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Xylanase Z (XynZ) is a prime part of cellulosome of Clostridium thermocellum. The several domains of the cellulosome complexes are frequently tethered to one another by intrinsically messy regions. This is multidomain xylanase comprising of N-terminus feruloyl esterase followed by substrate binding, dockerin and the catalytic domains. This study design was established to check the expression, activities and other characteristics of different variants of Xylanase Z produced by the C-terminal (XynZ-CB), N-terminal (XynZ-BC) and both terminals (XynZ-BCB) binding domain to catalytic domain in different arrangements. XynZ variants of C. thermocellum with binding domain committed with catalytic domain of C-terminal (XynZ-CB) and N-terminal (XynZ-BC) and both terminals (XynZ-BCB) binding domain and one with catalytic domain at both terminals of the binding domain (XynZ-CBC) were checked in E. coli in different levels like 15, 30, 30, and 33% of cell proteins, respectively. The specific xylanase Z activities of XynZ-CB, XynZ BC, XynZ-BCB and XynZ-BCB were 72, 68, 67 and 200 U mg−1, though their equimolar basis activities were 4176, 3944, 5092 and 19,200 U μM−1 compared to xylan (birchwood). The over-all activities of XynZ-CB, XynZ-BC, XynZ-BCB and XynZ-CBC produced in fermentation culture were 3678, 3579, 3800 and 5400 U L−1 OD600−1, respectively. The results showed that overall yield of XynZ-C remained manifold higher than all other variants due to combined effect of its specific activity and expression level while the activity of variant XynZ-CBC was increased four folds on equimolar basis as compared to its native form. The fruit saccharification increased with the treatment of xylanase and cellulase.

Xylanase Z of Clostridium thermocellum exists as a complex in the cellulosome with N-terminus feruloyl esterase, a carbohydrate binding module (CBM6) and a dockerin domain. To study the role of the binding modules on the activity of XynZ, different variants with the CBM6 attached to the catalytic domain at its C-terminal (XynZ-CB) and N-terminal (XynZ-BC), and the CBM22 attached at N-terminus (XynZ-B′C) were expressed in Escherichia coli at levels around 30% of the total cell proteins. The activities of XynZ-BC, XynZ-CB and XynZ-B′C were 4200, 4180 and 20,700 U μM−1 against birchwood xylan, respectively. Substrate binding studies showed that in case of XynZ-BC and XynZ-CB the substrate birchwood xylan remaining unbound were 51 and 52%, respectively, whereas in the case of XynZ-B′C the substrate remaining unbound was 39% under the assay conditions used. The molecular docking studies showed that the binding site of CBM22 in XynZ-B′C is more exposed and thus available for substrate binding as compared to the tunnel shape binding pocket produced in XynZ-BC and thus hindering the substrate binding. The substrate binding data for the two constructs are in agreement with this explanation.

The goal of this work was to produce high levels of endoglucanase in Escherichia coli for its potential usage in different industrial applications. Endoglucanase gene was amplified from genomic DNA of Bacillus subtilis JS2004 by PCR. The isolated putative endoglucanase gene consisted of an open reading frame of 1,701 nucleotides and encoded a protein of 567 amino acids with a molecular mass of 63-kDa. The gene was cloned into pET-28a(?) and expressed in E. coli BL21 (DE3). Optimum temperature and pH of the recombinant endoglucanase were 50 C and 9, respectively which makes it very attractive for using in bio-bleaching and pulp industry. It had a KM of 1.76 lmol and Vmax 0.20 lmol/min with carboxymethylcellulose as substrate. The activity of recombinant endoglucanse was enhanced by Mg 2? ,C a 2? , isopropanol and Tween 20 and inhibited by Hg 2? ,Z n 2? ,C u 2? ,N i 2? and SDS. The activity of this recombinant endoglucanase was signifi- cantly higher than wild type. Therefore, this recombinant enzyme has potential for many industrial applications involving biomass conversions, due to characteristic of broad pH and higher temperature stability.