Purification of delta-aminolevulinate dehydratase from genetically engineered yeast

Saccharomyces cerevisiae transformed with a multicopy plasmid carrying the yeast structural gene HEM2, which codes for delta-aminolevulinate dehydratase, was enriched 20-fold in the enzyme. Beginning with cell-free extracts of transformed cells, the dehydratase was purified 193-fold to near-homogeneity. This represents a 3900-fold purification relative to the enzyme activity in normal, untransformed yeast cells. The specific activity of the purified enzyme was 16.2 mumol h-1 per mg protein at pH 9.4 and 37.5 degrees C. In most respects the yeast enzyme resembles mammalian enzymes. It is a homo-octamer with an apparent Mr of 275,000, as determined by centrifugation in glycerol density gradients, and under denaturing conditions behaved as a single subunit of Mr congruent to 37,000. The enzyme requires reduced thiol compounds to maintain full activity, and maximum activity was obtained in the presence of 1.0 mM-Zn2+. It is sensitive to inhibition by the heavy metal ions Pb2+ and Cu2+. The enzyme exhibits Michaelis-Menten kinetics and has an apparent Km of 0.359 mM. Like dehydratases from animal tissues, the yeast enzyme is rather thermostable. During the purification process an enhancement in total delta-aminolevulinate dehydratase activity suggested the possibility that removal of an inhibitor of the enzyme could be occurring.     

Production of 5-aminolevulinic acid by Propionibacterium acidipropionici TISTR442

Propionibacterium acidipropionici TISTR442 produced the highest amount of 5-aminolevulinic acid (ALA) when cultivated in medium supplemented with glycine at 18g/l. ALA production correlated with ALA synthase activity, whereas ALA dehydratase activity was maintained at a low level. ALA yield reached 405mg/l after prolonged cultivation for 1 month.     

克雷伯杆菌生产1,3-丙二醇关键酶发酵条件研究

研究了克雷伯杆菌生产1,3-丙二醇关键酶(甘油脱氢酶、1,3-丙二醇氧化还原酶、甘油脱水酶)的发酵条件。研究各种碳源、无机氮源、有机氮源及无机盐对产酶的影响,应用均匀设计、神经网络和遗传算法优化发酵培养基组成,结果为(gL-1):甘油30、KCl 1.6、NH4Cl 6.7、CaCl2 0.28、酵母浸膏2.8。在温度37C、初始pH 7.0、摇床转速200rmin-1和接种量5%条件下,发酵24h,无细胞抽提液中甘油脱氢酶、1,3-丙二醇氧化还原酶和甘油脱水酶活力(U·mg-1)分别为9.16、18.72、0.48,发酵34h,发酵液中1,3-丙二醇(1,3-PD)最大浓度为7.96gL-1。代谢过程中关键酶酶活与1,3-PD峰值出现时间不一致,且提早于1,3-PD峰值的出现。此外,结果表明优化后的培养基去除了多种微量元素,克雷伯杆菌生产1,3-丙二醇关键酶可以在微氧条件下进行。     

gldABC基因与dhaT基因串联表达载体的构建

以肺炎克雷伯氏菌As1．1736的基因组DNA为模板，通过基因扩增仪（PCR）扩增得到了目的基因（g1dABC）并将该基因克隆到pMD19-T Simple载体。通过对该基因的序列分析，甘油脱水酶（g1dABC）结构基因的全长为2816bp。将限制性内切酶处理后的含有自身核糖体结合位点的dhaT基因插入到质粒pMD19-T Simple／g1dABC中g1dABC基因的下游，形成重组克隆质粒pMD19-T Simple／g1dABC-dhaT，并进一步将g1dABC与dhaT串联基因亚克隆到表达载体pET28a（＋）上。     

Isolation of α-Terpineol Dehydratase from Pseudomonas gladioli

An enzyme which converts the citrus by-product limonene to α-terpineol was purified 13-fold. After cell disruption of P. gladioli, α-terpineol dehydratase (α-TD) remained associated with a heterogeneous group of particulate material. α-TD was partially solubilized by extraction with 10 mM HEPES buffer, pH 7.0 containing 2.0% (w/v) Triton X-100 and 0.5M sodium trichloroacetate. Two soluble forms existed in 1.0% (w/v) Triton X-100 with apparent molecular weights of 94,500 and 206,500 daltons.     

5-氨基乙酰丙酸脱水酶缺失对大肠杆菌生长的影响

5-氨基乙酰丙酸(ALA)脱水酶直接关系到ALA的积累,研究该酶突变对细胞的影响对生物法生产ALA具有重要意义,然而目前这方面认识比较混乱.本研究通过无痕敲除Ecoli MG1655菌株的ALA脱水酶的结构基因hemB,发现ALA脱水酶酶活大幅降低,且突变株生长受到极大的影响,但是仍然有10%的残余酶活支持细胞微弱且不...     

dhaT基因克隆及其与gldABC基因串联表达载体的构建

以肺炎克雷伯氏菌As1.1736的基因组DNA为模板,通过PCR扩增得到了目的基因(dhaT)并将该基因克隆至pMD19-T Simple载体。基因的序列分析表明,dhaT基因全长为1164bp,编码387个氨基酸。将含有自身核糖体结合位点的dhaT基因片段插入到pMD19-T Simple/gldABC质粒的gldABC基因下游,形成重组克隆质粒pMD19-T Simple/gldABC-dhaT,并进一步将gldABC与dhaT串联基因亚克隆到表达载体pET28a(+)上。     

过量表达苏氨酸脱水酶对谷氨酸棒杆菌合成L–异亮氨酸的影响

考察过量表达解除反馈抑制的苏氨酸脱水酶对谷氨酸棒杆菌发酵生产L-异亮氨酸的影响.将解除反馈抑制的苏氨酸脱水酶基因ilvA（F383V）连接大肠杆菌/谷氨酸棒杆菌穿梭质粒pXMJ19,过量表达于L-异亮氨酸生产菌株Corynebacterium glutamicum YILW.经5 L发酵罐分批补料发酵产酸实验,与出发菌株C.glutamicum YILW相比,耗糖高峰期滞后,L-异亮氨酸产量增加了10.3%,副产物L-蛋氨酸、L-赖氨酸含量分别降低了33.3%2、6.5%,发酵液中没有L-苏氨酸的累积,乳酸的累积量增加了41.7%.对发酵稳定期的代谢流分布研究表明,过量表达解除反馈抑制的苏氨酸脱水酶使HMP途径流量增加了31.7%,L-异亮氨酸生物合成途径的代谢流提高了8.5%.     

巴斯德梭菌甘油脱水酶基因的克隆表达、纯化及酶学性质的研究

用聚合酶链反应(PCR)技术,从巴斯德梭菌(C lostridium pasteurianum)扩增出甘油脱水酶基因(dhaBCE),在大肠杆菌中高效表达,SDS-PAGE(聚丙烯酰胺凝胶)电泳结果显示,分别有相对分子质量为66、21、16 kD三条特异性蛋白表达条带。用金属镍亲和层析及S-300H凝胶层析将重组蛋白进行分离纯化,所得纯酶的比活为4.26 U/mg。重组酶的最适反应温度42~44℃,最适作用pH=9.10~9.50,它对三个底物结构类似物的米氏常数(Km)值分别是:1,2-丙二醇0.38 mmol/L,甘油0.29 mmol/L,1,2-乙二醇2.0 mmol/L;对辅酶B12的Km值为0.22μmol/L。