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Recombinant Lichenase from Clostridium Thermocellum Binds Glucomannan but Not to Lichenan: Analysis by Affinity Electrophoresis

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Recombinant Lichenase from Clostridium Thermocellum Binds Glucomannan but Not to Lichenan: Analysis by Affinity Electrophoresis Annals of Microbiology, 58 (4) 723-725 (2008) Recombinant lichenase from Clostridium thermocellum binds glucomannan but not to lichenan: Analysis by affinity electrophoresis Shadab AHMED1, Carlos M.G.A. FONTES2, Arun GOYAL1* 1Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, 781 039, Assam, India; 2CIISA-Faculdade de Medicina Veterinária, Rua Prof. Cid dos Santos, 1300 477, Lisbon, Portugal Received 19 May 2008 / Accepted 12 September 2008 Abstract - A family 26, glycoside hydrolase (GH26) named lichenase of the molecular size 30 kDa from Clostridium thermocellum previously cloned into an expression vector pET21a was over-expressed in Escherichia coli BL-21 cells and was purified to homogene- ity by a single step purification method using immobilised metal ion affinity-chromatography. The purified enzyme showed no activity towards soluble substrates such as carboxymethyl cellulose, hydroxymethyl cellulose, laminarin, galactomannan or glucomannan. However, unlike other members of the family GH26, the enzyme lichenase showed high activity towards lichenan and β-glucan and was highly specific endo acting β-1,3-1,4-glucanase. Quantitative affinity-gel electrophoresis on native-PAGE showed that lichenase binds only glucomannan (with a Ka of 41.3 C-1) and not lichenan or β-glucan. Key words: glycoside hydrolase, Clostridium thermocellum, recombinant lichenase, Escherichia coli. INTRODUCTION non catalytic family 11 carbohydrate binding module (CBM11) and a C-terminal dockerin (Goyal et al., 2005). The crystal struc- The biological process of cell wall degradation involves a wide ture of CBM11 demonstrated that it binds the ligands that are array of microbial enzymes comprising glycoside hydrolases also the substrates for lichenase (Goyal et al. 2004). The crystal (GHs) and esterases. Henrissat (1991) has grouped glycoside structure of lichenase also has been determined and interest- hydrolases into families based on primary sequence similarities. ingly, it was shown that lichenase belonging to mannanase fam- There are 112 glycoside hydrolase families listed on the web- ily shows no activity towards mannans but displays high activity site (http://www.cazy.org/fam/acc_GH.html) based on primary towards lichenan (Goyal et al., 2005). Here, we report the data sequence similarities. Interestingly, even though the members on quantitative analysis of recombinant lichenase of Clostridium of each family share similar protein structures, the substrate thermocellum binding to glucomannan. specificities displayed by different members of same family vary considerably. One of the largest glycoside hydrolase families is glycoside hydrolase family 26 (GH26), which encompasses MATERIALS AND METHODS mainly mannan endo-1,4-beta-mannosidases. Although two members of this family have been reported to display ß-1,3- Over-expression and purification of lichenase. The DNA xylanase activity (Araki et al., 2000; Okazaki et al., 2002). fragment encoding lichenase amplified by PCR was cloned into Almost all the GH26 mannanases characterised till date display NheI and XhoI restricted pET21a expression vector to generate very narrow substrate specificity, hydrolyzing mannan and glu- recombinant plasmid (Goyal et al., 2005). Escherichia coli cells comannan. The enzyme randomly hydrolyses ß-1,4-D-linkages BL-21 (Novagen) harbouring the recombinant plasmid containing in mannans, galacto-mannans, glucomannans and galactogluco- the gene of lichenase were cultured at 37 °C in Luria Bertani mannans, exhibiting no activity against other ß-glycans such as broth to mid-exponential phase (A550nm 0.6), at which point barley ß-glucans or soluble derivatives of cellulose (Braithwaite isopropyl 1-thio-ß-D-galacto-pyranoside (IPTG) was added to et al., 1995). In the present study the GH module (GH26) used a final concentration of 1 mM and the cultures were incubated and expressed belongs to a bi-functional cellulase that displays a for further 6 h to induce the expression of lichenase. The media modular architecture containing an N-terminal family 26 catalytic were supplemented with 100 mg/L ampicillin. The recombinant module (GH26), an internal family 5 catalytic module (GH5), a protein encoded by this plasmid contains a C-terminal His6 tag. The His6-taggd recombinant protein was purified from cell- * Corresponding author. Phone: 91-(361) 258 2208; free extracts by immobilised metal ion affinity chromatography Fax: 91-(361) 2690762; E-mail: [email protected] (IMAC) as described previously (Dias et al., 2004). The purified 724 S. AHMED et al. enzyme was analysed by sodium dodecylsulphate-polyacrylamide