Faculty Publications
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Item Polysulfone/N-phthaloylchitosan novel composite membranes for salt rejection application(2011) Padaki, M.; Isloor, A.M.; Wanichapichart, P.N-phthaloylchitosan (CS) was synthesized by the reaction of chitosan with phthalic anhydride in dimethyl formamide. Different compositions of polysulfone (PSf) and N-phthaloylchitosan were used to prepare novel polysulfone/N-phthaloylchitosan (PSf/CS) composite membranes by phase inversion method. The composition ratios between the former and the latter were 80:20, 85:15, 90:10, and 95:5. Water flux results revealed that, PSf:CS 80:20 membrane is found to have greatest effective pore area while PSf:CS 95:05 membrane has the smallest value. The pore area is found to be larger with the increase in CS composition. In addition, its water swelling property increases with the increase of CS composition. Water flux results are in consistent with dielectric constant value. Use of known molecular weight of polyethylene glycol rejection study, revealed that, PSf:CS 95:05 membrane possessed the smallest pore size among these membranes. In conclusion, change of ratio between PSf and CS, considerably affects membrane pore size and hydrophilicity. For salt filtration, membrane PSf:CS 95:05 showed 93%, 76.11% and 70.12% rejection of MgSO4, Na2SO4, and NaCl, respectively. © 2011 Elsevier B.V.Item Preparation and characterization of sulfonated polysulfone and N-phthloyl chitosan blend composite cation-exchange membrane for desalination(2012) Padaki, M.; Isloor, A.M.; Wanichapichart, P.; A.F., A.F.Sulfonated polysulfone (sPSf) was prepared and used as a polymer matrix for cation-exchange membranes (CEM). The sulfonation reaction was carried out at room temperature and the degree of sulfonation was calculated by titration method. Blend composite membranes were prepared using different ratios of sPSf and modified chitosan (CS). Membrane properties were studied in terms of water flux, water swelling ratio, molecular weight cut off (MWCO), ion-exchange capacity (IEC) and contact angle measurement. Charge on the membrane was confirmed by ionic diffusion potential (DP). It was observed that, DP increased with the increase in the concentration of sPSf. The membrane sPSf:CS 60:40 showed 1000Da MWCO, 14.6mV diffusion potential (DP) and 0.083mM/g of IEC. Similarly sPSf:CS 90:10 showed 10,000Da MWCO, 71.7mV diffusion potential (DP) and 0.176mM/g of IEC. Moreover, membrane sPSf:CS 60:40 showed 93%, 89% and 69% for MgSO 4, Na 2SO 4 and NaCl rejection respectively. © 2012 Elsevier B.V.Item Permeation, Antifouling and desalination performance of TiO2 nanotube incorporated PSf/CS blend membranes(2013) Kumar, R.; Isloor, A.M.; A.F., A.F.; Abdul Rashid, S.A.; Ahmed, A.A.Polysulfone (PSf) and chitosan (CS) blend membranes were prepared by incorporating titanium dioxide nanotubes (TiO2NT) in different compositions. The proper blending of PSf and CS in the PSf/CS/TiO2 membranes was confirmed by ATR-IR spectroscopy. The influence of nanotubes on morphology of membranes was investigated by Field Emission Scanning Electron Microscopy (FESEM). The effect of nanotubes on hydrophilicity of the membranes was studied by water swelling and contact angle measurements. The distribution of TiO2NT on the membrane surface was determined by Transmission Electron Microscope (TEM) analysis. The permeation property of PSf/CS/TiO2NT membranes was carried out by measuring the time dependent pure water flux (PWF). Bovine serum albumin (BSA) protein rejection studies were performed to know the antifouling properties. The rheological percolation threshold of PSf/CS/TiO2NT solutions was measured by viscosity studies. The nanotubes incorporated PSf/CS membranes showed enhanced permeation and antifouling properties compared to PSf/CS and nascent PSf ultrafiltration membranes. Membranes prepared well above rheological percolation threshold showed drastic reduction in pore size and acted as nanofiltration (NF) membranes. © 2013 Elsevier B.V.Item Modification of PSf/PIAM membrane for improved desalination applications using Chitosan coagulation media(2013) Kumar, R.; A.F., A.F.; Kassim, M.A.; Isloor, A.M.Polysulfone (PSf)/poly (isobutylene-alt-maleic anhydride) (PIAM) nanofiltration membranes were modified by changing the coagulation bath with various concentrations of glutaraldehyde cross-linked Chitosan solutions. Further the membranes were treated with 0.1N NaOH solution in order to achieve the hydrolysis of PIAM. The morphological changes of the membranes were determined using scanning