Studies On Carbohydrate Binding Module 2a Of Cellulase A From Cellulomonas Fimi

Cellulose,the major structural component of plant cell walls,is a homopolymer ofβ-1,4-linked glucose residues.It has attracted renewed interest as a potential source of energy through its biodegradation and fermentation to biofuels.The biodegradation of cellulose involves the concerted action of three types of enzymes, endo-β-1,4-glucanases(EC 3.2.1.4),exo-β-1,D-glucanases,or cellobiohydrolases(EC 3.2.1.91),andβ-glucosidases(EC 3.2.1.21).The former two classes of enzymes function to hydrolyze insoluble cellulose into soluble oligosaccharides which then serve as substrates forβ-glucosidases to release free glucose.Many of these cellulolytic enzymes,and polysaccharide hydrolases in general,are modular proteins,being comprised of catalytic modules and one or more ancillary modules.Of the latter,the most common are the carbohydrate binding modules(CBM).CBMs are defined as contiguous amino acid sequences with a discreet fold that possess carbohydrate-binding activity.The Carbohydrate Active Enzymes(CAZy) database(http://www.cazy.org/) currently lists 52 CBM families classified by primary and secondary structural similarities.Of the CBMs that bind cellulose,much is known about members of family 2 in terms of their structure and function relationship.To date,Family 2 CBMs are found in a large number of microorganisms involving 73 species of bacteria,2 species of archaea,12 species of eukaryotes,and seven viruses. CBM2 has been divided into two subfamilies according to substrate specificities; CBM2a binds cellulose whereas CMB2b interacts specifically with xylan.Whereas both involve approx.100 amino acid residues that fold similarly into aβ-sandwich, significant differences define the binding face of the module.In CBM2a,the cellulose-binding surface is relatively hydrophobic being comprised of three tryptophan residues which have been demonstrated to be essential for binding to cellulose.Current studies showed that the CBMs appear to aid in both the adsorption of the enzymes to the insoluble cellulose substrate and the destabilization of the hydrogen-bonding network within the crystalline substrate.However,surface diffusion measurements using fluorescence recovery techniques indicated that this interaction of the CBM is dynamic,involving mobility of the module along the surface of the crystalline cellulose.So it is very important to understand this mobility and binding mechanism of proteins at insoluble surfaces with surface analysis techniques.Herein,we describe the cloning and expression of the partial gene sequence of cellulase A(CenA) from Cellulomnas fimi encoding its leader sequence and CBM2a.To facilitate covalent binding of the isolated CBM2a to AFM probes with the appropriate orientation for binding to cellulose,a mutants form of the CBM was engineered and produced involving the site-specific replacement of Thr87 with Cys.Based on CBM_CenA T87C,four mutants(CBM_CenAT87C+W81A; CBM_CenAT87C+N114W;CBM_CenAT87C+N114Y;CBM_CenA T87C+N114F) were engineered to test the character of binding to cellulose.Methods1.The complete cenA gene was cloned previously from C.fimi genomic DNA was used as the template DNA for PCR amplification of first 426 base pairs of cenA using the following upstream 5'-ATCCAGATCGAATTCATGTCCACCCGCAGAACC-3',and downstream 5'-AATTTACATAAGCTTTCAGCTGGTCGTCGGCACGGTGCC-3' primers, respectively(EcoRâ… and Hindâ…¢restriction sites,respectively,are underlined).2.Amplified ORFs were cleaned using High Pure PCR Product Purification kit, digested with EcoRâ… and Hindâ…¢and ligated with appropriately digested pET30a(+) plasmid DNA.3.Recombinant plasmids were transformed into competent E.coli DH5α. Individual constructs were isolated from transformants,screened for the correct size of insert by agarose gel electrophoresis,and they were sequenced to confirm nucleotide identity.The resulting isolated construct was named pACHJ-1.4.Thr87 was targeted for replacement with Cys by site directed mutagenesis of the truncated and cloned cenA gene to facilitate binding of the module to AFM probes.Mutagenesis was performed using pACHJ-1 as template for Pfu Turbo DNA polymerase according to the QuikChange Site-Directed Mutagenesis with the forward and reverse primers 5'-GCGTCGTGCAACGGCGGCCAGGTC-3' and 5'-GCCGTTGCACGACGCGGTGCCGTTCC-3',respectively,where the boldface denotes the changed nucleotides.Following PCR,Dpnâ… was added to a 50μl PCR reaction mixture and incubated for at least 1 h at 37℃to remove the original,methylated template.The resulting plasmid(pACHJ-2) was used to transform E.coli DH5αand clones were screened for the correct mutations by DNA sequencing.5.E.coli BL21(DE3) freshly transformed with either pACHJ-1 or pACHJ-2 was inoculated into LB broth supplemented with 50μg/mL Kan and incubated at 37℃.When the cultures reached mid-exponential phase(absorbance approx.0.6), freshly prepared 0.1-1 mM IPTG(final concentration) was added to induce gene expression.Incubation of cells at either 15℃,30℃,or 37℃was continued for 12 h and growth was monitored by absorbance measurements.6.The target protein was purified by a combination of affinity using Ni₂+NTA-agarose and anion exchange chromatographies using Source Q, respectively.The mutants were confirmed by SDS-PAGE and Western blot analysis with an anti-His₆ antibody. 