Search results

1

Polyphenol oxidase from wheat bran is a serpin

100%

Yamasaki Y., Konno H., Noda K.

|

2008

|

vol. 55

|

issue 2

325-328

EN

Polyphenol oxidase (PPO; EC 1.10.3.2) was isolated from wheat bran by a procedure that included ammonium sulfate fractionation, batch adsorption by DEAE-cellulofine, CM-cellulofine column chromatography, DEAE-cellulofine column chromatography, preparative isoelectric focusing, adsorption on the membrane of a Vivapure Q Maxi H spin column, and heat treatment. These procedures led to 150-fold purification with 4.2% recovery. The PPO was homogeneous by SDS/PAGE. The relative molecular weight of the PPO was estimated to be 37000 based on its mobility in SDS/PAGE. The isoelectric point of the PPO was 4.4. The Km values of the PPO for caffeic acid, chlorogenic acid, pyrocatechol, 4-methyl catechol and l-DOPA as substrates were 0.077, 0.198, 1.176, 1.667 and 4.545 mM. The PPO was strongly inhibited by tropolone. The Ki value for tropolone is 2.2 × 10-7 M. The sequence of the 15 N-terminal amino-acid residues was determined to be ATDVRLSIAHQTRFA, which was identical to those of serpin from Triticum aestivum and protein Z from Hordeum vulgare. The PPO strongly inhibited the activity of trypsin, which is an enzyme of serine proteases; 50% inhibition was observed with 1.5 × 10-7 M PPO. The Ki value for PPO is 2.3 × 10-8 M. The wheat bran PPO should be a very important protein for protecting wheat against disease, virus, insect and herbivore damages by both the activities of PPO and protease inhibitor.

2

Pullulanase from rice endosperm

100%

Yamasaki Y., Nakashima S., Konno H.

|

2008

|

vol. 55

|

issue 3

507-510

EN

Pullulanase (EC 3.2.1.41) in non-germinating seeds was compared with that in germinating seeds. Moreover, pullulanase from the endosperm of rice (Oryza sativa L., cv. Hinohikari) seeds was isolated and its properties investigated. The pI value of pullulanase from seeds after 8 days of germination was almost equal to that from non-germinating seeds, which shows that these two enzymes are the same protein. Therefore, the same pullulanase may play roles in both starch synthesis during ripening and starch degradation during germination in rice seeds. The enzyme was isolated by a procedure that included ammonium sulfate fractionation, DEAE-cellulofine column chromatography, preparative isoelectric focusing, and preparative disc gel electrophoresis. The enzyme was homogeneous by SDS/PAGE. The molecular weight of the enzyme was estimated to be 100 000 based on its mobility on SDS/PAGE and 105 000 based on gel filtration with TSKgel super SW 3000, which showed that it was composed of a single unit. The isoelectric point of the enzyme was 4.7. The enzyme was strongly inhibited by β-cyclodextrin. The enzyme was not activated by thiol reagents such as dithiothreitol, 2-mercaptoethanol or glutathione. The enzyme most preferably hydrolyzed pullulan and liberated only maltotriose. The pullulan hydrolysis was strongly inhibited by the substrate at a concentration higher than 0.1%. The degree of inhibition increased with an increase in the concentration of pullulan. However, the enzyme hydrolyzed amylopectin, soluble starch and β-limit dextrin more rapidly as their concentrations increased. The enzyme exhibited α-glucosyltransfer activity and produced an α-1,6-linked compound of two maltotriose molecules from pullulan.

3

A novel α-glucosidase from the moss Scopelophila cataractae

100%

Yamasaki Y., Nakashima S., Konno H.

|

2007

|

vol. 54

|

issue 2

401-406

EN

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 α-glucosidase (EC 3.2.1.20) in BS and LS hydrolyzed amylopectin. The Km value of BS for maltose was 0.427. The Km 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 α-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 Km 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 α-glucosidases. The two enzymes also hydrolyzed β-limit dextrin, which has many α-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 α-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 α-glucosidase. These results show that α-glucosidases should be capable of complete starch digestion by themselves in cells of S. cataractae.

Refine search results

3 Acta Biochimica Polonica

2 2008

1 2007

3 Konno H.

3 Yamasaki Y.

2 Nakashima S.

1 Noda K.

Polyphenol oxidase from wheat bran is a serpin

Pullulanase from rice endosperm

A novel α-glucosidase from the moss Scopelophila cataractae