RESUMEN
A novel raw starch degrading alpha-cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19), produced by Klebsiella pneumoniae AS-22, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The specific cyclization activity of the pure enzyme preparation was 523 U/mg of protein. No hydrolysis activity was detected when soluble starch was used as the substrate. The molecular weight of the pure protein was estimated to be 75 kDa with SDS-PAGE and gel filtration. The isoelectric point of the pure enzyme was 7.3. The enzyme was most active in the pH range 5.5-9.0 whereas it was most stable in the pH range 6-9. The CGTase was most active in the temperature range 35-50 degrees C. This CGTase is inherently temperature labile and rapidly loses activity above 30 degrees C. However, presence of soluble starch and calcium chloride improved the temperature stability of the enzyme up to 40 degrees C. In presence of 30% (v/v) glycerol, this enzyme was almost 100% stable at 30 degrees C for a month. The K(m) and k(cat) values for the pure enzyme were 1.35 mg ml(-1) and 249 &mgr;M mg(-1) min(-1), respectively, with soluble starch as the substrate. The enzyme predominantly produced alpha-cyclodextrin without addition of any complexing agents. The conditions employed for maximum alpha-cyclodextrin production were 100 g l(-1) gelatinized soluble starch or 125 g l(-1) raw wheat starch at an enzyme concentration of 10 U g(-1) of starch. The alpha:beta:gamma-cyclodextrins were produced in the ratios of 81:12:7 and 89:9:2 from gelatinized soluble starch and raw wheat starch, respectively.
RESUMEN
Production of cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae pneumoniae AS-22 was optimized in shake flasks using a statistical experimental design approach. Effect of various components in the basal medium, like carbon, nitrogen, phosphorus, and mineral sources as well as initial pH and temperature, were tested on enzyme production. The optimum concentrations of the selected media components were determined using statistical experimental designs. Two level fractional factorial designs in five variables, namely, dextrin, peptone, yeast extract, ammonium dihydrogen orthophosphate, and magnesium sulphate concentrations were constructed. The optimum medium composition thus found consisted of 49.3 g/L dextrin, 20.6 g/L peptone, 18.3 g/L yeast extract, 6.7 g/L ammonium dihydrogen orthophosphate, and 0.5 g/L magnesium sulphate. The maximum CGTase activity obtained was 21.4 U/mL in 28 h of incubation. The cell growth and CGTase production profiles were studied with the optimized medium in shake flasks and in 1-L fermenters. It was observed that the enzyme production was growth associated both in shake flask and in fermenter, although it was slower in shake flask. The maximum CGTase activity obtained in the fermenter was 32.5 U/mL in 16 h. The optimized medium resulted in about 9-fold increase in the enzyme activity as compared to that obtained in the basal medium in shake flask as well as in fermenter.
Asunto(s)
Glucosiltransferasas/biosíntesis , Klebsiella pneumoniae/enzimología , Carbono/metabolismo , División Celular , Fermentación , Concentración de Iones de Hidrógeno , Modelos Estadísticos , Nitrógeno/metabolismo , Fósforo/metabolismo , Temperatura , Factores de TiempoRESUMEN
A novel raw starch degrading cyclomaltodextrin glucanotransferase (CGTase; E.C. 2.4.1.19), produced by Bacillus firmus, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The molecular weight of the pure protein was estimated to be 78,000 and 82,000 Da, by SDS-PAGE and gel filtration, respectively. The pure enzyme had a pH optimum in the range 5.5-8.5. It was stable over the pH range 7-11 at 10 degrees C, and at pH 7.0 at 60 degrees C. The optimum temperature for enzyme activity was 65 degrees C. In the absence of substrate, the enzyme rapidly lost its activity above 30 degrees C. K(m) and kcat for the pure enzyme were 1.21 mg/ml and 145.17 microM/mg per minute respectively, with soluble starch as the substrate. For cyclodextrin production, tapioca starch was the best substrate used when gelatinized, while wheat starch was the best substrate used when raw. This CGTase could degrade raw wheat starch very efficiently; up to 50% conversion to cyclodextrins was obtained from 150 g/l starch without using any additives. The enzyme produced alpha-, beta- and gamma-cyclodextrins in the ratio of 0.2:9.2:0.6 and 0.2:8.6:1.2 from gelatinized tapioca starch and raw wheat starch with 150 g/l concentration respectively, after 18 h incubation.