Characterization of Mannanase from Bacillus sp. and its Biotechnological Applications

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
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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.