北京棒杆菌新型天冬氨酸激酶双突变株Y198N/D201M的构建及酶学性质表征

利用定点突变提高天冬氨酸族氨基酸合成途径中的首个关键别构酶天冬氨酸激酶(aspartate kinase,AK)活力,并解除其受到代谢产物赖氨酸(Lys)与苏氨酸(Thr)的协同反馈抑制。在获得北京棒杆菌单体AK并分析结构的基础上,选取ATP周围的关键残基位点Tyr198和Asp201进行饱和定点突变,采用高通量筛选方法成功获得了酶活力提高到18.26倍的双突变体Y198N/D201M,动力学结果显示,K_m值由野生型(wild type,WT)的3.58 mmol/L减小到2.37 mmol/L,与底物亲和力增强;n值由WT的1.91减小到1.58,正协同性降低。酶学性质研究表明:双突变体Y198N/D201M最适反应温度由WT的25℃增至28℃,最适pH值由WT的8.0降至7.5,半衰期由WT的4.66 h延长至5.32 h。突变体Y198N/D201M较WT,在各个抑制剂浓度下活性抑制作用表现出不同程度的减弱,甚至在5 mmol/L和10 mmol/L Lys+Met、Thr+Met和Lys+Thr+Met条件下有激活作用。     

北京棒杆菌天冬氨酸激酶G377定点突变及酶学性质表征

采用饱和定点突变和高通量筛选技术,对北京棒杆菌天冬氨酸激酶（aspartate kinase,AK）AK Gly377位点进行突变,并筛选获得高活力突变体G377F,分离纯化后对G377F AK酶学性质进行表征。结果表明：突变体AK最适pH值为9.0,较突变前野生型（wide type,WT）最适pH值为8.5有所提高;最适反应温度与WT一致,均为25 ℃;半衰期由WT的2.6 h提高到5.3 h;pH耐受性在5 h内保持其活力50%以上,与WT 3 h内酶活力保持能力相同。G377F对金属离子和有机溶剂均表现出良好的抗性。其中,Lys的抑制作用在一定程度上被解除,当Lys和Met同时存在时对AK具有明显的激活作用。动力学研究显示：G377F AK Vmax较WT提高9.3 倍;n值为1.0明显低于WT的2.7,说明AK正协同性降低,趋向于米氏酶。     

北京棒杆菌Corynebacterium pekinense天冬氨酸激酶P184M酶动力学分析及酶学性质表征

目的研究北京棒杆菌天冬氨酸激酶(aspartate kinase,AK)184位点脯氨酸(proline,P)突变为蛋氨酸(DL-methionine,M)对酶动力学及酶学性质的影响。方法对北京棒杆菌AK家族同源序列比对,选择出高度保守且远离底物结合位点的Pro184位点,对其进行饱和定点突变,成功构建突变体P184M。结果动力学和酶学性质研究表明:P184M AK Vmax比WT(wide type)提高了5.06倍;n值为0.19,突变体由WT的正协同性变为负协同性,同时Km(mol/L)值增大;突变体AK最适pH为6.5,低于野生型最适pH 7.0;最适反应温度28℃,低于WT的30℃,适宜温度区间变窄;半衰期由WT的2.1 h降为1.2 h;金属离子、有机溶剂和抑制剂Thr+Met、Lys+Met、Thr+Lys+Met对突变株AK均表现出激活作用。结论突变体天冬氨酸激酶P184M的酶动力学性质和酶学性质改变显著,为构建高产蛋氨酸菌株提供了一定的参考依据。     

天冬氨酸激酶突变体G277K中AK基因的克隆表达及酶学性质表征

通过序列比对和晶体结构分析发现,天冬氨酸激酶（aspartokinase,AK）的G277位点高度保守,并与抑制剂Thr通过氢键相连,对其进行定点突变和酶学性质表征。结果表明：突变体G277K的AK最适反应条件是30 ℃、pH 8.5;动力学实验结果显示突变体AK的正协同效应降低,趋向于米氏酶,参数S0.5、nH、酶比活力、Vmax分别是7.05 mmol/L,1.24、964.3 U/mg、32.143 U/（mg·min）;热稳定性实验显示,其30 ℃半衰期为2.3 h,3 h后酶活力丧失80%左右;Ni2+在低浓度时表现出显著的激活效应;有机溶剂丙三醇、异丙醇和二甲基亚砜的体积分数为1%时,对AK的酶活力有很好的激活作用,表明突变体AK对一些有机溶剂有一定的抗性;低浓度的赖氨酸和蛋氨酸对AK有激活作用。     

北京棒杆菌单体天冬氨酸激酶T379N/A380C/T65I/D173G突变体酶学性质表征及工程菌构建

选取天冬氨酸激酶抑制剂赖氨酸(Lys)的结合位点苏氨酸(Thr) 379和丙氨酸(Ala) 380、底物天冬氨酸(Asp)的结合位点Thr65、既是底物也是ATP结合位点的催化活性中心Asp 173,对4个关键残基位点进行定点饱和突变.采用高通量筛选方法成功构建突变体T379N/A380C/T65I/D 173G,与野...     

