腈水合酶催化动力学及失活动力学研究

对丙烯腈水合酶在丙烯酰胺生产中的一些动力学参数做了探讨.研究表明:ρ(丙烯腈)>30 g/L时将引起底物抑制;底物不加限制的情况下,反应速率与菌悬液的加入量成正比关系;温度升高使反应速度明显加快,在28℃时的反应速率为在16℃时的反应速率的2.3倍;同时,温度的升高又使酶的失活加快,用阿累尼乌斯方程对温度与反应速率进行拟合可以得出催化反应的表观活化能50.6 kJ/mol,失活反应的表观活化能为12.65 kJ/mol.     

Novel catalytic activity of nitrile hydratase from Rhodococcus sp. N771

Nitrile hydratase (NHase) from Rhodococcus sp. N771 is a non-heme iron enzyme catalyzing the hydration of various nitriles to the corresponding amides. We report a novel catalytic activity of NHase. When NHase was incubated with an large excess of commercially available isovaleronitrile, the charge transfer band from the sulfur ligand to the Fe atom shifted from 710 nm to 820 nm, but recovered within 4 min. Similar UV-Vis absorption changes were observed after the addition of isobutylisonitrile (iBuNC), a major impurity in commercially available isovaleronitrile, suggesting that NHase catalyzes the conversion of iBuNC to other compounds. The reaction product was identified as isobutylamine (iBuNH(2)) by liquid chromatography tandem mass spectrometry. NHase also converts t-butylisonitrile and 1,1,3,3,-tetramethylbutylisonitrile to the corresponding amines. Kinetic analysis of the conversion of iBuNC to iBuNH(2) showed a K(m) value comparable to that for nitriles, while the V(max) value was more than 10(5) times smaller than that for methacrylonitrile. This is the first report suggesting that NHase is a bifunctional enzyme catalyzing a reaction other than the hydration of nitriles.     

羧基丁腈橡胶对酚醛树脂磨具的性能影响

文章研究了在酚醛树脂磨具结合剂中为增韧改性,掺入不同比例的羧基丁腈橡胶制成弹性抛光片,测试其塑料洛氏硬度、抛光陶瓷时两者的磨耗。同时用普通丁腈橡胶和羧基丁腈橡胶同样比例增韧酚醛树脂制成抛光片,对比测试两者的力学性能、磨耗比和电镜图。结果表明：随着羧基丁腈橡胶的增加,其洛氏硬度呈线性降低,抛光陶瓷时,抛光片的磨耗呈现曲线形变化,先开始降低,但增加到20%以上,保持平稳,直到增加到50%以上,磨耗迅速增加,但增加80%以上后,又开始降低;而陶瓷的磨耗则缓慢降低。但在增韧酚醛树脂时,羧基丁腈橡胶粉比普通丁腈橡胶粉增韧效果明显。     

Factors affecting the production of nitrile hydratase by thermophilic Bacillus smithii SC-J05-1

Ammonium present in the medium was found to control the production of nitrile hydratases (NHase) by a moderate thermophile, Bacillus smithii SC-J05-1. The enzyme production is initiated in the absence of ammonium and suppressed in the presence of ammonium. In addition to cobalt ions as the coordinated metal ion, manganese ions are also required for NHase production.     

顶空-气相色谱法测定聚丙烯腈类食品接触材料中3种腈类化合物迁移量规律

目的 建立顶空-气相色谱法测定聚丙烯腈类食品接触材料中乙腈、丙烯腈和丙腈等3种腈类化合物迁移量的分析方法。方法 通过优化模拟物体积、水基模拟物中盐及其用量和分流比、载气流速、升温程序、平衡温度与时间等色谱条件,实现了对3种腈类化合物迁移量的常规、快速分析,研究丁腈橡胶、聚丙烯腈-苯乙烯、聚丙烯腈-丁二烯-苯乙烯3种材质阳性产品在不同模拟物、不同时间和不同温度条件下3种腈类化合物迁移量规律。结果 3种腈类化合物在0.005～0.100 mg/L浓度范围内,线性相关系数均大于0.9960,方法定量限为1.57～3.77μg/kg,低、中、高浓度的回收率84.3%～108.9%,相对标准偏差值为1.7%～9.7%。结论 丁腈橡胶在水模拟物中容昐迁移出丙烯腈,而聚丙烯腈-丁二烯-苯乙烯和聚丙烯腈-苯乙烯产品在50%乙醇模拟物中容昐迁移出丙烯腈, 3种腈类化合物均随模拟时间的延长和温度的升高,越容昐迁移到食品模拟物中。     

AS/ABS产品中3种腈类化合物迁移量模型和风险评估模型建立

目的 建立AS/ABS 产品中乙腈、丙烯腈和丙腈等3种腈类化合物迁移量模型和风险评估模型的分析方法。方法 考察AS/ABS阳性产品在不同模拟物、不同时间和不同温度(20、40、70 ℃)条件下3种腈类化合物迁移量规律研究。结果 随着温度的升高和时间的延长, 腈类化合物的迁移量上升速率越快; 温度在20~70 ℃时, 水中丙烯腈和丙腈Ap范围值为7.9~14.1; 丙烯腈和丙腈化合物动力学参数活化能Ea分别为133.86 KJ/moL和134.07 KJ/moL。结论 AS中腈类化合物迁移到食品模拟物中的暴露量处于低风险水平, 建立AS产品中腈类化合物迁移模型和致癌风险评估评估数学模型。     

Observations on nitrile production during autolysis of kale and swedes,and their stability during incubation with rumen fluid

A method was modified for quantitatively measuring total nitriles produced from glucosinolate degradation during autolysis of kale and swede plant tissue. Nitrile production from New Zealand Medium Stem kale was less at pH 7.5 (0.28 mol mol^?1 glucosinolate) than at pH 5.7 (0.57 mol mol^?1 glucosinolate). Lower levels were produced from Maris Kestrel kale, due both to a lower glucosinolate content and to reduced conversion of glucosinolate to nitriles. The amount of nitrile obtained on conversion of glucosinolate tended to be reduced by growing New Zealand Medium Stem kale in soil of low SO₄-S concentration, due to a reduction in plant glucosinolate content. Nitrile production from swede tissue was 0.3–0.5 mol mol^?1 glucosinolate. Nitriles produced from swedes were rapidly degraded upon incubation with rumen fluid (4 h), whereas nitriles produced from kale remained stable for incubation periods up to 23 h. The high rate of conversion of glucosinolate to nitriles coupled with their stability in rumen fluid suggests that, in addition to goitrogenic properties, nitrile formation also deserves attention in the nutrition of ruminant animals fed kale diets. Of the four brassica diets tested, New Zealand Medium Stem kale is considered the most likely to cause nutritional problems (depressions in intake and reproduction) from nitrile production, and a method of testing this is suggested.