ABSTRACT
An anaerobic microbial isolate Bacillus species, designated B. thuringiensis GU-2, was isolated from soil as a specific γ-cyclodextrin (CD) producer strain in alkaline medium under anaerobic conditions. The optimum pH and temperature for bacterial growth and γ-CD production were estimated to be pH 8.5 and 37°C in the presence of 1.0% starch substrate, respectively. A high purity yield >95% of γ-CD from the total CD yield in the reaction mixture was obtained from starch that was supposed to be converted by gamma-cyclodextrin glycotransferase, tentatively named as γ-CGTase. The maximum γ-CGTase activity was estimated at 2.45U/mL under optimized condition. This is the first report demonstrating the generation of a specific γ-cyclodextrin (CD) producer strain by the action of a γ-CGTase under anaerobic conditions.
Subject(s)
Bacillus thuringiensis/enzymology , gamma-Cyclodextrins/metabolism , Bacillus thuringiensis/genetics , Bacillus thuringiensis/isolation & purification , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biocatalysis , Fermentation , Glucosyltransferases/chemistry , Glucosyltransferases/genetics , Glucosyltransferases/metabolism , Hydrogen-Ion Concentration , Kinetics , Molecular Typing , Phylogeny , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Soil MicrobiologyABSTRACT
An alkalophilic extracellular chitosanase (ACTase) was characterized from the culture supernatant of Bacillus cereus GU-02. Kinetic properties of ACTase produced from B. cereus GU-02 after cultivation in anaerobic condition, alkaline medium (pH 10) at 37°C for 3 days were investigated. ACTase was found to be stable in alkaline pH range from 8 to 10. Interestingly, optimum pH and temperature were estimated to be 10 and 37°C, respectively, where ACTase showed chitosan degrading activity (87%), which was enhanced by 15% in the presence of calcium ions (8 mM). The ACTase produced from B. cereus GU-02 was partially purified from the culture supernatant, and its enzymatic activity was kinetically characterized. The Vmax and Km were estimated with a chitosan (degree of deacetylation, DD 92% as substrate) as 0.038 U/min/µg protein and 0.327 µM, respectively. A combination of the TLC and MALDI-TOF MS results showed that the chitosan oligosaccharides obtained from the hydrolysis of high molecular weight chitosan (HMWC) by ACTase of the B. cereus GU-2 comprise oligomers with degree of polymerization (DP) mainly from dimers to pentamers. High production of ACTase and chitooligosaccharides may be useful for various industrial and biological applications.
Subject(s)
Bacillus cereus/enzymology , Bacterial Proteins/chemistry , Glycoside Hydrolases/chemistry , Bacterial Proteins/biosynthesis , Bacterial Proteins/isolation & purification , Carbohydrate Conformation , Chitosan/chemistry , Glycoside Hydrolases/biosynthesis , Glycoside Hydrolases/isolation & purification , Hydrogen-Ion Concentration , Hydrolysis , KineticsABSTRACT
Extracellular polysaccharide (EPS) was isolated from defatted micro-algae Dunaliela tertiolecta and defined as linear (1â4)-α-D-glucan based on monosaccharide composition, enzymatic and spectroscopic analyses. Optimization and characterization of acidic and enzymatic hydrolyses of EPS have been performed for its potential use as a renewable biorefinery material. The hydrolytic methods were improved to assess the effect of substrate specificity, reaction time, pH, ionic strength and temperature on efficiency of glucose production. EPS was effectively converted into glucose within one-step enzymatic or acidic hydrolysis under optimized conditions. Over 90% recovery of glucose was achieved for both hydrolytic approaches. High potential production of EPS and high yield conversion of this substrate to glucose may allow further exploration of microalga D. tertiolecta as a potential biomass producer for biotechnological and industrial exploitation of bioethanol.
