Heterologous Expression And Biochemical Characterization Of Myceliophthora Thermophila α-glucosidases

Isomaltooligosaccharides（IMOs）is defined as functional oligosaccharides with 2 to 5 glucosyl units conmected by mainly α-（1→6）linkages and occanionally by α-（1→2）andα-（1→3）linkages.The main functional components of IMOs include isomaltose,panose and isomaltotriose.IMOs is an important type of prebiotic,which can stimulate the proliferation of intestinal beneficial bacteria and enhance human immunity,and has been widely used in the food industry.The transglucosidase activity of GH31 α-glucosidases is employed to catalyze the synthesis of IMOs using the the malt syrup prepared from starch as the substrate.At present,the GH31 α-glucosidase（AgdA）derived from Aspergillus niger is widely used for IMOs synthesis.The low conversion efficiency,insufficient temperature stability,high price,and unstable supply of GH31 α-glucosidase are considered to be the main limiting factor for IMOs synthesis.Continutous mining for new GH31 α-glucosidases and achieving their efficient heterologous production is critical for enhancing the supply and quality of IMOs.In the present thesis,α-glucosidase derived from several typical thermophilic fungi were explored and two α-glucosidases（MT31αl and MT31α2）from Myceliophthora thermophila were selected for heterologous expression and biochemical analysis.In order to achieve efficient expression of these two α-glucosidases,we further studied the heterologous expression of MT31α1 and MT31α2 using Trichoderma reesei as the host.The main findings of this study are as follows:1.Database mining and analysis of GH31 family α-glucosidases derived from several typical filamentous fungi.Genomic mining revealed 5 GH31 α-glucosidases in A.niger while in Aspergillus nidulans,Aspergillus fumigatus and Aspergillus oryzae,there are 4,2 and 3 GH31α-glucosidases,respectively.Compared with AgdA of A.niger,AgdB of A.nidulans shows stronger transglycosidase activity and weaker hydrolysis activity.In order to exploreα-glucosidase with better thermostability,we further analyzed the genomes of several thermophilic fungi.In M.thermophila,Talaromyces emersonii,Chaetomium thermophilus and Thermomyces lanuginosa,the number of putative GH31 α-glucosidases were identified as 6,6,5 and 1,respectively.Since different α-glucosidases have different substrate and product specificities,phylogenetic analysis of these α-glucosidases was performed to identify the homogous protein of AgdA of A.niger and AgdB of A.nidulans.The results showed that the homologous proteins of A.niger AgdA were found in A.oryzae,A.fumigatus,A.nidulans,T.emersonii,and C.thermophiles.Their sequence identities with AgdA were between 58.15%and 78.33%.The homologous proteins of AgdB from A.nidulans were found in A.oryzae,A.fumigatus,A.nidulans,T.lanuginose,C.thermophilus and M.thermophila,and their sequence identity with AgdB of A.nidulans ranged from 54.25%to 81.77%.The homologous proteins[MT31α1（XP₀03658875.1）and MT31α2（XP₀03661084.1）]of AgdA and AgdB from M.thermophile were selected for further sequence analysis and biochemical characterization.Conserved domain analysis showed that both MT31α1 and MT31α2 possess the conserved catalytic domain of GH31 family and NtCtMGAM_N domain.Sequence alignment showed that both MT31α1 and MT31α2 had two conserved regions of GH31α-glucosidases,region A and region B,which contains the catalytic residues.The nucleophiles and acid-base catalysts of MT31α1 and MT31α2 are Asp495 and Asp662,Asp432 and Asp617,respectively.2.Heterologous expression and biochemical properties analysis of MT31αl and MT31α2.Pichia pastoris expression system has been widely used for expressing various proteins in industry in view of its high expression level,simple operationand post-translational modification capability.In the present study,MT31αl and MT31α2 were