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首页> 外文期刊>Applied Microbiology >Characterization of a Mannose-6-Phosphate Isomerase from Thermus thermophilus and Increased l-Ribose Production by Its R142N Mutant
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Characterization of a Mannose-6-Phosphate Isomerase from Thermus thermophilus and Increased l-Ribose Production by Its R142N Mutant

机译：嗜热栖热菌甘露糖6-磷酸异构酶的表征及其R142N突变体增加的1-核糖产量

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An uncharacterized gene from Thermus thermophilus , thought to encode a mannose-6-phosphate isomerase, was cloned and expressed in Escherichia coli . The maximal activity of the recombinant enzyme for l-ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu~(2+). Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion of l-ribulose to l-ribose, a potential starting material for many l-nucleoside-based pharmaceutical compounds. The active-site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 142 was correlated with an increase in l-ribulose isomerization activity. The R142N mutant showed the highest activity among mutants modified with Ala, Glu, Tyr, Lys, Asn, or Gln. The specific activity and catalytic efficiency ( k _(cat)/ K_(m) ) for l-ribulose using the R142N mutant were 1.4- and 1.6-fold higher than those of the wild-type enzyme, respectively. The k _(cat)/ K_(m) of the R142N mutant was 3.8-fold higher than that of Geobacillus thermodenitrificans mannose-6-phosphate isomerase, which exhibited the highest activity to date for the previously reported k _(cat)/ K_(m) . The R142N mutant enzyme produced 213 g/liter l-ribose from 300 g/liter l-ribulose for 2 h, with a volumetric productivity of 107 g liter~(?1) h~(?1), which was 1.5-fold higher than that of the wild-type enzyme.
机译：嗜热栖热菌的一个未鉴定基因被认为可以编码6-磷酸甘露糖异构酶，并在大肠杆菌中表达。在存在0.5 mM Cu〜（2+）的条件下，在pH 7.0和75°C下观察到重组酶对1-核糖异构化的最大活性。在所有评估的戊糖和己糖中，该酶表现出最高的活性，可将L-核糖转化为L-核糖，L-核糖是许多基于L-核苷的药物化合物的潜在原料。根据同源性模型预测的活性位点残基分别替换为Ala，第142位的残基与1-核糖异构化活性的增加相关。在用Ala，Glu，Tyr，Lys，Asn或Gln修饰的突变体中，R142N突变体显示出最高的活性。使用R142N突变体的1-核糖的比活性和催化效率（k_（cat）/ K_（m））分别比野生型酶高1.4倍和1.6倍。 R142N突变体的k_（cat）/ K_（m）比热树突土杆菌甘露糖-6-磷酸异构酶高3.8倍，迄今为止，以前报道的k_（cat）/ K_具有最高活性（米）。 R142N突变酶从300克/升的升核糖中发酵2小时可产生213克/升的升核糖，其容积生产率为107克升·（？1）h〜（？1），是后者的1.5倍。比野生型酶要强

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