DNA shuffling is a wildly used strategy of directed evolution.It can quickly test the effects of multiple mutations, however, the actual beneficial mutations among them and their structural basis are often unclear.β-Glucosidase is the rate-limiting enzyme for efficient degradation of cellulose, and good thermostability is a key factor affecting its practical catalytic efficiency.In this work, the beneficial mutations of a thermostable β-glucosidase mutant Bgl3-6511 (containing 60 single mutations and has a T50 value 4.6 ℃ higher than wild-type) were identified through sequence alignment, site-directed mutagenesis, and thermostability assay.Results showed that six single point mutations, Y50F, R56K, V65I, T67A, and P143A, increased the T50 of the enzyme by 2.9, 4.2, 1.5, 2.8, 3.2, and 1.2 ℃, respectively.At the same time, 5 single mutations decreased T50 by 1.0-3.4 ℃ were identified.A combination of beneficial mutations gave rise to mutant M6 (Y50F/R52H/R56K/V65I/T67A/P143A) with a T50 increase of 13.4 ℃, indicating that the harmful mutations in 6511 indeed hampered the thermostability optimization.Structural analysis and molecular dynamic simulations showed that the beneficial mutations mainly increased/enhanced hydrogen bonds and Π-Π interactions, and made the local loops more rigid.In summary, this work identified the beneficial mutations in Bgl3-6511, analyzed their structural basis, and obtained a more thermostable mutant M6.These results may be useful for the protein engineering of other enzymes.
XU Benhong
,
JIANG Yiwen
,
LUO Jingshi
,
YANG Xiangpeng
,
LI Guang
,
YUAN Shan
,
LIU Yuhuan
,
CAO Lichuang
. Identification and structural analysis of beneficial mutations for thermostability of β-glucosidase Bgl3[J]. Food and Fermentation Industries, 2025
, 51(15)
: 1
-8
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DOI: 10.13995/j.cnki.11-1802/ts.040962
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