The enzyme α-galactosidase (α-D-galactoside galactohydrolase; EC 3.2.1.22) catalyzes the hydrolysis of α-1,6-linked galactose residues in oligosaccharides and polymeric galactomannan. The α-galactosidases are of particular interest in view of their many potential biotechnological and medical applications. These enzymes have found wide use in various industries such as food and feed, sugar and paper and pulp for the removal of raffinose and stachyose. They are also important medically for blood group conversion and in the treatment of Fabry disease. Most of the research on α-galactosidases has focused on their isolation from various microbial sources. In the last decade, cloning of novel α-galactosidase genes and their heterologous expression has gained momentum. The present review focuses on the production of α-galactosidases from bacteria, fungi and yeast, and discusses their properties. Recent progress on cloning and heterologous expression in various hosts is summarized with special emphasis on their application in various fields.

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80 degrees C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70 degrees C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and alpha-D-glucose-1-phosphate. The Km for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the kcat was 5.4 s(-1). In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-beta-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s(-1)) kcat followed by 6-deoxy-D-glucose (17 s(-1)) and 2-deoxy-D-glucose (16 s(-1)). The natural substrate, D-glucose with the kcat of 8.0 s(-1) had the highest (1.1 x 10(4) M(-1) s(-1)) kcat/Km compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, alpha-D-glucose-1-phosphate, at higher concentrations.

The overall amino-acid sequence of MmcBC in Pelotomaculum thermopropionicum was substantially homologous (33%) to fumarase A in Escherichia coli, although its possible subunit structure was different from known fumarases and it lacked the fumarate-lyase signature sequence. Here, MmcBC in E. coli is expressed and characterized. The purified enzyme catalyzed reversible conversion of fumarate to L-malate at an optimum temperature of 70 degrees C. Its molecular size was 64.2 kDa, indicating that it consisted of one MmcB and one MmcC. EPR spectra revealed that it had an oxygen-sensitive [4Fe-4S] cluster. We propose that MmcBC represents a novel group of prokaryotic fumarases.

E. RAJASHEKHARA, E.R. SURESH AND s. ETHIRAJ. 1996. A heat-resistant mold identified as a strain of Neosartorya jischeri was isolated from microbiologically spoiled papaya fruits. The optimum heat activation temperature and time for the ascospores of the test mold was found to be 80°C for 15-30 min. The decimal reduction times (D-values) at 85", 87" and 89°C in phosphate buffer (pH 7.0) as heating medium were 35.25, 11.1 and 3.90 min respectively and hence the calculated z-value was 4.0"C. In grape and mango juices as heating media, the D,,oc and the DsjOc values were increased as the "Brix level raised from 10 to 45. In commercial fruit juices of mango, orange, pineapple and mango-pineapple blend as heating media DRsOc values were greater than those observed for phosphate buffer.

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