Thermostable Lipase Variants Constructed Via B-factor Saturation Mutagenesis Method

Pitch or lipophilic extractives could be efficiently removed from thermomechanical pulp using lipase A from Burkholderia sp. ZYB002 in our previous work. However, the temperature of thermomechanical pulp is nearly to 80℃ in pulp and paper industry. Consequently, it is necessary to increase the thermal tolerant of LipA using protein engineering technology. In this paper, several potential key amino acid residues responsible for thermostability of LipA were screened using the criteria of B-factor value and identified unsing site-directed mutagenesis technology. Here, we breifly introduced the methods and the following experiment results:9 3D structural data of LipA from B. cepacia deposited in the PDB database were evaluated and only one structure data (PDB file:3LIP) meets requirements for B-factor value analysis. Twenty amino acid residues responsible for thermostability of 3LIP were ranked according to B-factor value. Several corresponding amino acid residues of LipA from Burkholderia sp. ZYB002 were selected using the amino acid sequence alignment between 3LIP and LipA, including F221, V220, L218, V223 and E35.Then, series of NNK degenerate primers were synthesized and the corresponding five small mutation gene libraries were then constructed and screened, respectively. From the above five mutantion libraries, series of mutants with higher thermostability were successfully obtained, including LipA-F221D, LipA-V220E, LipA-L218E, LipA-V223D and LipA-E35P. The half-life at 55℃ of the above mutants increased by 3-fold,2.2-fold, 1.7-fold,1.5-fold and 1.8-fold, respectively.Lastly, three recombinant LipA mutants, LipA-V220P, LipA-F221P and LipA-V223S were futuer purified using Hi Trap DEAE FF anion exchange column chromatography and His TrapTM HP affinity chromatography. The relative molecular weight of the purified LipA was about 34 kDa.