北京棒杆菌E31天冬氨酸激酶突变体T361D酶学性质表征

对北京棒杆菌(Corynebacterium pekinense)中天冬氨酸激酶(aspartate kinase,AK)进行定点改造,为改善突变株的酶学性质,提高突变株的酶活力,削弱或解除AK的反馈抑制作用。对高度保守的T361位点进行定点突变及高通量筛选,以大肠杆菌BL21为载体,使突变体T361D在载体中实现高效表达,经分离及纯化后进行酶学性质和动力学的研究。结果表明:与野生型(wild type,WT)相比,突变体T361D Vmax提高7.63倍,最佳反应p H值降低为7.0,最佳反应温度升至30℃,半衰期延长1 h;突变体T361D在底物抑制剂赖氨酸(Lysine,Lys)存在下表现出激活作用,对金属离子Na~+、K~+和有机溶剂甲醇、异丙醇均表现出良好的抗性。试验为获得高产天冬氨酸族氨基酸菌株提供了参考和依据。     

新型天冬氨酸激酶突变株构建及酶学性质表征

通过定点随机突变技术,提高天冬氨酸代谢途径中首个关键限速酶天冬氨酸激酶(aspartate kinase,AK)的催化活力,减弱或解除代谢产物对其协同反馈抑制,并通过Discovery Studio等软件对其空间结构进行分析,借助分子动力学模拟对其机制进行分析.首先选择ATP附近关键残基位点,在前期T379N/A380...     

天冬氨酸激酶代谢调控的研究进展

在微生物发酵合成天冬氨酸族氨基酸的代谢途径中,天冬氨酸激酶（aspartokinase,AK）是限制碳源和氮源流速的限速酶,是决定能否获得高产天冬氨酸族氨基酸的关键之一。本文介绍了AK的重要性、不同来源AK的整体结构、效应结合位点的特点和调控机制研究进展,并对AK代谢调控的发展做出展望,为进一步明确AK代谢调控机制,促进天冬氨酸族氨基酸产业化发展提供参考和依据。     

易错PCR技术提高源于/Sulfolobus acidocaldarius麦芽寡糖基海藻糖合成酶活性

利用易错PCR技术结合高通量筛选技术对源于Sulfolobus acidocaldarius ATCC 33909的MTSase进行定向进化,获得一个酶活提高的突变体酶D-6。基因分析表明,突变体酶发生了2个位点突变:G432D/G586D,其中突变位点G586D位于Aβ8与AEα14的loop环上。将野生型MTSase和突变体酶D-6进行蛋白质纯化后,测定其酶学性质,结果表明:突变体酶D-6的比活力为野生型MTSase的1.22倍;野生型的Km值为4.74 mmol/L,而突变体D-6为2.77 mmol/L,表明其对底物亲和性较野生型有所提高。     

嗜热杜邦菌α-淀粉酶的定向进化及高效表达

目的:对嗜热杜邦菌来源α-淀粉酶进行分子改造,以提高其耐热性和产酶水平。方法:基于易错PCR技术构建嗜热杜邦菌来源α-淀粉酶（Td-amy）的随机突变文库,高通量筛选耐热性和比酶活提高的突变体,通过定点突变及同源结构模拟对突变体进行分析,并将其在毕赤酵母中表达。结果:筛选得到一个正向突变体（mTd-amy）。该突变体最适温度（60 ℃）较野生型（55 ℃）提高了5 ℃,比酶活（466.3 U/mg）较野生型（227.9 U/mg）提高至2.0倍。经序列对比,mTd-amy有四个氨基酸发生了变化,分别为Ala4Val、Ala122Val、Lys194Arg和Ala468Asp,定点突变结果表明Ala122Val和Ala468Asp位点为影响其比酶活和最适反应温度的关键。进一步将突变体mTd-amy在毕赤酵母中高效表达,经高密度发酵其酶活达64696 U/mL。结论:定向进化获得了嗜热杜邦菌来源α-淀粉酶的正向突变体,该突变体的最适温度和比酶活力均明显提高,为α-淀粉酶的分子改造以及工业化应用等提供了理论参考。     

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...     

