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Item Coconut pith, available in abundance, especially in tropical countries, can be an excellent new substrate for the production of cellulase enzyme by solid substrate cultivation of Trichoderma viride NCIM 1051. The effect of type of pretreatment, type and level of nutrient medium, inoculum volume, average substrate particle size, and time of fermentation on cellulase enzyme production by T. viride in coconut pith solid culture was studied. The hydrogen peroxide-pretreated coconut pith was found to be a better substrate. Reese and Mandels' mineral solution mixed with coconut pith in the ratio of 10:1 (v/w; ml g-1) supported maximum cellulase activity. The effect of inoculum volume on enzyme production was only marginal. An average substrate particle size of 375 ?m resulted in better enzyme production. The highest filter paper activity and carboxymethylcellulase activities of 4.27 and 12.05 IU g-1, respectively, were obtained in 7 days of fermentation, and the maximum cellobiase activity that could be obtained was 1.8 IU g-1 in 8 days. © 1994.(Solid substrate fermentation of coconut coir pith for cellulase production) Muniswaran, P.K.A.; Charyulu, N.C.L.N.1994Item Coconut coir pith, available in abundance especially in tropical countries, was studied as a substrate for the production of cellulase[1,4(1,3;1,4)???D?glucan 4?glucanohydrolase, EC 3.2.1.4] and ??D?glucosidase(??D?glucoside glucohydrolase, EC 3.2.1.21) in solid state fermentation. The effects of fermentation time, nutrient level, substrate particle size and inoculum size have been examined for optimal production of these enzymes by the fungal strain Aspergillus niger NCIM 1005. The highest filter paper activity (FPA) of 4.11 IU g?1, carboxyl methyl cellulose (CMCase) activity of 15·55 IU g?1 and cellobiase activity of 9·31 IU g?1 were obtained after 7 to 8 days of fermentation. Reese and Mandel's mineral solution in the substrate to mineral solution ratio of 1:10 (w/v) supported high cellulase and cellobiase activities. An inoculum size of 20–50% (v/v) based on the volume of mineral medium and substrate average particle size of 375 ?m were optimum for enzyme production. Copyright © 1994 SCI(Production of cellulases from coconut coir pith in solid state fermentation) Muniswaran, P.K.A.; Selvakumar, P.; Charyulu, N.C.L.N.1994Item Production of novel cell-associated tannase from newly isolated Serratia ficaria DTC(2010) Belur, P.D.; Gopal, M.; Nirmala, K.R.; Nainegali, N.Five strains of tannic acid degrading bacteria were isolated and identified by phenotypic characterization. All the five isolates showed cell-associated activity, whereas only three showed extracellular activity. Serratia ficaria DTC, showing the highest cell-associated activity (0.29 U/l), was selected for further shake-flask studies. Tannase synthesis was growth associated and reached the peak in the late stationary phase of growth. Organic nitrogen sources enhanced the tannase production. Peak tannase production of 0.56 U/l was recorded in the medium having the initial pH of 6. The pH and temperature optima of the enzyme were found to be 8.9 and 35°C, respectively. This is the first report of cell-associated activity in the case of bacterial tannase. Cell-associated tannase of Serratia ficaria DTC could be industrially important from the perspective of its activity at broad temperature and pH ranges, and its unusually high activity at pH 8.9. © The Korean Society for Microbiology and Biotechnology.Item Optimization of culture medium for novel cell-associated tannase production from bacillus massiliensis using response surface methodology(2012) Belur, P.D.; Goud, R.; Goudar, D.C.Naturally immobilized tannase (tannin acyl hydrolase, E.C. 3.1.1.20) has many advantages, as it avoids the expensive and laborious operation of isolation, purification, and immobilization, plus it is highly stable in adverse pH and temperature. However, in the case of cell-associated enzymes, since the enzyme is associated with the biomass, separation of the pure biomass is necessary. However, tannic acid, a known inducer of tannase, forms insoluble complexes with media proteins, making it difficult to separate pure biomass. Therefore, this study optimizes the production of cell-associated tannase using a "protein-tannin complex" free media. An exploratory study was first conducted in shake-flasks to select the inducer, carbon source, and nitrogen sources. As a result it was found that gallic acid induces tannase synthesis, a tryptose broth gives higher biomass, and lactose supplementation is beneficial. The medium was then optimized using response surface methodology based on the full factorial central composite design in a 3 l bioreactor. A 2 3 factorial