Cloning And Expression Of The Novel Inulinase Genes From Two Marine Yeast Strains

Inulin consists of linear chains of β-2,1-linked D-fructofuranose moleculesterminated by a glucose residue through a sucrose-type linkage at the reducing end.Inulin mainly exsits in jerusalem artichoke, yacon, chicory tubes, and arctium.Inulinases are fructofuranosyl hydrolases that target on the β-2,1linkage of inulin andhydrolyze it into fructose and glucose. They can be divided into exo-inulinase andendo-inulinase. The exo-inulinase catalyzes the removal of the terminal fructoseresidues from the non-reducing end of the inulin molecule while the endo-inulinasehydrolyzes the internal linkages in inulin to yield inulotriose, inulotetraose, andinulopentaose. In this study, the full-length of the inulinase gene was cloned andcharacterized from C. aureus HYA and C. membranifaciens subsp.flavinogenieW14-3, respectively. At the same time, the cloned HYAINU1gene from C. aureusHYA was expressed in P. pastoris and S. cerevisiae, respectively.The marine yeast C. aureus HYA had inulinases activity.The HYAINU1genefrom C. aureus HYA was cloned and characterized (Accession number: JX073660).The gene had an open reading frame (ORF) of1653bp long encoding an inulinase.The coding region of the gene was not interrupted by any intron. It encoded551amino acid residues of a protein with a putative signal peptide of23amino acids andthe calculated molecular mass of59.5kDa. The protein sequence deduced from theinulinase structural gene contained the inulinase consensus sequences(WMNDPNGL),(RDP), ECP, FS and Q.It also had two conserved putativeN-glycosylation sites. After analysis of the promoter of the HYAINU1gene, it wasfound that it had one TATA box and several sequences such as5′-SYGGRG-3′and5′-CGG-3′. It has been reported that Mig1, the main effector in glucose repression, isa C2H2zinc finger protein that is able to bind the promoters of a variety of genesrepressed by glucose and the binding site in the promoter should have the sequence5′-SYGGRG-3′. It also has been documented that the transcriptional activator, Zn(II)2Cys6protein is able to bind the promoters of a variety of genes induced by thesubstrates and the binding site in the promoter has the sequence5′-CGG-3′. Thismeans that expression of the HYAINU1gene cloned from C. aureus HYA can berepressed by glucose and fructose and induced by the substrates, such inulin andsucrose.The inulinase gene without the signal sequence was subcloned into pPICZaAexpression vector and expressed in P. pastoris X-33. After the expressed fusionprotein was analyzed by SDS-PAGE and Western Blot, a specific band withmolecular mass of about60kDa was found. Enzyme activity assay verified therecombinant protein as an inulinase. A maximum inulinase activity of16.3±0.24U/mL was obtained from the culture supernatant of P. pastoris X-33harboring the inulinase gene. The optimal temperature and pH for action of theenzyme were50°C and5.0, respectively. A large amount of monosaccharides weredetected after the hydrolysis of inulin with the purified recombinant inulinase. Theresults suggested that the recombinant inulinase produced by P. pastoris X-33carrying the HYAINU1gene had exo-inulinase activity.The HYAINU1gene with the signal sequence was subcloned into pMIRSC11expression vector and expressed in Saccharomyces sp. W12d (a diploid uracilauxotroph strain of the high ethanol producing yeast Saccharomyces sp. W0). Thetransformant MHYAINU1-69obtained produced19.2±0.3U/mL of extracellularinulinase within72h of cultivation. During the150-mL fermentation,11.9%(v/v,20°C)ethanol was produced within120h and the inoculum size and inulin concentrationwere10.0%(v/v) and25.0%(w/v), respectively. During the3-L fermentation,13.1±0.6%(v/v,20°C) of ethanol was produced from inulin.The marine yeast strain W14-3was isolated from seawater of China Eastern Seaand identified to be Candida membranifaciens subsp. flavinogenie W14-3. The strainW14-3was found to have inulinases activity and produce riboflavin.The inulinasegene from the strain W14-3was cloned by degenerate PCR and genomic walking.The cloned gene had an open reading frame (ORF) of1536bp long encoding aninulinase (Accession number: KC576811). It encoded512amino acid residues with the calculated molecular mass of57.8kDa. After analysis of the5′-flanking region ofthe inulinase gene, it was found that in its promoter there were two sequences of5′-SYGGRG-3′and several sequences of5′-CGG-3′, which had the same function asconsensus sequences of the promoter of the HYAINU1gene from C. aureus HYA,suggesting that expression of the inulinase gene cloned from C. membranifacienssubsp. flavinogenie W14-3can be repressed by glucose and fructose and induced bythe substrates, such inulin and sucrose.