ß-1,3-1,4 glucans such as lichenan and ß-glucan. However, gel electrophoresis (SDS-PAGE) by the method as described by lichenase showed no activity towards other soluble polysac- Laemmli (1970). The samples were run on 12% polyacrylamide charides such as carboxymethyl cellulose, laminarin or ß-1,4 gels after boiling for 5 min. The gels were stained with Coomassie cellulose and also no activity towards insoluble polysaccharides Brilliant Blue R-250. such as avicel. The capacity of lichenase enzyme to bind a range of differ- Affinity gel electrophoresis of lichenase. The ability of ent polysaccharides was evaluated. Interestingly, on analysis lichenase to bind various soluble polysaccharides such as by affinity-electrophoresis on native-PAGE showed that lichen- lichenan, carboxymethyl cellulose (CMC) and glucomannan ase binds glucomannan, but does not show any enzyme activ- was evaluated by affinity native gel electrophoresis. Native ity towards this substrate. It displays an example of divergent polyacrylamide gels were prepared consisting of 7.5% (w/v) evolution where the acquisition of altered different specificities acrylamide in 25 mM Tris/250mM glycine buffer. The substrate (of glucanase) by mutations of the gene (for mannanase) from glucomannan was added in varying concentrations viz. 0, 0.01, a common ancestor has occurred. The quantitative analysis 0.02, 0.04 and 0.5% to the polyacrylamide mixture before by affinity electrophoresis was performed using lichenase with polymerisation. For binding analysis of lichenase with lichenan varying concentration of glucomannan in native gel (Fig. 1). and CMC, the native gels contained 0.2% lichenan and 0.2% The enzyme movement in the gel was retarded with increase CMC. Approx. 10 μg of lichenase enzyme and BSA (as non in concentration of glucomannan from 0.01 to 0.05% (Fig. 1). interacting negative control) were loaded to the gels and sub- The relative migrating distance of protein in presence of affinity jected to electrophoresis at 2 mA/lane for approximately 2 h at ligand (r), was plotted against glucomannan concentration (Fig. room temperature. The value of association constant (Ka) was 2). With increasing the concentration of glucomannan the value calculated using method described by Takeo (1984) by plotting of r decreased. (Fig. 2, Inset). GH26 displayed weaker binding 1/r versus concentration of glucomannan. with glucomannan with a value of association constant, Ka of 41.3 C-1. The catalytic module lichenase did not bind or interact with either carboxymethyl cellulose or lichenan when analysed RESULTS AND DISCUSSION by affinity gel electrophoresis. These data show that lichenase from C. thermocellum has limited or narrower substrate specifi- Affinity-electrophoresis of lichenase city as compared to other similar glycoside hydrolases of same Lichenase from Clostridium thermocellum is a family 26 glyco- family. side hydrolase and shares considerable amino acid homology with various mannan specific enzymes of this family (http:// www.cazy.org/fam/acc_GH.html), that generally contains man- CONCLUSIONS nanases, but it was interesting to discover that being a member of mannanase family, lichenase does not show any activity The lichenase family 26 glycoside hydrolase (GH26) is a cata- towards mannan, galactomannan or glucomannan. The enzyme lytic module of bifunctional cellulase with lichenan and ß-glucan lichenase in contrast to the substrate specificity displayed by hydrolysing activities. As reported earlier the function of GH26 most of other GH26 enzymes, shows high activity towards enzyme is predominantly to display endo-mannase activity, FIG. 1 - Quantitative binding analysis of lichenase with glucomannan. Affinity gel-electrophoresis of lichenase with different concen- trations of glucomannan (A: 0%; B: 0.01%; C: 0.02%; D: 0.04%; E: 0.05%) in 7.5% native-PAGE. Lane 1: BSA, lane 2: GH26. Ann. Microbiol., 58 (4), 723-725 (2008) 725 REFERENCES 1 15 10 Araki T., Hashikawa S., Morishita T. (2000). Cloning, sequencing 1/r 5 and expression in Escherichia coli of the new gene encoding beta-1,3-xylanase from a marine bacterium, Vibrio sp. strain 0 0 0.010.020.030.040.05 XY-214. Appl. Environ. Microbiol., 66: 1741-1743. Glucomannan (%) 0.5 Braithwaite K.L., Black G.W., Hazlewood G.P., Ali B.R., Gilbert Rr H.J. (1995). A non-modular endo-beta-1,4-mannanase from Pseudomonas fluorescens subspecies cellulosa. Biochem. J., 305: 1005-1010. Dias F.M.V., Vincent F., Pell G., Prates J.A.M., Centeno M.S.J., 0 Tailford L.E., Ferreira L.M.A., Fontes C.M.G.A., Davies G.J., 0 0.02 0.04 0.06 Gilbert, H.J. (2004). Insights into the molecular determi- nants of substrate specificity in glycoside hydrolase family Glucomannan (%) 5 revealed by the crystal structure and kinetics of Cellvibrio mixtus mannosidase 5A. J. Biol. Chem., 279: 25517-25526. FIG. 2 - The plot of the relative migrating distance of protein Goyal
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