The nature of the carbohydrate binding module determines the catalytic efficiency of xylanase Z of Clostridium thermocellum

doi:10.1016/j.jbiotec.2013.09.010

Journal of Biotechnology

Volume 168, Issue 4, December 2013, Pages 403-408

https://doi.org/10.1016/j.jbiotec.2013.09.010 Get rights and content

Highlights

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Variants of xynlanase Z of Clostridium thermocellum in combination with CBM6 and CBM22 were expressed in E. coli.
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The construct with CBM22 showed 5-fold higher activity as compared to the one containing CBM6.
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Location of the binding site inside a tunnel like structure of the CBM6 construct seemed to restrict the substrate binding.
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The construct with CBM22 produced a more open structure, facilitating substrate binding and release of the product.

Abstract

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.

Introduction

Carbohydrate binding modules (CBMs) are the non-catalytic part of many polysaccharide targeting enzymes such as cellulases and hemicellulases. Based on their sequence similarity, CBMs have been classified into 64 families (www.cazy.org), around half of which have members that bind to the cell wall polymers (Najmudin et al., 2010, Herve et al., 2010). Xylanases (E.C.3.2.1.8), involved in random cleavage of xylan to produce xylose, xylobiose, xylotriose and xylooligosacchrides (Shallom and Shoham, 2003), contain CBMs that belong to different families. For instance, xylanase 10B (Xyn10B) of Polyplastron multivesiculatum contains family 22 CBM which facilitates its binding to both xylan and cellulose (Devillard et al., 2003) while XynA of Thermotoga maritima has family 9 CBM through which it binds to amorphous and crystalline cellulose (Notenboom et al., 2001). Xyn5 of Paenibacillus sp., however, contains two CBMs that belong to two different families i.e., family 22 and family 9 through which binding to substrate avicel is ensured (Ito et al., 2003). Irrespective of the family type, the most obvious and defined role of CBMs is to mediate attachment and potentiate the catalytic activity of the enzyme by bringing its catalytic module in close and prolong vicinity with the target substrate (Guillen et al., 2010). Truncated or proteolytically cleaved CBMs may thus alter the catalytic efficiencies of the appended catalytic module and hence the polysaccharide hydrolysis (Kim et al., 2010).

XynZ of Clostridium thermocellum is a multimodular, bifunctional enzyme that contains a feruloyl esterase module, a family 6 CBM, a dockerin module and a glycosyl hydrolase family 10 (GH10) xylanase domain. These are all linked together through short linker peptides (Grepinet et al., 1988, Blum et al., 2000). Previously we reported that the variant XynZ-C [consisting only of the catalytic domain (C)] shows significantly higher catalytic activity as well as thermostability as compared to XynZ-BDC [containing CBM6 binding module (B), dockerin (D) and catalytic (C) domains] (Sajjad et al., 2010). Here we compare the role of CBM6 and another binding module CBM22 (from XynC of C. thermocellum) on the activities as well as structural and other properties of the enzyme variants.

Section snippets

Plasmids, bacterial strains and growth media

InsT/Aclone PCR product cloning kit containing pTZ57R/T vector (MBI Fermentas, Ontario, Canada) was employed for initial cloning of the PCR products. Taq DNA polymerase, restriction endonucleases, T4 DNA ligase, etc. were also acquired from MBI, Fermentas. Escherichia coli strain DH5α was used for vector propagation while E. coli BL21 CodonPlus (RIPL) and pET28a(+) vector system (Novagen, Madison, USA) were employed for expression studies. Culture media used for bacterial growth were either the

Xylanase expression and activity yields

Each of the XynZ variants, i.e., XynZ-BC, XynZ-CB and XynZ-B′C were expressed in E. coli in soluble form. The expression level as determined by densitometric analysis of the SDS-PAGE gel (Fig. 2) was about 30% of the total cell proteins in each case. Positioning of CBM6 at either N- or C-termini of the catalytic domain did not alter the expression level. The enzyme variants were partially purified up to 80% (Fig. 2, lanes 4–6) by incubating the cell lysate supernatant at 65 °C for 30 min with

Acknowledgement

This work is part of the project funded by Ministry of Science and Technology, Government of Pakistan, Pakistan.

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