The Composition And Synergistic Mechanism Of A Konjac-Degrading Enzyme System From Aspergillus Fumigatus HBHF5

Konjac mannan oligosaccharides is a kind of functional oligosaccharide,which has a broad prospect of application in the food,feed and medicine industry.KMOs was prepared from Konjac glucomannan(KGM)using enzymatic hydrolysis,which has the advantages of green,safe and efficient.However,the enzymatic hydrolysis efficiency is not feasible to increase with the substrate concentrations,due to the high viscosity the polymer creates in aqueous media.In this study,the thermophilic fungi were isolated from Daqu,which showed high activity in KGM degradation.We focus on the composition of mannan-degrading enzymes utilizing transcriptomics and proteomics.Simultaneously,the key genes for glycogen degradation were heterologously expressed,characterized and synergistic.The results showed the following:1.The thermophilic fungi isolated from Daqu were identified and screened to assess their degradative potential.In this study,a total of 20 thermophilic fungi were isolated from high-temperature Daqu.The morphological and molecular identification revealed that the isolates belonged to 7 different species,such as Theimia ramosa,Rhizomucor pusillus,Rhizomucor tauricus,Rasamsonia compostiticola,Thermopuscus crustaceus,Aspergillus fumigatus and Rhizopus microsporus.Further study also found that the A.fumigatus HBHF5 reduced the viscosity values of the KMG solution by 35.8%,which released the direct reducing sugars(DRS)and total reducing sugars(TRS)up to 1.88 mg/ml and 0.96 mg/ml,respectively.A total of 239 genes encoding carbohydrate-active enzymes(CAZymes)were identified for A.fumigatus HBHF5 under cultured with wheat bran,among which were starch-degrading enzymes,cellulase-degrading enzymes and hemicellulose-degrading enzymes.In summary,the A.fumigatus HBHF5 strain was selected for further study.2.The transcriptome and proteome analyses of A.fumigatus HBHF5For the in-depth study of the enzyme system of A.fumigatus HBHF5 for Konjac glucomannan degradation.Comparative transcriptome and proteomic analyses were conducted on the strain HBHF5 by using Konjac glucomannan or glucose as the sole carbon source.Transcriptome sequencing of A.fumigatus HBHF5 was analyzed and results demonstrated that a total of 9449 expressed genes,including 644 up-regulated genes and 588 down-regulated genes.A total of 629 unique proteins were identified by label-free quantification proteomics.,of which 156 were up-regulated and 348 were down-regulated.Further analysis revealed that these up-regulated expressed genes encoding CAZymes,such as cellulase,mannanase,xylanase,pectinase and glucosidase.3.Enzymic characterization of the key hydrolase genes for konjac glucomannan degradationWe probed the functional analysis of the 13 key hydrolase genes from the results of the transcriptome and proteomic analyses.It was found that the maximum activity of all these key hydrolase enzymes performed better above 60?,which belonged to the thermophilic enzyme.The mannanase(AfMan5 A and AfMan5B)possessed optimum p H and temperature of 5.0-6.0 and60?,respectively.Simultaneously,the two enzymes could maintain 70% of their original activity at broad p H4.0-8.0 and be thermostable at 60?.There was no evidence of a synergistic effect between the two mannanases in the konjac glucomannan degradation.The pectinase AfPLy A,AfPLy C,and AfPGL showed the optimal temperature were 60?,60? and 80?,respectively.The pectinase AfPLy Cand AfPGL were acidic pectinases with the optimum activity at p H 5.0-6.0.However,the pectate lyase(AfPly A)was alkaline pectic Lyase,which optimum p H was 8.0,and could act in a wide range of p H(2.0-12.0)and temperatures.There is almost no synergetic effect between AfPLy C or AfPly A with mannanase(AfMan5B)in the konjac glucomannan degradation.However,there was a synergistic interaction between AfPGL mannanase(AfMan5B),which approximately 12-24% increase in degradability.The cellulase AfCel5 A,AfCel5 B,AfEn and Afhp showed the optimal temperature were 75?,80?,60? and 80?,respectively.Meanwhile,these enzymes also presented a relatively good thermostability,such as AfCel5 A and AfCel5 B retained an enzymatic activity greater than 85% at 70 ? for 60 min.All five recombinant cellulases were acidic enzymes,which had an optimum enzyme activity between p H 4.0-6.0 and were stable in a wide p H range of 4.0-10.0.The most significant synergistic effects were noticed for the konjac glucomannan degradation in combination AfCel5 B with AfMan5 B,and the total reducing sugar content increased by about 300%.After synergistic AfEn or swollenin protein and AfMan5 B,the reducing sugar yield was increased by 10-30%.The optimum temperature of Chitinase AfChi and Tannase AfTan were 50? and 40? respectively.In regard to the stability of AfChi,it was more stable at p H 4.0-10.0 with a residual activity greater than 80% and was also thermally stable at 45? with 80% residual activity after 60 min.AfTan was also stable at p H3.0-6.5 and retained more than 90% activity,and retained over 60% of its maximum activity at40? for 30 min.The tannase Aftan also showed a synergistic effect on the degradation of KONJAC Mannan with AfMan5 B.In summary,we investigated the konjac glucomannan-degradation potential of the 20 thermophilic fungi isolated from Daqu.It was found that A.fumigatus HBHF5 successively degraded the konjac glucomannan through sequential secretion of enzymes,such as cellulase,pectinase,mannanase and swollenin.this study not only successfully refined the existing theory of konjac glucomannan-degradation but also play a guiding role in producing efficiency of KMOs.