Mechanochemical Coupling of Catalysis and Motion in a Cellulose-Degrading Multienzyme Nanomachine.

The cellulosome is a megadalton-size protein complex that functions as a biological nanomachine of cellulosic fiber degradation. We show that the cellulosome behaves as a Brownian ratchet that rectifies protein motions on the cellulose surface into a propulsion mechanism by coupling to the hydrolysis of cellulose chains. Movement on cellulose fibrils is unidirectional and results from "macromolecular crawl" composed of dynamic switches between elongated and compact spatial arrangements of enzyme subunits. Deletion of the main exocellulase Cel48S eliminates conformational bias for aligning the subunits to the long fibril axis, which we reveal as crucial for optimum coupling between directional movement and substrate degradation. Implications of the cellulosome acting as a mechanochemical motor suggest a distinct mechanism of enzymatic machinery in the deconstruction of cellulose assemblies.

Processive Enzymes Kept on a Leash: How Cellulase Activity in Multienzyme Complexes Directs Nanoscale Deconstruction of Cellulose.

Biological deconstruction of polymer materials gains efficiency from the spatiotemporally coordinated action of enzymes with synergetic function in polymer chain depolymerization. To perpetuate enzyme synergy on a solid substrate undergoing deconstruction, the overall attack must alternate between focusing the individual enzymes locally and dissipating them again to other surface sites. Natural cellulases working as multienzyme complexes assembled on a scaffold protein (the cellulosome) maximize the effect of local concentration yet restrain the dispersion of individual enzymes. Here, with evidence from real-time atomic force microscopy to track nanoscale deconstruction of single cellulose fibers, we show that the cellulosome forces the fiber degradation into the transversal direction, to produce smaller fragments from multiple local attacks ("cuts"). Noncomplexed enzymes, as in fungal cellulases or obtained by dissociating the cellulosome, release the confining force so that fiber degradation proceeds laterally, observed as directed ablation of surface fibrils and leading to whole fiber "thinning". Processive cellulases that are enabled to freely disperse evoke the lateral degradation and determine its efficiency. Our results suggest that among natural cellulases, the dispersed enzymes are more generally and globally effective in depolymerization, while the cellulosome represents a specialized, fiber-fragmenting machinery.

Molecular advancements on over-expression, stability and catalytic aspects of endo-ß-mannanases.

The hydrolysis of mannans by endo-ß-mannanases continues to gather significance as exemplified by its commercial applications in food, feed, and a rekindled interest in biorefineries. The present review provides a comprehensive account of fundamental research and fascinating insights in the field of endo-ß-mannanase engineering in order to improve over-expression and to decipher molecular determinants governing activity-stability during harsh conditions, substrate recognition, polysaccharide specificity, endo/exo mode of action and multi-functional activities in the modular polypeptide. In-depth analysis of the available literature has also been made on rational and directed evolution approaches, which have translated native endo-ß-mannanases into superior biocatalysts for satisfying industrial requirements.

Zn2+ stapling of N and C-terminal maintains stability and substrate affinity in GH26 endo-mannanase.

Metal binding sites are present in one-third of proteins and are crucial for biological functions and structural maintenance. GH26 endo-mannanase (ManB-1601) from Bacillus sp. harbors a Zn2+ binding site which connects N (H1, H23) and C (E336)-terminal residues. Present study reveals how native circularization of ManB-1601 through Zn2+ coordination regulates the structure-function. We generated individual Zn2+ coordinating mutants and characterized them using biochemical and biophysical approaches. Contribution of individual Zn2+ coordination towards maintaining ManB-1601 stability and rigidity was in the following order H23>H1 > E336. Elimination of E336 and H23-Zn2+ coordination affected substrate hydrolysis to a greater degree than H1-Zn2+ coordination. Metal quantification of mutant proteins indicated that H23A did not contain Zn2+. Molecular dynamic simulation studies revealed disruption of H23-Zn2+ coordination leads to increased flexibility of N and C-terminal, active site loops and consequent drifting of substrate away from the active site region. Finally, mechanistic understanding on the functioning of Zn2+ site in ManB-1601 is developed wherein 1) H23 by anchoring Zn2+ ion majorly regulates the structure-function properties, 2) H1 provides thermo-stability, 3) E336 contributes towards maintaining substrate hydrolysis.

Salt bridges are pivotal for the kinetic stability of GH26 endo-mannanase (ManB-1601).