7.The ability of purified CBM2a to bind to micro-crystalline cellulose was tested using Avicel in 50 mM sodium phosphate buffer,pH 7.0 at 4℃.The amount of CBM2a bound was determined by difference using UV absorbance spectroscopy to measure protein concentrations in both the starting material and supernatants following incubation for 8 h and removal of the insoluble cellulose and adsorbed protein by centrifugation.8.Based on CBM_CenA T87C,four mutants were engineered using proper primers, respectively,to attach to AFM involving to test the character of binding to cellulose.(CBM_CenAT87C+W81A;CBM_CenAT87C+N114W; CBM_CenAT87C+N114Y;CBM_CenA T87C+N114F)Results1.Cloning of gene sequence encoding CBM2a from CenABased on the nucleotide sequence of cenA,appropriate oligonucleotide primers were synthesized for PCR amplification of the N-terminal 142 amino acids of the encoded protein which would include its signal pcptide,the entire CBM2a.The amplified product was cloned into pET30a(+) in frame with coding for a cleavable N-terminal His₆ tag,and the construct,pACHJ-1,was transformed into E.coli DH5α. The nucleotide sequence was confirmed by DNA sequencing prior to subsequent use.2.Production of CBM2aInitially,the CBM2a construct pACHJ-1 was transformed into E.coli BL21 (DE3) for induction-expression trials.Cells were grown to mid-exponential phase and then induced for cbm2a expression with varying concentrations of IPTG(0.1 mM to 1 mM) and incubation at either 15℃,30℃,or 37℃.Continuing growth was monitored over a 12 h period by both turbidity measurements while CBM2a production was followed by SDS-PAGE and Western immunoblot analysis. Relatively poor expression of cbm2a from pACHJ-1 was observed when induction was conducted at either 15℃or 37℃,even for extended periods of time with varying IPTG concentrations.Optimum induction conditions were established to involve the addition of 0.5 mM IPTG for 2.5 h at 30℃.3.Purification of CBM2aImmobilized metal ion affinity chromatography with Ni²⁺-NTA agarose was used to isolate and purify His₆-tagged CBM2a.Crude protein samples were applied to the resin at pH 8 and after an initial wash,a step gradient of increasing imidazole served to elute the CBM with minimal contamination as determined by SDS-PAGE.The extent of the contaminating protons co-eluting with CMB2a was observed to increase if cell pellets were not used immediately but instead stored for extended periods of time(more than 3 days).After removal of the imidazole and other buffer components by dialysis,the CBM2a preparation was further purified by anion-exchange chromatography on Source Q.The CBM2a was retained by the resin at pH 8.5 and it was recovered in 50 mM NaCl after the application of a linear gradient to 1 M NaCl.After removal of the NaCl by dialysis,these preparations of CBM2a were found to be purified to greater than 98%homogeneity,as judged by SDS PAGE and densitometry.This production and purification protocol routinely provided an average of 0.9 mg purified CBM2a per L of cell culture.Western immunoblot analysis using and anti-hexa-His tag polyclonal antibody confirmed the identity of the engineered CBM2a.4.Cellulose binding activity of CBM2a.Under the conditions,a direct relationship was observed between the amount of CBM2a applied to the cellulose and that adsorbed.Depending upon initial protein concentrations,the efficiency of binding was found to be between 78%and 88%. Interestingly,this binding was not observed to be dependent on temperature as similar results were obtained after incubation at 30℃.The binding of CBM2a was found to be virtually irreversible.The isolated CBM2a did not elute from the insoluble cellulose in water at either 4℃or 30℃. Even elution with 5%SDS led to the recovery of no more than 25%of the adsorbed module.This situation was thus analogous to that observed involving CenA derivative that lacked a complete linker peptide(Pro-Thr box);binding of this protein was considered to be 'quassi-irreversible'. 5.Site-directed mutagenesisBased on the predicted three-dimensional structure of CBM2a,Thr87 was replaced with Cys to facilitate binding of the module to AFM probes.As expected,the Thr87→Cys mutant CBM2a retained the same binding capacity to Avicel as compared to wild type CBM2a.Thus,74%of the applied mutant CBM2a was retained by Avicel.Other four mutants(CBM_CenAT87C+W81A;CBM_CenAT87C+ N114W;CBM_CenAT87C+N114Y;CBM_CenAT87C+N114F) were successfully constructed,expressed and purified.Sufficient concentrations of the mutant module were obtained in homogeneous form by this protocol to permit its further characterization and delineation of its mechanism of action by AFM and other sensitive analytical techniques.Conclusions1.The gene encoding CBM2a and leader sequence of CenA from Cellulomonas fimi was first cloned and expressed.The target protein was purified.Optimum induction and expression conditions were established.2.The binding of CBM_CenA to insoluble cellulose is virtually irreversible,depending upon initial protein concentrations,the efficiency of binding was found to be between 78%and 88%.This binding was not observed to be dependent on temperature.3.The Thr87→Cys mutant CBM2a retained the same binding capacity to Avicel as compared to wild type CBM2a.4.Based on CBM_CenAT87C,four mutants(CBM_CenAT87C+W81A; CBM_CenAT87C+N114W;CBM_CenAT87C+N114Y;CBM_CenA T87C+N114F) were constructed,expressed and purified to test the character of binding to cellulose by Atomic Force Microscope.