Our stock cultures were screened for microorganisms that can produce galacto-oligosaccharide (Gal-OS) from lactose. Of the 574 strains of bacteria and yeasts tested, Sterigmatomyces elviae CBS8119, Rhodotorula minuta IFO879, and Sirobasidium magnum CBS6803 were found to be efficient producers of Gal-OS from lactose and S. elviae CBS8119 was selected as a representative, high-level producing strain. With toluenetreated resting S. elviae CBS8119 cells, 135 mg of Gal-OS per ml was produced from 360-mg/ml lactose. During this reaction, the by-product glucose was found to inhibit Gal-OS production. Therefore, in order to remove the glucose from the reaction mixture, a culture method in which cell growth followed the enzymatic reaction was devised, which increased the yield of Gal-OS considerably because of the consumption of glucose for cell growth. Under such conditions, 232 mg of Gal-OS per ml was produced from 360-mg/ml lactose after incubation at 30؇C for 60 h. The structure of the major product was identified as O-␤-D-galactopyranosyl-(134)-O-␤-D-galactopyranosyl-(134)-D-glucopyranose (4-galactosyl-lactose) by 13 C nuclear magnetic resonance spectroscopy.

A thermostable ␤-galactosidase which catalyzed the production of galacto-oligosaccharide from lactose was solubilized from a cell wall preparation of Sterigmatomyces elviae CBS8119. The enzyme was purified to homogeneity by means of chromatography on DEAE-Toyopearl, Butyl-Toyopearl, Chromatofocusing, and p-aminobenzyl 1-thio-␤-D-galactopyranoside agarose columns. The molecular weight of the purified enzyme was estimated to be about 170,000 by gel filtration with a Highload-Superdex 200pg column and 86,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its isoelectric point, determined by polyacrylamide gel electrofocusing, was 4.1. The optimal temperature for enzyme activity was 85؇C. It was stable at temperatures up to 80؇C for 1 h. The optimal pH range for the enzyme was 4.5 to 5.0, it was stable at pH 2.5 to 7.0, and its activity was inhibited by Hg 2؉. The K m values for o-nitrophenyl-␤-D-galactopyranoside and lactose were 9.5 and 2.4 mM, respectively, and the maximum velocities for these substrates were 96 and 240 mol/min per mg of protein, respectively. In addition, this enzyme possessed a high level of transgalactosylation activity. Galacto-oligosaccharides, including tri-and tetrasaccharides, were produced with a yield, by weight, of 39% from 200-mg/ml lactose.

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