Study On Laccase Production In Liquid Fermentation Of Trametes Versicolor Induced By GHK-Cu And Its Mechanism

Laccase（Benzene diol:Oxyoxidoreductase,EC 1.10.3.2）is a kind of copper-containing polyphenol oxidases.Due to its ability to catalyze the oxidation of various phenolic and non-phenolic compounds,laccase has been widely used in different area,such as food,pharmacy,pulping and environment.However,the low expression level of laccase and the high production cost limited its production and application in large scale.Adidition of inducer was one of efficient strategies to enhance laccase production.It is essential to develop high-efficient and non-toxic inducers.In this study,a Cu²⁺-chelated tripeptide（GHK-Cu）was used as the inducer for laccase production by Trametes versicolor in liquid fermentation.For this purpose,the medium composition was optimized for the laccase production induced by GHK-Cu.The high-efficient induction mechanism of GHK-Cu in laccase synthesis was explored.With the help of transcriptome method,the expression regulation of GHK-Cu on the related genes in laccase synthesis pathway of T.versicolor was also investigated.The main conclusions of this study are as follows:（1）Using single-factor experiment and response surface methodology,the medium composition was optimized for the liquid fermentation of T.versicolor induced by GHK-Cu.The optimal medium composition was obtained as follows:GHK-Cu 290μM,glucose 4.21 g/L,NH₄Cl 0.31 g/L,KH₂PO₄ 0.18 g/L,Ca Cl₂ 0.047 g/L,FeSO₄.7H₂O0.043 g/L,MgSO₄.7H₂O 0.05 g/L.The maximal response value in the model was830.233 U/L.The actual laccase activity of 850.04 U/L was achieved in the liquid fermentation of T.versicolor with the optimal medium,which was 12.77 times higher than that of 66.59 U/L in the optimized medium without inducer.The scaling-up of T.versicolor fermentation with the optimal medium was carried out in 5-L stirring reactor.The laccase activity reached the maximum of 1113.57 U/L after 5 days culture in reactor,which was 1.31 times that in shake flask culture.These results indicated that GHK-Cu could greatly enhance the laccase production of T.versicolor in liquid fermentation and this strategy could be applied for the large-scale laccase production in reactor.（2）At the same inducer concentration of 290μM,the laccase production induced by GHK-Cu was 92.5%higher than that induced by CuSO₄.The existing form of Cu²⁺in GHK-Cu can be one of the reasons for its enhanced induction effect.Compared to the control without inducer,the increased membrane permeability and reduced cell wall thickness happened to the hyphae of the T.versicolor with the addition of inducers containing Cu²⁺.In addition,the damage of GHK-Cu to cell wall and membrane was weaker than that of CuSO₄.The maximum extracellular consumption rate and the maximum intracellular accumulation rate of copper in GHK-Cu group were 0.35mg/L/h and 0.11 mg/g/h,respectively.These two rates were lower in CuSO4 group,which were 0.15 mg/L/h and 0.06 mg/g/h,respectively.It indicated that it was more favorable for the assimilation and utilization of Cu²⁺by T.versicolor when the GHK-Cu was used as the source of Cu²⁺.After 6-day liquid culture,the expression of laccase gene Tv Lac2 in GHK-Cu group was 28.15 times higher than that in control,while its expression in CuSO₄ group was only 14.88 times higher than that in control.The expression of Tv Lac3 and Tv Lac4 reached their peak values in both inducer groups after5 days culture,which was great improved compared to the control.In addition,their expression in GHK-Cu group were higher than those in CuSO₄ group.These results illustrated that GHK-Cu can promote the expression of laccase genes and it exhibited better induction performance compared to CuSO₄.（3）The analysis of T.versicolor transcriptome showed that the total differential gene reached the maximum on Day 5 in both inducer groups compared to the control,where there were 3794 differential genes in CuSO₄ group and 5573 differential genes in the GHK-Cu group.Based on KEGG enrichment analysis,it could be found that the up-regulated genes in inducer groups were significantly enriched in the pathways mainly involved in the metabolism and synthesis of amino acids.The pathways significantly enriched with the down-regulated genes included the pathway for the synthesis of secondary metabolites.However,the addition of GHK-Cu up-regulated the expression of genes involved in DNA synthesis,ribosome synthesis,a large number of RNA transport and m RNA surveillance pathway,which didn’t occur in in CuSO₄ group and could be closely related to the laccase synthesis promoted by GHK-Cu.In both inducer groups,the expression of genes related to metal ion transport and transfer proteins were significantly up-regulated,where the up-regulated level of related genes in GHK-Cu group was significantly higher than that in CuSO₄ group.The expression of genes19411270 and 19416261 belonging to ion channel-forming bestrophin family protein and gene 19411942 related to metal binding domain was up-regulated in GHK-Cu group,but no change occurred in CuSO₄ group.Using GO functional enrichment analysis,it was proved that the pathways significantly enriched with the up-regulated genes included GO:0005507-copper ion binding in both inducer groups.The down-regulated genes significantly enriched in inducer groups involved the genes related to the synthesis of cell wall and membrane,including GO:0031224-intrinsic component of membrane,GO:0016021-integral component of membrane,GO:0005618-cell wall and GO:0009277-fungal-type cell wall.Therefore,the addition of inducers containing Cu²⁺was disadvantage to cell wall synthesis and membrane intergridity of T.versicolor.During the culture period,laccase genes Tv Lac1-Tv Lac7 were induced by GHK-Cu,and their expression levels were greatly increased.However,CuSO₄ had no significant up-regulation effect on the expression of Tv Lac2 and Tv Lac6.It indicated that GHK-Cu was a high-efficient inducer for laccase synthesis of T.versicolor.