title: Oligo-1,6-glucosidase from a thermophile, Bacillus thermoglucosidasius KP1006, was efficiently produced by combinatorial expression of GroEL in Escherichia coli.
abstract: To improve the production of oligo-1,6-glucosidase from the obligately thermophilic Bacillus thermoglucosidasius KP1006 in Escherichia coli, the combined expression of oligo-1,6-glucosidase with various chaperone proteins of Hsp (heat-shock protein) 60 team proteins (GroES and GroEL) or Hsp70 team proteins (GrpE, DnaK and DnaJ) from the same thermophile was examined. This attempt was based on the facts that, (i) among glycosyl hydrolases of Family 13, bacillary oligo-1,6-glucosidases share highest homology with yeast alpha-glucosidase, and (ii) this yeast enzyme interacts with GroEL. In B. thermoglucosidasius Hsp60 team proteins, in particular, GroEL brought about a remarkable rise in expression of B. thermoglucosidasius oligo-1,6-glucosidase, while Hsp70 team proteins had no significant effect. The effect of B. thermoglucosidasius GroEL on oligo-1,6-glucosidase expression was supported by the finding that thermally inactivated B. thermoglucosidasius oligo-1,6-glucosidase was revived by B. thermoglucosidasius GroEL. Although the molecular mass of B. thermoglucosidasius oligo-1,6-glucosidase (66 kDa) exceeds the major range of substrates for GroEL proteins, the GroEL molecules probably recognized the alpha/beta motifs contained in the N-terminal domain and the subdomain of the oligo-1,6-glucosidase. Here we show that (i) the production of B. thermoglucosidasius oligo-1,6-glucosidase in E. coli was improved 3.8-fold by Hsp60 team proteins, (ii) the system can function for the expression of other glycosyl hydrolases of Family 13 that have defects in expression and (iii) the combinatorial expression of thermostable proteins with GroEL from the same thermophile in E. coli can increase the production of thermostable enzymes, preventing problems derived from differences in protein biogenesis.
authors.affiliation: Laboratory of Microbial Physiology and Applied Microbiology, Department of Applied Biochemistry, Kyoto Prefectural University, Shimogamo, Sakyo, Kyoto 606-8522, Japan. kwatanab@kpu.ac.jp
authors.forename: Kunihiko
authors.initials: K
authors.lastname: Watanabe
authors.forename: Hisashi
authors.initials: H
authors.lastname: Fujiwara
authors.forename: Kazuyo
authors.initials: K
authors.lastname: Inui
authors.forename: Yuzuru
authors.initials: Y
authors.lastname: Suzuki
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D018834:Chaperonin 60 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D009834:Oligo-1,6-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D017386:Sequence Homology, Amino Acid
chemical_list: D018834:Chaperonin 60; D009834:Oligo-1,6-Glucosidase