TY - JOUR
T1 - High level expression of a recombinant xylanase by Pichia pastoris cultured in a bioreactor with methanol as the sole carbon source
T2 - Purification and biochemical characterization of the enzyme
AU - Cayetano-Cruz, Maribel
AU - Pérez de los Santos, Ara Itzel
AU - García-Huante, Yolanda
AU - Santiago-Hernández, Alejandro
AU - Pavón-Orozco, Patricia
AU - López y López, Victor Eric
AU - Hidalgo-Lara, María Eugenia
N1 - Publisher Copyright:
© 2016 Elsevier B.V.
PY - 2016/8/15
Y1 - 2016/8/15
N2 - The xylanase gene xyn11A from Cellulomonas uda was expressed in Pichia pastoris under the control of an inducible promoter AOX1. The recombinant xylanase was named Xyn11AAOX1. The P. pastoris clone (C9) showing the highest xylanase activity was selected to evaluate the production of Xyn11AAOX1 in liquid cultures in a bioreactor. The culture was carried out by fed-batch fermentation using two strategies, one-stage method using methanol, and two-stage method using glucose and methanol as carbon sources. Interestingly, after 48 h of fermentation using one-stage method, a dry cell weight of 34 g/L and total protein concentration of 1.16 g/L were obtained, where Xyn11AAOX1 was the major enzyme secreted into the culture medium. Xyn11AAOX1 was purified from the culture supernatant of P. pastoris/pPICZαB - xyn11A and showed an estimated molecular mass of 45 kDa. The optimal temperature and pH were 50 °C and 6.5, respectively. The KM and Vmax values were 4.5 mg/mL and 5000 U/mg protein, respectively. This is the first report on cultivating P. pastoris with methanol as the sole carbon source in a minimal salt medium in which the recombinant enzyme was obtained as the major enzyme secreted into the culture supernatant within a short fermentation time.
AB - The xylanase gene xyn11A from Cellulomonas uda was expressed in Pichia pastoris under the control of an inducible promoter AOX1. The recombinant xylanase was named Xyn11AAOX1. The P. pastoris clone (C9) showing the highest xylanase activity was selected to evaluate the production of Xyn11AAOX1 in liquid cultures in a bioreactor. The culture was carried out by fed-batch fermentation using two strategies, one-stage method using methanol, and two-stage method using glucose and methanol as carbon sources. Interestingly, after 48 h of fermentation using one-stage method, a dry cell weight of 34 g/L and total protein concentration of 1.16 g/L were obtained, where Xyn11AAOX1 was the major enzyme secreted into the culture medium. Xyn11AAOX1 was purified from the culture supernatant of P. pastoris/pPICZαB - xyn11A and showed an estimated molecular mass of 45 kDa. The optimal temperature and pH were 50 °C and 6.5, respectively. The KM and Vmax values were 4.5 mg/mL and 5000 U/mg protein, respectively. This is the first report on cultivating P. pastoris with methanol as the sole carbon source in a minimal salt medium in which the recombinant enzyme was obtained as the major enzyme secreted into the culture supernatant within a short fermentation time.
KW - Enzyme production
KW - Fed-batch culture
KW - Methanol
KW - Pichia pastoris
KW - Purification
KW - Recombinant DNA
UR - http://www.scopus.com/inward/record.url?scp=84967152694&partnerID=8YFLogxK
U2 - 10.1016/j.bej.2016.04.014
DO - 10.1016/j.bej.2016.04.014
M3 - Artículo
SN - 1369-703X
VL - 112
SP - 161
EP - 169
JO - Biochemical Engineering Journal
JF - Biochemical Engineering Journal
ER -