Bronnenmeier Karin, Kundt Kerstin, Riedel Kathrin, Schwarz Wolfgang H, Staudenbauer Walter L
Institute for Microbiology, Technical University Munich, Arcisstra�e 21, D-80290 M�nchen, Federal Republic of Germany.
Microbiology (Reading). 1997 Mar;143 ( Pt 3):891-898. doi: 10.1099/00221287-143-3-891.
The nucleotide sequence of the celY gene coding for the thermostable exo-1,4-beta-glucanase Avicelase II of Clostridium stercorarium was determined. The gene consists of an ORF of 274Z bp which encodes a preprotein of 914 amino acids with a molecular mass of 103 kDa. The signal-peptide cleavage site was identified by comparison with the N-terminal amino acid sequence of Avicelase II purified from C stercorarium. The celY gene is located in close vicinity to the celZ gene coding for the endo-1,4-beta-glucanase Avicelase I. The CelY-encoding sequence was isolated from genomic DNA of C. stercorarium with the PCR technique. The recombinant enzyme produced in Escherichia coli as a LacZ'-CelY fusion protein could be purified using a simple two-step procedure. The properties of CelY proved to be consistent with those of Avicelase II purified from C. stercorarium. Sequence comparison revealed that CelY consists of an N-terminal catalytic domain flanked by a domain of 95 amino acids with unknown function joined to a type III cellulose-binding domain. The catalytic domain belongs to the recently proposed family L of cellulases (family 48 of glycosyl hydrolases).
测定了编码嗜热栖热梭菌耐热外切 -1,4-β-葡聚糖酶微晶纤维素酶II的celY基因的核苷酸序列。该基因由一个2742 bp的开放阅读框组成，编码一个含有914个氨基酸、分子量为103 kDa的前体蛋白。通过与从嗜热栖热梭菌纯化的微晶纤维素酶II的N端氨基酸序列进行比较，确定了信号肽切割位点。celY基因位于编码内切 -1,4-β-葡聚糖酶微晶纤维素酶I的celZ基因附近。利用PCR技术从嗜热栖热梭菌的基因组DNA中分离出编码CelY的序列。在大肠杆菌中作为LacZ'-CelY融合蛋白产生的重组酶可以通过简单的两步法进行纯化。CelY的性质被证明与从嗜热栖热梭菌纯化的微晶纤维素酶II的性质一致。序列比较显示，CelY由一个N端催化结构域组成，两侧是一个具有未知功能的95个氨基酸的结构域，该结构域与一个III型纤维素结合结构域相连。催化结构域属于最近提出的纤维素酶L家族（糖基水解酶家族48）。
