Studies on Plant and Microbial β-Amylases

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It has long been known that ungerminated barley contains only β-amylase while α-amylase appears at germination and that there are two types of β-amylases, active and inactive. Many scientists have so far paid much attention to the activation mechanism of the inactive β-amylase. However, their papers were found to have some problems left unresolved. One of the main reasons seems to be due to the failure to isolate inactive β-amylase in a native state and no experiments on it in vitro. Therefore, I attempted to isolate salt-soluble inactive β-amylase and two types of inactive enzymes, heteropolymer type (MW. 280, 000) and homopolymer type (MW. 160, 000) were isolated. From the activation of these enzymes in vitro, it was shown that both reductive cleavage of disulfide bonds and proteolytic cleavage of peptide bonds are necessary for the complete activation of all the types of inactive β-amylases in ungerminated barley. On the other hand, the activation of these amylases during germination was found to proceed mainly through protein disulfide reductase and malt proteinase. On the basis of these results, it was concluded that active β-amylase synthesized in the early stages of barley ripening is polymerized by disulfide bonds and/or peptide bonds into inactive enzymes in the later stages of ripening and that, during germination, the inactive enzymes are again depolymerized into active β-amylase which hydrolyzes starch with α-amylase to supply energy for germination. It is interesting that the reversible reactions between the sulfhydryl and disulfide groups are concerned with the activation and inactivation of β-amylase in barley. In the 1970s the distribution of fl-amylase was reported in microorganisms. We also isolated several microorganisms producing β-amylase from soil. Bacillus cereus BQ10, one of the isolates, was treated by UV irradiation for enhancement of β-amylase productivity. The BQ10-S1, UV-mutant was found to produce about 30 times more β-amylase than the wild strain. Furthermore, a rifampin-resistant, asporogenous mutant, BQ10-S1 Spo-, was obtained by NTG treatment. The amount of β-amylase produced by this asporo-genous mutant reached about 500 times that of the wild strain. The molecular weight of the enzyme was 60, 000 and the optimum pH and temperature were around neutral pH and 55°C. The gene of β-amylase was isolated and cloned in E. coli, and all the base sequences and amino acid sequences were determined. The homology of the amino acid sequences between other plants and microbial β-amylases was compared. About 50% homology was found among those from B. polymyxa, B. circulars, Cl. thermosulfurogenes, etc., and about 30% among those of soybean, barley, sweet potato, etc. Amino acid residues at the catalytic site of β-amylase, which have long remained unelucidated, were examined with B. cereus crystallized β-amylase in enzymatic and X-ray crystallographic studies and found to be not sulfhydryl but two glutamic acid residues. The hydrolytic activity of raw starch by BQ10-S1 Spo-was also found to be due to a "starch-binding domain (SB)" which was not found in plant β-amylases.
日本応用糖質科学会の論文

Studies on Plant and Microbial β-Amylases

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日本応用糖質科学会 | 論文

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