Soluble Overexpression And Molecular Modification For Improved Thermostability Of Bacillus Acidopullulyticus Pullulanase

Pullulanase is a debranching enzyme that can degrade the α-1,6-linkages of pullulan, amylopectin, and other branched polysaccharides. In the grain industry, pullulanase is often used with glucoamylase or β-amylase to produce high glucose and maltose syrups. The reverse synthesis action of pullulanase was verified at high substrate concentration. The reversibility of hydrolysis can be used to obtain polysaccharides with specific structures and functions, which have potential applications in pharmaceutical industry. Currently, starch saccharification is typically performed at 60?C for about 48–60 h, which need the pullulanase must have the ability to maintain high activity and stability at the high temperature. Screening of thermotolerant pullulanase to reduce the use cost and improve process in starch saccharification is an important development direction.In this thesis, we successfully expressed Bacillus acidopullulyticus pullulanase gene(Pul A, Accession No. Ax203843.1) in different expression systems. The difference of expression level and secretion efficiency between these expression systems was compared to screen the suitable expression host. Various strategies had been used to obtain soluble overexpression of pullulanase in Escherichia coli. The amino acids and modules responsible for the thermostability of B. acidopullulyticus pullulanase(Pul A) were selected based on the combination of structural biology, bioinformatics and homologous sequence alignment analysis. These sites are mutated according to rational design results using site directed mutagenesis method. The differences of enzyme properties between wide-type enzyme and mutants are analyzed to select the thermostable mutants. The main research results are listed as follows:1. A combination of vectors and hosts was performed to improve the soluble expression level of Pul A. The expression of Pul A in p ET-20b(+) or p GEX4T2 had an extremely low solubility, and the protein was predominantly in IB form. The GST tag had no obvious effect on Pul A soluble expression. The soluble Pul A in p ET-22b(+) and p ET-28a(+) was 29.7 ± 0.6 U×m L-1 and 27.3 ± 0.8 U×m L-1, respectively, which was 6.8 fold and 6.2 fold that of p ET-20b(+). The Tac promoter was used to replace the T7 promoter in p ET-22b(+) to test the effect of T7 promoter on Pul A solubility. The pullulanase expression predominantly remained insoluble and in IB trapped form in p ET-22b(+)-Tac. These results suggested that the basal expression at initial stage of bacterial growth led to insoluble inclusion bodies of Pul A in p ET-20b(+) and p GEX4T2. Rosetta(DE3) was tested as a host to remedy the codon bias, while no significant improvement on the soluble expression was observed.2. An N-terminal domain truncation was adopted to facilitate Pul A variant expression and secretion. The activities of M1(ΔCBM41) and M5(ΔCBM41ΔX25) variants were 2.9-and 2.4-fold that of wild-type enzyme, respectively. The enhanced expression of soluble protein is the main reason for these improved activities. After fermentation, about 56.6 and 93.4% of the total activity of Pel B-M1 and Pel B-M5 were transferred to theperiplasm, respectively, followed by cell lysis and leakage of the partial enzyme into the extracellular medium. The effect of X25 domain on soluble expression was not obvious. X45 domain may play a role in promoting the correct conformation of the catalytic domain, and the target protein is mainly in the form of insoluble inclusion bodies after the deletion of X45. The optimal temperature and p H for purified preparations of M1, M3, and M5 were similar to those of the WT enzyme. The hydrolysis efficiency of M1, M3, and M5 on larger molecular weight substrates was decreased compared to wild-type enzyme. The deletion of CBM41 and/or X25 domain did not affect the enzyme application in starch saccharification.3. Four data driven rational design methods(BFITTER, proline theory, Po PMu Si C-2.1, and sequence consensus approach) were adopted to identify the key residue potential links with thermostability, and 39 residues of Bacillus acidopullulyticus pullulanase were chosen as mutagenesis targets. Single mutagenesis followed by combined mutagenesis resulted in the best mutant E518I-S662R-Q706 P, which exhibited an 11-fold half-life improvement at 60°C and a 9.5°C increase in Tm. The optimum temperature of the mutant increased from 60 to 65°C. Fluorescence spectroscopy results demonstrated that the tertiary structure of the mutant enzyme was more compact than that of the wild-type enzyme. Structural change analysis revealed that the increase in thermostability was most probably caused by a combination of lower stability free-energy and higher hydrophobicity of E518 I, more hydrogen bonds of S662 R, and higher rigidity of Q706 P compared with the wild-type enzyme.4. An chimeric enzyme Pul A-GTPB was obtained by replacing a module of Pul A with a new module of G. thermoleovorans pullulanase(GTPB). Chimeric mutation had no effect on cell growth and soluble expression. The specific activity of Pul A-GTPB was only 43% of the wild-type enzyme. The optimum substrate of wild-type and chimeric mutant was pullulan. They had no hydrolysis activity to the highly branched glycogen. But the affinity of Pul A-GTPB on pulluan was decreased, and the catalytic efficiency was 51% of the wild-type enzyme. The hydrolysis activity of Pul A-GTPB on dextrin and soluble starch was significantly higher than that of the wild type. The optimum p H of Pul A-GTPB increased from 5.0 to 6.5, and the stability in acid solution is lower than that of wild-type. The optimum temperature of Pul A-GTPB increased from 60 to 70°C, the residual activity increased from 55% to 95% at 60°C for 30 min, the half-life increased from 34.9 min to 168.4 min, and the Tm increase from 65°C to 72°C.