Ravi Kiran Purama

Screening and optimization of nutritional factors for higher dextransucrase production by Leuconostocmesenteroides NRRL B-640 using statistical approach

To improve dextransucrase production from Leuconostocmesenteroides NRRL B-640 culture medium was screened and optimized using the statistical design techniques of Plackett-Burman and response surface methodology (RSM). Plackett-Burman design with six variables viz. sucrose, yeast extract, K2HPO4, peptone, beef extract and Tween 80 was performed to screen the nutrients that were significantly affecting dextransucrase production. The variables sucrose, K2HPO4, yeast extract and beef extract showed above 90% confidence levels for dextransucrase production and were considered as significant factors for optimization using response surface methodology. 2(4)-central composite design was used for RSM optimization. The experimental results were fitted to a second-order polynomial model which gave a coefficient of determination R2=0.95. The optimized composition of 30g/l sucrose, 18.9g/l yeast extract, 19.4g/l K2HPO4 and 15g/l beef extract gave an experimental value of dextransucrase activity of 10.7U/ml which corresponded well with the predicted value of 10.9U/ml by the model.

An overview of purification methods of glycoside hydrolase family 70 dextransucrase

The enzyme dextransucrase (sucrose:1, 6-α-D-glucan 6-α-glucosyltransferase, EC 2.4.1.5) catalyses the synthesis of exopolysaccharide, dextran from sucrose. This class of polysaccharide has been extensively exploited in pharmaceutical industry as blood volume expander, as stabiliser in food industry and as a chromatographic medium in fine chemical industry because of their nonionic nature and stability. Majority of the dextrans are synthesized from sucrose by dextransucrase secreted mainly by bacteria belonging to genera Leuconostoc, Streptococcus and Lactobacillus. Bulk of the information on purification of extracellular dextransucrase has been generated from Leuconostoc species. Various methods such as precipitation by ammonium sulphate, ethanol or polyethylene glycol, phase partitioning, ultrafiltration and chromatography have been used to purify the enzyme. Purification of dextransucrase is rendered difficult by the presence of viscous dextran in the medium. However, processes like ultra-filtration, salt and PEG precipitation, chromatography and phase partitioning have been standardized and successfully used for higher scale purification of the enzyme. A recombinant dextransucrase from Leuconostoc mesenteroides B-512F with a histidine tag has been expressed in E. coli cells and purifi ed by immobilized metal ion chromatography. This review reports the available information on purifi cation methods of dextransucrase from Leuconostoc mesenteroides strains.

Enhanced production of a novel dextran fromLeuconostoc mesenteroides NRRL B-640 by Response Surface Methodology

In our earlier study dextran produced byLeuconostoc mesenteroides NRRL B-640 was reported to possess novel food gelling and thickening properties (Puramaet al., 2009). In the present study response surface methodology based experimental designs were applied to enhance the production of this novel dextran byLeuconostoc mesenteroides NRRL B-640. Eleven medium components were examined for their significance on dextran production using Plackett-Burman factorial design. Sucrose, peptone and beef extract were found to have significant effect on the dextran production. The combined effect of these nutrients on dextran production were studied using a 23 full-factorial central composite design, a second-order polynomial was established to identify the relationship between the output i.e. dextran produced and the three medium components. The optimal concentration of variables for maximum dextran production were 5%, w/v sucrose, 2.5%, w/v peptone, and 2.5%, w/v beef extract. The maximum concentration of dextran obtained by predicted model was 12.0 mg/ml that was in perfect agreement with the experimental determined value (12.2±0.2 mg/ml). This value of dextran concentration was over 70 percent higher as compared to un-optimized medium that gave 7.0±0.2 mg/ ml of dextran.

Stabilization of dextransucrase from Leuconostoc mesenteroides NRRL B-640

Stabilization of dextransucrase from Leuconostoc mesenteroides NRRL B-640 with various stabilizers at different temperatures was studied. Dextransucrase was stable at lower temperatures (10–30°C) and lost the activity at above 30°C. The salts such as CaCl2, CoCl2 and MgCl2 enhanced the dextransucrase activity. A 22% higher dextransucrase activity was obtained by 4 mM CoCl2. The dextransucrase activity was lost by 50% at 1 mM EDTA. Urea denatured the enzyme and caused 45%, 90% and 98% loss of activity in 30 min when treated with 1 M, 3 M, and 5 M urea concentrations, respectively. Amongst the stabilizers Tween 80, glycerol, PEG-8000, dextran (500 kDa) and glutaraldehyde, Tween 80 provided the maximum stability at 30°C. In the presence of Tween 80 the enzyme lost only 8% activity at 30°C in 20 h but, it lost 65% of activity with out any stabilizer. The enzyme lost 92% of activity with in 4 days at 30°C and lost only 25% of activity at −20°C after 14 days.

Functional characterization and mutation analysis of family 11, Carbohydrate-Binding Module (CtCBM11) of cellulosomal bifunctional cellulase from Clostridium thermocellum

The non-catalytic, family 11 carbohydrate binding module (CtCBM11) belonging to a bifunctional cellulosomal cellulase from Clostridium thermocellum was hyper-expressed in E. coli and functionally characterized. Affinity electrophoresis of CtCBM11 on nondenaturing PAGE containing cellulosic polysaccharides showed binding with β-glucan, lichenan, hydroxyethyl cellulose and carboxymethyl cellulose. In order to elucidate the involvement of conserved aromatic residues Tyr 22, Trp 65 and Tyr 129 in the polysaccharide binding, site-directed mutagenesis was carried out and the residues were changed to alanine. The results of affinity electrophoresis and binding adsorption isotherms showed that of the three mutants Y22A, W65A and Y129A of CtCBM11, two mutants Y22A and Y129A showed no or reduced binding affinity with polysaccharides. These results showed that tyrosine residue 22 and 129 are involved in the polysaccharide binding. These residues are present in the putative binding cleft and play a critical role in the recognition of all the ligands recognized by the protein.

Structural and biochemical properties of lichenase from Clostridium thermocellum

The recombinant enzyme lichenase of size 30 kDa was over-expressed using E. coli cells and purified by immobilized metal ion affinity chromatography (IMAC) and size exclusion chromatography. The enzyme displayed high activity towards lichenan and β-glucan. The enzyme showed no activity towards carboxymethyl cellulose, laminarin, galactomannan or glucomannan. Surprisingly, affinity-gel electrophoresis on native-PAGE showed that the enzyme binds only glucomannan and not lichenan or β-glucan or other manno-configured substrates. The enzyme was thermally stable between the temperatures 60°C and 70°C. Presence of Cu2+ ions at a concentration of 5 mM enhanced enzyme activity by 10% but higher concentrations of Cu2+ (>25 mM) showed a sharp fall in the enzyme activity. Heavy metal ions Ni2+, Co2+ and Zn2+ did not affect the activity of the enzyme at low concentrations (0–10 mM) but at higher concentrations (>10 mM), caused a decrease in the enzyme activity. The crystals of lichenase were produced and the 3-dimensional structure of native form of enzyme was previously solved at 1.50 Å. Lichenase displayed (β/α)8-fold a common fold among many glycoside hydrolase families. A cleft was identified that represented the probable location of active site.