Efficient Expression And Stabilization Of Bacillus Subtilis Laccase

Laccase（EC 1.10.3.2）is a multi-copper oxidase that can oxidize various substrates,including phenols,non-phenols,and inorganic compounds.Laccase has been widely used in food processing,papermaking,textile,and bioremediation.In order to improve the fermentation production level and application effect of laccase,the secretory expression of Bacillus subtilis laccase（Cot A）in Escherichia coli was enhanced by ribosomal binding site（RBS）replacement,rational design of N-terminal coding region,co-expression of cleavage protein,and optimization of culture conditions,and its thermal stability was improved based on rational design.The main results are as follows:（1）Intracellular expression of Cot A in E.coli.The Cot A gene synthesized based on E.coli codon bias was cloned into p ET24a-Cot A and transformed into E.coli BL21（DE3）,yielding the recombinant strain Cot A-RBS.The induction was conducted by adding isopropyl beta-D-thiogalacyranoside（IPTG）at 20℃for 28 h,and the intracellular Cot A activity of Cot A-RBS reached 448.96 U·L^-1.RBS calculator and RBS library calculator designed five RBSs with different translation initiation rates（7695.43-882376.36）.The recombinant strain Cot A-RBS5carrying RBS5（the lowest translation initiation rate）produced the highest intracellular enzyme activity(1198.12 U·L^-1),1.67 times higher than before optimization.Based on the statistical model characterizing the relationship between the N-terminal coding region sequence parameters and expression level,the coding sequence of the first ten residues of Cot A was optimized,yielding the recombinant strain Cot A-RBS5-N1.Its intracellular enzyme activity reached 1632.3 U·L^-1,1.36 times higher than before optimization.Finally,by optimizing IPTG addition,Cu²⁺concentration,cell concentration before induction,and inducing temperature,the intracellular Cot A reached 7132.99 U·L^-1,3.37 times higher than before optimization.（2）Extracellular expression of Cot A in E.coli.It was shown that the extracellular enzyme activity of Cot A-RBS5-N1 reached 2616.31 U·L^-1 at 24 h under the above optimal inducing expression condition.Because Cot A was fused with secretory signal peptides and the cell grew slowly,the recombinant strain of Cot A-RBS5-N1 may be cleaved in the late stage of the fermentation.In order to improve the extracellular expression level of Cot A,the effects of sorbitol,glycine,Triton X-100,and Tween-80 on the extracellular synthesis of Cot A were investigated.The results showed that the addition of 10 g·L^-1 sorbitol could increase the extracellular enzyme activity of Cot A-RBS5-N1 to 5540.83 U·L^-1 after 20 h induction,1.12times higher than that before optimization.Based on this condition,the plasmid p CDF-Duet-E encoding cleavage protein E was transformed into Cot A-RBS5-N1 to obtain the recombinant strain Cot A-RBS5-N1-E,and the addition conditions of protein E inducer arabinose were optimized.When the OD₆₀₀ of Cot A-RBS5-N1-E arrived at 6,the activity of extracellular Cot A reached 6488.75 U·L^-1 by adding 2 mmol·L^-1 arabinose.（3）Molecular modification for enhanced thermal stability of Cot A.Based on the previously Rosetta Cartesian＿ddg script by our group,we scanned the proline of Cot A and analyzed the expression and thermal stability of the 15 mutants with the highest decrease in folding free energy.The results showed that the residual enzyme activity of mutant G321P increased from 73%（wild-type Cot A）to 85%after 30 min of water bath at 80℃.Meanwhile,the specific activity of G321P was 51.64 U·mg^-1,which was not significantly lower than that of Cot A(53.96 U·mg^-1).Based on Rosetta supercharge,24 sites on the surface of Cot A molecule were virtually mutated（replacing surface polar or basic amino acid residues with acidic amino acids）,and the static charge on the surface of Cot A reached-46.The mutants with high folding energy were removed by Rosetta Cartesian＿ddg analysis,and a combined mutant Cot A-Mut containing 9-point mutations was designed.Thermal stability analysis showed that the residual activity of Cot A-Mut treated at 80℃for 30 min was increased to 91%,but its specific activity was 3.22 U·mg^-1.Molecular dynamics analysis showed that the increases in hydrogen bond and ion interaction might account for the enhanced thermostability of G321P and Cot A-Mut,respectively.