Effects of Asn-33 glycosylation on the thermostability of Thermomyces lanuginosus lipase.
OBJECTIVE
The study was to examine whether glycosylation could improve the thermostability of recombinant Thermomyces lanuginosus lipase (Tll) expressed in Pichia pastoris.


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.


CONCLUSIONS
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.
CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China.
J
J
Zhu
H
H
Liu
J
J
Zhang
P
P
Wang
S
S
Liu
G
G
Liu
L
L
Wu
D001216:Asparagine / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006031:Glycosylation; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D055550:Protein Stability; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D004718:Saccharomycetales / Q000737:chemistry* / Q000201:enzymology; D013696:Temperature
D005656:Fungal Proteins; D011994:Recombinant Proteins; D001216:Asparagine; D008049:Lipase
Thermomyces lanuginosus lipase; glycosylation; mutation; thermostability