Purification and properties of a novel thermostable galacto-oligosaccharide-producing beta-galactosidase from Sterigmatomyces elviae CBS8119

A thermostable beta-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-beta-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 degrees C. It was stable at temperatures up to 80 degrees 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 Hg2+. The Km values for o-nitrophenyl-beta-D-galactopyranoside and lactose were 9.5 and 2.4 mM, respectively, and the maximum velocities for these substrates were 96 and 240 mumol/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.

[1]  G. McFeters,et al.  Induction and Characterization of β-Galactosidase in an Extreme Thermophile , 1972, Journal of bacteriology.

[2]  J. Segrest,et al.  [5] Molecular weight determination of glycoproteins by polyacrylamide gel electrophoresis in sodium dodecyl sulfate , 1972 .

[3]  N. Asp,et al.  Oligosaccharide formation during hydrolysis of lactose with Saccharomyces lactis lactase (Maxilact®): Part 2—Oligosaccharide structures , 1980 .

[4]  K. Schleifer,et al.  Cloning and characterization of β-galactoside and β-glucoside hydrolysing enzymes of Thermotoga maritima , 1993 .

[5]  R. Rella,et al.  Thermostable β-galactosidase from the archaebacterium Sulfolobus solfataricus Purification and properties , 1990 .

[6]  R. Got,et al.  Isolement, purification et quelques caractéristiques physicochimiques de deux β‐hexosidases du suc digestif d'Helix pomatia , 1968 .

[7]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[8]  J. Bourne,et al.  Formation of oligosaccharides during enzymatic lactose hydrolysis and their importance in a whey hydrolysis process: Part II: Experimental , 1987, Biotechnology and bioengineering.

[9]  R. Goodman,et al.  beta-Galactosidase from Bacillus stearothermophilus. , 1976, Canadian journal of microbiology.

[10]  Takeshi Kobayashi,et al.  Overproduction of thermostable β-galactosidase in Escherichia coli, its purification and molecular structure , 1992 .

[11]  T. Toba,et al.  Oligosaccharide structures formed during the hydrolysis of lactose by Aspergillus oryzae β-galactosidase , 1985 .

[12]  R. E. Huber,et al.  A quantitation of the factors which affect the hydrolase and transgalactosylase activities of beta-galactosidase (E. coli) on lactose. , 1976, Biochemistry.

[13]  J. Cabezas,et al.  β-galactosidase and β-glucosidase activities of the same enzyme from rabbit liver , 1977 .

[14]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[15]  D. Hoover,et al.  BIFIDOBACTERIA : their potential for use in American dairy products , 1991 .

[16]  N. Asp,et al.  Oligosaccharide formation during hydrolysis of lactose with Saccharomyces lactis lactase (MaxilactR): Part 1—Quantitative aspects , 1979 .

[17]  A. Kuroda,et al.  Effects of Administration of TOS and Bifidobacterium breve 4006 on the Human Fecal Flora , 1983 .

[18]  J. Bourne,et al.  Formation of oligosaccharides during enzymatic lactose: Part I: State of art , 1987, Biotechnology and bioengineering.

[19]  R. Shinke,et al.  Purification and properties of a β-galactosidase with high galactosyl transfer activity from Cryptococcus laurentii OKN-4 , 1990 .

[20]  D. Grogan.  Evidence that β-Galactosidase of Sulfolobus solfataricus Is Only One of Several Activities of a Thermostable β-d-Glycosidase , 1991 .

[21]  M. Rosa,et al.  A constitutive beta -galactosidase from the extreme thermoacidophile archaebacterium Caldariella acidophila: properties of the enzyme in the free state and in immobilized whole cells. , 1980 .

[22]  D. Cowan,et al.  β-Galactosidase from a strain of the anaerobic thermophile, Thermoanaerobacter , 1989 .

[23]  K. Nakanishi,et al.  Purification and Properties of β-Galactosidases from Bacillus circulans , 1984 .