Jiang Fei, Kongsaeree Puapong, Charron Rose, Lajoie Curtis, Xu Haowen, Scott Gary, Kelly Christine
Cell Genesys, Inc., South San Francisco, California, USA.
Biotechnol Bioeng. 2008 Feb 15;99(3):540-9. doi: 10.1002/bit.21590.
A method for the production and concentration of the lignin-degrading enzyme, manganese peroxidase (rMnP), was developed using the yeast Pichia pastoris in high cell density, fed-batch cultivations. A gene encoding manganese peroxidase (mnp1) from the white-rot fungus Phanerochaete chrysosporium was cloned into a protease deficient (pep4-) strain of the methylotrophic yeast P. pastoris. Heme is an important cofactor for active rMnP production, and amendment of yeast cultures with heme increased active rMnP concentrations. In both shake-flasks and fed-batch bioreactors, the relationship between heme concentration and rMnP activity was logarithmic, with increasing heme concentrations resulting in progressively lesser increases in enzyme activity. Scale-up from shake-flasks to 2 L fed-batch cultivations increased rMnP activities from 200 U/L to 2,500 U/L, with addition of 0.1 g/L heme (added heme per liquid volume) at the beginning of the fed-batch phase resulting in higher enzyme activities than addition at the beginning of the batch phase. A combination of centrifugation, acetone precipitation, dialysis, and freeze drying was found to be effective for concentrating the rMnP from 2,500 U/L in the P. pastoris bioreactor culture to 30,000 U/L in 0.1 M potassium phosphate buffer pH 6. The rMnP recovery yield was 60% and the purity was 1-4%. By using 0.1 g/L heme during the fed-batch cultivation, the heme content of the final enzyme preparation could be reduced by 97%, and had sufficiently high rMnP activity and low enough color to be suitable for pulp bleaching experiments.
一种利用巴斯德毕赤酵母进行高细胞密度补料分批培养来生产和浓缩木质素降解酶——锰过氧化物酶(rMnP)的方法得以开发。将来自白腐真菌黄孢原毛平革菌的编码锰过氧化物酶(mnp1)的基因克隆到甲基营养型酵母巴斯德毕赤酵母的蛋白酶缺陷型(pep4-)菌株中。血红素是活性rMnP产生的重要辅因子,用血红素对酵母培养物进行补充可提高活性rMnP的浓度。在摇瓶和补料分批生物反应器中,血红素浓度与rMnP活性之间的关系呈对数关系,随着血红素浓度的增加,酶活性的增加逐渐减少。从摇瓶放大到2 L补料分批培养,rMnP活性从200 U/L提高到2500 U/L,在补料分批阶段开始时添加0.1 g/L血红素(每液体体积添加的血红素)比在分批阶段开始时添加导致更高的酶活性。发现离心、丙酮沉淀、透析和冷冻干燥相结合对于将rMnP从巴斯德毕赤酵母生物反应器培养物中的2500 U/L浓缩到pH 6的0.1 M磷酸钾缓冲液中的30000 U/L是有效的。rMnP的回收率为60%,纯度为1 - 4%。通过在补料分批培养期间使用0.1 g/L血红素,最终酶制剂的血红素含量可降低97%,并且具有足够高的rMnP活性和足够低的颜色,适合用于纸浆漂白实验。