Purification, Immobilization, and Some Properties of Glucose Isomerase from Streptomyces flavogriseus

Abstract

Glucose isomerase (EC 5.3.1.5) produced from Streptomyces flavogriseus was purified by fractionation with (NH4)2SO4 and chromatography on diethylaminoethyl (DEAE)-cellulose and DEAE-Sephadex A-50 columns. The purified enzyme was homogeneous as shown by ultracentrifugation and sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Benzyl DEAE-cellulose, triethylaminoethylcellulose, and DEAE-cellulose were effective in the immobilization of partially purified glucose isomerase. Several differences in properties were found between purified soluble enzyme, immobilized enzyme (DEAE-cellulose-glucose isomerase), and heat-treated whole cells. Glucose and xylose served as substrate for the enzyme. Whole cells had the highest Km values for glucose and xylose; the soluble enzyme had the lowest values. The optimum temperature for activity of the soluble and immobilized enzymes was 700C; that for whole cells was 750C. The pH optimum for the three enzyme preparations was 7.5. Magnesium ion or C02+ was required for enzyme activity; an addition effect resulted from the presence of both Mg2e and Co2+. The enzyme activity was inhibited by Hg2+, Ag+, or Cu2. The conversion ratio of the enzyme for isomerization was about 50%. The soluble and immobilized enzymes showed a greater heat stability than whole cells. The soluble enzyme was stable over a slightly wider pH (5.0 to 9.0) range than the immobilized enzyme and whole cells (pH 5.5 to 9.0). The molecular weight of the enzyme determined by the sedimentation equilibrium method was 171,000. A tetrameric structure for the enzyme was also indicated. After operating at 700C for 5 days, the remaining enzyme activity of the immobilized enzyme and whole cells, which were used for the continuous isomerization of glucose in a plug-flow type of column in the presence of Mg2+ and C02+, was 75 and 55%, respectively. Elimination of C02+ decreased operational stability.

Purification, characterization and immobilization of glucose isomerase from Streptomyces albaduncus

“…In the present study, a promising enzyme activity was observed over the range of 6.5 to 8, with optimum level at pH 7 which is in agreement with that reported by Kawai et al (1994). While, slight alkaline condition, pH 7.5 and 8, are the optimum for the activity of GI enzyme produced by Streptomyces flavogriseus and Streptomyces phaeochromogenes NRRL B-3559, respectively (Standberg and Smiley, 1971;Chen and Anderson, 1979).…”

“…In the present study, the optimum temperature of the enzymatic activity of GI produced by S. albaduncus is 70°C. The same optimum temperature was reported by Chen and Anderson (1979). However, higher optimum temperature (80°C) for GI activity was observed in the study of Azin et al (1997) and Srivastava et al (2010).…”

“…The enzyme homogeneity in the final product was confirmed by formation of single protein band of estimated molecular weight 54 kDa in the SDS-PAGE. Kawai et al (1994) purified GI enzymes from culture supernatant of Bifidobacterium adolescent with estimated molecular weight of 53 kDa, while the GI enzyme produced by Streptomyces flavogriseus has a smaller molecular weight of 43 kDa (Chen and Anderson, 1979).…”

“…After 6 h, the autolyzate was centrifuged and filtered. The filtrate was used as a crude enzyme preparation (Chen and Anderson, 1979).…”

“…This process was carried out as described by Chen and Anderson (1979). Suspension of 500 mg (dry weight) of DEAE-Cellulose in 0.05 M sodium phosphate buffer (pH 7.0) was stirred gently at 4°C.…”