electron microscope. The blending of PSf/PIAM membrane, the incorporation of Chitosan (CS) molecules in membrane matrix and the hydrolysis of PIAM in the membrane upon alkali treatment were studied by Attenuated Total Reflectance Infra Red (ATR-IR) spectroscopy. The hydrophilicity of modified membranes was measured using the contact angle analyzer. The pressure and time dependent pure water flux of modified PSf/PIAM/CS membranes were measured and compared with PSf/PIAM membranes (after alkali treatment). The antifouling property of membranes was determined using Bovine Serum Albumin (BSA) protein rejection studies. The modified membranes showed improved hydrophilicity and reduced pore size. The order of rejection of membranes for various electrolytes was Na2SO4>MgSO4>NaCl at minimum pressure of 0.2MPa TMP. The antifouling property of modified membranes increased with an increase in the composition of Chitosan in coagulation bath and membrane M-0.8 showed a maximum fouling resistance ratio of 74%. © 2013 Elsevier B.V.Item Preparation and characterization study of PPEES/chitosan composite membrane crosslinked with tripolyphosphate(Elsevier, 2014) Shenvi, S.; A.F., A.F.; Isloor, A.M.A novel composite membrane was prepared using chitosan (CH) as the active layer supported on a Poly(1,4-phenylene ether ether sulfone) (PPEES) membrane. The chitosan layer was ionically cross linked with sodium tripolyphosphate (TPP). The composite nature of PPEES/CH membranes was confirmed by Scanning Electron Microscopy (SEM). Infrared (IR) spectroscopy results and SEM-EDX analysis confirmed the crosslinking of chitosan surface with TPP. The membranes exhibited higher crosslinking density in acidic media than in basic media. The changes in the hydrophobic nature of PPEES membrane surface due to deposition of chitosan active layer followed by crosslinking were studied by their contact angle measurement, water flux and water uptake studies. The membranes showed rejection up to 55% and 21% towards MgSO4 and NaCl respectively at pH=5 and a flux recovery ratio of 73%. © 2014 Elsevier B.V.Item Expression studies of Bacillus licheniformis chitin deacetylase in E. coli Rosetta cells(Elsevier B.V., 2017) Raval, R.; Simsa, R.; Raval, K.Chitin, the biopolymer of the N-acetylglucosamine, is the most abundant biopolymer on the planet after cellulose. However owing to its crystalline nature, its deacetylated derivative; chitosan is industrially more potent. This conversion on an enzymatic scale can be made using chitin deacetylase. The metagenomics library constructed from the soil exposed to chitin and chitosan yielded chitin modifying enzymes, one of them being chitin deacetylase (CDA) utilized for the present study. The gene was amplified and expressed using the pET 22b vector in E. coli Rosetta cells. The effect of two additives; chitin and glycerol on the CDA activity were studied. The inclusion of glycerol in the medium improved the biomass by 50% from the initial value of 1.25 g/l to 2.5 g/l. The activity of CDA increased from 90 ?mol/min/ml to 343 ?mol/min/ml. The CDA activity reported in the present paper is the highest observed for any strain. The addition of glycerol to the media not only helped improve the yield of the chitin deacetylase but also imparted value addition to the waste of the biofuel industry. © 2017 Elsevier B.V.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 Cloning, expression, purification and characterization of chitin deacetylase extremozyme from halophilic Bacillus aryabhattai B8W22(Springer Science and Business Media Deutschland GmbH, 2021) Pawaskar, G.M.; Raval, K.; Rohit, P.; Shenoy, R.P.; Raval, R.Chitin deacetylase (CDA) (EC 3.5.1.41) is a hydrolytic enzyme that belongs to carbohydrate esterase family 4 as per the CAZY database. The CDA enzyme deacetylates chitin into chitosan. As the marine ecosystem is a rich source of chitin, it would also hold the unexplored extremophiles. In this study, an organism was isolated from 40 m sea sediment under halophilic condition and identified as Bacillus aryabhattai B8W22 by 16S rRNA sequencing. The CDA gene from the isolate was cloned and overexpressed in E. coli Rosetta pLysS and purified using a Ni–NTA affinity chromatography. The enzyme was found active on both ethylene glycol chitin (EGC) and chitooligosaccharides (COS). The enzyme characterization study revealed, maximum enzyme velocity at one hour, optimum pH at 7 with 50 mM Tris–HCl buffer, optimum reaction temperature of 30 ºC in standard assay conditions. The co-factor screening affirmed enhancement in the enzyme activity by 142.43 ± 