The Response Of CAZyme To Lignocellulose And The Expression And Biotransformation Of Lignocellulose Degrading Enzyme In Thermophilic Trametes Trogii

Trametes trogii can secrete a variety of carbohydrate active enzymes(CAZyme)to participate in the degradation of natural lignocellulose,and is also an important source of laccase and other lignocellulose degrading enzymes.In the previous study,we screened a strain of T.trogii S0301,which has the characteristics of fast growth,high utilization rate of lignocellulose and outstanding laccase properties.However,the response of CAZyme to lignocellulose has not been studied,and the utilization of lignocellulose degrading enzyme gene resources is limited.In view of this,our research based on the strain of T.trogii S0301 analyzed the response of CAZyme to lignocellulose during carbon source conversion process under the condition of co-culture of glucose and natural lignocellulose.In addition,the expression of?-glucosidase Ttbgl3 and laccase lac13 and the transformation of glycosides by tbgl3were studied.1.Transcriptome analysis of cazy enzyme response to lignocellulose in T.trogii S0301In the previous study,602 genes of CAZyme were predicted from the genome sequence of T.trogii S0301,but the response to lignocellulose was not clear.In order to study the response of CAZy enzyme to lignocellulose in T.trogii S0301 and the secretion pattern of lignocellulose degrading enzymes,T.trogii S0301 was co-cultured with glucose and natural lignin.Glucose was basically depleted in the medium on the5th day,and then the mycelium began to using natural lignocellulose,along with?-glucosidase,EG,cellulase,xylanase,Mn P,Li P and other lignocellulose utilization enzyme activity increases rapidly.The analysis of transcriptome sequencing showed that the number of genes encoding AA,CBM and GH gene families increased from 31to 44,21 to 27 and 50 to 121,respectively,of which the most significant increase was the genes of GH Family.From the 2th d to the 5th d,the genes of lignin degrading enzymes such as Mn P were highly expressed,and the genes encoding cellulase and hemicellulase were also rapidly induced.On the 8th day,the expression of Mn P decreased rapidly,and the remaining peroxidase and oxidase(alcohol oxidase,chitooligosaccharide oxidase,galactose oxidase,heme peroxidase,GMC oxidoreductase,glyoxal Oxidase)continued to accumulate,synergistically degrading lignocellulose(LPMOs,?-glucosidase and cellobiose hydrolase of GH family)with cellulose and hemicellulase.The simultaneous increase of extracellular lignocellulose degrading enzyme activity detected in LG medium was consistent with the result of transcriptome analysis,which revealed the mode of simultaneous degradation of lignocellulose in T.trogii S0301.In addition,many transcription factors(zbtb,zf-c2h2,homeobox,etc.)are involved in this process to regulate the synergistic action of many enzymes.2.Prokaryotic expression of Ttbgl3 gene and the transformation of glycosidesLignocellulose-degrading enzymes represented by?-glucosidase show high application prospects in food bioprocessing and pharmaceutical industry.In this study,the?-glucosidase gene Ttbgl3 that was significantly upregulated by lignocellulose in the GH3 family was selected based on the results of transcriptome analysis.The expression of Ttbgl3 recombinase was highest under the induction conditions of 0.1m M IPTG,20°C,and 12 hours of culture.The analysis of the properties of the enzyme showed that when p NPG was used as the substrate,the optimal temperature of Ttbgl3was 50°C,the optimal p H was 6.0,and the stability was better at 50°C and p H 5-10.Fe³⁺can increase the activity of Ttbgl3 to 119.6%.Ttbgl3 can maintain more than 50%of the enzyme activity in other organic solvents except for 10%SDS which completely inhibiting the activity of Ttbgl3.In addition,low concentration(20 m M)glucose has a significant inhibitory effect on the recombinant enzyme Ttbgl3.The K_mvalue of Ttbgl3is 1.04 m M and the V_max value is 263.16?M/mg/min.Conversion analysis of glycoside compounds showed that Ttbgl3 can hydrolyze compounds containing?-glucosidic bonds such as Gastrodin,Aesculin and Daidzin,and also hydrolyze compounds containing?-glucuronide glycosides like Baicalin.After optimization of enzyme concentration and transformation time.we finally determined the optimal conversion parameters for three natural compounds:Gastrodin 500?g/m L,Trbgl3 60 U/m L,incubation at 37°C for 8h,the conversion of p-hydroxybenzyl conversion is 0.17 m M/h;Aesculin 500?g/m L,Trbgl3 5 U/m L,incubation at 37°C for2h,the conversion rate of Aesculetin is 0.96 m M/h;Daidzin 500?g/m L,Trbgl3 5 U/m L,and incubation at 37°C for 0.5h,the conversion rate of Daidzein is 1.47 m M/h.It is speculated that the two residues of His85 and Lys467 are the enzyme binding sites of the substrate containing?-glucoside bond,and Glu377 and Thr424 are the enzyme binding sites of the substrate containing?-glucuronide bond by homology modeling and molecular docking.3.Expression of laccase isozyme Lac13 of T.trogii S0301 by Pichia pastorisSeveral laccase isoenzyme genes have been cloned from T.trogii S0301 in the preliminary research.In this study,plasmids(p PICZ?-Lac13a and p PICZ?-Lac13b)for removing and retaining the signal peptide of Lac13 were constructed.,the laccase activity can be detected by Pichia pastoris heterologous expression system after the removal of its own signal peptide.The conditions of Cu²⁺concentration,p H and temperature were optimized.When the induction conditions were 1%methanol,0.5m M Cu²⁺p H 6.0 and 25°C,the laccase showed the darkest color change and the highest activity,which was purified by anion column.In conclusion,the transcriptome sequencing and extracellular enzyme activity of T.trogii S0301 in co culture of glucose and natural lignin revealed the response process of T.trogii S0301 to lignocellulose and the secretion pattern of lignocellulose degrading enzyme.With the prokaryotic expression,property analysis and transformation of glycosides of Ttbgl3 in T.trogii S0301,it was found that Ttbgl3 has dual-enzyme activity and can transform a variety of glycoside compounds efficiently.In addition,the laccase isozyme Lac13 was successfully expressed in the Pichia pastoris expression system.These results lay the foundation for further exploring the mechanism of efficient utilization of lignocellulose by Trametes trogii and the development of lignocellulose gene resources.