Heterologous Expression Of Hemicellulases From Aspergillus Niger BE-2 And Its Application

Enzymatic hydrolysis of hemicelluloses-the main composition of lignocelluloses,to fermentable monosacchrides(xylose and arabinose)or high-value added products(xylooligosaccharides and ferulic acid)is of great significance for bio-refineryof lignocelluloses.The degradation of hemicelluloses completely needs the main-chain enzymes(xylanases and xylosidase)and side-chain enzymes(arabinosidase,acetyl xylan esterase,glucuronidase,and feruloyl esterase)to work synergistically due to its complex structure.Nowadays,the cost for production of hemicellulases is very high and its synergistic mechanism among various hemicellulases has not been illuminated completely,which imposes restrictions on the conversion of hemicelluloses with high efficiency.In this study,the genes that encoding acetyl xylan esterase(CE1 families,AnAxe),feruloyl esterase A(CE1 families,AnFaeA),and xylanase(GH11 families,AnXyn11 A)have been cloned from Aniger BE-2 and expressed in P.pastoris with the purpose of establishing the complete hemicellulases system,thus the cost of hemicellulase will be decreased.Then,the complete hemicellulases system was to be used in hydrolyzing bamboo and wheat bran synergistically for illustrate the mechanism of synergistic action of hemicellulases.As a result,the efficient conversion of hemicelluloses will be realized.The main results are as follows:(1)The gene encoding AnAxe was cloned from A.niger BE-2 by PCR.The full length of AnAxe was 912 bp,coded a polypeptide with 304 amino acids.The recombinant expression vector(pPIC9K-AnAxe)was constructed and electro-transformed into P.pastoris GS115.After the induction of 7 days with 1%methanol,the enzymatic activity of AnAxe toward pNPA was 87 IU/mL.The properties of AnAxe and the synergistic action among these hemicelluases have been characterized and studied.The optimum temperature and pH of AnAxe was 40? and 7,respectively.There still remained high residual enzymatic activity below 50?.Metal ions and EDTA has limited influence on AnAxe.(2)When AnXyn11A worked alone,the maximal release of reducing sugars was 11.2%,but when adding AnAxe(5 IU),the reducing sugars released from bamboo was 19.6%,which showed that the remove of acetyl groups could promote the xylan hydrolysis rate of xylanase.On the basis of the addictive of AnXyn11A and AnAxe,the AnXln3D,AnAxh62A and AnGus67 have been used simultaneously for studying the synergy degree(SD)among them.Results exhibited that,the addictive of AnXln3D,AnAxh62A and AnGus67 could improve the yields of reducing sugars,and the SD was 1.30,1.18,1.15,respectively.(3)The gene encoding AnFaeA has been cloned from A.niger BE-2.Its full length was 903 bp and coded a polypeptide with 301 amino acids.The amino acids sequence was uploaded to NCBI Blast for homologous alignment analysis,which showed that the similarity of AnFaeA and other feruloyl esterases(from A.niger CBS513.88 and A.usamii)was 99%and 98%respectively.The 3-D structure of AnFaeA protein was predicted to be ?-? hydrolase by Swiss-Model,and Ser154-Asp215-His268was projected to be catalytic residues after the alignment with other feruloyl esterase.The recombinant vector(pPIC9K-AnFaeA)was constructed and electo-transfomed to P.pastoris GS115.After the culture of 7 days and induced by 1%methanol,the enzymatic activity toward MFA was 21 IU/mL.The optimum pH and temperature of AnFaeA was 5.0 and 45?,the AnFaeA was thermostable when the temperature was below 55 ?.(4)The full length of AnXynllA was 696 bp encoding a polypeptide with 232 amino acids.Swiss-Model was used to predict the 3-D structure of AnXyn11A,which showed that the structure of AnXyn11A was similar to most xylanase from GH11 families,that was classical hand structure with 2 ? sheets and 1 a helix.The plasmid pPIC9 was used as the vector for the construction of pPIC9-AnXyn11A,then transformed into P.pastoris GS115 and the positive transformants were screened and cultured for 7days at the induction of 1%methanol.The enzymatic activity of AnXyn11A was 240 IU/mL.The optimum pH and temperature were 5.0 and 60?,respectively,and was stable below 50?.(5)The synergistic action among AnFaeA and AnXyn11A in degrading wheat bran for high value-added products was studied.The release of ferulic acid increased to 70%when AnFaeA and AnXyn11A worked synergistically,compared to AnFaeA worked along(only 16.8%ferulic acid released),the result showed that there was significant synergistic effect between AnXyn11A and AnFaeA.In the same time,when AnFaeA(100 IU)and AnXyn11A(300 IU)worked together,the XOS released from wheat bran was twice than AnXyn11A(300IU)worked alone.In summary,the efficient expression of AnAxe,AnFaeA and AnXyn11A was conducted in this study.Moreover,the synergy interaction of AnAxe with other hemicellulases in degrading bamboo was performed and exhibited significant results.Finally,high efficiency coproduction of ferulic acid and XOS was realized in the cooperation of AnFaeA and AnXyn11A.