Cloning and recombinant expression of a cellulase from the cellulolytic strain Streptomyces sp. G12 isolated from compost.
Cloning and recombinant expression of a cellulase from the cellulolytic strain Streptomyces sp. G12 isolated from compost.
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来自纤维素分解菌株链霉菌的纤维素酶的克隆和重组表达。 G12 从堆肥中分离出来。
DOI：
10.1186/1475-2859-11-164
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发表时间：
2012-12-26
影响因子：
6.4
通讯作者：
Faraco V
中科院分区：
文献类型：
--
作者：
Amore A;Pepe O;Ventorino V;Birolo L;Giangrande C;Faraco V
使用木质纤维素材料生产第二代乙醇将带来几个好处，例如最大限度地减少粮食生产和燃料生产的土地利用之间的冲突，提供比传统农业原料更便宜的原材料，允许比第一代乙醇更低的温室气体排放。然而，纤维素生物燃料的生产还没有达到具有竞争力的水平，主要是因为纤维素酶的生产成本很高。因此，本研究旨在发现新的纤维素分解微生物和酶。从蔬菜加工工业废弃物的原料堆肥中分离到不同的细菌，对它们在含有羧甲基纤维素的固体基质上的纤维素分解活性进行了筛选。根据放线菌群的表型特征和在含有羧甲基纤维素的固体培养基上的纤维分解活性，筛选出4株放线菌。经16S rRNA测序鉴定为链霉菌属，命名为链霉菌属(Streptomyces sp.)菌株G12。用蛋白质组学方法对链霉菌G12产生的纤维素酶活性进行了研究，鉴定出两种内切葡聚糖酶。对其中一种酶的编码基因CelStrep进行了克隆和测序。分子分析表明，该基因有一个开放阅读框，编码379个氨基酸残基的蛋白质，其中包括一个37个氨基酸残基的信号肽。与其他纤维素酶的氨基酸序列比较表明，CelStrep酶属于12糖苷水解酶家族。在大肠杆菌中进行了CelStrep的异源重组表达，并从培养上清液中纯化了具有活性的重组酶，并对其进行了鉴定。其催化羧甲基纤维素水解符合米氏动力学，Km值为9.13 mg/ml，Vmax为3469 μM -1。该酶在60℃和50℃下的半衰期分别为24 h和96 h，在40℃下96 h的半衰期保持在80%左右。G12，从工业废渣堆肥中分离、鉴定了可能影响其纤维分解活性的酶，克隆和重组了链霉菌编码基因。G12纤维素酶CelStrep的特性表明，该酶表现出相应的耐热性，提高了其转化纤维素的潜力。
The use of lignocellulosic materials for second generation ethanol production would give several advantages such as minimizing the conflict between land use for food and fuel production, providing less expensive raw materials than conventional agricultural feedstock, allowing lower greenhouse gas emissions than those of first generation ethanol. However, cellulosic biofuels are not produced at a competitive level yet, mainly because of the high production costs of the cellulolytic enzymes. Therefore, this study was aimed at discovering new cellulolytic microorganisms and enzymes. Different bacteria isolated from raw composting materials obtained from vegetable processing industry wastes were screened for their cellulolytic activity on solid medium containing carboxymethylcellulose. Four strains belonging to the actinomycetes group were selected on the basis of their phenotypic traits and cellulolytic activity on solid medium containing carboxymethylcellulose. The strain showing the highest cellulolytic activity was identified by 16S rRNA sequencing as belonging to Streptomyces genus and it was designated as Streptomyces sp. strain G12. Investigating the enzymes responsible for cellulase activity produced by Streptomyces G12 by proteomic analyses, two endoglucanases were identified. Gene coding for one of these enzymes, named CelStrep, was cloned and sequenced. Molecular analysis showed that the celstrep gene has an open reading frame encoding a protein of 379 amino acid residues, including a signal peptide of 37 amino acid residues. Comparison of deduced aminoacidic sequence to the other cellulases indicated that the enzyme CelStrep can be classified as a family 12 glycoside hydrolase. Heterologous recombinant expression of CelStrep was carried out in Escherichia coli, and the active recombinant enzyme was purified from culture supernatant and characterized. It catalyzes the hydrolysis of carboxymethylcellulose following a Michaelis–Menten kinetics with a KM of 9.13 mg/ml and a vmax of 3469 μM min-1. The enzyme exhibits a half life of around 24 h and 96 h at 60°C and 50°C, respectively and shows a retention of around 80% of activity after 96 h at 40°C. In this manuscript, we describe the isolation of a new cellulolytic strain, Streptomyces sp. G12, from industrial waste based compost, the identification of the enzymes putatively responsible for its cellulolytic activity, the cloning and the recombinant expression of the gene coding for the Streptomyces sp. G12 cellulase CelStrep, that was characterized showing to exhibit a relevant thermoresistance increasing its potential for cellulose conversion.
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影响因子：
2.9
作者：
Cheng, Ya-Shan;Ko, Tzu-Ping;Guo, Rey-Ting
通讯作者：
Guo, Rey-Ting
影响因子：
5
作者：
Deng, Yu;Fong, Stephen S.
通讯作者：
Fong, Stephen S.
DOI：
10.1111/j.1749-6632.1996.tb40558.x
发表时间：
1996-01-01
期刊：
RECOMBINANT DNA BIOTECHNOLOGY III: THE INTEGRATION OF BIOLOGICAL AND ENGINEERING SCIENCES
影响因子：
--
作者：
Irdani, T;Perito, B;Mastromei, G
通讯作者：
Mastromei, G
DOI：
10.1111/j.1365-2672.1995.tb03160.x
发表时间：
1995-10-01
期刊：
JOURNAL OF APPLIED BACTERIOLOGY
影响因子：
--
作者：
LIN, SB;STUTZENBERGER, FJ
通讯作者：
STUTZENBERGER, FJ
影响因子：
2.9
作者：
BEGUIN, P
通讯作者：
BEGUIN, P