野生和 EMS 变异克雷伯氏菌 JCM1665 合成的罗丹锰的特征

IF 4.8 Q1 AGRICULTURE, MULTIDISCIPLINARY
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引用次数: 0

摘要

氰化物中毒仍然是全球军民关注的一个重大问题。采用标准技术,纯化并鉴定了由氧合克雷伯氏菌 JCM 1665 野生菌株(KOJCM1665)和两个选定突变菌株(KOJCM1665c 和 KOJCM1665d)产生的氰化物解毒酶--罗丹锰。野生菌和所选突变体的细胞外络合锰产量分别为 6.2 ± 0.6、26.7 ± 0.7 和 18.3 ± 0.3 U/mL。纯化后,野生型、KOJCM 1665c 和 KOJCM 1665d 的络锰活性总回收率和折合率分别为 49%、66% 和 58%,以及 2.6%、2.6% 和 2.3%。三种酶制剂的原生分子量分别为 35.1、34.9 和 34.8 kDa,而所有酶的亚基分子量均为 35 kDa。在 50 °C 和 pH 值为 6.0 时,酶的活性达到最佳。三种酶制剂以 KCN 为底物的实际千米数分别为 1.03 × 10-3、0.95 × 10-3 和 0.80 × 10-3 M,而以第二种底物(Na2S2O3)为底物的千米数分别为 0.59 × 10-3、0.49 × 10-3 和 0.69 × 10-3 M。底物特异性研究表明,该酶偏爱硫代硫酸钠作为底物。Na+ 和 K+ 等金属离子对酶活性的抑制作用明显更大。该研究证明了增强 K. oxytoca JCM 1665 过度表达胞外络合锰的潜力,而该酶的理化特性也有望成为提高氰化物生物修复过程效率的目标。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Characterization of rhodanese synthesized by the wild and EMS-mutated Klebsiella oxytoca JCM1665

Characterization of rhodanese synthesized by the wild and EMS-mutated Klebsiella oxytoca JCM1665

Cyanide poisoning remains a significant concern for both civilian and military personnel worldwide. Rhodanese, a cyanide detoxify enzyme produced by the wild (KOJCM1665) and two selected mutant strains (KOJCM1665c and KOJCM1665d) of Klebsiella oxytoca JCM 1665 was purified and characterized using a combination of standard techniques. Extracellular rhodanese yield of the wild and selected mutants were 6.2 ± 0.6, 26.7 ± 0.7 and 18.3 ± 0.3 U/mL respectively. The overall levels of recovery of rhodanese activity and fold after purification were 49 %, 66 %, and 58 % and 2.6, 2.6, and 2.3 % for the wild, KOJCM 1665c, and KOJCM 1665d, respectively. The native molecular weights of the three enzyme preparations were 35.1, 34.9, and 34.8 kDa, while the subunit molecular weight was 35 kDa for all the enzymes. The optimum activity of the enzymes was observed at 50 °C and pH 6.0. The real km of the three enzyme preparations for KCN as a substrate were 1.03 × 10−3, 0.95 × 10−3, and 0.80 × 10−3 M, respectively, while the km for the second substrate (Na2S2O3) were 0.59 × 10−3, 0.49 × 10−3, and 0.69 × 10−3 M, respectively. The substrate specificity study showed that the enzyme preferred sodium thiosulfate as the substrate. Metal ions such as Na+ and K+ had significantly greater inhibitory effects on enzyme activity. This study demonstrated the potential of enhancing K. oxytoca JCM 1665 to overexpress extracellular rhodanese, and the physicochemical properties of the enzyme hold significant promise as a target for improving the efficiency of cyanide bioremediation processes.

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来源期刊
CiteScore
5.40
自引率
2.60%
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审稿时长
69 days
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