Bifunctional xylanases

Apart from producing multiple isoforms, to effectively degrade the complex substrate such as plant cell wall, many microorganisms develop a cell associsated multprotein complexs, called celluloseme or xylosome, which contains cellulase, xylanase and cellulose binding factors. Another strategy is to induce the bifunctional or multifunctionalization of certain enzymes to hydrolyze different kinds of substrates.  The natural diversity of enzymes provides some candidates that have evolved bifunctional xylanase-cellulase complex. Flint et al (1993) reported bifunctional xylanase-cellulase in Rumiinococcus flavefaciens 17. Sequencing of  Rumiinococcus flavefaciens 17 DNA fragment revealed that both the activites are encoded by a single 2406 bp open reading frame corresoponding to the XynD gene. Thus is an example of a bifunctional polysaccharidase having two separate catalytic domains within the same polypeptide chain that can act on different polymeric substrates. Pohlschroder et al (1994) reported the presence of cellulase-xylanase multicomplexs system, comprising at least seven diverse protein complexes in Clostridium papyrosolvens C7. Two of these seven complexes are having xylanase activity in addition to cellulases activity. Another cellulose-xylanase complex has been reported by Murashima et al (2003). Both the enzymes in this complex proved to work simultaneoulsly for synergistic degradation of corn cell wall. Furthermore, a novel bifunctional enzyme with xylanase and beta-(1,3-1,4)-glucanase activities has been reported by Chen et al in Aspergillus niger A-25.The purified XynIII was shown to hydrolyze birchwood xylan, oat spelt xylan, lichenin, and barley beta-glucan, but not CMC, avicel cellulose, or soluble starch, which indicates that both the activities are catalyzed by the same active site. Whereas, bifunctional multimodular enzyme bearing two independent xylanase and alpha-L- arabinofuranosidease domains separated by Ser/Gly-rice linker has been reported from lignocellululolytic actinomycete Streptomyces chattanoogensi CECT-3336 (Hernandez et al  2001). Bifuctinal xylanase-deacetylase (14), xylanase-esterase(90) and Bifuctional xylanase-xylanase (15) has also recently been reported.

            In addition to their important role in recycling of Carbon by aiding the efficient microbial degradation of plant cell wall, these bifuctional enzymes has number of prospective  application where their bifuctionality or polyfuctionality have an edge over the hydrolytic enzymes with single catalytic site. Polyfunctional recombinant chimeric proteins are undoubtedly more valuable than a single enzyme for applications such as, bioethnol production  which requires complete saccharification of plant cell wall to fermentable sugars. A critical factor concerning the cost of process is the efficient and cheap cellulase and xylanases to achieve breakdown in single step. Furthermore, due to the advantages of polyfuctional enzymes over individual enzymes regarding reaction kinetics and enzyme producton as well as novel properties and reactivity (45,63,57), their have been increasing research efforts in this area.  Mesta et al 2001, designed an active chimeric enzyme, XYN3A4, by fusing two different catalytic domains exhibiting the same endoxylanases activity, XYN3A and XYN4, which originated from two different fungal endoxylanase genes, xyn3 and xyn4, respectively. Chimeric enzyme exhibited a better affnity and an improved rate of hydrolysis of the xylan substrate than its respective counterparts, XYN3A and XYN4. Whereas, Hong et al 2006, demonstrated that cellulase (TM1751) and xylanase (TM0061) from Thermotoga maritima can be fused end-to-end, via overlapping PCR, to creat a bifunctional enzyme.  Their results further suggest that correct folding and protein interaction are important cretieron in stabilizing protein structure and affect activity of chimera. Moreover, the specific enzyme activities of the fusion protein also reported to be dependent on how the fusion has been made, such as  the sequential order of the enzymes and the length and composition of the connecting region (31).

            Fan et al 2009 recently reported two highly active trifunctional hemicellulases constructed by linking the catalytic portion of a xylanase with an arabinofuranosidase and a xylosidase, using either flexible peptide linkers or linkers containing a cellulose-binding domain. The multifunctional enzymes retain the parental enzyme properties and exhibit synergistic effects in hydrolysis of natural xylans and corn stover. Similarly Waeonukul et al 2009 recently reported, cloning, sequencing, and expression of the gene encoding a multidomain endo-beta-1,4-xylanase from Paenibacillus curdlanolyticus B-6, and characterization of the recombinant enzyme. Sequence analysis indicated that Xyn10A is a multidomain enzyme comprising nine domains in the following order: three family 22 carbohydrate-binding modules (CBMs), a family 10 catalytic domain of glycosyl hydrolases (xylanase), a family 9 CBM, a glycine-rich region, and three surface layer homology (SLH) domains. Xyn10A could effectively hydrolyze agricultural wastes and pure insoluble xylans, especially low substituted insoluble xylan. Xyn10A bound to various insoluble polysaccharides including Avicel, alpha-cellulose, insoluble birchwood and oat spelt xylans, chitin, and starches, and the cell wall fragments of P. curdlanolyticus B-6, indicating that both the CBM and the SLH domains are fully functioning in the Xyn10A. Removal of the CBMs from Xyn10A strongly reduced the ability of plant cell wall hydrolysis. Author suggested that the CBMs of Xyn10A play an important role in the hydrolysis of plant cell walls. Whereas, Dodd et al 2009, have reported Biochemical analysis of a bifunctional xylanase-ferulic acid esterase from a xylanolytic gene cluster in Prevotella ruminicola. The gene predicted to encode a bifunctional xylanase-ferulic acid esterase (xyn10D-fae1A) was expressed as recombinant protein in Escherichia coli. Biochemical analysis of purified Xyn10D-Fae1A revealed that this protein possesses both endo-beta-1,4-xylanase and ferulic acid esterase activities. Directed mutagenesis studies of Xyn10D-Fae1A mapped the catalytic sites for the two enzymatic functionalities to distinct regions within the polypeptide sequence. The fuctionallity of two catalytic domains for Xyn10D-Fae1A were further shown to be coupled.