A novel member of glycoside hydrolase family 88: overexpression, purification, and characterization of unsaturated beta-glucuronyl hydrolase of Bacillus sp. GL1.
Unsaturated beta-glucuronyl hydrolase of Bacillus sp. GL1 catalyzes the hydrolytic release of unsaturated glucuronic acids from oligosaccharides produced through the reactions of polysaccharide lyases such as gellan, xanthan, hyaluronate, and chondroitin lyases. An overexpression system for the enzyme was constructed in Escherichia coli cells involving regulation of the enzyme gene under the T7 promoter and terminator. The expression level of the enzyme in E. coli cells was 250-fold higher than that in Bacillus sp. GL1 cells. The enzyme expressed in E. coli cells was purified and characterized. The optimal pH and temperature, and substrate specificity of the purified enzyme were similar to those of the native enzyme from Bacillus sp. GL1 cells, although the enzyme expressed in E. coli cells underwent self-assembly into polymeric forms through the formation of intermolecular disulfide bonds. Circular dichroism analysis indicated that the secondary structure of the enzyme was rich in alpha-helices. Genes showing high identity (over 40% identity) with that of the enzyme were found in the genomes of some pathogenic bacteria, such as Streptococcus pyogenes and Streptococcus pneumoniae, which cause serious diseases (e.g., meningitis and pneumonia). Therefore, the enzyme of Bacillus sp. GL1 and the streptococcal proteins form a new glycoside hydrolase family, 88.
Division of Food and Biological Science, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Shigetarou
S
Mori
Sae
S
Akao
Hirokazu
H
Nankai
Wataru
W
Hashimoto
Bunzo
B
Mikami
Kousaku
K
Murata
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D002240:Carbohydrate Sequence; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D004220:Disulfides; D006026:Glycoside Hydrolases; D011133:Polysaccharide-Lyases