Gene Cloning And Functional Characterization Of Thermophilic Cellulases And One Expansin From Talaromyces Leycettanus JCM12802

Cellulose is the most abundant natural renewable resource on Earth.It is of great economic value and environmental significance to degrade cellulose into small sugar molecules,which are further converted into more value-added products.However,cellulose is complex and compact in the structure,which hydrolysis requires the synergistic action of various enzymes.Considering the low efficiency of current cellulase additives,it is of importance to mine new cellulases and auxiliary proteins to facilitate the development of cellulases and improve their efficiency.Talaromyces leycettanus JCM12802 is a great producer of thermophilic glycoside hydrolases(GHs).In this study,two cellulases(TlCel5A and TlCel6A)belonging to GH5 and GH6 respectively were identified in T.leycettanus JCM12802 and successfully expressed in Pichia pastoris.The full-length Tlcel5 A and Tlcel6 A were 1464 bp(interrupted by four introns of 67,56,57,and 54 bp)and 1898 bp(interrupted by seven introns of 69,75,78,69,71,70,and 71 bp),respectively.The cDNAs were 1230 bp and 1395 bp,and encoded polypeptides of 409 and 464 amino acids,respectively.Multiple sequence alignment indicated that TlCel6 A showed the highest identity with T.leycettanus(CDF76448.1)(99%)compared to Tlcel6 A with only three amino acids at 371-373.Both enzymes have a putative signal peptide of 18 amino acids.The enzymes had acidic and thermophilic properties,showing optimal activities at pH 3.5?4.5 and 70?80°C.TlCel5 A and TlCel6 A acted against several cellulose substrates,3101.1 v.s.92.9 U/mg to barley ?-glucan,3905.6 U/mg v.s.109.0 U/mg to lichenan,and 840.3 and 0.1 U/mg to CMC-Na.When using Avicel,phosphoric acid swollen cellulose or steam-exploded corn straw as the substrate,combination of TlCel5 A and TlCel6 A showed significant synergistic action.Mutant TlCel6A-3A showed similar optimal pH but 20°C decreased optimal temperature.When using barley ?-glucan as the substrate,the specific activity of mutant TlCel6A-3A was 15 U/mg,5.2-fold lower than that of TlCel6A(92.9 U/mg).An expansin-encoding gene Tlexlx1,1196 bp in length,was also cloned from thermophilic T.leycettanus JCM12802.In comparison to most swollenins derived from fungi,deduced TlEXLX1 lacks a CBM domain at the N-terminus,consists of a 22-residue signal peptide at the N-terminus and a GH45 homologous domain.The gene contains 1 intron(83 bp)and encodes a polypepetide of 370 amino acids and a stop codon.Recombinant plasmids CBM-Tlexlx1 and Tlexlx1 harboring the N-terminal CBM domain of TlSWOl or not were constructed,and the gene products were produced in Pichia pastoris.Both the enzymes had high glucanase activities,7.2 and 17.2 U/mg to lichenan,4.4 and 9.4 U/mg to barley ?-glucan,and trick activity to Avicel.When using lichenan as the substrate,both enzymes showed optimal activities at 60°C and pH 6.0.TlEXLX1 had capability of destructing the smooth surface of Avicel,and showed synergy with commercial cellulase in cellulose degradation.In summary,two thermophilic cellulase genes and one expansin gene were cloned from T.leycettanus JCM12802 and expressed successfully in the P.pastoris GS115.Both cellulases are thermophilic enzymes with high activity and synergistic effects.Preliminary studies on the thermophilic mechanisms of the two cellulases and the synergies between endo-and exo-cellulases and cellulase and expansin(swollenin)not only enriches cellulase gene resources and its auxiliary proteins,but also reduces its production costs to promote industrial applications.