Mukesh Kapoor

COST-EFFECTIVE ENDO-MANNANASE FROM Bacillus sp. CFR1601 AND ITS APPLICATION IN GENERATION OF OLIGOSACCHARIDES FROM GUAR GUM AND AS DETERGENT ADDITIVE

The indigenous bacteria Bacillus sp. CFR1601 produced significant levels of endo-mannanase when grown on agro-wastes, namely, green gram husk and sunflower oil cake (25.6 IU/mL), used as sole carbon and nitrogen sources, respectively. Under immobilized cell system, synthetic supports (polyurethane foam, scotch brite, polyester; up to 33.2 IU/mL) were found marginally superior as compared to natural supports (cotton and silk; up to 28.2 IU/mL) for endo-mannanase production. Cooperative interactions between L-lysine HCl (0.3% w/v), Tween 60 (0.3% v/v), and sunflower oil cake (3.0% w/v) in central composite design response surface methodology ameliorated (1.61-fold) endo-mannanase titers to 48.0 IU/mL. Partially purified endo-mannanase was tested for its ability to produce oligosaccharides from guar gum. These oligosaccharides were tested in vitro for their ability to promote growth of Lactobacillus plantarum MTCC 5422 and Lactobacillus salivarius CHS 1E. Results indicated that low-molecular-weight degraded products from guar gum were (1) able to support the growth of tested strains [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; and (2) after purification and analysis by electron spray ionization–mass spectrometry (ESI-MS) were found to be composed of mainly disaccharide and tetrasaccharide. The compatibility of endo-mannanase with various detergents together with wash performance test confirmed its potential applicability for laundry industry.

Production, properties, and applications of endo-β-mannanases

The present review provides up-to-date information on the occurrence and methodologies used for producing and purifying endo-β-mannanases and a comprehensive comparison of their biochemical properties. The amalgamation of biochemical, molecular and structural biology approaches which have been used for understanding endo-β-mannanase families, catalytic mechanism, substrate binding, non-catalytic modules, trans-glycosylation, and multi-functional enzyme complexes has been given critical attention. A separate section entailing the state-of-the-art about thermostable endo-β-mannanases, which has emerged as an exciting field of both basic and applied research, is also deliberated. The remarkable progress made by endo-β-mannanases in various industrial sectors like food, feed, detergents, biofuel and oil drilling is also emphasized.

Recombinant endo-mannanase (ManB-1601) production using agro-industrial residues: Development of economical medium and application in oil extraction from copra

Expression of pRSETA manb-1601 construct in Hi-Control Escherichia coli BL21 (DE3) cells improved recombinant endo-mannanase (ManB-1601) production by 2.73-fold (1821±100U/ml). A low-cost, agro-industrial residue supplemented industrial medium for enhanced and economical production of ManB-1601 was developed in two mutual phases. Phase-I revealed the potential of various pre- (induction time: 5h, induction mode: lactose 0.5mM) and post-induction [peptone supplementation: 0.94%(w/v), glycerol 0.123%(v/v)] parameters for enhanced production of ManB-1601 and resulted in 4.61-fold (8406±400U/ml) and 2.53-fold (3.30g/l) higher ManB-1601 and biomass production, respectively. Under phase-II, economization of phase-I medium was carried out by reducing/replacing costly ingredients with solubilized-defatted flax seed meal (S-DFSM), which resulted in 3.25-fold (5926U/ml) higher ManB-1601 production. Industrial potential of ManB-1601 was shown in oil extraction from copra as enzyme treatment led to cracks, peeling, fracturing and smoothening of copra, which facilitated higher (18.75%) oil yield.

Metal-dependent thermal stability of recombinant endo-mannanase (ManB-1601) belonging to family GH 26 from Bacillus sp. CFR1601

A GH 26 endo-mannanase from Bacillus sp. CFR1601 was purified to homogeneity (Mw ∼39kDa, specific activity 10,461.5±100IU/mg). Endo-mannanase gene (manb-1601, 1083bp, accession No. KM404299) was expressed in Escherichia coli BL21 (DE3) and showed typical fingerprints of α/β proteins in the far-UV CD. A high degree of conservation among amino acid residues involved in metal chelation (His-1, 23 and Glu-336) and internal repeats (122-152 and 181-212) was observed in endo-mannanases reported from various Bacillus sp. Thermal inactivation kinetics suggested that metal ions are quintessential for stabilization of ManB-1601 structure as holoenzyme (Ea 87.4kcal/mol, ΔH 86.7kcal/mol, ΔS 186.6cal/k/mol) displayed better values of thermodynamic parameters compared to metal-depleted ManB-1601 (Ea 47kcal/mol, ΔH 45.7kcal/mol, ΔS 64.7cal/k/mol). EDTA treatment of ManB-1601 not only lead to transitions in both secondary and tertiary structure but also promulgated the population of conformational state that unfolds at lower temperature. ManB-1601 followed a three-state process for thermal inactivation wherein loss of tertiary structure preceded the concurrent loss of secondary structure and activity.

