Rixon J E, Ferreira L M, Durrant A J, Laurie J I, Hazlewood G P, Gilbert H J
Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, U.K.
Biochem J. 1992 Aug 1;285 ( Pt 3)(Pt 3):947-55. doi: 10.1042/bj2850947.
A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA constructed in pUC18 and expressed in Escherichia coli was screened for recombinants expressing 4-methylumbelliferyl beta-D-glucoside hydrolysing activity (MUGase). A single MUGase-positive clone was isolated. The MUGase hydrolysed cellobiose, cellotriose, cellotetraose, cellopentaose and cellohexaose to glucose, by sequentially cleaving glucose residues from the non-reducing end of the cello-oligosaccharides. The Km values for cellobiose and cellohexaose hydrolysis were 1.2 mM and 28 microM respectively. The enzyme exhibited no activity against soluble or insoluble cellulose, xylan and xylobiose. Thus the MUGase is classified as a 1,4-beta-D-glucan glucohydrolase (EC 3.2.1.74) and is designated 1,4-beta-D-glucan glucohydrolase D (CELD). When expressed by E. coli, CELD was located in the cell-envelope fraction; a significant proportion of the native enzyme was also associated with the cell envelope when synthesized by its endogenous host. The nucleotide sequence of the gene, celD, which encodes CELD, revealed an open reading frame of 2607 bp, encoding a protein of M(r) 92,000. The deduced primary structure of CELD was confirmed by the M(r) of CELD (85,000) expressed by E. coli and P. fluorescens subsp. cellulosa, and by the experimentally determined N-terminus of the enzyme purified from E. coli, which showed identity with residues 52-67 of the celD translated sequence. The structure of the N-terminal region of full-length CELD was similar to the signal peptides of P. fluorescens subsp. cellulosa plant-cell-wall hydrolases. Deletion of the N-terminal 47 residues of CELD solubilized MUGase activity in E. coli. CELD exhibited sequence similarity with beta-glucosidase B of Clostridium thermocellum, particularly in the vicinity of the active-site aspartate residue, but did not display structural similarity with the mature forms of cellulases and xylanases expressed by P. fluorescens subsp. cellulosa.
对构建于pUC18中并在大肠杆菌中表达的荧光假单胞菌纤维素亚种DNA的基因组文库进行筛选，以寻找表达4-甲基伞形酮基β-D-葡萄糖苷水解活性（MUG酶）的重组体。分离出一个MUG酶阳性克隆。该MUG酶通过依次从纤维寡糖的非还原端切割葡萄糖残基，将纤维二糖、纤维三糖、纤维四糖、纤维五糖和纤维六糖水解为葡萄糖。纤维二糖和纤维六糖水解的Km值分别为1.2 mM和28 μM。该酶对可溶性或不溶性纤维素、木聚糖和木二糖无活性。因此，MUG酶被归类为1,4-β-D-葡聚糖葡糖水解酶（EC 3.2.1.74），并被命名为1,4-β-D-葡聚糖葡糖水解酶D（CELD）。当由大肠杆菌表达时，CELD位于细胞包膜部分；当由其内源宿主合成时，相当一部分天然酶也与细胞包膜相关。编码CELD的基因celD的核苷酸序列显示有一个2607 bp的开放阅读框，编码一个Mr为92,000的蛋白质。通过大肠杆菌和荧光假单胞菌纤维素亚种表达的CELD（85,000）的Mr，以及从大肠杆菌中纯化的酶的实验测定的N端，证实了CELD推导的一级结构，其与celD翻译序列的52-67位残基相同。全长CELD的N端区域结构与荧光假单胞菌纤维素亚种植物细胞壁水解酶的信号肽相似。在大肠杆菌中删除CELD的N端47个残基可使MUG酶活性溶解。CELD与嗜热栖热梭菌的β-葡萄糖苷酶B表现出序列相似性，特别是在活性位点天冬氨酸残基附近，但与荧光假单胞菌纤维素亚种表达的纤维素酶和木聚糖酶的成熟形式没有结构相似性。
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