氨基酸对植物乳杆菌KLDS1.0391生长及细菌素合成的影响

为探究氨基酸对植物乳杆菌生长及细菌素合成的影响，调节化学成分确定培养基中的氨基酸组成，培养植物乳杆菌KLDS1.0391，采用高效液相色谱法测定乳酸含量，通过实时聚合酶链式反应（real-time polymerase chain reaction，real-time PCR）分析细菌素和氨基酸合成相关基因的表达。结果表明，天冬氨酸、组氨酸、异亮氨酸、亮氨酸、脯氨酸、苏氨酸、酪氨酸、天冬酰胺及谷氨酰胺的缺失导致该菌生长能力和细菌素合成量均显著降低（P＜0.05）；谷氨酸、甘氨酸、丙氨酸、半胱氨酸、精氨酸、苯丙氨酸、丝氨酸、色氨酸及缬氨酸的缺失仅影响菌体生长，对细菌素合成无明显影响；而赖氨酸胁迫（缺失及过量）对菌体的生长影响很小，但却显著影响细菌素的合成。色谱结果显示，赖氨酸、酪氨酸、异亮氨酸、亮氨酸、甲硫氨酸、天冬氨酸、天冬酰胺、脯氨酸及苏氨酸单缺失后乳酸含量升高。real-time PCR结果表明，细菌素合成相关基因plnEF、plnD及plNC8HK因赖氨酸的缺失表达显著下调（P＜0.05），而上述基因在外源赖氨酸质量浓度达到2.0 g/L前，上调水平随赖氨酸添加量的增大而增大。氨基酸合成关键基因dapG、yclM 因赖氨酸缺失表达显著上调，相反，上述基因在赖氨酸质量浓度为2.0 g/L时表达量显著下调（P＜0.05）。由上述研究结果推测，当赖氨酸缺失时，菌体通过上调基因dapG 和yclM 的表达以合成多种蛋白质保证其正常生长代谢，赖氨酸可正向诱导该菌细菌素合成，其可能作为细菌素合成底物发挥作用。     

Free amino acid composition in primary and secondary inflorescences of 11 broccoli (Brassica oleracea var italica) cultivars and its variation between growing seasons

Eleven broccoli cultivars were grown in the field in spring/summer (April–July) and summer/winter (September–January). Free amino acid composition was determined by HPLC in primary and secondary inflorescences separately. A total of 17 amino acids were identified: L ‐alanine (Ala), L ‐arginine (Arg), L ‐asparagine (Asn), L ‐aspartic acid (Asp), glycine (Gly), L ‐glutamic acid (Glu), L ‐glutamine (Gln), L ‐histidine (His), L ‐isoleucine (Ile), L ‐leucine (Leu), L ‐methionine (Met), L ‐phenylalanine (Phe), L ‐serine (Ser), L ‐threonine (Thr), L ‐tryptophan (Trp), L ‐tyrosine (Tyr) and L ‐valine (Val). The major amino acid was L ‐glutamine, which represented on average between 39.5% (in cvs Durango and Green Valiant) and 55.5% (in cv Shogun) of the total amino acid content among cultivars, followed by L ‐glutamic acid with a variation between 12.1% (in cv Shogun) and 17.4% (in cv Marathon). A few amino acids represented less than 1% each (Gly, Leu, Met, Phen, Thr, Trp and Tyr). For most of the amino acids there were significant differences between cultivars, whilst only a few amino acids showed significant variations between inflorescences. Season also induced significant differences in the content of most of the identified amino acids. The cultivar with the highest total free amino acid content (323.9 mmol kg⁻¹ DW) on average of both seasons (391.3 in spring/summer and 256.4 in summer/winter) was Shogun, whilst the others were above the minimum of 177.3 mmol kg⁻¹ DW found in cv SK3. There was a general tendency for higher total amino acids levels in spring/summer than in summer/winter, but it was clear that this effect was dependent on the cultivar. © 2000 Society of Chemical Industry     

苹果酒发酵过程中酵母对氨基酸利用的研究

对酵母J00的氨基酸利用特性进行了研究，确定出该酵母对各种氨基酸和NH_4~+的需要范围。通过调整苹果浓缩汁中的氨基酸含量，并分析氨基酸对酵母发酵代谢产物的影响，发现当果汁中氨基酸及NH_4~+的组成为：NH_4~+(120mg／L)、组氨酸(30mg／L)、苯丙氨酸(30mg／L)、缬氨酸(35mg／L)、异亮氨酸(30mg／L)、亮氨酸(10mg／L)、天门冬氨酸(160mg／L)、苏氨酸(1000mg／L)、谷氨酸(45mg／L)、甘氨酸(5mg／L)、丙氨酸(25mg／L)、蛋氨酸(15mg／L)、酪氨酸(15mg／L)、赖氨酸(45mg／L)、精氨酸(25mg／L)、天门冬酰胺(30mg／L)时，各种氮源的比例比较合适，使酵母发酵过程较优，可增加乙醇产率，提高总糖利用率，同时可以达到降低高级醇生成量的目的。     

