Mannans are the major constituents of the hemicellulose fraction in softwoods and show widespread distribution in plant tissues. The major mannan-degrading enzymes are β-mannanases, β-mannosidases and β-glucosidases. In addition to these, other enzymes such as α-galactosidases and acetyl mannan esterases, are required to remove the side chain substituents. The mannanases are known to be produced by a variety of bacteria, fungi, actinomycetes, plants and animals. Microbial mannanases are mainly extracellular and can act in wide range of pH and temperature because of which they have found applications in pulp and paper, pharmaceutical, food, feed, oil and textile industries. This review summarizes the studies on mannanases reported in recent years in terms of important microbial sources, production conditions, enzyme properties, heterologous expression and potential industrial applications.

A novel extracellular thermo-alkali-stable laccase from Bacillus tequilensis SN4 (SN4LAC) was purified to homogeneity. The laccase was a monomeric protein of molecular weight 32 KDa. UV-visible spectrum and peptide mass fingerprinting results showed that SN4LAC is a multicopper oxidase. Laccase was active in broad range of phenolic and non-phenolic substrates. Catalytic efficiency (k cat/K m) showed that 2, 6-dimethoxyphenol was most efficiently oxidized by the enzyme. The enzyme was inhibited by conventional inhibitors of laccase like sodium azide, cysteine, dithiothreitol and β-mercaptoethanol. SN4LAC was found to be highly thermostable, having temperature optimum at 85°C and could retain more than 80% activity at 70°C for 24 h. The optimum pH of activity for 2, 6-dimethoxyphenol, 2, 2′-azino bis[3-ethylbenzthiazoline-6-sulfonate], syringaldazine and guaiacol was 8.0, 5.5, 6.5 and 8.0 respectively. Enzyme was alkali-stable as it retained more than 75% activity at pH 9.0 for 24 h. Activity of the enzyme was significantly enhanced by Cu2+, Co2+, SDS and CTAB, while it was stable in the presence of halides, most of the other metal ions and surfactants. The extracellular nature and stability of SN4LAC in extreme conditions such as high temperature, pH, heavy metals, halides and detergents makes it a highly suitable candidate for biotechnological and industrial applications.

Degradation of residual lignin in kraft pulp by chemical bleaching is implicated in causing environmental pollution. The use of thermo- and alkali-tolerant bacterial laccases is considered to be important biological alternative to chemical processing. Laccases from Bacillus species have shown promise in this respect but their intracellular/spore bound presence make their industrial application economically unfeasible. We report here on a novel extracellular active thermo-alkali-stable laccase (SN4 laccase)  which is active at 90 °C and pH 8.0 using 2,6-dimethoxyphenol as substrate from Bacillus tequilensis SN4. SN4 laccase retained 27 % activity for 5 min at 100 °C and more than 80 % activity for 24 h at 70 °C. The enzyme is also stable at a higher pH (9.0–10.0). Enzyme production was optimized by submerged fermentation. Relatively high yields (18,356 nkats ml−1) of SN4 laccase was obtained in a medium containing 650 μM MnSO4, 350 μM FeSO4, and 3.5 % ethanol. A 764-fold increase in laccase activity was observed under optimal conditions. In addition, reduction in kappa number and increase in brightness of softwood pulp by 28 and 7.6 %, respectively, were observed after treatment with SN4 laccase without a mediator. When N-hydroxybenzotriazole was used as a mediator, the kappa number was decreased to 47 % and brightness was increased to 12 %.

Crystals tructuresa re knownf or severalg lycosylh ydrolase family 10 (GH10) xylanases. However, none of them is from an alkalophilic organism that cang rowi na lkalineconditions. We have determined thec rystal structures at 2.2Å of aGH10extracellular endoxylanase (BSX)fromanalkalophilic Bacillus sp.NG-27, for then ativea nd thec omplex enzyme with xylosaccharides. Thei ndustriallyi mportant enzyme is optimally active ands tablea t3 43 Ka nd at ap Ho f8 .4.C omparisono ft he structureo fB SX with thoseo fo ther thermostable GH10 xylanaseso ptimally active at acidic or closet o neutralp Hs howedt hatt he solventexposeda cidica mino acids, Aspa nd Glu, arem arkedlye nhancedi nB SX,w hile solvent-exposedA sn was noticeably depleted.T he BSXc rystal structurew henc omparedw ithp uta tive three-dimensionalh omology models of otherextracellular alkalophilic GH10 xylanasesfromalkalophilicorganisms suggests that aprotein surfacerichinacidicresiduesmay be an importantfeature common to thesealkalithermostableenzymes.A comparison of thesurface features of BSXand of halophilic proteins allowedustopredict theactivityofBSX at high salt concentrations,w hich we verified throughe xperiments.Thisoffered us importantl essons in the polyextremophilicity of proteins,where understandingthe structural features of aprotein stable in oneset of extremec onditionsp rovidedc lues aboutt he activity of thep rotein in othere xtreme conditions.T he work brings to thef oret he role of then aturea nd compositiono fs olvent-exposedr esiduesi nt he adaptation of enzymest op olyextreme conditions,a si nB SX.Keywords: alkali thermostable; GH10 xylanase;s olvent-exposed acidic residues; solvent-exposed basic residues; polyextremophilicity; alkalophilic organism Supplemental material: see www.proteinscience.org Xylanases (EC3 .2.1.8) are xylan-degradinge nzymes belongingtoglycosyl hydrolases that catalyze thehydrolysisofinternal b -1,4bondsofxylan backbones. Xylan is themajor hemicellulose component of the plant cell wall, and its hydrolysis by xylanases hasp otentiale conomical and environmentally friendly applications ( Shalloma nd Shoham 2003). Xylanases arem ainlyu sedi nt he paper Reprint requests to:S uryanarayanarao Ramakumar,D epartment of Physics,I ndian Institute of Science, Bangalore 5600 12,I ndia;e -mail: ramak@physics.iisc.ernet.in; fax: +91(080)2360-2602; or Va nga Siva Reddy,P lant Transformation Group, InternationalC entre for Genetic Engineering andB iotechnology, NewD elhi 1100 67, India; e-mail: vsreddy@icgeb.res.in; fax: +91(011)2616-2316.Article publishedonline aheado fp rint.A rticle and publicationd ate area th ttp://www.proteinscience.org/cgi

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