Nhân dòng gen, biểu hiện và tinh sạch xylanase tái tổ hợp từ chủng aspergillus niger dsm 1957 trong pichia pastoris gs115

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Tác giả: Thị Tuyên Đỗ, Đình Thi Quyền

Ngôn ngữ: vie

Ký hiệu phân loại: 589.23 [Unassigned]

Thông tin xuất bản: Công nghệ Sinh học, 2013

Mô tả vật lý: 285-291

Bộ sưu tập: Metadata

ID: 559251

A gene coding an endo-Beta-1,4-xylanase (XlnA) from Aspergillus niger DSM 1957 was cloned and sequenced. The cDNA sequence of 978 bps encodes for a putative endoxylanase of 326 aa with a predicted molecular mass of 36 kDa. The cDNA was overexpressed in Pichia pastoris GS 115 under the control of an AOX1 promoter. Conditions were first optimized to offer the highest production yield of the recombinant protein. A series of methanol induction concentrations (0.5 to 2,5 percent) was tested. The induction by 0.5 percent of methanol showed the highest production level of the recombinant xylanase. The xylanase activity reached to 21.5 V/ml culture supernatant, after 144 h of cell growth in YP medium induced with 0.5 percent of methanol. The culture supernatants were collected and used for enzyme activity assay. The recombinant xylanase was purified from the culture supernatant by the affinity chromatography with ProBond resin. The molecular mass of the purified XlnA, determined by SDS-PAGE, was 36 kDa, with the specific activity of 246.9 V/mg toward 0.5 percent xylan. The recombinant XlnA (rXlnA) was stable over a temperature range of 25-40°C. The activity of rXlnA was declined to 66 percent activity when it was heated at 50°C for 240 min. Residual activities of about 45 percent was observed after prolonged heating at 60°C for 60 min. However, the rXlnA enzyme heated at 80°C for only 2 min showed no activity. The rXlnA was stable and highly specific toward xylan and is expected to show high potential for downstream biotechnological applications.
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