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Li Y.-Y.,Guangdong VTR Bio Technology Co. | Li Y.-Y.,Guangdong Feed Additive Research and Development Center | Zhong K.-X.,Guangdong VTR Bio Technology Co. | Zhong K.-X.,Guangdong Feed Additive Research and Development Center | And 8 more authors.
Protein Expression and Purification | Year: 2015

A gene encoding xylanase 2 mutant from Trichoderma reesei (T2C/T28C, named mxyn2) was cloned into the Pichia pastoris X33 strain using the vector pPICZαA. Recombinant Mxyn2p was functionally expressed in P. pastoris X33 and secreted into the supernatant. Real time qPCR demonstrated that an increase in gene copy number correlated with higher levels of expression. Supernatant from methanol induced cells was concentrated by ultrafiltration with a 10 kDa cut off membrane, and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. Recombinant Mxyn2p protein had the highest activity at 75 °C, while recombinant protein encoded by the "wild type" xylanase gene xyn2, also expressed in Pichia, was 20 °C lower. The Mxyn2p enzyme retained more than 70% of its activity after incubation at 80 °C for 10 min. The effects of the optimal pH and temperature for higher expression levels in P. pastoris were also determined, 6.0 and 22 °C, respectively. The maximum xylanase activity of Mxyn2p was 13,000 nkat/mg (9.88 g/l) in fed-batch cultivation after 168 h induction with methanol in a 50 l bioreactor. © 2014 Elsevier Inc. Source


Wang J.-R.,Guangdong VTR Bio Technology Co. | Wang J.-R.,Guangdong Feed Additive Research and Development Center | Li Y.-Y.,Guangdong VTR Bio Technology Co. | Li Y.-Y.,Guangdong Feed Additive Research and Development Center | And 8 more authors.
International Journal of Molecular Sciences | Year: 2014

A gene encoding Rhizopus oryzae lipase containing prosequence (ProROL) was cloned into the pPICZαA and electrotransformed into the Pichia pastoris X-33 strain. The lipase was functionally expressed and secreted in Pichia pastoris with a molecular weight of 35 kDa. The maximum lipase activity of recombinant lipase (rProROL) was 21,000 U/mL, which was obtained in a fed-batch cultivation after 168 h induction with methanol in a 50-L bioreactor. After fermentation, the supernatant was concentrated by ultrafiltration with a 10 kDa cut off membrane and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. The optimum pH and temperature of the rProROL were pH 9.0 and 40 °C, respectively. The lipase was stable from pH 4.0 to 9.0 and from 25 to 55 °C. The enzyme activity was enhanced by Ca2+ and inhibited by Hg2+ and Ag+. The lipase showed high activity toward triglyceride-Tripalmitin (C16:0) and triglyceride-Trilaurin (C12:0). © 2013 by the authors; licensee MDPI, Basel, Switzerland. Source

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