J Zhu1, H Liu, J Zhang, P Wang, S Liu, G Liu, L Wu. 1. CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China.
Abstract
AIMS: The study was to examine whether glycosylation could improve the thermostability of recombinant Thermomyces lanuginosus lipase (Tll) expressed in Pichia pastoris. METHODS AND RESULTS: The Tll gene was synthesized and transformed into Pichia pastoris GS115.The recombinant Tll protein was expressed and purified, and its glycosylation site was identified by LCMS/MS as Asn-33. Two nonglycosylated mutants were constructed and the variant proteins were also expressed and purified. Effects of temperature on activities of the wild-type Tll and variants were analysed. The glycosylated Tll exhibited better thermostability than nonglycosylated variants. CONCLUSIONS: Our experiments have demonstrated the improvement of Tll thermostability by Asn-33 glycosylation. SIGNIFICANCE AND IMPACT OF THE STUDY: This work has deepened our understanding in the mechanism of Tll thermostability and will guide us to directional improvement of lipases and even other industrial enzymes.
AIMS: The study was to examine whether glycosylation could improve the thermostability of recombinant Thermomyces lanuginosus lipase (Tll) expressed in Pichia pastoris. METHODS AND RESULTS: The Tll gene was synthesized and transformed into Pichia pastoris GS115.The recombinant Tll protein was expressed and purified, and its glycosylation site was identified by LCMS/MS as Asn-33. Two nonglycosylated mutants were constructed and the variant proteins were also expressed and purified. Effects of temperature on activities of the wild-type Tll and variants were analysed. The glycosylated Tll exhibited better thermostability than nonglycosylated variants. CONCLUSIONS: Our experiments have demonstrated the improvement of Tll thermostability by Asn-33 glycosylation. SIGNIFICANCE AND IMPACT OF THE STUDY: This work has deepened our understanding in the mechanism of Tll thermostability and will guide us to directional improvement of lipases and even other industrial enzymes.
Authors: Abhijit N Shirke; Danielle Basore; Samantha Holton; An Su; Evan Baugh; Glenn L Butterfoss; George Makhatadze; Christopher Bystroff; Richard A Gross Journal: Appl Microbiol Biotechnol Date: 2016-01-13 Impact factor: 4.813