基因工程α-半乳糖苷酶的制备及其性质研究

Fermentation and Purification of Recombinant α-Galactosidase from Pichia pastoris

在获得可分泌表达α 半乳糖苷酶基因工程毕赤酵母菌株的基础上 ,尝试了基因工程α 半乳糖苷酶在 5L发酵罐中的表达以及从发酵液中纯化α 半乳糖苷酶的研究。在 4L无机盐培养基中接种 0 .4LpPIC9K Gal GS115培养物 ,最终得到 3 .5L发酵液。离心所得上清中总蛋白含量为 2 .1g L。根据发酵液中目的蛋白含量高、杂质少等特点 ,设计了如下的纯化流程 :离心→超滤→阳离子交换层析→脱盐→浓缩。纯化后电泳银染结果呈单一蛋白带 ,总回收率 41%。通过测定米氏常数等生化性质对重组酶进行鉴定后 ,完成了人B型红细胞的酶解实验。结果表明 ,从发酵液中纯化的α 半乳糖苷酶可将B型红细胞改造成O型红细胞。本研究同时在数量和质量上为α 半乳糖苷酶在众多领域的广泛应用奠定了基础。

In order to obtain an adequate supply of α-galactosidase for research and practical use, the fermentation, purification and identification of the recombinant coffee bean α-galactosidase were carried out. Baffled flasks containing 100mL BMGY were inoculated with the pPIC9K-Gal/GS115 strain and allowed to grow at 30℃, 250～300r/min until a maximum optical density at 600nm (OD 600) between 2.0 to 6.0 was attained. Entire 400mL seed culture was transferred aseptically to the 5-liter fermenter, which contained 4 liter sterilized basal salts medium and 4% glycerol. The batch culture grew at 30℃, pH 5.0 until the glycerol was completely consumed, and a glycerol feed was initiated to increase the cell biomass prior to induction with methanol. The culture was centrifuged at 8000×g and the supernatant was collected. Following ultrafiltration, the retentate was balanced in 20mmol/L sodium formicate buffer, pH 3.8 and loaded onto a cation-exchange column, HiTrap SP. The column was washed with the same buffer and bound proteins were eluted with 1mol/L NaCl. The fractions containing recombinant α-galactosidase were pooled and concentrated with PEG20 000. Subsequently, the biochemical properties of the enzyme were determined with typical methods. At last, the fresh human blood A and B erythrocytes were incubated with the purified α-galactosidase at 26℃ for 2～4 hours. Hemagglutinins were assayed by the standard method. After an elapsed fermentation times (EFT) of 18h, the fed-batch phase was initiated to increase the cell biomass. A cellular yield of nearly 200 g/liter wet cells was achieved when induction was initiated. 72h later, the α-galactosidase activity against artificial substrate PNPG (PNP-α-galactopyranoside) achieved 36 000u per liter culture. The crude fementation supernatant contained few impurities as detected by SDS-PAGE. The supernatant was purified by cation-exchange chromatography, the target α-galactosidase was eluted with 40% 1mol/L NaCl and showed a 41kD band on SDS-PAGE. After concentration, the final recovery was about 41%. The Michaelis constant of the recombinant α-galactosidase was determined as 0.275 mmol/L, which slightly lower than the nature enzyme and suggested a higher affinity with specific substrate. When human blood type B erythrocytes pretreated with 100u/mL recombinant α-galactosidase reacted with bood type B antiserum, no hemagglutination occurred. This suggested that the B antigens had been removed by the enzyme successfully. These results demonstrated that the recombinant α-galactosidase could be produced in large-scale and made it possible to explore the application of α-galactosidase in more fields.