Hazlewood G P, Davidson K, Laurie J I, Romaniec M P, Gilbert H J
Department of Biochemistry, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, UK.
J Gen Microbiol. 1990 Oct;136(10):2089-97. doi: 10.1099/00221287-136-10-2089.
Genomic DNA from Butyrivibrio fibrisolvens strain A46 was digested with EcoRI and ligated into lambda gt11. Two recombinant phages isolated from the gene bank hydrolysed carboxymethylcellulose and were shown to contain the same 2.3 kb EcoRI restriction fragment, which was cloned into pUC12 to generate pBA46. Escherichia coli JM83 harbouring pBA46 expressed an endoglucanase (EGA) which hydrolysed a range of other substrates including barley beta-glucan, Avicel, filter paper and p-nitrophenyl beta-D-cellobioside. Nucleotide sequencing of the B. fibrisolvens strain A46 DNA cloned in pBA46 revealed a single open reading frame (ORF) of 1296 bp, encoding a protein of 48,863 Da. Confirmation that the ORF coded for EGA was obtained by comparing the N-terminal sequence of the purified endoglucanase with that deduced from the nucleotide sequence. EGA contains a typical prokaryotic signal peptide at its N-terminus and shows some homology with the Bacillus family of cellulases. The enzyme does not contain distinct functional domains, which are prevalent in cellulases from Pseudomonas fluorescens subsp. cellulosa and Cellulomonas fimi.
用EcoRI消化溶纤维丁酸弧菌A46菌株的基因组DNA，并将其连接到λgt11中。从基因文库中分离出的两个重组噬菌体可水解羧甲基纤维素，且显示含有相同的2.3 kb EcoRI限制性片段，该片段被克隆到pUC12中以产生pBA46。携带pBA46的大肠杆菌JM83表达一种内切葡聚糖酶（EGA），该酶可水解一系列其他底物，包括大麦β-葡聚糖、微晶纤维素、滤纸和对硝基苯基β-D-纤维二糖苷。对克隆于pBA46中的溶纤维丁酸弧菌A46菌株DNA进行核苷酸测序，发现一个1296 bp的单一开放阅读框（ORF），编码一个48,863 Da的蛋白质。通过比较纯化的内切葡聚糖酶的N端序列与从核苷酸序列推导的序列，证实该ORF编码EGA。EGA在其N端含有一个典型的原核信号肽，与芽孢杆菌属纤维素酶家族有一些同源性。该酶不包含在荧光假单胞菌纤维素亚种和纤维单胞菌的纤维素酶中普遍存在的独特功能域。
