A digestive beta-glucosidase from the silkworm, Bombyx mori: cDNA cloning, expression and enzymatic characterization.
A digestive beta-glucosidase cDNA was cloned from the silkworm, Bombyx mori. The B. mori beta-glucosidase cDNA contains an open reading frame of 1473 bp encoding 491 amino acid residues. The B. mori beta-glucosidase possesses the amino acid residues involved in catalysis and substrate binding conserved in glycosyl hydrolase family 1. Southern blot analysis of genomic DNA suggested the B. mori beta-glucosidase to be a single gene. Northern blot analysis of B. mori beta-glucosidase gene confirmed larval midgut-specific expression. The B. mori beta-glucosidase mRNA expression in larval midgut was detectable only during feeding period, whereas its expression was downregulated during starvation. The B. mori beta-glucosidase cDNA was expressed as a 57-kDa polypeptide in baculovirus-infected insect Sf9 cells, and the recombinant beta-glucosidase was active on cellobiose and lactose, but not active on salicin, indicating that the B. mori beta-glucosidase possesses the characteristics of the Class 2 enzyme. The enzyme activity of the purified recombinant beta-glucosidase expressed in baculovirus-infected insect cells was approximately 665 U per microg of recombinant B. mori beta-glucosidase. The purified recombinant B. mori beta-glucosidase showed the highest activity at 35 degrees C and pH 6.0, and were stable at 50 degrees C at least for 10 min. Treatment of recombinant virus-infected Sf9 cells with tunicamycin, a specific inhibitor of N-glycosylation, revealed that the recombinant B. mori beta-glucosidase is N-glycosylated, but the carbohydrate moieties are not essential for enzyme activity.
Byeon
Gyeong Min
GM
College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Korea.
Lee
Kwang Sik
KS
Gui
Zhong Zheng
ZZ
Kim
Iksoo
I
Kang
Pil Don
PD
Lee
Sang Mong
SM
Sohn
Hung Dae
HD
Jin
Byung Rae
BR
D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000235:genetics* / Q000378:metabolism*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D001617:beta-Glucosidase