Ayumi Arai

A new enzyme, maltobionate α-d-glucohydrolase, from alkalophilic Bacillus sp. N-1053

A new enzyme, maltobionate alpha-D-glucohydrolase, was purified to apparent homogeneity from a cell-free extract of alkalophilic Bacillus sp. N-1053 about 930-fold with a yield of 18% and some of its properties were investigated. The enzyme showed optimum activity at about pH 7.0, and was stable over the range of pH 6.0-9.5. The molecular weight was estimated to be 152,000 and 71,000 by HPLC gel filtration on TSKgel G3000SWXL and SDS-polyacrylamide gel electrophoresis, respectively. The enzyme hydrolyzed maltobionate more effectively than disaccharides such as maltose and maltitol or trisaccharides such as maltotrionate, maltotriose and maltotriitol, but showed no activity toward polysaccharides such as amylose, amylopectin and soluble starch. The reaction products from 1 mol of maltobionate were found to be 1 mol of beta-D-glucose and 1 mol of D-gluconate. The Km value for maltobionate was 1.63 mM and the Vmax/Km value for maltobionate was the largest among the substrates tested. The enzyme activity was almost completely inhibited by Hg2+, Ag+, iodine and N-bromosuccinimide, and also inhibited by p-nitrophenyl alpha-D-glucoside, maltose and maltitol.

Molecular cloning and nucleotide sequence of the pyruvate kinase gene of an actinomycete Microbispora thermodiastatica

Abstract The gene for the thermostable pyruvate kinase of Microbispora thermodiastatica IFO 14046, a moderate thermophilic actinomycete, was cloned in Escherichia coli. This gene consists of an open reading frame of 1422 nucleotides and encodes a protein of 474 amino acids with molecular mass of 50 805 Da. The open reading frame was confirmed as the pyruvate kinase gene by comparison with the N-terminal amino acid sequence of the purified pyruvate kinase from M. thermodiastatica.

A novel enzymic determination of maltobionate

Abstract A novel enzymic determination of maltobionate with three enzymes (a new enzyme, maltobionate α- d -glucohydrolase [EC 3.2.1.-], gluconate kinase [EC 2.7.1.12], and 6-phosphogluconic dehydrogenase [EC 1.1.1.44]) is described. It fulfilled the need for an accurate, specific, and rapid assay of maltobionate, as shown by the procedure, the linearity of calibration curve, and the analytical precision, and was simple as compared to an HPLC approach. There was a good correlation ( r = 0.998) between the results obtained by the enzymic and HPLC methods. As an application, the concentrations of maltobionate in the culture broth of Pseudomonas taetrolens IFO 3460 were determined by the proposed method, affording the maltobionate production from maltose in the course of cultivation of this strain.