초록
<P>Our previous studies demonstrated that the N-glycans in <I>Rhizopus chinensis</I> lipase (RCL) was important for its secretion. In order to improve the secretion of <I>Rhizopus oryzae</I> lipase (ROL) under the control of the <I>GAP</I> promoter in <I>Komagataella phaffii</I>, two extra N-glycosylation sites were introduced in ROL according to the position of the N-glycosylation sites of RCL by sequence alignment. The results indicated that the secretion level of ROL was strongly improved by N-glycosylation engineering, and the highest value of extracellular enzyme activity was increased from 0.4 ± 0.2 U/mL to 207 ± 6 U/mL in a shake flask. In the 7-L fermenter, the extracellular enzyme activity of the mutant (2600 ± 43 U/mL) and the total protein concentration (2.5 ± 0.2 g/L) were 218- and 6.25-fold higher than these of the parent, respectively. This study presents a strategy for constitutive recombinant expression of ROL using the <I>GAP</I> promoter combined with N-glycosylation engineering, providing a potential enzyme for application in the food industry.</P><P><B>Graphic Abstract</B><BR><IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jafcau/2017/jafcau.2017.65.issue-29/acs.jafc.7b01884/production/images/medium/jf-2017-01884z_0007.gif'></P>