一种热稳定型甘露醇脱氢酶的表征，该酶来自嗜热热热菌neapolitana DSM 4359，在甘露醇生产中具有潜在的应用前景

Q2 Chemical Engineering Journal of Molecular Catalysis B-enzymatic Pub Date : 2016-12-01 DOI:10.1016/j.molcatb.2016.10.010

Marwa Yagoub Farag Koko, Hinawi Abdo Mustafa Hassanin, Rebaone Letsididi, Tao Zhang, Wanmeng Mu

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引用次数: 7

摘要

甘露醇-2脱氢酶(MtDH)基因(E.C. 1.1.1.67)从热toga neapolitana DSM 4359中克隆，并在大肠杆菌BL21中表达。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)中，纯化酶的预测清晰带为36 kDa，天然分子分子量为135 kDa。d -果糖还原为d -甘露醇的Km和Vmax值分别为20 mM和200 U mg-1。还原方向kcat为180 s−1,kcat/Km为9 mM−1 s−1。该酶的最适pH为6.5，最适温度为90℃，相对活性为100%。纯化后的酶在75°C时相当稳定，在90°C孵育1小时后活性只有初始活性的一半。(TnMtDH)对木糖醇、肌醇、山梨醇、rahmanose、甘露糖和木糖，以及NADPH和NADP+均无活性。一些二价金属的存在增强了酶的活性。利用该酶可在高温条件下生产无其他糖构象的甘露醇。

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Characterization of a thermostable mannitol dehydrogenase from hyperthermophilic Thermotoga neapolitana DSM 4359 with potential application in mannitol production

Mannitol-2-dehydrogenase (MtDH) (E.C. 1.1.1.67) gene was cloned from Thermotoga neapolitana DSM 4359 and expressed in Escherichia coli BL21. The purified enzyme showed a predicted clear band of 36 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), native molecular mas was 135 kDa. K_m and V_max values for reduction of D-fructose to D-mannitol were 20 mM and 200 U mg-1 respectively. k_cat for reduction direction was 180 s⁻¹ and k_cat/K_m were 9 mM⁻¹ s⁻¹. The enzyme showed optimal pH at 6.5 and the optimum temperature was 90 °C with 100% relative activity. The purified enzyme was quite stable at 75 °C and had half of initial activity after 1 h of incubation at 90 °C. (TnMtDH) showed no activity with xylitol, inositol, sorbitol, rahmanose, mannose and xylose, and with NADPH and NADP⁺ as co factors. The presence of some divalent metals in the reaction enhanced the enzyme activity. The enzyme might be utilizing to produce mannitol without other sugar conformation under high temperature.

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来源期刊

Journal of Molecular Catalysis B-enzymatic 生物-生化与分子生物学

CiteScore

2.58

自引率

0.00%

发文量

审稿时长

3.4 months

期刊介绍： Journal of Molecular Catalysis B: Enzymatic is an international forum for researchers and product developers in the applications of whole-cell and cell-free enzymes as catalysts in organic synthesis. Emphasis is on mechanistic and synthetic aspects of the biocatalytic transformation. Papers should report novel and significant advances in one or more of the following topics; Applied and fundamental studies of enzymes used for biocatalysis; Industrial applications of enzymatic processes, e.g. in fine chemical synthesis; Chemo-, regio- and enantioselective transformations; Screening for biocatalysts; Integration of biocatalytic and chemical steps in organic syntheses; Novel biocatalysts, e.g. enzymes from extremophiles and catalytic antibodies; Enzyme immobilization and stabilization, particularly in non-conventional media; Bioprocess engineering aspects, e.g. membrane bioreactors; Improvement of catalytic performance of enzymes, e.g. by protein engineering or chemical modification; Structural studies, including computer simulation, relating to substrate specificity and reaction selectivity; Biomimetic studies related to enzymatic transformations.