A novel alpha-glucosidase from the moss Scopelophila cataractae.
Scopelophila cataractae is a rare moss that grows on copper-containing soils. S. cataractae protonema was grown on basal MS medium containing copper. A starch-degrading activity was detected in homogenates of the protonema, after successive extraction with phosphate buffer and buffer containing 3 M LiCl. Buffer-soluble extract (BS) and LiCl-soluble extract (LS) readily hydrolyzed amylopectin to liberate only glucose, which shows that alpha-glucosidase (EC 3.2.1.20) in BS and LS hydrolyzed amylopectin. The K(m) value of BS for maltose was 0.427. The K(m) value of BS for malto-oligosaccharide decreased with an increase in the molecular mass of the substrate. The value for maltohexaose was 0.106, which is about four-fold lower than that for maltose. BS was divided into two fractions of alpha-glucosidase (BS-1 and BS-2) by isoelectric focusing. The isoelectric points of these two enzymes were determined to be 4.36 (BS-1) and 5.25 (BS-2) by analytical gel electrofocusing. The two enzymes readily hydrolyzed malto-oligosaccharides. The two enzymes also hydrolyzed amylose, amylopectin and soluble starch at a rate similar to that with maltose. The two enzymes readily hydrolyzed panose to liberate glucose and maltose (1 : 1), and the K(m) value of BS for panose was similar to that for maltotriose, whereas the enzymes hydrolyzed isomaltose only weakly. With regard to substrate specificity, the two enzymes in BS are novel alpha-glucosidases. The two enzymes also hydrolyzed beta-limit dextrin, which has many alpha-1,6-glucosidic linkages near the non-reducing ends, more strongly than maltose, which shows that they do not need a debranching enzyme for starch digestion. The starch-degrading activity of BS was not inhibited by p-chloromercuribenzoic acid or alpha-amylase inhibitor. When amylopectin was treated with BS and LS in phosphate buffer, pH 6.0, glucose, but not glucose-1-phosphate, was detected, showing that the extracts did not contain phosphorylase but did contain an alpha-glucosidase. These results show that alpha-glucosidases should be capable of complete starch digestion by themselves in cells of S. cataractae.
Yamasaki
Yoshiki
Y
Research Institute for Bioresources, Okayama University, Okayama, Japan. yosikiy@rib.okayama-u.ac.jp
Nakashima
Susumu
S
Konno
Haruyoshi
H
D019068:Bryopsida / Q000201:enzymology*; D002021:Buffers; D007526:Isoelectric Point; D007700:Kinetics; D018021:Lithium Chloride; D006005:Phosphorylases / Q000378:metabolism; D012995:Solubility; D013213:Starch / Q000378:metabolism; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000378:metabolism; D000520:alpha-Glucosidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D001614:beta-Amylase / Q000378:metabolism
D002021:Buffers; D013213:Starch; D006005:Phosphorylases; D000516:alpha-Amylases; D001614:beta-Amylase; D000520:alpha-Glucosidases; D018021:Lithium Chloride