Directed evolution of an aminoalcohol dehydrogenase for efficient production of double chiral aminoalcohols

The aminoalcohol dehydrogenase (AADH) of Rhodococcus erythropolis MAK154, which can be used as a catalyst for the stereoselective reduction of (S)-1-phenyl-1-keto-2-methylaminopropane to d-pseudoephedrine (dPE), is inhibited by the accumulation of dPE in the reaction mixture, limiting the yield of dPE. To improve this weak point of the enzyme, random mutations were introduced into aadh, and a mutant enzyme library was constructed. The mutant library was screened with a color detectable high-throughput screening method to obtain the evolved enzymes showing the activity in the presence of a high concentration of dPE. Two mutant enzymes showed higher tolerability to dPE than the wild type enzyme. Each of these enzymes had a single amino acid substitution in a different position (G73S and S214R), and a third mutant enzyme carrying both of these amino acid substitutions was constructed. Escherichia coli transformant cells, which express mutant AADHs, showed activity in the presence of 100mg/ml dPE. A kinetic parameter analysis of the wild type and mutant enzymes was carried out. As compared with the wild type enzyme, the mutant enzymes carrying the S214R amino acid substitution or both the S214R and G73S substitutions showed higher k(cat) values, and the mutant enzymes carrying the G73S amino acid substitution or both the G73S and S214R substitutions showed higher K(m) values. These results suggest that the Ser214 residue plays an important role in enzyme activity, and that the Gly73 residue participates in enzyme-substrate binding.     

Kinetic and mutation analyses of aspartate kinase from Thermus flavus

To reveal the catalytic mechanism of Thermus aspartate kinase, each of 29 amino acid residues that were highly conserved in the sequenced aspartate kinases, was replaced with alanine or leucine by PCR site-directed mutagenesis. Comparison of the kinetic parameters of these mutants with those of the wild-type aspartate kinase suggested that Thr47 was involved in binding aspartate and that Lys7 and Glu74 were involved in catalysis. Analysis of the effective concentrations of magnesium ion on the activity showed that the mutants with replacements at Ser41, Thr47, Asp154 and Asp182 required higher concentrations of magnesium ion. This suggests that these four residues play important roles in the binding of magnesium ions which are required for enzymatic activity.     

大米肽中压离子交换色谱分离及其免疫活性评价

为了建立一种以高效液相色谱检测大米肽中氨基酸含量的方法,以异硫氰酸苯酯（Phenyl isothiocyanate,PITC）为柱前衍生剂,实现大米肽中16种氨基酸（天冬氨酸（Asp）、谷氨酸（Glu）、丝氨酸（Ser）、甘氨酸（Gly）、组氨酸（His）、精氨酸（Arg）、苏氨酸（Thr）、丙氨酸（Ala）、脯氨酸（Pro）、酪氨酸（Tyr）、缬氨酸（Val）、蛋氨酸（Met）、异亮氨酸（Ile）、亮氨酸（Leu）、苯丙氨酸（Phe）、赖氨酸（Lys））能同时、快速、有效地分析检测。采用Athena AAA色谱柱为分析柱,以甲醇:乙腈:水=20:60:20和50 mmol/L乙酸钠（pH6.5）为流动相,进行梯度洗脱,流速为1.0 mL/min,柱温35 ℃,紫外检测器波长为254 nm。结果表明,16种氨基酸在0.125~2.5 μmol/mL范围内具有良好的线性关系,14种氨基酸线性关系的决定系数R²≥0.9999,另外2种氨基酸的决定系数R²分别为0.9998和0.9994,检出限为0.003%~0.018%,定量限为0.010%~0.059%,加标回收率在82.21%~103.59%,RSD均小于5%。该方法使得大米肽中16种氨基酸很好地分离,其中含量最高的氨基酸是谷氨酸（Glu）,含量范围为13.11%±0.39%~21.38%±0.48%,含量最低的氨基酸是组氨酸（His）或蛋氨酸（Met）,含量范围为1.59%±0.05%~2.27%±0.03%或1.26%±0.05%~2.32%±0.06%,其准确度和精密度佳,灵敏度和回收率高,适用于大米肽的氨基酸含量分析。