Studies on the Production, Optimization, Partial Purification and Characterization of the Thermotolerant Carboxymethyl Cellulase From A Bacillus Strain Isolated from Local

Abstract

CMCases are important enzymes that can hydrolyze lignocellulosic mass efficiently. Apart from this, CMCases are used in industries pertaining to textile, food processing, wine and brewery, pulp & paper, animal feed, agro based products, detergents, waste management, olive oil extraction and carotenoid extraction. The aim of this study is to explore the diversity of the indigenous thermophilic bacteria that are capable of degrading cellulosic biomass. A total of thirty-seven bacterial strains were isolated from stove ash samples. All the isolates were Gram positive and on the basis of physico-biochemical characterization identified and named as Bacillus sp TLW-1 to TLW-37. Plate screening was performed for ten different industrially important hydrolytic enzymes. Almost all the isolates were able to produce all the tested enzymes (except pectinase) with different profile. Positive CMCase producers were quantitatively screened by shake flask method. The isolate Bacillus sp. TLW-3 showed the highest CMCase production and was selected for further study. Isolate was identified on the basis of 16S rDNA sequencing as Bacillus licheniformis TLW-3. Sequence was submitted to NCBI (with KY440432 accession number). According to the growth curve, the isolate entered into log phase after 6 hrs of incubation and the log phase ended after 30 hrs incubation. Afterwards, the stationary phase began which ended after an incubation period of 72 hrs then the decline phase started. Highest enzyme production was found during the stationary phase. Optimization of production and growth conditions was performed by using two techniques i.e. OFAT and RSM. According to the OFAT approach, the highest growth and CMCase production were obtained in the medium containing 1% CMC, 1% peptone and 0.5% yeast extract with pH 7.0 at 50˚C and 150 rpm after the incubation period of 72 hrs. When compared with RSM optimization, the best growth was found at 70˚C in the medium having 0.5% peptone, 2.3% yeast extract and 2.52% CMC with pH 9.0 after 49.2 hrs of incubation. RSM optimized production conditions for highest CMCase production was found to be 1.8% peptone, 1.4% yeast extract and 1.6% CMC with pH 7.0 at 70˚C and 69.63 hrs of incubation period. Co-production of the enzyme was obtained in the medium containing 1% CMC and 1% starch as the sole carbon source. For the purification purpose, various precipitation and chromatography techniques were employed. CMCase was successfully (partially) purified with 8.250-fold and 13.75 % yield. Molecular weight of CMCase was estimated to be 28 kDa. Enzyme kinetics of partially purified CMCase demonstrated that enzyme was found active in the reaction mixture containing 1% CMC with pH 7.0 and at temperature 70˚C in the presence of Co+2, Ca+2 and Hg+2 ions. When the glucose was added in the reaction mixture enzyme activity was inhibited. The stability conditions of CMCase include, pH 6.0 and at temperature 4-50˚C; however, the stability was totally lost in the presence of the tested metal ions. The Vmax, Km and activation energy for CMCase were found to be 42.553 IU/ml/min, 9.587 mg/ml and 2.649 KJ/mole respectively. Endoglucanase gene was amplified, sequenced (11888 bp) and submitted to NCBI (with MF953225 accession number). Expasy-protparam online server was used for amino acid translation and in slicio physico-chemical characterization of the sequence. Secondary and tertiary structure of the protein revealed using SOPMA and ITASSER online server respectively.