INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY ISSN Print: 1560–8530; ISSN Online: 1814–9596 16–768/2017/19–2–355–362 DOI: 10.17957/IJAB/15.0290 http://www.fspublishers.org Full Length Article A Novel Application Potential of GH6 Cellobiohydrolase CtCel6 from Thermophilic Chaetomium thermophilum for Gene Cloning, Heterologous Expression and Biological Characterization Qinzheng Zhou, Jianchao Jia, Peng Ji and Chao Han* Department of Mycology, Shandong Agricultural University, Taian, Shandong 271018, China *For correspondence: [email protected] Abstract Chaetomium thermophilum is a thermophilic fungus expressed a series of glycoside hydrolases. Genome sequence analysis of C. thermophilium revealed that ctcel6 gene encoded a putative cellobiohydrolase which composed of 397 amino acid residues including a predicted signal peptide sequence. Ctcel6 gene was cloned, heterologously expressed in Pichia pastoris and purified by Ni2+ affinity chromatography. Sequence alignment indicated that CtCel6 enzyme belonged to glycoside hydrolase family 6 (GH6) and the molecular mass of purified recombinant enzyme CtCel6 was 42 kDa by SDS-PAGE analysis. Characterization of recombinant CtCel6 exhibited high hydrolysis activity and excellent thermostability. The optimum reaction temperature and pH was 70oC and pH 5, respectively. The bivalent metallic cations Mg2+ and Ca2+ significantly enhanced the activity of CtCel6. The specific activity of CtCel6 enzyme was 1.27 U/mg and Km value was 0.38 mM on β-D- glucan. The substrate specificity and hydrolysis products insisted that CtCel6 was an exo-/endo-type cellobiohydrolase. The biochemical properties of recombinant CtCel6 made it potentially effective for bioconversion of biomass and had tremendous potential in industrial applications such as enzyme preparation industry and feed processing industry. © 2017 Friends Science Publishers Keyword: Glycoside hydrolase family 6; Cellobiohydrolase; Heterologous expression; Thermostable enzyme; Bioconversion Introduction of cellulose chains to generate cellobiose. GH6 family members are mainly produced by bacterial and fungal Lignocellulosic biomass, the most abundantly renewable sources, and the hydrolysis mechanism is inverting. GH6 and available carbohydrate resource on the earth, has been family includes both endoglucanases and regarded as an available feedstock for biochemical and cellobiohydrolases, many GH6 endoglucanases have been biotechnological applications to produce biofuels and reported, such as Thermobifida fusca Cel6A (Ali et al., chemicals (Margeot et al., 2009). Since the global energy 2015), Thermobifida halotolerans GH6 endoglucanase (Yin crisis and environmental pollution are intensifying, it has et al., 2015), and Cellulosimicrobium funkei CelL (Kim et attracted extensive attention to the utilization of al., 2016). However, the study of GH6 cellobiohydrolases lignocellulosic biomass. Cellulose is the major composition has been reported rarely. of lignocellulosic biomass and the bioconversion of Chaetomium thermophilum is a thermophilic fungus cellulose needs a combined effect of three classes of living in the high temperature environment up to 60oC hydrolytic enzymes, including endoglucanases (EC 3.2.1.4), belonged to the phylum Ascomycota. By now, many cellobiohyrolases (EC 3.2.1.176; EC 3.2.1.91) and β- glycoside hydrolases have been isolated from C. glucosidases (EC 3.2.1.21) (Lynd et al., 2002; Sánchez, thermophilum, such as a GH55 β-1,3-glucanase 2009). Cellobiohyrolases are a group of glycoside (Papageorgiou and Li, 2015), a β-glucosidase (Xu et al., hydrolases, which hydrolyzed oligosaccharides of assorted 2011) and a cellobiohydrolase II (Wang et al., 2013). lengths generated by endoglucanases to cellobiose Generally, C. thermophilum glycoside hydrolases are (Ragauskas et al., 2006). According to the classification of thermostable and have a high optimal reaction temperature the carbohydrate-active enzymes database (CAZy), based on the previous researches. Thermostable enzymes cellobiohyrolases are assigned to 5 glycoside hydrolase have potential advantages in lignocelluloses conversation, families: GH5-7, GH9 and GH48 (Cantarel et al., 2009). In on account of effectively improving hydrolysis efficiency particular, the cellobiohydrolases of GH6 are widely and reducing the possible contamination at high temperature believed to act processively from the non-reducing terminal in industrial processes (Huy et al., 2016). To cite this paper: Zhou, Q., J. Jia, P. Ji and C. Han, 2017. A novel application potential of GH6 cellobiohydrolase CtCel6 from thermophilic Chaetomium thermophilum for gene cloning, heterologous expression and biological characterization. Int. J. Agric. Biol., 19: 355‒362 Zhou et al. / Int. J. Agric. Biol., Vol. 