Evaluation of a novel bifunctional xylanase–cellulase constructed by gene fusion

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Abstract

An artificial bifunctional enzyme, xylanase–cellulase, has been prepared by gene fusion. Three chimeric genes were constructed that encoded fusion proteins of different lengths. The fusion proteins exhibited both xylanase (XynX) and cellulase (Cel5Z::Ω) activity when cel5Z::Ω was fused downstream of xynX, but not when xynX was fused downstream of cel5Z::Ω. Activities of bifunctional enzymes decreased when a shorter xylanase peptide was fused. Three fusion enzymes were purified, and the molecular weights of the enzymes were estimated by CMC-SDS-PAGE and XYN-SDS-PAGE to be 149, 129, and 87 kDa, respectively. The fusion enzymes displayed optimum cellulase activity at pH 8.0 and 50 °C and optimum xylanase activity at pH 8.0 and 70 °C.

Introduction

Cellulose, a β-1,4-linked polymer of glucose, represents approximately half of the dry weight of plant cell walls, which also contain xyloglucan and xylan. Each year, 4 × 1010 metric tons of this polymer are produced by photosynthesis [1]. The interest in cellulose hydrolysis is focused on animal nutrition [2] and the potential development of environmentally benign processes for microbial conversion into fuel ethanol [3]. Plant cell wall hydrolysis enzymes include all enzymes that degrade these substances and the complex, co-operation of polysaccharide digestion of these enzymes. Therefore, the bifunctional fusion of such enzymes may ensure a more efficient enzyme capable of degrading cellulose.

The gene fusion technique has become an indispensable tool in biochemical research. The construction of recombinant fusion/chimeric proteins has allowed increased expression of soluble proteins and facilitated protein purification. Furthermore, a wide range of gene fusion applications has been reported in the filed of biotechnology. These include the immunoassays using chimeras between antibody fragments or antibody binding domains and enzymes or green fluorescent protein variants [4], [5], [6], [7], the selection and production of antibodies [8], and the engineering of bifunctional enzymes [9], [10].

Although the natural diversity of enzymes provides some candidates that have evolved bifunctional activity, most fusion enzymes have resulted from the in vitro fusion of individual enzymes. Fusion proteins are created by end-to-end fusions of whole genes that encode intact functional proteins or of functional domains [11], [12], [13]. As practical cases of the multistep sequential reaction were studied, the performance of the fusion enzyme was sometimes better than that achieved by successive action of individual enzymes, expanding the potential use of natural enzymes [14], [15]. Therefore, functional fusion of two enzymes may have several advantages over individual enzymes regarding reaction kinetics and enzyme production, as well as novel properties and reactivity [13], [16], [17].

Previously, we cloned and expressed the xylanase gene from Clostridium thermocellum [18] and the cellulase gene from Pectobacterium chrysanthemi PY35 [19]. We designed xylanase–cellulase bifunctional fusion proteins by an end-to-end fusion method. We also constructed the modified fusion enzymes of different lengths. Here, we report the performance of the resulting fusion enzymes as evaluated and compared with the performances of the co-expressed and independently expressed enzymes. To the best of our knowledge, this is the first report of a xylanase–cellulase bifunctional fusion protein.

Section snippets

Bacterial strains and growth conditions

C. thermocellum ATCC 27405 was used as a source of the xylanase gene. P. chrysanthemi PY35 was used for the cellulase gene. E. coli DH5α, BL21 (DE3), and recombinant E. coli cells were cultured in Luria–Bertani (LB) medium and, if necessary, ampicillin (50 μg/ml) was added to the medium as appropriate antibiotics. The complete nucleotide sequence of the xylanase and cellulase genes has been deposited in the GeneBank database under accession number M67438 (xynX, EC 3.2.1.91) and number AF208495 (

Construction of the fusion enzymes

To construct a fusion enzyme possessing both xylanase and cellulase activities, we selected the C. thermocellum xynX gene and enhanced P. chrysanthemi PY35 cel5Z::Ω gene as the constituents of this fusion enzyme. The xynX and cel5Z genes were fused by overlapping PCR [25]. Modified fusion enzymes, XynX′CD,CBD + Cel5Z::Ω and XynX″CD + Cel5Z::Ω, were digested to removal the S-layer-like domain (SLD) and cellulose binding domain (CBD) from the whole open reading frame (ORF) encoding xylanase. The 4.2 

Discussion

Fusion enzymes sometimes demonstrate superiority over two individual native enzymes catalyzing the same multi-step sequential reaction [14], [15]. In the case of plant cell wall hydrolyzing enzymes, plant polysaccharides are digested by co-operation of enzymes. Therefore, we designed a bifunctional enzyme capable of more efficient digestion. The experimental results shown in this paper clearly demonstrate that xylanase and cellulase can function as a bifunctional fusion enzyme following an

Acknowledgments

This work was supported by a Grant 2000-1-22100-004-5 from the Basic Research Program of KOSEF and the 21C Frontier Microbial Genomics and Application Center Program, Ministry of Science and Technology (Grant MG02-0101-003-1-0-1)(H.D.Y), Republic of Korea. J.M.A. and Y.K.K. are the recipient of a BK21 fellowship.

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