design augmented by 7 axial points (? = 1.682) and 2 replicates at the center point was implemented in 17 experiments. A mathematical model was also developed to show the effect of each medium component and their interactions on the production of cell-associated tannase. The validity of the proposed model was verified, and the optimized medium was shown to produce maximum cell-associated tannase activity of 9.65 U/l, which is 93.8% higher than the activity in the basal medium, after 12 h at pH 5.0, 30°C. The optimum medium consists of 38 g/l lactose, 50 g/l tryptose, and 2.8 g/l gallic acid. © The Korean Society for Microbiology and Biotexhnology.Item Production of propyl gallate in nonaqueous medium using cell-associated tannase of Bacillus massiliensis: Effect of various parameters and statistical optimization(2013) Aithal, M.; Belur, P.D.Enzymatic synthesis of propyl gallate in an organic solvent was studied using cell-associated tannase (E.C. 3.1.1.20) of Bacillus massiliensis. Lyophilized biomass showing tannase activity was used as a biocatalyst. The influence of buffer pH and strength, water activity, temperature, biocatalyst loading, gallic acid concentration, and 1-propanol concentration was studied by the one-factor-at-a-time method. Subsequently, response surface methodology was applied based on a central composite design to determine the effects of three independent variables (biocatalyst loading, gallic acid concentration, and 1-propanol concentration) and their mutual interactions. A total of 20 experiments were conducted, and a statistical model was developed, which predicted the maximum propyl gallate yield of 20.28 ?g/mL in the reaction mixture comprising 40.4 mg biocatalyst, 0.4 mM gallic acid, and 6.52 % (v/v) 1-propanol in 9.5 mL benzene at 30°C. The subsequent verification experiments established the validity of the model. Under optimal conditions, 25% conversion of gallic acid to propyl gallate was achieved on a molar basis. The absence of the need for enzyme purification and subsequent immobilization steps and good conversion efficiency makes this enzyme system an interesting one. Reports on the applications of bacterial whole cell systems for synthetic reactions in organic solvents are scarce, and perhaps this is the first report on bacterial cell-associated tannase-mediated esterification in a nonaqueous medium. © 2013 International Union of Biochemistry and Molecular Biology, Inc.Item Studies on the Site-specific PEGylation Induced Interferences Instigated in Uricase Quantification Using the Bradford Method(Springer Netherlands, 2016) Nanda, P.; JagadeeshBabu, P.E.Uricase from Bacillus fastidiosus was site-specifically PEGylated using methoxypolyethyleneglycol-maleimide (mPEG-mal) of different molecular weights (750 Da, 5 kDa, 10 kDa) via Thiol PEGylation strategy. The obtained monoPEGylated uricase conjugates were characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the molecular weight of single subunit of the conjugate was found to be 42.6, 48.1 and 56.3 kDa with respect to different molecular weights of m-PEG-mal. The influence of PEGylation on the quantification of uricase using protein quantification techniques like Bradford assay, RP-HPLC detection and Dumbroff method has been evaluated. A gradual decline in the absorbance value was observed with the advancement of the PEGylation reaction, indicating an interferences in the protein quantification due to PEGylation. The extent of interference highly dependence on mPEG-mal concentration and its chain length. The present study indicates that the quantification of PEGylation induced interferences caused in protein measured ought to be prudently measured at every discrete step of the PEGylation process. © 2016, Springer Science+Business Media New York.Item Anionic surfactant based reverse micellar extraction of l-asparaginase synthesized by Azotobacter vinelandii(Springer Verlag, 2017) Murugesan, S.; Iyyaswami, R.; Kumar, S.V.; Surendran, A.Abstract: l-Asparaginase synthesized by Azotobacter vinelandii via submerged fermentation in the presence of sucrose was successfully extracted using Reverse micellar extraction. Single step enzyme purification process was developed by varying the process variables which resulted in maximum specificity and extraction of l-asparaginase. The effect of different variables, including broth pH, addition of alcohol during the forward extraction and pH of the fresh stripping aqueous phase, addition of alcohol and electrolyte during backward extraction process were studied. Lower concentration of butanol resulted in maximum activity of the enzyme during forward extraction while enzyme activity was found to increase further with the addition of higher concentrations of ammonium sulphate during backward extraction. Chromatographic analysis of l-asparaginase peak at ~7.65 min was intense for the back extracted sample confirming the maximum purity of l-asparaginase obtained. Purity of l-asparaginase was increased to about 379.68 fold. Graphical abstract: [Figure not available: see fulltext.]