High-level expression of a novel thermostable and mannose-tolerant β-mannosidase from Thermotoga thermarum DSM 5069 in Escherichia coli

Hao Shi 0 1 Yingjuan Huang 0 1 Yu Zhang 0 1 Wenqian Li 0 1 Xun Li 0 1 Fei Wang 0 1 0 Jiangsu Key Lab of Biomass-Based Green Fuels and Chemicals , Nanjing 210037 , China 1 College of Chemical Engineering, Nanjing Forestry University , Nanjing 210037 , China Background: Mannan is one of the primary polysaccharides in hemicellulose and is widely distributed in plants. -Mannosidase is an important constituent of the mannan-degrading enzyme system and it plays an important role in many industrial applications, such as food, feed and pulp/paper industries as well as the production of second generation bio-fuel. Therefore, the mannose-tolerant -mannosidase with high catalytic efficiency for bioconversion of mannan has a great potential in the fields as above. Results: A -mannosidase gene (Tth man5) of 1,827 bp was cloned from the extremely thermophilic bacterium Thermotoga thermarum DSM 5069 that encodes a protein containing 608 amino acid residues, and was overexpressed in Escherichia coli BL21 (DE3). The results of phylogenetic analysis, amino acid alignment and biochemical properties indicate that the Tth Man5 is a novel -mannosidase of glycoside hydrolase family 5. The optimal activity of the Tth Man5 -mannosidase was obtained at pH 5.5 and 85C and was stable over a pH range of 5.0 to 8.5 and exhibited 2 h half-life at 90C. The kinetic parameters Km and Vmax values for p-nitrophenyl--D-mannopyranoside and 1,4--D-mannan were 4.360.5 mM and 227.271.59 mol min-1 mg-1, 58.341.75 mg mL-1 and 285.7110.86 mol min-1 mg-1, respectively. The kcat/Km values for p-nitrophenyl--D-mannopyranoside and 1,4--D-mannan were 441.350.04 mM-1 s-1 and 41.471.58 s-1 mg-1 mL, respectively. It displayed high tolerance to mannose, with a Ki value of approximately 900 mM. Conclusions: This work provides a novel and useful -mannosidase with high mannose tolerance, thermostability and catalytic efficiency, and these characteristics constitute a powerful tool for improving the enzymatic conversion of mannan through synergetic action with other mannan-degrading enzymes. - Background Mannans are complex polysaccharides representing one of the major components of hemicellulose, consisting of four types: linear mannan, glucomannan, galactomannan, and galactoglucomanan [1]. Each of these polysaccharides has a -1,4-linked backbone units including mannose or a combination of glucose and mannose residues, with the presence of -1,6-linked side-chain substitutions [2]. It was reported that the hydrolysis of these polysaccharides requires several mannan-degrading enzymes, primarily including -mannanase (EC 3.2.178), -mannosidase (EC 3.2.1.25) and -glucosidase (EC 3.2.1.21). Other enzymes such as -galactosidase and mannan esterase are required to remove -galactosyl and O-acetyl side-chain substituent. Among these enzymes, two types of mannandegrading enzymes are necessary [3]. One endotype, -mannanase, is responsible for the cleavage of -1,4linked mannose residues backbone randomly to generate mannooligosaccharides. Another exotype, -mannosidase, hydrlyses the nonreducing end of mannooligosaccharides to release mannoses [2]. It is known that -mannosidase is produced from plants, bacterial, fungi, invertebrates as well as some mammalian species [4,5]. Based on amino acid similarity and multi-domains, -mannosidases have been mainly classified into glycoside hydrolase family (GHF) 1, 2 and 5 (http://www.cazy.org/). These -mannosidases from different GHFs possess considerable industrial applications in many fields, such as food, feed and pulp/paper industries [6]. In addition, -mannosidases have important role in saccharification of hemicellulose for fuel and other chemicals production. In human, lack of -mannosidase can lead to -mannosidosis [7,8]. During the last two decades, thermostable enzymes from thermophilic or hyperthermophilic microorganisms have become the hotspots of researches in many fields [9]. he amino acid sequences of -mannosidases are abundantly available on the constantly updating databases. However, only a few -mannosidases especially from hyperthermophile have been cloned, purified and characterized [3,5,10]. It was found that the known hyperthermophilic -mannosidases from Pyrococcus furiosus, Thermotoga maritima, and Thermotoga neapolitana belonged to the GHF1, GHF2, and GHF2, respectively [11-13]. Thermotoga thermarum, isolated from continental solfataric springs at Lac Abbe (Djibouti, Africa), is an anaerobic hyperthermophilic bacteria that grows at 80C [14]. And it has many glycoside hydrolase genes based on the genomic sequence (GenBank accession number: CP002351). The biotechnology industry is essential in modern societies [15], which is reflected in the production of recombinant enzymes (including -mannosidases) and their applications. In this study, we described the cloning, expression and functional characterizations of a novel recombinant -mannosidase (Tth Man5) in E. coli. Results Amino acid sequence of Tth Man5 -mannosidase The Tth man5 gene isolated from the T. thermarum genome was 1,824 bp in length coding 608 amino acids and it was predicted as an endo--mannanase (Theth_0949) available at NCBI and CAZy sites (http://www.ncbi.nlm. nih.gov/, http://www.cazy.org/) (Lucas S etal, 2011). As shown in Figure 1, Tth Man5 displayed 33% identity to -mannosidase from Sorangium cellulosum So ce56, 32% identity to putative -mannosidase from Actinosynnema mirum DSM 43827 and 32% identity to the glycoside hydrolase from Streptomyces flavogriseus ATCC 33331. The results of alignments also revealed that Glu141, Glu237, Glu238, Glu292 and Glu591 were conserved amino acids among these GHF5 -mannosidases. According to the CAZy database, two glutamic acids are the acid/base and the nucleophile, respectively. Against the similar catalytic domain of GHF5 endoglucanase (EXPDB No: 1TVP_A) from Pseudoalteromonas haloplanktis, it was presumed that active amino acids of Tth Man5 mannosidase were Glu141 and Glu238 [16]. Over-expression and purification of Tth Man5 -mannosidase When using native gene from T. thermarum for expression, the protein production was very difficult to detected (data not shown). Thus, in order to increase the expression level of Tth Man5 -mannosidase in Escherichia coli, rare codons were replaced by optimal codons without change of amino acid sequence (data not shown). The mature protein without the signal peptide, allowing the insertion of a His6-tag at the C-terminus, was successfully expressed in E. coli BL21 (DE3), after induction with IPTG for 5 h at 37C. The recombinant protein in the cell-free extract was purified by a heat treatment followed by a nickel affinity column (Table 1). Finally, the purified recombinant enzyme displayed a single band on SDSPAGE with an estimated molecular weight (MW) of 70 kDa (Figure 2), which was consistent with the predicted MW of monomer (71, 725 Da). Size exclusion chromatography was also carried out using the AKTAFPLC system to compute (...truncated)