Avishek Majumder

Artificial intelligence based optimization of exocellular glucansucrase production from Leuconostoc dextranicum NRRL B-1146.

Two different artificial intelligence techniques namely artificial neural network (ANN) and genetic algorithm (GA) were integrated for optimizing fermentation medium for the production of glucansucrase. The experimental data reported in a previous study were used to build the neural network. The ANN was trained using the back propagation algorithm. The ANN predicted values showed good agreement with the experimentally reported ones from a response surface based experiment. The concentrations of three medium components: viz Tween 80, sucrose and K2HPO4 served as inputs to the neural network model and the enzyme activity as the output of the model. A model was generated with a coefficient of correlation (R2) of 1.0 for the training set and 0.90 for the test data. A genetic algorithm was used to optimize the input space of the neural network model to find the optimum settings for maximum enzyme activity. This artificial neural network supported genetic algorithm predicted a maximum glucansucrase activity of 6.92U/ml at medium composition of 0.54% (v/v) Tween 80, 5.98% (w/v) sucrose and 1.01% (w/v) K2HPO4. ANN-GA predicted model gave a 6.0% increase of enzyme activity over the regression based prediction for optimized enzyme activity. The maximum enzyme activity experimentally obtained using the ANN-GA designed medium was 6.75+/-0.09U/ml which was in good agreement with the predicted value.

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.

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.