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首页> 外文期刊>Applied and Environmental Microbiology >Molecular cloning and expression in Escherichia coli of a cellodextrinase gene from Bacteroides succinogenes S85.
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Molecular cloning and expression in Escherichia coli of a cellodextrinase gene from Bacteroides succinogenes S85.

机译：产自琥珀酸杆菌S85的纤维糊精酶基因的分子克隆和在大肠杆菌中的表达。

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摘要

A DNA fragment coding for a cellodextrinase of Bacteroides succinogenes S85 was isolated by screening of a pBR322 gene library in Escherichia coli HB101. Of 100,000 colonies screened on a complex medium with methylumbelliferyl-beta-D-cellobioside as the indicator substrate, two cellodextrinase-positive clones (CB1 and CB2) were isolated. The DNA inserts from the two recombinant plasmids were 7.7 kilobase pairs in size and had similar restriction maps. After subcloning from pCB2, a 2.5-kilobase-pair insert which coded for cellodextrinase activity was isolated. The enzyme was located in the cytoplasm of the E. coli host. It exhibited no activity on carboxymethyl cellulose, Avicel microcrystalline cellulose, acid-swollen cellulose, or cellobiose but hydrolyzed p-nitrophenyl-beta-D-cellobioside and p-nitrophenyl-beta-D-lactoside. The Km (0.1 mM) for the hydrolysis of p-nitrophenyl-cellobioside by the enzyme expressed in E. coli was similar to that reported for the purified enzyme from B. succinogenes. Expression of the cellodextrinase gene was subjected to catabolite repression by glucose and was not induced by cellobiose. The origin of the DNA insert from B. succinogenes was confirmed by Southern blot analysis. Western blotting (immunoblotting) using antibodies raised against the purified B. succinogenes cellodextrinase revealed a protein with a molecular weight of approximately 50,000 in E. coli clones which comigrated with the native enzyme isolated from B. succinogenes. These data indicate that the cellodextrinase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to those of the native enzyme.
机译：通过在大肠杆菌HB101中筛选pBR322基因文库，分离出编码琥珀酸杆菌属细菌S85的纤维糊精酶的DNA片段。在以甲基伞形基-β-D-纤维二糖苷为指示物的复合培养基上筛选的100,000个菌落中，分离出两个纤维糊精酶阳性克隆（CB1和CB2）。来自两个重组质粒的DNA插入片段大小为7.7kb，并具有相似的限制性酶切图。从pCB2亚克隆后，分离出编码纤维素糊精酶活性的2.5碱基对插入物。该酶位于大肠杆菌宿主的细胞质中。它对羧甲基纤维素，Avicel微晶纤维素，酸膨胀纤维素或纤维二糖没有活性，但水解了对硝基苯基-β-D-纤维二糖苷和对硝基苯基-β-D-乳糖苷。用大肠杆菌中表达的酶水解对硝基苯基纤维二糖苷的Km（0.1 mM）与从琥珀酸芽孢杆菌中纯化的酶报道的Km（0.1 mM）相似。纤维糊精酶基因的表达通过葡萄糖进行分解代谢物抑制，而不受纤维二糖诱导。通过Southern印迹分析证实了来自琥珀酸芽孢杆菌的DNA插入物的来源。使用针对纯化的琥珀酸双歧杆菌纤维糊精酶的抗体进行的蛋白质印迹（免疫印迹）显示，在大肠杆菌克隆中分子量约为50,000的蛋白质与从琥珀酸双歧杆菌分离的天然酶竞争。这些数据表明在大肠杆菌中表达的纤维糊精酶基因是完全功能性的，并编码具有与天然酶相似性质的酶。

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