The cyclomaltrodextrin glucanotransferase (CGTase) [1,4-alpha-D-glucan:4-alpha-D-(1,4-alpha-D-glucano)-transferase (cyclizing), EC 2.4.1.19] from Bacillus circulans E 192 has been purified to homogeneity by Cetavlon treatment, ammonium sulfate precipitation, DEAE Trisacryl M chromatography, Q Fast Flow chromatography, and affinity on beta-cyclodextrin-Sepharose 4B. Two isoenzymes were separated by FPLC on a Mono Q column. Their isoelectric points were estimated as 6.7 and 6.9 and they represented 13 and 87%, respectively, of the initial activity. Their molecular weight, pH, and temperature optima were estimated as 78,000, 5.5, and 60 degrees C, respectively. Kinetic parameters indicated that both enzymes had the same properties; they preferentially modified high-molecular-weight substrates to produce cyclodextrins. The apparent Vmax and Km values for soluble starch were 43 mumol of beta-cyclodextrin/min/mg of protein and 0.57% (w/v), respectively. Although this CGTase is not markedly thermostable, it is protected against heat denaturation by substrate, product, and/or calcium ions. The ratios of alpha-, beta-, and gamma-cyclodextrins produced have been determined as 1/7/2 in the initial phase of the reaction and 3/3/1 at equilibrium.

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Renin was purified from 47 kg of hog kidney to produce enough enzyme for enzymatic and physicochemical characterization. The procedure included extraction at pH 3.5 in the presence of protease inhibitors, two ammonium sulfate precipitations, ion exchange chromatography on Sepharose-hexamethylenediamino-pepstatin gel, gel filtration, and isoelectric focusing. Renin, 2.3 mg, with a specific activity of 1,100 GU/mg of protein was obtained with about 70,000-fold purification and 16% overall recovery. The purity criteria were: (1) a single band on sodium dodecyl sulfate (SDS)-gel electrophoresis, (2) same retardation factor on polyacrylamide gel electrophoresis for renin activity, protein and glycoprotein coloration. Renin was characterized by its stability at--20 degrees C, pH 6.5; its molecular weight on SDS-gel electrophoresis, 36,800; its relative mobility on polyacrylamide gel electrophoresis at pH 7.8; its isoelectric point, 5.15; its amino acid composition, which revealed that renin is a glycoprotein; and its Michaelis constant on tetradecapeptide substrate at pH 6.6, Km = 7.7 X 10(-6) M.

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SummaryThe exocellular DD-carboxypeptidase-transpeptidase that Streptomyces R61 excretes during growth has been produced in large fermentation units of 15 m3 total capacity. The yield from 15,000 liter culture filtrate was 1.080 g purified enzyme (92% purity) with a total recovery of 29% and at least a 2000-fold increased specific activity.