The nucleotide sequence of the β-glucosidase gene from Cellvibrio gilvus

Kashiwagi, Yutaka; Aoyagi, Chika; Sasaki, Takashi; Taniguchi, Hajime

doi:10.1016/0922-338x(93)90108-k

Cited by 16 publications

(15 citation statements)

References 25 publications

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“…The ␤-glucosidases from C. gilvus share conserved regions in ␤-glucosidases from different organisms. The nucleotide sequence of the ␤-glucosidase gene revealed that this enzyme belongs to the BGB group of ␤-glucosidases (15). The amino acid sequences of the C. gilvus ␤-glucosidase gene show significant similarity (about 40%) with those of a ␤-glucosidase gene from Agrobacterium tumefaciens (18).…”

mentioning

confidence: 83%

“…carrying C. gilvus (15,16) and A. tumefaciens (18) ␤-glucosidase genes, respectively. DNA Manipulation-Recombinant DNA techniques and methods for agarose gel electrophoresis were followed as described by Sambrook et al (22).…”

Section: Bacterial Strains and Plasmidsmentioning

confidence: 99%

“…The isolation and characterization of the cellulase, xylanase, and ␤-glucosidase systems of this organism (13)(14)(15) as well as the cloning, analysis, and manipulation of the genes coding these enzymes (16,17) have been investigated in our laboratory. The ␤-glucosidases from C. gilvus share conserved regions in ␤-glucosidases from different organisms.…”

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confidence: 99%

“…1 Although, the deletion of about 100 amino acid residues near the C-terminal region of the ␣-amylase gene did not affect enzyme activity (19), cyclomaltodextrin glucanotransferases lacking 30 amino acids (20) and an endoglucanase lacking 75 amino acids (21) from the C-terminal end showed no enzyme activity. Keeping in mind the importance of the C-terminal region and the estimated location of the catalytic center of Asp-291 in the N-terminal region of C. gilvus (15), the C-terminal region was selected for the construction of chimeric enzymes.…”

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Construction of Chimeric β-Glucosidases with Improved Enzymatic Properties

Singh

Hayashi

1995

Journal of Biological Chemistry

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The amino acid sequences of ␤-glucosidases from Cellvibrio gilvus and Agrobacterium tumefaciens show about 40% similarity. The pH/temperature optima and stabilities and substrate specificities of the two enzymes are quite different. C. gilvus ␤-glucosidase exhibits an optimum pH of 6.2-6.4 and temperature of 35°C, whereas the corresponding values for A. tumefaciens are 7.2-7.4 and 60°C, respectively. The substrate specificity of A. tumefaciens enzyme toward different aryl glycosides is broader than C. gilvus enzyme. To analyze these properties further, three chimeric ␤-glucosidases were constructed by substituting segments from the Cterminal homologous region of C. gilvus ␤-glucosidase gene with that of A. tumefaciens. The chimeric enzymes were characterized with respect to pH/temperature activity and stability and substrate specificity. Chimeric enzymes exhibited chromatographic behavior similar to that of C. gilvus enzyme. However, enzymatic properties of chimeras were admixtures of those of the two parents. The chimeric enzymes were optimally active at 45-50°C and pH 6.6 -7.0. K m values of chimeric enzymes for the various saccharides were admixtures of both parental enzymes. These results suggest that the two domains of C. gilvus and A. tumefaciens enzymes probably can fold independently. The homologous C-terminal region in ␤-glucosidase appears to play an important role in determining enzyme characteristics. Changes in the properties on substitution of segments in this region might be related to the enzyme specificity, and ␤-glucosidases with improved properties can be prepared by manipulating this region.The enzyme ␤-glucosidase (EC 3.2.1.21) catalyzes the hydrolysis of alkyl-and aryl-␤-D-glucosides (methyl-␤-D-glucoside and p-nitrophenyl-␤-D-glucoside) as well as glycosides containing only carbohydrate residues (Cellobiose). On the basis of substrate specificity, ␤-glucosidases can be classified as aryl-␤-glucosidases, cellobiases, and those hydrolyzing both aryl-␤-glucosides and oligosaccharides. The last group is often found in cellulolytic microorganisms (1, 2). On the basis of sequence homology, ␤-glucosidases have been divided into two subfamilies (2): BGA (␤-glucosidases and phospho-␤-glucosidases from bacteria to mammals) and BGB (␤-glucosidases from yeasts, molds, and rumen bacteria). It is one of the components of the cellulase enzyme complex required for the hydrolysis of cellulose to glucose by catalyzing the final step which converts cellobiose to glucose (3, 4).The study of these enzymes has been facilitated by the use of recombinant DNA technology (1,5,6). Although a number of cellulase genes including several ␤-glucosidases have been cloned and expressed in both Escherichia coli and Saccharomyces cerevisiae (7-10), their enzymological properties, especially structure-function relationships, have not been well understood, partially because most of the cellulases show little sequence homology. Analysis of structure-function relationships may be facilitated by the formation of chimeric genes/e...

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confidence: 83%

Section: Bacterial Strains and Plasmidsmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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Construction of Chimeric β-Glucosidases with Improved Enzymatic Properties

Singh

Hayashi

1995

Journal of Biological Chemistry

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“…E. coli JM109 was used for transformation. Plasmid pCG5 and pMU1 were the recombinant plasmids carrying Cellvibrio gilvus (Kashiwagi et al, 1993) and Ruminococcus albus ß-glucosidase genes, respectively. DNA manipulations: Plasmid DNA was prepared by using a kit (QIAprep Spin Miniprep, Qiagen, USA).…”

Section: Methodsmentioning

confidence: 99%