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Gaikaiwari R.P.,Hi Technology Biosciences India Ltd. | Wagh S.A.,Hi Technology Biosciences India Ltd. | Kulkarni B.D.,CSIR - National Chemical Laboratory
Separation and Purification Technology | Year: 2012

Tannin acyl hydrolase commonly called as tannase (EC 3.1.1.20) is a commercially important enzyme. Partially purified and concentrated tannase is required for commercial applications. Typical objectives of purification process comprise high fold-purification, recovery and concentration. These objectives may be potentially conflicting. Conventional methods of purification require multiple steps which are time consuming and may cause higher loss. Reverse micellar extraction (RME) using ionic surfactants provides an attractive option for concentration and purification of tannase which is a highly hydrophilic glycoprotein. This study presents an optimized methodology for RME and purification of Aspergillus allahabadi intracellular tannase. Fold-purification, percent recovery and extraction time were the objective while the type and concentration of surfactant, contact time, pH, ionic strength, and the ratio of organic to aqueous phase were the decision variables. Some of these parameters were also studied for their effect on back-extraction. Among the surfactants tested, CTAB-isooctane system was found to be suitable. Under optimized conditions, 12.7-fold purification, 81.2% recovery and 3-fold concentration of tannase with a process time of 45 min was obtained. Conventional purification methods provided a higher fold-purification albeit at a much lower enzyme recovery. Further, the conventional method requires a process time of several hours. © 2012 Elsevier B.V. All rights reserved. Source


Gaikaiwari R.P.,Hi Technology Biosciences India Ltd. | Wagh S.A.,Hi Technology Biosciences India Ltd. | Kulkarni B.D.,CSIR - National Chemical Laboratory
Bioresource Technology | Year: 2012

Reverse micellar extraction (RME) of enzyme provides an attractive option for conventional method with the potential to achieve purification and concentration in a single step with high yield. This study presents a methodology for optimization of RME with Pseudomonas lipase as model system. Fold-purification, percent recovery and extraction time were the objective functions while the type and concentration of surfactant, contact time, pH, ionic strength, and the ratio of organic to aqueous phase were the decision variables. Under optimized conditions, the AOT (Aerosol OT (bis 2-ethylhexyl) sodium sulfosuccinate)-isooctane system gave a 15-fold purification, 80% recovery and 2.5-fold concentration of the Pseudomonas lipase with process time of 45. min. © 2011 Elsevier Ltd. Source


Sohoni S.V.,Indian Institute of Technology Bombay | Nelapati D.,Indian Institute of Technology Bombay | Sathe S.,Indian Institute of Technology Bombay | Javadekar-Subhedar V.,Hi Technology Biosciences India Ltd. | And 2 more authors.
Bioresource Technology | Year: 2015

Nitrilases constitute an important class of biocatalysts for chiral synthesis. This work was undertaken with the aim to optimize nitrilase production in a host that is well-studied for protein production. Process parameters were optimized for high cell density fermentation, in batch and fed-batch modes, of Escherichia coli BL21 (DE3) expressing Pseudomonas fluorescens nitrilase with a T7 promoter based expression system. Effects of different substrates, temperature and isopropyl β- d-1-thiogalactopyranoside (IPTG) induction on nitrilase production were studied. Super optimal broth containing glycerol but without an inducer gave best results in batch mode with 32. °C as the optimal temperature. Use of IPTG led to insoluble protein and lower enzyme activity. Optimized fed-batch strategy resulted in significant improvement in specific activity as well as volumetric productivity of the enzyme. On a volumetric basis, the activity improved 40-fold compared to the unoptimized batch process. © 2015 Elsevier Ltd. Source

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