Evidence for Multiple Molecular Forms of Yeast β-Glucosidase in a Hybrid Yeast

A cellobiose-utilizing bacterium isolated from sugar cane bagasse and identified as a strain of Alcaligenes faecalis (ATCC 21400) produced an inducible #3-glucosidesplitting enzyme. The enzyme was purified by a series of streptomycin and ammonium sulfate fractionations and by Sephadex and diethylaminoethyl column chromatography. The final preparation was purified 130-fold, with a recovery of about 10% of the initial enzyme activity. The enzyme had a wide pH range, with optimal activity at pH 6.0 to 7.0. The enzyme was stable in solution at pH 6.5 to 7.8 when kept at 30 C for 2 hr, but it was destroyed by temperatures above 55 C. At 58 and 60 C, the time required to inactivate 90% of the initial activity was 16 and 6.5 min, respectively. An activation energy of 9,500 cal/mole and a Km of 1.25 X 10-4 M were obtained by using p-nitrophenyl ,B-glucoside as a substrate. The Ki value and hydrolysis of cellobiose by the enzyme indicated a high affinity of the enzyme for the cellobiose. The enzyme had its specificity on,3-glucosidic linkage and the rate of hydrolysis of glucosides depended upon the nature of the aglycon moiety. The inactivation studies showed the presence of sulfhydryl groups in the enzyme. The activity of the enzyme was easily destroyed by the Cu++ and Hg++ ions. The Michaelis-Menten relationship and the rate of heat inactivation indicated the presence of one type of noninteracting active site in the bacterial 3-glucosidase. Molecular weight of the enzyme was estimated by gel filtration (Sephadex G-200) and sucrose density gradient, and a value of 120,000 to 160,000 was obtained.

Section: Discussionmentioning

confidence: 99%

Purification and Characterization of β-Glucosidase of Alcaligenes faecalis

Han

Srinivasan

1969

“…We referred earlier to the fact that similar characteristics were displayed by the yeast ,B-glucosidases throughout most of the purification procedure. An exception was that Y-19 enzyme eluted from columns of DEAE cellulose at an applied salt concentration of 0.215 M, whereas Y-18 enzyme eluted at 0.230 (4). Perhaps the most decisive difference between the Y-18 and Y-19 enzymes is their serological specificity.…”

Section: Discussionmentioning

confidence: 99%

“…The enzymes appeared to be characteristic of those obtained from the parent yeast strains. Additional studies, however, suggested that the hydrolytic activity against 13-glucosides in the hybrid yeast might actually result from a population of hybrid molecules with varying compositions of two distinct polypeptides (4). If such were the case, mechanisms which regulate the synthesis of these hybrid enzymes would be unique among the variety of control mechanisms known to exist for other carbohydrase systems in yeasts.…”

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confidence: 99%

“…This report deals primarily with procedures 1 Presented in part at Annual Meetings of the American Society for Microbiology, Cleveland, Ohio, [5][6][7][8][9] May 1963 and Washington, D.C., [3][4][5][6][7] May 1964. The material in this paper is part of a thesis submitted by the senior author to the Graduate School of the University of Kansas in partial fulfillment of the requirements for the Ph.D. degree.…”

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confidence: 99%

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Purification and Characterization of Yeast β-Glucosidases

Fleming

Duerksen

1967

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Constitutive /3-glucosidases from Saccharomyces fragilis (Y-18) and S. dobzhanskii (Y-19) precipitated at the same concentration of ammonium sulfate. The partially purified enzymes had similar activation energies, molecular weights, affinities for certain natural and synthetic j3-glucosides, and optimal pH values for substrate hydrolysis, and they were stable over approximately the same pH range. The enzymes, however, could be clearly distinguished by other criteria. Affinities of the synthetic, sulfur-containing ,B-glucosides for Y-18 enzyme were many times greater than for Y-19 enzyme. The latter enzyme was more resistant to heat. The two enzymes eluted from diethylaminoethyl cellulose at different concentrations of sodium chloride. In precipitin tests, homologous enzyme-antisera systems were highly specific. The ,3-glucosidase synthesized by a hybrid, S. fragilis X S. dobzhanskii (Y-42), was unique. Characterization of this enzyme produced values which were intermediate to those for the enzymes from the parental yeast strains. Heatinactivation slopes and Lineweaver-Burk plots for the Y-42 enzyme were anomalous. It is suggested that hydrolytic activity in Y-42 preparations is due to a spectrum of hybrid enzyme molecules composed of varying amounts of two distinct polypeptides. It is further suggested that these polypeptides may be identical to those synthesized by the parental Y-18 and Y-19 yeast strains.

“…Y-14 was originally isolated by Wickerham and Burton (6). Y-1057 A l Presented in part at the annual meeting of the American Society for Microbiology, Los Angeles, Calif., [1][2][3][4][5] May 1966. The material in this paper is part of a thesis submitted by the senior author to the Graduate School of the University of Kansas in partial fulfillment of the requirements for the Ph.D.…”

Section: Methodsmentioning

confidence: 99%

Purification of β-Glucosidase from Saccharomyces lactis Strains Y-14 and Y-1057A

Marchin

Duerksen

1968

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Two strains of Saccharomyces lactis (Y-14 and Y-1057A), medium (B m ) and low (B 1 ) constitutive producers of β-glucosidase, were grown in enriched medium. β-Glucosidase was extracted by autolysis and purified by ammonium sulfate precipitation, gel filtration, and calcium phosphate gel adsorption-elution. The kinetics of release, purification, and stability of β-glucosidase from strains Y-14 and Y-1057A were compared with the enzyme from strain Y-123. The ability of glycerol, sorbitol, and mannitol to stabilize the β-glucosidases is presented. A lower molecular weight, labile form of the Y-14 enzyme is demonstrated. Differences in the initial specific activites of β-glucosidase among the three strains are discussed.