Synthesis And Folding Optimization Of Carbohydrate Binding Module From Trichoderma Reesei Cellobiohydrolase

Cellulase is an important enzyme system for lignocellulosic transformation,which hydrolyzes glycosidic bonds at different locations to form glucose.Carbohydrate binding module(CBM)is an important part of cellulase.The CBM has no catalytic activity,but it can shorten the distance between the enzyme and the substrate,thereby improving the catalytic efficiency of the enzyme.The physicochemical properties of CBM are closely related to the catalytic efficiency of cellulase.In order to improve the physicochemical properties of CBM,it is necessary to obtain the effects of different sugar structures and glycosylation sites on CBM.However,naturally derived CBM is a mixture of difficult-to-isolate glycosylated isoforms.To determine the effect of specific glycation on CBM,a variety of glycosylated CBM isoforms with high purity was required.Chemical synthesis based on solid phase peptide synthesis(SPPS)is a more precise and flexible method to prepare glycosylated CBM isoforms with high purity and systematic differences in sugar structure.For the synthesis of glycosylated CBM isoforms,folding is the last and critical step.The folding of CBM is usually carried out overnight,which is time-consuming and labor-intensive,and is not suitable for the preparation of CBM in large quantities.Therefore,the optimization of CBM oxidative folding is a necessary step to improve the efficiency of its synthesis.In this study,the family 1 CBM from Trichoderma reesei Family 7 cellobiohydrolase(Tr Cel7A)was synthesized.The CBM sequence is TQSHYGQCGGIGYSGPTVCASGTTC QVLNPYYSQCL,with 36 amino acids,2 disulfide bonds,and three O-glycosylation sites of Thr1,Ser3 and Ser14.Ten glycosylated CBM isoforms were synthesized.The sugar amino acids used in the synthesis process are Fmoc-Thr(Ac4Manα)-OH,Fmoc-Thr(Ac4Manα1-2Ac3Manα)-OH,Fmoc-Ser(Ac4Manα)-OH,Fmoc-Ser(Ac4Manα1-2Ac3Manα)-OH.From the synthesis of the primary structure to the completion of folding,the total yield is 9-30%.Furthermore,the GSH/GSSG ratio,temperature,p H,thiol/disulfide and their concentration,peptide concentration used in the CBM folding process were systematically changed and optimized.To monitor the folding progress,ultra-performance liquid chromatography and mass spectrometry were used to analyze isomer changes and disulfide bond formation.The results showed that it took 90 min to complete the reaction at room temperature when the concentration of CBM was 67 μM.By optimizing the conditions,it was found that the optimal temperature for CBM folding was 25-35 oC,the optimal p H of folding buffer was 9,and the optimal ratio of GSH/GSSG was 6:1-8:1.Adjustment of the temperature,p H and the reducing agent/oxidant ratio can speed up the folding,but it still took 40 minutes to complete the reaction.The use of the L-cysteine/L-cystine can further improve the CBM folding rate.When the folding conditions were: L-cysteine: L-cystine = 8: 1,L-cysteine + L-cystine = 9m M,p H = 9,it only took 10 minutes to complete the reaction,which is nearly 9 times faster than the commonly used folding method.In addition,although the yield of folding at a concentration of 334 μM was reduced by 12%,the preparation efficiency was increased by5-fold,which greatly reduced the time cost.This study provides not only a reference for the efficient preparation of CBM,but also an idea for the performance modification of CBM.