Hitherto, how salt bridges contribute towards the structure-function of endo-mannanases has not been demonstrated. In the present study, we revealed that ManB-1601 (GH26 endo-mannanase from Bacillus sp.) has eight salt bridges which are highly conserved among GH26 endo-mannanases from Bacillus spp. Disruption of salt bridges do not alter overall structure, optimum pH and temperature of ManB-1601. Among the salt bridges, elimination of K95/E156 and E171/R221 pair decreased the substrate affinity and catalytic efficiency of ManB-1601. Differential scanning calorimetry and isothermal equilibrium denaturation studies suggested that salt bridges do not markedly contribute towards thermal (∆Tm 0 to -5 °C) and conformational stability (∆∆G -0.37 to -1.25 Kcal mol-1) of ManB-1601. Interestingly, salt bridges were found to prominently contribute towards kinetic stability of ManB-1601 as salt bridge mutants exhibited drastic reduction in half-life of enzyme inactivation (T1/2) at 66 °C (1.1 to 6-fold) and 70 °C (4.09 to 22.5-fold). Molecular dynamic simulations studies showed that salt bridges contribute towards maintaining the biological activity against thermal denaturation by rigidifying the active site. Our study on ManB-1601 suggest that even in the case salt bridges do not confer thermal and conformational stabilities they may serve as crucial structural elements for enzyme functioning by contributing towards kinetic stability.

How substrate subsites in GH26 endo-mannanase contribute towards mannan binding.

Comprehensive knowledge on the role of substrate subsites is a prerequisite to understand the interaction between glycoside hydrolase and its substrate. The present study delineates the role of individual substrate subsites present in ManB-1601 (GH26 endo-mannanase from Bacillus sp.) towards interaction with mannans. Isothermal titration calorimetry of catalytic mutant (E167A/E266A) of ManB-1601 with mannobiose to mannohexose revealed presence of six substrate subsites in ManB-1601. The amino acids present in substrate subsites of ManB-1601 were found to be highly conserved among GH26 endo-mannanases from Bacillus spp. Qualitative substrate binding analysis of subsite mutants by native affinity gel electrophoresis suggested that -3, -2, -1, +1 and + 2 subsites have a major role while, -4 subsite had minor role towards mannan binding. Affinity gels also pointed out the pivotal role of -1 subsite towards glucomannan binding. Quantitative substrate binding analysis using fluorescence titration revealed that -1 and -2 subsite mutants had 27- and 30-fold higher binding affinity (KD) for carob galactomannan when compared with catalytic mutant. The -1 subsite mutant also had highest KD values for glucomannan (13.6-fold) and ivory nut mannan (5-fold) among all mutants. The positive subsites contributed more towards binding with glucomannan (up to 10-fold higher KD) and ivory nut mannan (up to 4.3-fold higher KD) rather than carob galactomannan (up to 4-fold higher KD). Between distal subsites, -3 mutant displayed 10-fold higher KD for both carob galactomannan and glucomannan while, -4 mutant did not show any noticeable change in KD values when compared to catalytic mutant.

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 ß-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).

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

A comparative study on immobilization of recombinant endo-ß-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 ß-manno-oligosaccharides.

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.

A psychrotolerant strain of Serratia marcescens (MTCC 4822) produces laccase at wide temperature and pH range.

A psychrotolerant bacterial strain of Serratia marcescens, originally isolated from a glacial site in Indian Himalayan Region (IHR), has been investigated for laccase production under different culture conditions. The bacterial strain was found to grow between 4 to 45°C (opt. 25°C) and 3 to 14 pH (opt. 5 pH) on prescribed growth medium, coinciding with production of laccase in laccase producing medium. However, the production of laccase was more consistent toward alkaline pH. Laccase enzyme was partially purified using gel filtration chromatography. The molecular mass of laccase was determined ~53 kDa on native PAGE. The Km and Vmax values were determined to be 0.10 mM and 50.00 µM min(-1), respectively, with ABTS. Inoculum size (4.0% v/v at 1.5 O.D.) resulted in significantly higher production of laccase. Carbon and nitrogen sources also affected the laccase production significantly. All the carbon sources enhanced laccase production, xylose being the best enhancer (P < 0.01). Among nitrogen sources, organic sources were found to act as inhibitors (P < 0.01), and among the in-organic sources only sodium nitrate enhanced the laccase production. Low molecular weight organic solvents significantly (P < 0.01) enhanced laccase production up to 24 h of incubation with a decline in later incubation period. Production of laccase by the psychrotolerant bacterium in wide range of temperature and pH is likely to have inference in biotechnological processes.