successfully expressed in Pichia pastoris GS115 and purified by ammonium sulfate precipitation and nickel affinity chromatography.Biochemical analysis showed that the optimum temperature of MT31α1 and MT31α2 were 65℃ and 60℃,respectively,and the optimum pH were 4.5 and 6.5,respectively.Therostability analysis revealed that MT31αl and MT31α2 retained more than 60%of their activities after incubating at 55℃ and 50℃,respectively,for 24 h.Moreover,both enzymes were found to be stable at pH 3 to 11.Besides,most tested metal ions and detergents had only very slight effect on the the activity of MT31α1 and MT31α2 whereas Ag+,Fe3+,Hg2+ and SDS inactivated MT31α1 and MT31α2.Kinetic analysis showed that MT31α1 showed much higher（about 150 folds）pNPG hydrolysis activity than MT31α2.Using maltose as substrate kinetic analysis showed that high concentrations of maltose showed inhibition on the hydrolysis activity and its hydrolysis activity is higher than its transglycosidase activity.3.Transglycosylation products analysis of MT31αl and MT31α2.1H NMR analysis of the reaction mixture of MT31α1 with 30%maltose substrate showed that a new anomeric carbon signal appeared at δ 4.99 ppm,which was the reporter signal of α-（1→6）linkages indicating the formation of IMOs.HPLC analysis showed that MT31α1 initially synthesized panose by transferring glucosyl unit to the non-reducing end of maltose with the formation of α-（1→6）linkages.As the reaction continued,maltose was gradually consumed and glucose was accumulated and used as the acceptor substrate for the synthesis of isomaltose and isomaltotriose.In the final product mixtures,the content of main IMOs comppouents（isomaltose,panose and isomaltotriose）was determined to be 40.86%±1.95,which is higher than commercial IMO-50（35%）.Thus,MT31α1 showed a high potential for industrial IMOs synthesis.1H NMR spectrum of the reaction mixture of MT31α2 with 30%maltose substrate demonstrated a new anomeric signal at δ 5.33 ppm,suggesting the formation of α-（1→3）linkages in the generated product.HPLC analysis showed that MT31α2 synthesized two transglycosylated products with relatively high abundance using maltose as substrate.The structures of the two products were determined to be α-D-Glcp-（1→3）-α-D-Glcp-（1→4）-D-Glcp and α-D-Glcp-（1→3）-α-DGlcp-（1→3）-α-D-Glcp-（1→4）-D-Glcp by LC-MS and NMR analysis.Therefore,MT31α2 can synthesize oligosaccharides with α-（1→3）linkages.4.Heterologous expression of MT31α1 and MT31α2 in Trichoderma reesei.Trichoderma reesei with great protein secretion ability is the principal strain for industrial production of cellulase and hemicellulase.In addition,it is considered to be generally recognized as safe（GRAS）by the US FDA.In order to explore the efficient expression of α-glucosidase,we further attempted to heterologously express MT31α1 and MT31α2 in Trichoderma reesei.For this purpose,the expression of MT31α1 and MT31α2 were under the control of the highly expressed tcu promoter.In addition,the secretion of MT31α1 and MT31α2 were dirved by their own or CBHI signal peptide,obtaining T.reesei-α1-ZI,T.reesei-αl-CBH1,T.reesei-α2-7I and T.reesei-α2-CBHl strains.The expression of MT31αl and MT31α2 was analyzed by SDS-PAGE,protein concentration determination and pNPG activity determination of the transformants.The results showed that the enzyme activity and protein content of T.reesei-α2-ZI and T.reesei-α2-CBH1 increased with the increase of fermentation time.Their enzyme activities reached 0.06 and 0.04 U/mL at 96 h,respectively.The enzyme activity and expression of MT31α1 were not detected in T.reesei-α1-CBH1.For T.reesei-α1-ZI,although its protein expression is relatively low as shown by SDS-PAGE and protein concentration determination,its enzyme activity is the highest,reaching 0.16 U/mL at 96 h.The expression and secretion of MT31α1 and MT31α2 in T.reesei warrant to be further studied.