Faculty Publications
Permanent URI for this communityhttps://idr.nitk.ac.in/handle/123456789/18736
Publications by NITK Faculty
Browse
11 results
Search Results
Item 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 Multistrain probiotic production by co-culture fermentation in a lab-scale bioreactor(Wiley-VCH Verlag info@wiley-vch.de, 2016) Jangra, M.; Belur, P.D.; Oriabinska, L.B.; Dugan, O.M.Most commercial probiotic products intended for pharmaceutical applications consist of combinations of probiotic strains and are available in various forms. The development of co-culture fermentation conditions to produce probiotics with the correct proportion of viable microorganisms would reduce multiple operations and the associated costs. The aim of this study was to develop a fermentation medium and process to achieve biomass comprising the desired proportion of two probiotic strains in co-culture. Initially, a quantification medium was developed, and the method was optimized to allow the quantification of each strain's biomass in a mixture. The specific growth rates of Lactobacillus delbrueckii spp. bulgaricus and Lactobacillus plantarum were determined in media with different carbon sources. The inoculum volume was optimized to achieve equal proportion of biomass in co-culture fermentation in test tubes. Next, fermentation was carried out in a 3-L bioreactor. A biomass concentration of 2.06 g/L, with L. delbrueckii spp. bulgaricus and L. plantarum in the ratio of 47%:53% (by weight), was achieved with concomitant production of 12.69 g/L of lactic acid in 14 h. The results show that with careful manipulation of process conditions, it is possible to achieve the desired proportion of individual strains in the final biomass produced by co-culture fermentation. This process may serve as a model to produce multistrain probiotic drugs at industrial scale. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.Item Therapeutic potential of benfotiamine and its molecular targets(Verduci Editore s.r.l g.lombardi@verduci.it, 2018) Raj, V.; Ojha, S.; Howarth, F.C.; Belur, P.D.; Subramanya, S.B.OBJECTIVE: The water-soluble vitamin, thiamine forms an important part of the diet because of its role in the energy metabolism. The protective effects of thiamine against diabetic vascular complications have been well documented. However, slower absorption and reduced bioavailability is a major limiting factor for its clinical use. To overcome this issue, lipid-soluble derivatives of thiamine (allithiamines) was developed. Among the many synthetic lipophilic derivatives of thiamine, benfotiamine (BFT) is regarded as the first choice based on its safety and clinical efficacy data. BFT facilitates the action of thiamine diphosphate, a cofactor for the enzyme transketolase. The activation of transketolase enzyme accelerates the precursors of advanced glycation end products (AGEs) towards the pentose phosphate pathway thereby reducing the production of AGEs. The reduction in AGEs subsequently decreases metabolic stress which benefits vascular complications seen in diabetes. The effects of BFT on the AGE-dependent pathway is well established. However, several studies have shown that BFT also modulates pathways other than AGE such as arachidonic acid (AA), nuclear transcription Factor ?B (NF-?ß), protein kinase B, mitogen-activated protein kinases (MAPK) and vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathways. In the present review, we have comprehensively reviewed all the molecular targets modulated by BFT to provide mechanistic perspective to highlight its pleiotropic effects. © 2018 Verduci Editore s.r.l. All rights reserved.Item Enhancement of n-3 polyunsaturated fatty acid glycerides in Sardine oil by a bioimprinted cross-linked Candida rugosa lipase(Elsevier Inc. usjcs@elsevier.com, 2018) Sampath, C.; Belur, P.D.; Iyyaswami, R.Considering the advantages of bioimprinting and carrier free immobilization, cross-linked enzyme aggregates (CLEA) were prepared by using bioimprinted Candida rugosa lipase (CRL) with Bovine serum albumin (BSA), Polyethyleneimine and glutaraldehyde. Effect of various factors such as CRL-Oleic acid ratio, CRL-BSA ratio, CRL- Polyethyleneimine