| Feruloyl esterase and xylanase are important enzymes for the complete degradation of hemicellulose. They can used as green biological catalyst in many fields, such as in animal feed, energy, food and pharmaceutical industries. However, high temperatrue is commonly encountered or needed in industry, which greatly limits the application of many mesophilic enzymes. Thus, the modification of mesophilic enzymes becomes meaningful.In this paper, the effects of native disulfide bridges in AuFaeA, a feruloyl esterase A from Aspergillus usamii E001, on its structure and function were studied. The thermostability modification of AuFaeA was implemented by introducing an extra disulfide bridge rationally and directed evolution. The full-length cDNA and complete DNA sequences of a novel glucoside hydrolase(GH) family 10 xylanase from Aspergillus oryzae CICC40186(AoXyn10B) were cloned, and its gene Aoxyn10 B was expressed in Pichia pastoris GS115. In addition, AoXyn10 B and a modified feruloyl esterase AuFaeAA126C-N152 C with higher thermostability were coexpressed in P. pastoris GS115, and the synergistic action between them was studied preliminarily. The main research results are listed as follows:(1) Three native disulfide bridges in AuFaeA were separately eliminated, producing three variants AuFaeAC29 T, AuFaeAC91 T and AuFaeAC234 T. Then, the AuFaeA and these three variants were expressed in P. pastoris GS115, respectively. The expressed recombinant AuFaeA(reAuFaeA), reAuFaeAC29 T, reAuFaeAC91 T and reAuFaeAC234 T were with the specific activities of 50.2, 10.5, 5.3 and 6.1 U·mg-1. The temperature optimum of reAuFaeA was 45℃, and its half-lives at 50, 55 and 60℃ were 50, 15 and 8 min, respectively. The temperature optima of reAuFaeAC29 T, reAuFaeAC91 T and reAuFaeAC234 T were separately 20, 20 and 10℃ lower than that of reAuFaeA. Additionally, the thermostabilities of these three variants were decreased significantly. The half-lives of them at their temperature optima were only 10, 7 and 9 min, respectively. The catalytic efficiency(kcat/Km) values of these variants towards substrate pNPF were 283, 143 and 153 mmol·L-1·min-1, respectively, which were greatly lower than that(2235 mmol·L-1·min-1) of reAuFaeA. All the results indicated that the native disulfide bridges in AuFaeA played important roles in its structure and function.(2) Based on the prediction of MODIP and DbD softwares, an extra disulfide bridge C126-C152 was introduced into AuFaeA. The designed variant AuFaeAA126C-N152 C was expressed in P. pastoris GS115 with a specific activity of 59.7 U·mg-1, which was slightly higher than that(50.2 U·mg-1) of reAuFaeA. The temperature optimum of reAuFaeAA126C-N152 C was 51℃, 6℃ higher than reAuFaeA. Its half-lives at 55 and 60℃ were 188 and 40 min, which were 12.5- and 5-folds longer than those(15 and 8 min) of reAuFaeA, respectively. In addition, it’s worth mentioning that the kcat/Km of reAuFaeAA126C-N152 C was still maintained.(3) According to the B-factor, ΔΔG values and structural analysis, four amino acid positions Ser33, Asn92, Glu203 and Asp245 in AuFaeA were selected for iterative saturated mutagenesis(ISM). The saturated mutant libraries were constructed using P. pastoris expression system. After screening, 6, 9, 2 and 8 thermostable variants were screened out, respectively, for each position. Among the Ser33 variants, S33 E was the best one with the temperature optimum of 49℃. Its half-lives at 50 and 55℃ were 82 and 18 min. Its Tm value was 41.7℃. Among the Asn92 variants, S33E/N92 R was the best one with the temperature optimum of 51℃. Its half-lives at 50 and 55℃ were 198 and 35 min. Its Tm value was 44.5℃. Among the Glu203 variants, S33E/N92R/E203 T was the best one with the half-life of 25 min at 55 ℃. Among the Asp245 variants, S33E/N92R/D245 E was the best one with the temperature optimum of 49℃. Its half-lives at 50 and 55℃ were 90 and 31 min. The majority of variants were not much thermostable as expected by ISM method, but the best one S33E/N92 R was obviously improved in its thermostability than AuFaeA.(4) The full-length cDNA(1689 bp) and complete DNA(2038 bp) sequences of a gene Aoxyn10 B were cloned from the total RNA and genome DNA of A. oryzae CICC40186, respectively. The amino acid sequence of AoXyn10 B precursor consists of 21-aa signal peptide and a 452-aa AoXyn10 B that contains three parts: a 317-aa catalytic domain(CD), a 38-aa family 1 carbohydrate-binding module(CBM1) and a 97-aa linker between CD and CBM1. The modeled three dimentional(3D) structure of AoXyn10 B consists principally of the(β/α)8 barrel fold. Two conserved glutamic acid(Glu131 and Glu238) are located in the hydrophobic cleft of AoXyn10 B. The AoXyn10 B was expressed in P. pastoris GS115 with the specific activity of 577 U·mg-1. The reAoXyn10 B displayed the maximum activity at pH 5.5 and 60℃. It was stable at a pH range of 4.0~7.0, and at 50℃ or below. Its Km and Vmax values, towards birchwood xylan, were determined to be 1.7 mg·mL-1 and 817 μmol·min-1·mg-1, respectively. Its activity was not affected by an array of metal ions or EDTA, but was inhibited by Mn2+ and Ba2+.(5) The recombinant expression plasmid pPICZαA-AufaeAA126C-N152 C was constructed, and then linearized, followed by transforming it into the engineered P. pastoris containing Aoxyn10 B. Therefore, the AuFaeAA126C-N152 C and AoXyn10 B were coexpressed. After preliminary extraction, purification and freeze drying, the activities of reAuFaeAA126C-N152 C and reAoXyn10 B were up to 11.59 and 46.4 U·mg-1, respectively. When the reAuFaeAA126C-N152 C and reAoXyn10 B were synergistically acted on the destarched wheat bran(DSWB) at 50℃, the released ferulic acid(FA) amount reaches the maximum, which was 8.06-fold more than that released by reAuFaeAA126C-N152 C alone. However, the reAuFaeA and reAoXyn10 B were at 45℃, and their synergistic released FA amount was 8.64-fold more than that released by reAuFaeA alone. Futhermore, the FA amount released by reAuFaeAA126C-N152 C and reAoXyn10 B, reacting towards DSWB at 50℃ for 9 h, was up to the maximum 1.35 mg, which was 1.12-fold more than that(1.20 mg) by reAu FaeA and reAoXyn10 B at 45℃ for 10 h. The results demonstrated that the synergistic effect between thermostable feruloyl esterase and xylanase was better than that between thermolabile ones. The thermostable feruloyl esterase has a promising application prospect. |
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