. © 2017, Springer-Verlag Berlin Heidelberg.Item Production of oxalate oxidase from endophytic Ochrobactrum intermedium CL6(Journal of Pure and Applied Microbiology micro_drkhan@yahoo.com 54, Near Post Office, Thana Street, Shahjahanabad Bhopal 462 001, 2018) Kumar, K.; Belur, P.D.Four oxalate degrading endophytic bacteria were isolated from oxalate rich Colocasia esculenta tubers. Based upon the oxalate oxidase (EC 1.2.3.4) activity produced in nutrient medium, one bacterium was selected and identified as Ochrobactrum intermedium by 16S rDNA sequencing. Studies on effect of nutritional and non-nutritional parameters showed that oxalate oxidase production is inducible, requires Manganese ions in the medium, and very low fill-up volume is beneficial. Shake flask fermentation carried out with medium comprising Sucrose, Ammonium chloride, Sodium oxalate along with basal salts gave 0.5 UmL-1 oxalate oxidase activity and 0.454 Umg-1specific activity after 65h of fermentation. © 2018 The Author(s).Item Expression of Bacillus licheniformis chitin deacetylase in E. coli pLysS: Sustainable production, purification and characterisation(Elsevier B.V., 2019) Bhat, P.; Pawaskar, G.-M.; Raval, R.; Cord-Landwehr, S.; Moerschbacher, B.; Raval, K.Chitosan obtained by enzymatic deacetylation of chitin using chitin deacetylase (CDA) holds promise primarily due to the possibility to yield chitosan with non-random patterns of acetylation and more environmentally friendly process compared to chemical deacetylation. In the present study, a sustainable bioprocess is reported for over-expression of a bacterial CDA in E. coli pLysS cells. A Bacillus licheniformis CDA gene is identified in the genome of the bacterium, cloned, and expressed, yielding enzymatically active recombinant protein. For enzyme production, a growth medium is formulated using carbon and nitrogen sources, which do not compete with the human food chain. The maximum enzyme activity of 320 ± 20 U/mL is achieved under optimized conditions. The CDA productivity is improved by about 23 times in shake flask culture by optimizing operating conditions and medium components. The CDA is purified and the enzyme kinetic values i.e. Km, Vmax and Kcat are reported. Also the effect of cofactors, temperature, and pH on the enzyme activity is reported. Further, economic yield is proposed for production of CDA through this bioprocess. © 2019 Elsevier B.V.Item L-asparaginase production using solid-state fermentation by an endophytic talaromyces pinophilus isolated from rhizomes of curcuma amada(Journal of Pure and Applied Microbiology micro_drkhan@yahoo.com 54, Near Post Office, Thana Street, Shahjahanabad Bhopal 462 001, 2020) Krishnapura, P.R.; Belur, P.D.In recent times, exploration of endophytes for L-asparaginase production is gradually gaining momentum. This work deals with studies on the production of L-asparaginase from Talaromyces pinophilus, an endophytic fungus isolated from the rhizomes of Curcuma amada. L-asparaginase production was carried out by Submerged Fermentation (SmF) followed by Solid-state Fermentation (SSF). A liquid medium was designed and optimized using Plackett-Burman Design and Response Surface Methodology (RSM), under SmF. Additionally, optimal concentrations of various metal salts were incorporated in the optimized liquid medium, by one-factor-at-a-time experiments. To further enhance L-asparaginase production, SSF was carried out using Polyurethane Foam (PUF) as inert support impregnated with the optimized liquid medium. Effects of PUF cube volume, mass of PUF, moisture content, initial medium pH, and incubation temperature on the enzyme production in SSF were optimized by one-factor-at-a-time approach.L-asparaginase production enhanced from 80.8 U/mL in the unoptimized medium to 94.4 U/mL in the optimized medium under SmF. Enzyme production further increased to 120.3 U/mL under SSF by using PUF soaked in the optimized liquid medium. This study highlights the benefits of carrying out SSF with PUF, using the same liquid medium optimized for SmF - a novel approach to enhance the enzyme yield (in our case an increase of about 27% was observed). To the best of our knowledge, this is the first report on the production of L-asparaginase by both SmF and SSF, from an endophyte Talaromyces pinophilus isolated from the rhizomes of Curcuma amada. © The Author(s) 2020. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which permits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.