Over-expression of the gene (bglBC1) from Bacillus circulans encoding an endo-beta-(1-->3),(1-->4)-glucanase useful for the preparation of oligosaccharides from barley beta-glucan.
An endo-beta-(1-->3),(1-->4)-glucanase gene (bglBC1) from Bacillus circulans ATCC21367 was modified by substituting its native promoter with a strong promoter, BJ27X, to increase expression of the gene when cloned into B. subtilis RM125 and B. megaterium ATCC14945. A 771-bp endo-beta-(1-->3),(1-->4)-glucanase open reading frame was inserted into a new shuttle plasmid, pBLC771, by ligating the ORF and pBE1, the latter of which contained the strong promoter, BJ27X. B. subtilis , transformed with the recombinant plasmid pBLC771, produced an extracellular endo-beta-(1-->3),(1-->4)-glucanase that was 130 times (7176 mU ml(-1)) more active than that of the gene donor cells (55 mU ml(-1)), while the enzyme from the transformed B. megaterium was 7 times (378 mU ml(-1)) more active than that of the gene donor cells. M(r) of the enzyme was 28 kDa, with proteolytic processing of the enzyme being observed only in B. subtilis cells. The major products of water-soluble beta-glucan hydrolyzed by over-produced endo-beta-(1-->3),(1-->4)-glucanase were tri- and tetra-oligosaccharides which can be developed as useful products such as anti-hypercholesterolemic, anti-hypertriglyceridemic, and anti-hyperglycemic agents.
Kim
Ki-Hoon
KH
Department of Biomedical Laboratory Science and Biohealth Products Research Center, Inje University, Gimhae, 621-749, Republic of Korea.
Kim
Yea-Oon
YO
Ko
Bong-Sun
BS
Youn
Hyun-Joo
HJ
Lee
Dong-Seok
DS
D000284:Administration, Oral; D000818:Animals; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D002784:Cholesterol / Q000097:blood; D008076:Cholesterol, HDL / Q000097:blood; D003921:Diabetes Mellitus, Experimental / Q000097:blood / Q000139:chemically induced / Q000188:drug therapy*; D044644:Endo-1,3(4)-beta-Glucanase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D009844:Oligosaccharides / Q000008:administration & dosage* / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000096:biosynthesis / Q000737:chemistry; D013311:Streptozocin; D016896:Treatment Outcome; D047071:beta-Glucans / Q000008:administration & dosage / Q000737:chemistry*
D008076:Cholesterol, HDL; D009844:Oligosaccharides; D011994:Recombinant Proteins; D047071:beta-Glucans; D013311:Streptozocin; D002784:Cholesterol; D044644:Endo-1,3(4)-beta-Glucanase