Gene Cloning, Expression, And Characterization Of Lac3 From Trichoderma Asperellum

Laccases act as a green tool in many processes of the biotechnology industries for its extensive range for substrate catalysis and the advantage that they only require oxygen for catalysis. Three laccase-producing filamentous fungal strains were screened from soil by guaiacol chromogenic reaction, and were identified as Trichoderma asperellum LS-10, Trametes sp. LS-10C and Alternaria tenuissima LS-19 by mycelia morphology and rDNA-ITS sequence analysis, respectively. As the strain LS-10C had higher laccase activity but with a long fermentation period, and the LS-19 strain’s had very weak laccase production ability, while the LS-10 strain showed shorter fermentation time and rapid laccase production speed and other characteristics was chosen to study on optimization of fermentation conditions in flask and its enzyme properties. Three laccase genes were cloned, and two of them were heterologous expressed in Pichia pastoris successfully. The main research results were listed as follows:(1) Screening, identification and optimization of fermentation conditions of laccase-producing filamentous fungal strains, and enzyme propertiesThe Trichoderma asperellum LS-10 was chosen to fermentate. The culture medium and fermentation conditions were optimized by single factor and orthogonal design in flask. The results showed that the optimal condition for laccase production with this new isolated strain was (g/L):sucrose 25, wheat bran juice 80, ammonium tartrate 10, bean pulp 18, KH2PO42, MgSO4·7H2O 0.6, CaCl2·2H2O 0.8, CuSO4·5H2O 0.25(1 mmol/L) and the initial pH 5.5-6.5, furthermore, a laccase production level of 622.85 U/L was obtained under the above fermentation conditions. The highest activity of crude fermented liquid enzyme was studied on respectively the optimal temperature, the optimum pH, thermal stability and pH stability, the results showed that the optimal reaction temperature of laccase activity was 40℃。 The laccase activity was high between 35-45 ℃, above 45℃ or below 35℃, the activity of enzyme was low. Addition of metal ions showed that metal ion Al3+(1 mmol/L) could significantly promoted laccase activity, and low concentration (1 mmol/L) of Fe3+、Mn2+、Na+、K+、Ca2+、 Mg2+、Zn2+ could stimulate, while high concentration (5 mmol/L) would inhibit the laccase activity. Moreover, Fe2+could completely inhibit the laccase activity. Addition of SDS (0.5 mmol/L) would completely inhibit enzyme activity, too. The inhibition of EDTA was not obvious. Experimental results showed that the laccase was stable below 50 ℃, when incubated at 80 ℃ for 90 min the laccase activity would completely lost.(2) Gene cloning and bioinformatics analysis of laccaseAccording to the predicted laccase conservative sequence information of Trichoderma asperellum on JGI website, six pairs of primer of laccase genes were designed, and three laccase genes were amplificated successfully.Sequence analysis showed that the full-length and cDNA of lacl、 lac2 and lac3 were 1769 bp and 1701 bp,2303 bp and 1803 bp,1918 bp and 1773bp, respectively. Sequence analysis of the intron/exon showed that lacl, lac2 and lac3 gene contained one, seven and one introns, respectively. Bioinformatics analysis showed that Lac1, Lac2 and Lac3 was composed of 600,566 and 590 amino acids, respectively, and its molecular weight was 62.05,66.34 and 64.90 kDa, respectively, the theoretical isoelectric point was 4.35,5.43 and 6.25, respectively. Signal peptide analysis showed that Lacl and Lac2 had signal peptide, while Lac3 had no signal peptide. Signature characteristics analysis showed all of them had typical laccase conservative amino acid sequences, such as L1-L4 domains, SDS-gate and C-terminal DSGL/I/V domain with 3,4 and 4 Copper ion binding sites, respectively. Discover Studio 2.5 was used to homology modeling.(3) Fermentation of Pichiapastoris integrantsThe laccase gene lacl and lac3 were inserted into the expression vector pPIC9K and transformed into Pichia pastoris GS115 successfully. A number of transformants were screened and identified. The recombinant strains were fermented induced by methanol. The fermentation results showed the culture conditions were initial pH 5.0, cultivated temperature 29℃, shaker rotate speed 220 rpm, induced by Cu2+ concentration 0.03 mmol/L and a final methanol concentration of 0.5% with 45/250 mL liquid volume in shaker flask, and the highest laccase activity of Lac1 and Lac3 reached 2.81 U/L and 2.56 U/L induced at 24 h respectively.