Gene Cloning And Expression Of GH 26 Family β-mannanase And Its Application

β-mannanase(EC 3.2.1.78), the key member of hemicellulase, can randomly cleave the internal β-1,4-glycosidic linkages of mannan backbone, which has been broadly applied in food, medicine, pulp, feed and some other industries. While commodified β-mannanases are still restricted to its poor tolerance to extreme environment and low catalytic activities, developing β-mannanases with excellent properties has become an urgent matter.Using the Aspergillus usamii E001 total RNA as template, a c DNA gene(Auman26A) encoding a β-mannanase mature peptide(abbreviated to Au Man26A) was obtained by reverse transcription PCR and nested PCR techniques. The full length of Auman26 A sequence is 954 bp, encoding 317 amino acids. Subsequently, the constructed recombinant expression vector p PIC9KM-Auman26 A was linearized with Sal Ⅰ and transformed into P. pastoris GS115 by electroporation. After induction by methanol, one transformant expressing the highest re Au Man26 A activity towards locust bean gum of 481.9 U/m L was screened by geneticin G418.The re Au Man26 A was purified by a single combination of ultrafiltration and Sephadex G-75 gel filtration. The specific activity of the purified re Au Man26 A was 2717.6 U/mg. SDS-PAGE analysed that re Au Man26 A displayed a disperse and inhomogeneous protein band with the apparent molecular weight at a range of 66.4-97.2 k Da, which was much larger than the theoretical value of re Au Man26A(36.1 k Da). The optimal temperature of re Au Man26 A was 40℃. It was highly thermostable after incubating at 80℃ and 90℃ for 60 min, and the residual activities were 53.8% and 33.1%, respectively. The optimal p H of re Au Man26 A was 5.5, and it remained stable at a range of p H 5.0-7.0. Its activities were activated or inhibited by EDTA, Fe2+ and Fe3+, and the residual activities were 100.8%, 54.7% and 39.8%, while other metal ions had no significant influence on re Au Man26 A activity. It diaplayed the relative activity towards locust bean gum, konjac flour and guar gum were 100.0%、56.4% and 28.5%, but no activities were detected towards soluble starch, birchwood xylan and CMC-Na. The Km and Vmax of re Au Man26 A towards locust bean gum were 15.25 mg/m L and 7841.9 U/mg, respectively.Bioinformatics analysis demonstrated that there were four potential N-linked glycosylation sites(Asn3-Gln4-Thr5, Asn124-Val125-Ser126, Asn134-Gly135-Thr136 and Asn305-Asp306-Thr307) in Au Man26 A, which may contribute to the increased molecular weight and thermostability of re Au Man26 A. Therefore, five deglycosylated mutant enzymes N3 E, N124 D, N134 Q, N305 Q and NA were constructed by site-directed mutagenesis, respectively. The results showed that the specific activities of N3 E, N124 D, N134 Q and N305 Q were 2082.9、2709.7、1367.0 and 2638.2 U/mg and the sugar contents were 27.4%、23.7%、19.3% and 29.2%, but no activity was detected of NA. SDS-PAGE showed that the dispersion degree and molecular weight of deglycosylated mutant enzymes, except N305 Q, were lower than those of re Au Man26 A, among which N134 Q appeared the lowest value. Hence, we speculated that Asn134-Gly135-Thr136 displayed the highest degree of glycosylation, followed by Asn3-Gln4-Thr5 and Asn124-Val125-Ser126, and no glycosylation was formed in Asn305-Asp306-Thr307. The half-life(t1/290) of N3 E, N124 D, N134 Q, N305 Q and re Au Man26 A at 90℃ were 10.5、20.0、9.0、21.5 and 20.0 min, respectively. Those results suggested that Asn3-Gln4-Thr5 and Asn134-Gly135-Thr136 played a vital role in maintaining thermostability of re Au Man26 A.Using the the hydrolysis rate of konjac powder as index, the parameters of hydrolyzing konjac powder to produce glucomannan-oligosaccharides(GMOS) with re Au Man26 A were optimized individually using the ‘one-factor-at-a-time’ method. Under the optimized reaction conditions of enzyme amount 60 U/g konjac powder, 30 g/L konjac powder concentration at 40°C for 5 h, the hydrolysis rate of konjac powder reached 53.3% and reducing sugar concentration was 10.45 mg/m L. Furthermore, the main components of the enzymatic hydrolysate analysed by HPLC were glucomannan-oligosaccharides, among which the contents of glucomannan-oligosaccharides and reducing monosaccharide were 90.17% and 9.83%, respectively. As far as I know, it’s the first report on cloning and expressing heterologously GH 26 family β-mannanase gene from A. usamii and explore the effects of glycosylation sites on β-mannanase.