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首页> 外文期刊>Protein Expression and Purification >Characterization and cloning of chitin deacetylases from Rhizopus circinans
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Characterization and cloning of chitin deacetylases from Rhizopus circinans

机译：圆根霉几丁质脱乙酰基酶的鉴定和克隆

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Chitin deacetylase catalyzes hydrolysis of the acetamido groups of N-acetylglucosamine of chitin in fungal cell walls. Here a chitin deacetylase secreted by Rhizopus circinans was purified to homogeneity and partially characterized. The enzyme exhibits an apparent molecular weight of similar to 75 kDa. At 37 degrees C it shows optimal activity at pH 5.5-6. Its pH stability and thermal stability are good. Mn2+ and Mg2+ slightly enhance the activity of the enzyme and Cu2+ strongly inhibits it. An R. circinans cDNA library was constructed and screened with a homologous probe synthesized by RT-PCR or with synthetic primers derived from the N-terminal amino-acid sequence of the native purified chitin deacetylase. Three chitin deacetylase cDNAs (RC, D2, and I3/2) were isolated from the cDNA library and sequenced. These cDNAs exhibit features characteristic of chitin deacetylase sequences: the presence of a polysaccharide deacetylase domain, a metal-binding triad, the conserved catalytic residues, and high homology with various chitin deacetylase genes. The cDNAs were cloned in a Pichia pastoris expression system and produced as polyhistidine-tagged proteins. Only one recombinant enzyme (called RC) was active under the tested conditions. It was purified to homogeneity in a single step and further characterized. The protein showed an apparent molecular mass of similar to 75 kDa and, like the native enzyme, showed optimal activity at pH 5.5-6 at 37 degrees C. It was strongly inhibited by Cu2+. The isolation of several chitin deacetylase cDNAs from the same microorganism is discussed. (C) 2008 Elsevier Inc. All rights reserved.
机译：几丁质脱乙酰基酶催化真菌细胞壁中几丁质的N-乙酰氨基葡糖的乙酰氨基水解。在这里，由圆根霉分泌的几丁质脱乙酰基酶被纯化至同质并部分表征。该酶的表观分子量约为75 kDa。在37摄氏度时，它在pH 5.5-6时表现出最佳活性。其pH稳定性和热稳定性均良好。 Mn2 +和Mg2 +稍微增强了酶的活性，而Cu2 +则强烈抑制了该酶。构建了圆形马氏酵母cDNA文库，并用通过RT-PCR合成的同源探针或衍生自天然纯化的几丁质脱乙酰基酶N末端氨基酸序列的合成引物进行筛选。从cDNA文库中分离出三个几丁质脱乙酰基酶cDNA（RC，D2和I3 / 2）并进行测序。这些cDNA具有几丁质脱乙酰酶序列特征：多糖脱乙酰酶结构域，金属结合三联体，保守的催化残基以及与各种甲壳质脱乙酰酶基因的高度同源性。将该cDNA克隆到巴斯德毕赤酵母表达系统中，并以多组氨酸标记的蛋白形式产生。在测试条件下，只有一种重组酶（称为RC）具有活性。一步将其纯化至均质并进一步表征。该蛋白质的表观分子量约为75 kDa，与天然酶一样，在37°C的pH 5.5-6下表现出最佳活性。它被Cu2 +强烈抑制。讨论了从同一微生物中分离几种几丁质脱乙酰基酶cDNA的方法。 （C）2008 Elsevier Inc.保留所有权利。

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