19, No. 2, 2017 Currently, many microorganism-derived enzymes are mycelia as described in the manufacturer’s instructions produced by fermentation processes in vitro (Villatte et al., (TransGen, China). Based on the RNA template, reverse 2001). Pichia pastoris, as a convenient production system of transcription was carried out using reverse transcription kit yeast that could heterologously express proteins in high (TransGen, China). The ctcel6 gene was identified from the amounts, has been wildly used as a heterogeneous preview research of genome of C. thermophilum expression system (Li et al., 2014; Zhao et al., 2015). To (http://ct.bork.embl.de/) (Amlacher et al., 2011). The gene satisfy the strong demand for the thermostable hydrolase sequence was amplified using the primers as follows: 5’- production in enzyme preparation industry, heterologous CCTACGTAGCCCCCAACCCAACCCA-3’, and 5’- expression using P. pastoris has become the primary way GGCCTAGGTTAGTGGTG which significantly improves the protein expression level GTGGTGGTGGTGGAACGGAGGGTTGGCA-3’. The (Formighieri and Melis, 2016). However, as a significant designed primers contained a C-terminal 6x His-tagged part of glycoside hydrolases of thermophilic C. sequence for the purification of expressed product and the thermophilum, there are few reports about the thermostable compatible restriction sites of SnaBI and AvrП, respectively. GH6 cellobiohydrolases from C. thermophilum. PCR reaction program was as follows: 94oC, 5 min; (94oC, In this study, a novel GH6 cellobiohydrolase gene 30 s; 53oC, 30 s; 72oC, 60 s) ⅹ 30 cycles; 72oC, 10 min. The ctcel6 was first cloned from C. thermophilum. The gene amplified product was connected to pMD18-T vector, and ctcel6 was heterologously expressed in P. pastoris system, the recombinant plasmid was transformed to E. coli T1. The and the corresponding recombinant enzyme CtCel6 was screened positive transformants were gene sequenced to purified and characterized. The thermostable of CtCel6 was ensure that the cloned gene was correct. investigated. Moreover, we found it could effectively The signal peptide sequence of CtCel6 protein was degrade crystalline cellulose and soluble cellulose at high predicted using the online software SignalP 4.1 temperature and the hydrolysis products were mainly (http://www.cbs.dtu.dk/services/SignalP/). The characteristic cellobiose. The effect of cations and the substrate specific analysis of CtCel6 amino acid sequence was carried out activity of recombinant CtCel6 were also determined. These using the online ProtParam biochemistry characterizations suggested that recombinant (http://web.expasy.org/protparam/). Multiple sequences CtCel6 had a great prospect of commercial application in alignment was based on ClustalW2 bioconversion of lignocellulosic biomass. (http://www.simgene.com/ClustalW). Glycosylation sites analysis was performed using NetNGlyc 1.0 Materials and Methods (http://www.cbs.dtu.dk/services/NetNGlyc/) for N-linked glycosylation sites and NetOGlyc 4.0 Materials (http://www.cbs.dtu.dk/services/NetOGlyc/) for O-linked glycosylation sites, respectively. Chaetomium thermophilum HSAUP072651 was isolated from bovine feces at Tengchong (Yun’nan, China). The Construction of Expression System strain was deposited in China General Microbiological Culture Collection Center (CGMCC; Beijing, China) with The cloned gene ctcel6 was digested with SnaBI and AvrП, accession No. 3.17990. Escherichia coli T1 (TransGen, as well as pPIC9K vector. Then, the digested ctcel6 was China) was used for nucleotide sequenced. Pichia pastoris ligated to pPIC9K vector to produce the secretion strain GS115 (Invitrogen, USA) was used as heterologous expression plasmid pPIC9K/ctcel6, which was confirmed expression system. The pMD18-T vector (Takara, China) by DNA sequencing and restriction analysis. The and the pPIC9K vector (Invitrogen, USA) were used for recombinant plasmid pPIC9K/ctcel6 was preserved and clone system and expression system, respectively. Primers prepared for the next step. were synthesized by Sangon Bitech Co. Ltd. (Shanghai, China). RNA extraction kit and reverse transcription kit Transformation and Expression of the Recombinant were purchased from TransGen Company. Sodium Enzyme in Pichia pastoris carboxymethyl cellulose (CMC-Na), β-D-glucan, pectin, chitin, xylan, amylose and saccharose were purchased from The recombinant expression plasmid pPIC9K/ctcel6 was Sigma-Aldrich. Phosphoric acid swollen cellulose (PASC) linearized using the restriction enzyme SacI. After digestion was prepared from Avicel according to the method of Wood process, pPIC9K/ctcel6 transformed to P. pastoris GS115. (1988). Cello-oligosaccharides (Megazyme, Wicklow, Transformants were screened on MD and MM plates