Shigeo Suzuki

Purification and some properties of Bacillus macerans cycloamylose (cyclodextrin) glucanotransferase

Bacillus macerans cycloamylose (cyclodextrin) glucanotransferase (EC 2.4.1.19) was purified by the technique of starch adsorption and DEAE-cellulose column chromatography, and then crystallized from an ammonium sulfate solution containing mM calcium chloride. The crystals of the enzyme were rod-shaped and showed a single band by disc-gel electrophorsis. The purified enzyme was dissociated into two subunits by sodium dodecyl sulfate-disc electrophoresis. The subunits had no enzyme activity. Details of each purification step and some properties of the enzyme are described in this paper.

Isolation and action pattern of maltohexaose producing amylase from Aerobacter aerogenes

During studies on the fine structure of amylopectin and N~igeli amylodextrin [1 ], we discovered that an unusual hydrolase activity was contained in a puUulanese preparation obtained from Aerobacter aerogenes by the method of Wallenfels et al. [2]. This hydrolase produced large amounts ofmaltohexaose (G6) from amylose, amylopectin and whole starch. Using crude enzyme extract, we could easily get more than 35% of G 6 from amylopectin. After examining more than 30 strains of Aerobacter aerogenes and cloacae, both of which organisms had the G6-producing activity, we obtained one strain of A. aerogenes which had strong and stable enzyme activity. The enzyme was separated completely from other starch-hydrolyzing activity by ammonium sulfate precipitation and DEAE-cellulose column chromatography. We report here the experimental data and evidence which support an exo action pattern of the new enzyme, analogous to that of t3-amylase and Pseudomonas stutzeri amylase, the latter as reported by Robyt et al. [3].

Purification and some properties of a novel maltohexaose-producing exo-amylase from Aerobacter aerogenes.

Maltohexaose producing amylase (EC 3.2.1.-) is the fourth known exo-amylase, the three previously known being glucoamylase, beta-amylase and Pseudomonas stutzeri maltotetraose producing amylase. The enzyme after release from Aerobacter aerogenes cells by 0.1% sodium lauryl sulfate extraction was purified by ammonium sulfate precipitation, DEAE-Sephadex column chromatography and Sephadex G-100 gel filtration to 80-fold of the original sodium lauryl sulfate extract activity, It gave a single band on disc electrophoresis, and the molecular weight by gel filtration was 54 000. This amylase showed maximal activity at 50 degrees C and pH 6.80. The pH stability range was relatively wide, the enzyme retaining more than 90% of its initial activity in the range of 6.50-9.0. 80% of the activity was retained after 15 min at 50 degrees C. This enzyme produced maltohexaose from starch, amylose and amylopectin by exo-attack, but did not act on alpha- or beta-cyclodextrin, pullulan or maltohexaitol. Also the enzyme acted on beta-limit dextrins of amylopectin and glycogen to form branched oligosaccharides. The unusual reaction of this enzyme on beta-limit dextrin is discussed from the standpoint of the stereochemistry of 1,4-alpha- and 1,6-alpha-glucosidic bonds. This is the anomalous amylase for which it is recognized that 1,6-alpha-glucosidic linkages in the substrates can mimic the effect of 1,4-alpha-bonds, as previously observed in pseudo-priming reactions of E. coli phosphorylase.

Multiple forms of invertase of potato tuber stored at low temperature

Abstract Five types of invertase in cold treated potato tuber are separable by DEAE-cellulose column chromatography. Invertase I and II are different from each other on several grounds (pH, foaming, heat stability and K m ).

Phosphofructokinase of Solanum tuberosum tuber

Abstract Potato tuber phosphofructokinase was purified 19·.6-fold by a combination of ethanol fractionation and DEAE-cellulose column chromatography. The enzyme was very unstable; its pH optimum was 8·0. Km for fructose-6-phosphate, ATP and Mg2+ was 2·1 × 10−4 M, 4·5 × 10−5 M and 4·0 × 10−4 M respectively. ITP, GTP, UTP and CTP can act as phosphate donors, but are less active than ATP. Inhibition of enzyme activity by high levels of ATP was reversed by increasing the concentration of fructose-6-phosphate; the affinity of enzyme for fructose-6-phosphate decreased with increasing concentration of ATP. 5′-AMP, 3′,5′-AMP, 3′-AMP, deoxy AMP, UMP, IMP, CMP, GMP, ADP, CDP, GDP and UDP did not reverse the inhibition of enzyme by ATP. ADP, phosphoenolpyruvate and citrate inhibited phosphofructokinase activity but Pi did not affect it. Phosphofructokinase was not reactivated reversibly by mild change of pH and addition of effectors.

Isoenzymes of phosphoglucoisomerase in sweet potato

Abstract Isoenzymes (I, II and III) of phosphoglucoisomerase in sweet potato were demonstrated by DEAE-cellulose chromatography. Some properties of the two main types (I and III) were examined. Phosphoglucoisomerase III was very heat-sensitive compared with phosphoglucoisomerase I, and the Km of phosphoglucoisomerase I was smaller than that of phosphoglucoisomerase III with fructose-6-phosphate as substrate.

Preparation of Maltooligosaccharaides by the Improved Carbon Column Chromatography

Fractionation of maltooligosaccharides and isomaltooligosaccharides was carried out by the improved carbon column chromatography using a concentration gradient system of 3% n-butanol and 10% n-propanol . Excellent resolution of the oligosaccharides series was obtained. In the case of the maltooligosaccharides series, each oligosccharide was separated with wide window up to DP 12 and the similar results were obtained for the isomaltooligosaccharides up to DP 9 . Deactivation of carbon had strong influence on the resolution of oligosaccharides .