ratio, glutaraldehyde loading, cross-linking time etc., on lipase activity recovery and aggregate yield were studied and optimized. This immobilized lipase (CRL-CLEA) was used for the selective hydrolysis of ester linkages of non-PUFA glycerides, with an aim to concentrate EPA and DHA glycerides in the Sardine oil. Imprinting with oleic acid in the presence of ethanol and Tween 60, and further immobilization with co-aggregates and cross-linking agent showed 10.4 times higher degree of hydrolysis compared to free enzyme. As result, 2.83-fold increase of n-3 PUFA content in deacidified oil was obtained by using CRL-CLEA. The resultant oil had negligible di- and triglycerides content, proving higher efficiency in hydrolysing ester bonds of fatty acids, other than n-3 PUFA. Reusability studies showed CRL-CLEA could be reused up to 5 runs without a substantial reduction in its performance. Improvement in degree of hydrolysis, thermostability, efficiency of hydrolysis and reusability were achieved due to bioimprinting and subsequent immobilization of CRL in the form of CLEA. © 2017 Elsevier Inc.Item A novel fibrinolytic serine metalloprotease from the marine Serratia marcescens subsp. sakuensis: Purification and characterization(Elsevier B.V., 2018) Krishnamurthy, A.; Belur, P.D.This study demonstrates the purification and characterization of a fibrinolytic serine metalloprotease from the marine Serratia marcescens subsp. sakuensis (KU296189.1). The purified enzyme (1033 U/mg) had a molecular weight of 43 KDa, with optimum pH and temperature being 7 and 55 °C. The in vitro half-life of the fibrinolytic enzyme at 37 °C was found to be 19 h. The kinetic constants, Km and Vmax of the purified enzyme determined using fibrin as substrate was 0.66 mg/mL and 158.73 U/mL. The Kcat and catalytic efficiency of the enzyme was found to be 12.21 min?1 and 18.32 mL/(mg min) respectively. The fibrinolytic enzyme did not show any proteolytic activity towards blood plasma proteins like haemoglobin, ?-globulins and transferrin. In vitro studies revealed that the fibrinolytic enzyme displayed 38% clot lysis for a period of 3 h which was higher than that displayed by streptokinase and heparin. A total of seven peptide sequences were obtained after the LC-MS/MS-TOF analysis, out of which only four sequences showed 67% homology with the sequences of the other proteases. All these results suggest its novelty and potential application in thrombolytic therapy. © 2018 Elsevier B.V.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 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.Item Production, Purification and Characterization of Extracellular Tannase from a Newly Isolated Yeast, Geotrichum cucujoidarum(Journal of Pure and Applied Microbiology, 2022) Thangavelu, N.; Hugar, P.; Belur, P.D.With an aim to isolate a tannase positive organism, the microbial mat growing on the stored areca extract leachate surface was screened. Once the tannase positive organism was isolated, it was identified by ITS/18S rRNA gene sequencing. Further, the enzyme was purified and examined for its biochemical properties. A potent extracellular tannase-producing yeast was isolated and was identified as Geotrichum cucujoidarum. After the shake flask studies, the enzyme activity of 4.42 U/ml and specific activity of 29.86 U/mg were achieved in a medium with tannic acid as an inducer. Later, ethanol (70%) precipitation followed by purification through FPLC using SEC 650 column resulted in 166.37 U/mg specific activity and a recovery of 50.54%. The purified enzyme was a monomer with a molecular weight of 63 kDa. The optimum pH and the temperature of the enzyme were found to be 5.0 and 30°C, respectively. The Michaelis-Menten constant (Km) was found to be 2.9 mM, and the turn over number (kcat) and catalytic efficiency (kcat/km) of the purified tannase were 102 S-1 and 35.17 mM-1S-1 respectively. Temperature and pH stability profiles of the enzyme, influence of various metal ions, chelators and surfactants on enzyme activity and kinetic constants of enzyme shows that the tannase produced from Geotrichum cucujoidarum is unique and is a potential candidate for further studies. © The Author(s) 2022.