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Characterization of Mannanase from Bacillus sp. and its Biotechnological Applications

Praveen Kumar, Srivastava (2015) Characterization of Mannanase from Bacillus sp. and its Biotechnological Applications. PhD thesis, University of Mysore.

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Abstract

Mannans/hetromannans are important constituents of plant cell wall hemicelluloses. Endo-mannanases play a pivotal role in mannan degradation along with other mannan degrading enzymes. Mannanases have immense potential in various industrial sectors which include food and feed, coffee, and fruit juice clarification, pulp bleaching, oil drilling, fabric cleaning and bio-energy. An endo-mannanase producing Bacillus sp. CFR1601 was isolated from decaying plant litter and identified using standard microbiological and molecular techniques. In SmF, green gram husk and sunflower oil meal supported highest endo-mannanase production (25.6 IU/ml). Bacillus sp. CFR1601 cells Immobilized on synthetic supports were found to be marginally superior for endomannanase production (up to 33.2 IU/ml) as compared to natural supports (up to 28.2 IU/ml). Combination of L-lysine HCl, tween-60, and sunflower oil cake in central composite design (CCD) of response surface methodology (RSM) ameliorated (1.61- fold) endo-mannanase titres to 48 IU/ml. In SSF, enhanced endo-mannanase production from Bacillus sp. CFR1601 was attained when defatted coconut residue was supplemented with sesame oil meal, tween-80, and inoculated with 12h old (O.D600nm≈ 3.6) bacterial cells. CCD of RSM resulted in 4.04-4.39 fold (800-870 IU/g) improvement in endo-mannanase yield as compared to un-optimized growth conditions. Aqueous two phase system consisting of a combination of PEG 3350 and Na2SO4 resulted in partitioning of endo-mannanase towards bottom phase (3.8-fold purification and 95.4% recovery). Second stage ATPS further improved the purification fold (12.32). The enzyme was purified to homogeneity (Mw~39 kDa, specific activity 10,461 ± 100 IU/mg) and identified to be a member of GH-26. Endo-mannanase gene (manb-1601, 1083 bp, accession No. KM404299) was expressed in E. coli BL21 (DE3), and the expressed enzyme (ManB-1601) (666 IU/ml) was found to be a typical α/β protein. A high degree of conservation among amino acid residues involved in metal chelation (His-1, 23 and Glu-336) and internal repeats (122 to 152 and 181 to 212) was observed in ManB-1601 as well as other endo-mannanases reported from various Bacillus sp. Thermal inactivation kinetics suggested that metal ions are quintessential for stability of ManB- 1601 as holoenzyme (Ea 87.4 kcal/mol, �H 86.7 kcal/mol, �S 186.6 cal/k/mol) displayed better values of thermodynamic parameters compared to metal-depleted ManB-1601 (Ea 47 kcal/mol, �H 45.7 kcal/mol, �S 64.7 cal/k/mol). EDTA treatment of ManB-1601 led to subtle transitions in both secondary and tertiary structure and also viii promulgated the population of conformational state that unfolds at relatively lower temperature. The enzyme followed a three-state process for thermal inactivation wherein loss of tertiary structure preceded the concurrent loss of secondary structure and activity. ManB-1601 was optimally active at moderately high temperature (550C) and neutral pH (pH 7) and showed excellent stability over a broad pH range. The apparent Km, Vmax, and Vmax/Km values of ManB-1601 were 6.5 mg/ml, 5000 IU/ml/min, and 769.2 μmol/min/mg, respectively. Enzyme was stable in the presence of organic solvents, certain heavy metals, and displayed tolerance towards proteases. Partially purified endomannanase generated low molecular weight degraded products from guar gum (consisting of DP 2 and 4 oligosaccharides) which supported the growth of Lactobacillus spp. [increased O.D600nm up to 2.3-fold and decrease in pH (<6.3) due to production of short chain fatty acid (SCFA)] when used as sole carbon source. Hydrolysis of locust bean gum by ManB-1601 produced mannooligosaccharides (MOS) of degree of polymerization 2-5 along with mannose as revealed by ESI-MS. In 1H and 13C NMR, chemical shifts of DP 2 revealed presence of both α-1, 6-galactosyl-mannopyranose and un-substituted mannobiose while chemical shifts of DP 3 indicated absence of any unsubstituted moiety. The terminal galactosyl residue in oligosaccharides suggested presence of hitherto less known ability in ManB-1601 of cleaving mannosidic linkages near galactose substitution which are otherwise difficult due to steric hindrance. Lactobacillus casei var rhamnosus, L. casei, L. fermentum, and L. plantarum when grown on DP 2 and 3 MOS showed up to 51-fold and up to 3.46-fold higher CFU/ml and A600nm, respectively, when compared to positive control. However, growth parameters were lower than positive control when DP 5 MOS were used as sole carbon source. ManB-1601 hydrolysis of guar gum (GG) led to the production of partially hydrolyzed guar gum (PHGG) with high flow behaviour index and markedly reduced average degree of polymerization, average molecular weight and apparent viscosity. FTIR spectra of GG and PHGG indicated similarity in basic chemical structure. The compatibility of endomannanase with various detergents along with wash performance test corroborated its potential in laundry industry.

Item Type: Thesis (PhD)
Uncontrolled Keywords: Mannans, hetromannans, plant cell wall hemicelluloses, Mannanases, decaying plant litter
Subjects: 500 Natural Sciences and Mathematics > 10 Plants > 01 Plant Cell
500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 16 Enzyme Chemistry
Divisions: Protein Chemistry and Technology
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 19 May 2016 06:51
Last Modified: 19 May 2016 06:51
URI: http://ir.cftri.com/id/eprint/12174

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