Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 24, Issue 23, Pages -Publisher
MDPI
DOI: 10.3390/ijms242316562
Keywords
lipase; thermostability; B-factor; site-directed mutagenesis; N-terminus; C-terminus
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This study constructed five variants of lipase through site-directed mutagenesis and found that substituting residues from the 25-loop could enhance the stability of both the N-terminus and C-terminus, resulting in improved thermostability.
(1) Lipases are catalysts widely applied in industrial fields. To sustain the harsh treatments in industries, optimizing lipase activities and thermal stability is necessary to reduce production loss. (2) The thermostability of Thermomyces lanuginosus lipase (TLL) was evaluated via B-factor analysis and consensus-sequence substitutions. Five single-point variants (K24S, D27N, D27R, P29S, and A30P) with improved thermostability were constructed via site-directed mutagenesis. (3) The optimal reaction temperatures of all the five variants displayed 5 degrees C improvement compared with TLL. Four variants, except D27N, showed enhanced residual activities at 80 degrees C. The melting temperatures of three variants (D27R, P29S, and A30P) were significantly increased. The molecular dynamics simulations indicated that the 25-loop (residues 24-30) in the N-terminus of the five variants generated more hydrogen bonds with surrounding amino acids; hydrogen bond pair D254-I255 preserved in the C-terminus of the variants also contributes to the improved thermostability. Furthermore, the newly formed salt-bridge interaction (R27 & mldr;E56) in D27R was identified as a crucial determinant for thermostability. (4) Our study discovered that substituting residues from the 25-loop will enhance the stability of the N-terminus and C-terminus simultaneously, restrict the most flexible regions of TLL, and result in improved thermostability.
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