A Novel Strategy for Thermostability Improvement of Trypsin Based on N-Glycosylation within the ��-Loop Region

Abstract

The Ω-loop is a nonregular and flexible structure that plays an important role in molecular recognition, protein folding, and thermostability. In the present study, molecular dynamics simulation was carried out to assess the molecular stability and flexibility profile of the porcine trypsin structures. Two Ω-Loops (fragment 57-67 and fragment 78-91) were confirmed to represent the flexible region. Subsequently, glycosylation site-directed mutations (A73S, N84S, and R104S) were introduced within the Ω-loop region and its wing chain based on its potential N-glycosylation sites (Asn-Xaa-Ser/Thr consensus sequences) and structure information to improve the thermostability of trypsin. The result demonstrated that the halflife of the N84S mutant at 50°C increased by 177.89 min when compared with that of the wildtype enzyme. Furthermore, the significant increase in the thermal stability of the N84S mutant has also been proven by an increase in the Tm values determined by circular dichroism. Additionally, the optimum temperatures of the wild-type enzyme and the N84S mutant were 75°C and 80°C, respectively. In conclusion, we obtained the thermostability-improved enzyme N84S mutant, and the strategy used to design this mutant based on its structural information and N-linked glycosylation modification could be applied to engineer other enzymes to meet the needs of the biotechnological industry.

Thermal stabilization of the deglycating enzyme Amadoriase I by rational design

“…Enhanced thermal stability is generally an essential requirement for those enzymes that are used as biocatalysts in industrial processes occurring at high temperatures. To this end, an enzyme can be modified by enhancing glycosylation 46 , 47 , or by introducing disulfide bridges 48 , salt-bridges 49 , or hydrogen bonds 50 . In particular, the introduction of disulfide bonds provide considerable stability to proteins by locking their fold in a well-defined local or global conformation 51 , 52 .…”

Enhancing the Thermal Resistance of a Novel Acidobacteria-Derived Phytase by Engineering of Disulfide Bridges

“…Therefore, the resistance of phytase to high temperature is a necessary property. To improve its thermal resistance, an enzyme could be modified by enhancing glycosylation [16,44,47], or adding disulfide bridge [35,38], salt-bridges [21,49], or hydrogen bonds [44,45,49,50], as well as other rational design strategies [3,41,46]. Directed evolution of enzymes is also utilized [22,36,37,51].…”

Enhanced trypsin thermostability in Pichia pastoris through truncating the flexible region

“…In recent years, a number of effective approaches, such as enzyme immobilization, protein engineering, and use of cosolvents, have been successfully utilized to improve the thermostability of trypsin [ 5 – 7 ]. For example, Pazhang et al reported that polyols (sorbitol and glycerol) stabilized trypsin by decreasing the polypeptide chain fluctuations.…”