7.13% and 146.88 ± 4.09% with substrate EGC and COS, respectively, in the presence of 2 mM Mg2+. This activity was decreased with the inclusion of EDTA and acetate in the assay solutions. The enzyme was found to be halotolerant; the relative activity increased to 116.98 ± 3.87% and 118.70 ± 0.98% with EGC and COS as substrates in the presence of 1 M NaCl. The enzyme also demonstrated thermo-stability, retaining 87.27 ± 2.85% and 94.08 ± 0.92% activity with substrate EGC and COS, respectively, upon treatment at 50 ºC for 24 h. The kinetic parameters Km, Vmax, and Kcat were 3.06E?05 µg mL?1, 3.06E + 01 µM mg?1 min?1 and 3.27E + 04 s?1, respectively, with EGC as the substrate and 7.14E?07 µg mL?1, 7.14E + 01 µM mg?1 min?1 and 1.40E + 06 s?1, respectively, with COS as the substrate. The enzyme was found to be following Michaelis–Menten kinetics with both the polymeric and oligomeric substrates. In recent years, enzymatic conversion of chitosan is gaining importance due to its known pattern of deacetylation and reproducibility. Thus, this BaCDA extremozyme could be used for industrial production of chitosan polymer as well as chitosan oligosaccharides for biomedical application. © 2021, The Author(s).Item A marine chitinase from Bacillus aryabhattai with antifungal activity and broad specificity toward crystalline chitin degradation(Taylor and Francis Ltd., 2022) Subramani, A.K.; Raval, R.; Sundareshan, S.; Sivasengh, R.; Raval, K.Chitinases convert chitin into chitin oligomers and are also known antifungal agents. Chitin oligomers have numerous industrial applications. However, chitin’s crystalline nature requires pretreatment before breakdown into oligomers. In the study, a novel marine bacterium Bacillus aryabhattai is isolated from the Arabian Sea. Bacterial growth in different crystalline chitin substrates like chitin powder, chitin flakes, and colloidal chitin confirmed the chitinase presence in bacterium could act upon insoluble crystalline chitin with the fractional release of oligomers. The domain architecture analysis of the chitinase confirmed the presence of two N-terminal LysM domains which help enzyme action on crystalline chitin. Statistical optimization of media and Process parameters revealed glycerol, yeast extract, magnesium chloride, and manganese sulfate as significant media components along with colloidal chitin. The optimum process parameters such as pH 7, temperature 40 °C, inoculum size 12.5% (v/v), and inoculum age 20 hours enhanced the specific enzyme activity to ±146.2 U/mL, ±114.9 U/mL and ±175.4 U/mL against chitin powder, chitin flakes and colloidal chitin respectively, which is five to six times higher than basal level activity. The antifungal activity of chitinase against plant pathogenic fungi like Candida albicans and Fusarium oxysporum revealed a zone of inhibition with 14 mm diameter. © 2022 Taylor & Francis Group, LLC.Item Engineering a recombinant chitinase from the marine bacterium Bacillus aryabhattai with targeted activity on insoluble crystalline chitin for chitin oligomer production(Elsevier B.V., 2024) Subramani, A.K.; Ramachandra, R.; Thote, S.; Govindaraj, V.; Vanzara, P.; Raval, R.; Raval, K.Chitin, an abundant polysaccharide in India, is primary by-product of the seafood industry. Efficiently converting chitin into valuable products is crucial. Chitinase, transforms chitin into chitin oligomers, holds significant industrial potential. However, the crystalline and insoluble nature of chitin makes the conversion process challenging. In this study, a recombinant chitinase from marine bacteria Bacillus aryabhattai was developed. This enzyme exhibits activity against insoluble chitin substrates, chitin powder and flakes. The chitinase gene was cloned into the pET 23a plasmid and transformed into E. coli Rosetta pLysS. IPTG induction was employed to express chitinase, and purification using Ni-NTA affinity chromatography. Optimal chitinase activity against colloidal chitin was observed in Tris buffer at pH 8, temperature 55°C, with the presence of 400 mM sodium chloride. Enzyme kinetics studies revealed a Vmax of 2000 μmole min−1 and a Km of 4.6 mg mL−1. The highest chitinase activity against insoluble chitin powder and flakes reached 875 U mg−1 and 625 U mg−1, respectively. The chitinase demonstrated inhibition of Candida albicans, Fusarium solani, and Penicillium chrysogenum growth. Thin Layer Chromatography (TLC) and LC-MS analysis confirmed the production of chitin oligomers, chitin trimer, tetramer, pentamer, and hexamer, from chitin powder and flakes using recombinant chitinase. © 2024 Elsevier B.V.