Ozaki K, Shikata S, Kawai S, Ito S, Okamoto K
Tochigi Research Laboratories, Kao Corporation, Tochigi, Japan.
J Gen Microbiol. 1990 Jul;136(7):1327-34. doi: 10.1099/00221287-136-7-1327.
A gene for alkaline cellulase from the alkalophilic Bacillus sp. KSM-635 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. Although the recombinant plasmid contained two HindIII inserts of 2.6 kb and 4.0 kb, the inserts were found to be contiguous in the Bacillus genome by hybridization analysis. Nucleotide sequences of a 2.4 kb region which was indispensable for the production of cellulase, and the flanking, 1.1 kb region, were determined. There was an open reading frame (ORF) of 2823 bp in the 3498 bp sequence determined, which encoded 941 amino acid residues. Two putative ribosome-binding sites and a sigma 43-type, promoter-like sequence were found upstream from an initiation codon in the ORF. The deduced amino-terminal sequence resembles the signal peptide of extracellular proteins. A region of amino acids, 249 to 568, of the deduced amino acid sequence of the cellulase from this organism is homologous with those of alkaline and neutral enzymes of other micro-organisms, but nine amino acid residues were found to be conserved only in the alkaline enzymes.
将嗜碱芽孢杆菌KSM - 635的碱性纤维素酶基因克隆到pBR322的HindIII位点，并在大肠杆菌HB101中表达。虽然重组质粒含有两个分别为2.6 kb和4.0 kb的HindIII插入片段，但通过杂交分析发现这些插入片段在芽孢杆菌基因组中是相邻的。测定了纤维素酶产生所必需的2.4 kb区域及其侧翼1.1 kb区域的核苷酸序列。在所测定的3498 bp序列中有一个2823 bp的开放阅读框（ORF），编码941个氨基酸残基。在该ORF的起始密码子上游发现了两个假定的核糖体结合位点和一个σ43型启动子样序列。推导的氨基末端序列类似于细胞外蛋白的信号肽。该生物体纤维素酶推导氨基酸序列中249至568位的氨基酸区域与其他微生物的碱性和中性酶的相应区域同源，但发现有九个氨基酸残基仅在碱性酶中保守。