Structural Characterization and in Vitro Fermentation of β-Mannooligosaccharides Produced from Locust Bean Gum by GH-26 endo-β-1,4-Mannanase (ManB-1601)

Size exclusion chromatography of β-mannooligosaccharides (β-MOS) mixtures, obtained from ManB-1601 hydrolysis of locust bean gum, resulted in separation of oligosaccharides with various degrees of polymerization (DP 2, 3, and 5). The oligosaccharides were structurally [ESI-MS, FTIR, XRD, TGA, and NMR (1H and 13C)] and functionally (in vitro fermentation) characterized. DP2 oligosaccharide was composed of two species, (A) mannopyranose β-1,4 mannopyranose and (B) α-1,6-galactosyl-mannopyranose, while DP3 oligosaccharide showed the presence of only one species, i.e. α-d-galactosyl-β-d-mannobiose. ManB-1601 was capable of cleaving near the branch points in the substrate, resulting in oligosaccharides with galactose at the terminal position apart from attacking unsubstituted β-1,4-glycosidic linkages. DP2 and DP3 improved the growth of three out of seven species of Lactobacillus while DP5 resulted in poor growth of all Lactobacillus spp. under in vitro conditions. DP2, DP3, and DP5 were found to inhibit the growth of Escherichia coli, Listeria monocytogenes and Salmonella typhi.

Cross-linked enzyme aggregates (CLEAs) and magnetic nanocomposite grafted CLEAs of GH26 endo-β-1,4-mannanase: Improved activity, stability and reusability

A comparative study on immobilization of recombinant endo-β-1,4-mannanase (ManB-1601), using cross-linked aggregated form (MB-C) and novel chitosan magnetic nanocomposites of MB-C (MB-Mag-C) was carried out. FT-IR and Raman spectroscopy were used to confirm the surface modifications while, scanning electron and atomic force microscopy were performed to demonstrate the surface topology and magnetic nature of MB-C and MB-Mag-C. Among MB-C and MB-Mag-C, the former showed better activity and stability in broad range of pH, thermo-stability and kinetic parameters while, the latter showed higher temperature optima and solvent stability. MB-C and MB-Mag-C when compared with free enzyme showed up to 73.2% higher activity (pH 4-9), up to 95.6% higher stability (pH 3-10, 9h incubation at room temperature), up to 15°C higher optimal temperature, higher stability (up to 83%) in the presence of solvents and up to 1.62-fold higher deactivation energy (Ed). Immobilized enzymes were able to repeatedly hydrolyze locust bean gum till 12 cycles and generated predominantly di-, tri- and tetra- species of β-manno-oligosaccharides.

Low molecular weight α-galactosidase from black gram (Vigna mungo): Purification and insights towards biochemical and biophysical properties

A hitherto unknown low molecular weight form of α-galactosidase (VM-αGal-P) from germinating black gram (Vigna mungo) seeds was purified (324 U/mg specific activity, 1157-fold purification, ~45 kDa) using ion-exchange (DEAE-cellulose, CM-sepharose), gel filtration (Sephadex G-75) and affinity (Con-A Sepharose 4B) chromatography but with poor yield (0.75%). Partially purified enzyme (VM-αGal) (146.3 U/mg specific activity, 522.5-fold purification) was used for further studies. VM-αGal showed optimal activity at pH 5 and 55 °C. Hg2+ and SDS completely inhibited VM-αGal activity. The Km, Vmax and catalytic efficiency (kcat/Km) of VM-αGal for pNPG and raffinose was 0.99, 17.23 mM, 1.66, 0.146 μmol ml-1 min-1, and 0.413, 0.0026 s-1 mM-1, respectively. VM-αGal was competitively inhibited by galactose (Ki 7.70 mM). Thermodynamic parameters [activation enthalpy (ΔH), activation entropy (ΔS) and free energy (ΔG)] of VM-αGal at 45-51 °C showed that VM-αGal was in a less energetic state and had susceptibility towards denaturation. Temperature-induced structural unfolding studies of VM-αGal probed by fluorescence, and far-UV CD spectroscopy revealed significant loss in tertiary structure and a steep decline in β-sheet content at 45-65 °C, and above 55 °C, respectively. VM-αGal improved the nutritional quality of soymilk by hydrolyzing raffinose family oligosaccharides (26.5% and 18.45% decrease in stachyose and raffinose, respectively).