Establishment Of High-throughput Screening Based On Microtiter Plate And Application In Pullulanase Directed Evolution

Pullulanase transfrom amylopectin into amylose by hydrolysis of α-1,6-linkages, greatly improving the utilization of starch and applications in the food industry. This research focused on the high-throughput screening technology and studied the base parameters of MTP. Based on microtiter plate, a high-throughput culture method and an efficient high-throughput detection method for pullulanase activity were established. The mutation library of pullulanase was screening with HTS established efficiently and a mutant strain was achieved. The main results are as follows:1. The property of thermostability, prevention of contamination, gas permeability, evaporation of PP, PTFE and e PTFE membrane were tested. Membrane of PTFE and e PTFE is more suitable for the sandwich cover. We also tested the property of kLa, and feasibility parallelism of MTP. The kLa measured for 24-well MTP was similar to shake flask, and higher than that of 48-well. However, the growth state of E.coli in MTP is superior to shake flask. The researchers should choose appropriate MTP and culture volume according to the aerobic nature of microorganisms. The kLa of 96-well MTP is poor and not suitable for aerobic microorganisms.2. The pullulanase gene(Gen Bank Accession No: AX203843) was heterologous expression in E.coli BL21(DE3) and fermentation conditions in shake flask was studied. Optimized culture conditions were as follow: TB/SB medium, initial OD595 0.025, 5.5 h after inoculation induced with 0.1 mmol·L-1 IPTG, 0.16 mol·L-1 glycine, and 0.1 mol·L-1 Na Cl at 20 ?C. After 20 h induction, enzyme activity in the supernatant was 5.1 U·m L-1.3. The culture condition was scale-down to MTP successfully and a high-throughput culture method based on microtiter plate was established. An efficient high-throughput detection method for pullulanase activity were established as well. The high-throughput screening method for pullulanase was established by coupling of high-throughput culture method and high-throughput detection method.4. Random mutagenesis on pullulanase gene was performed through error-prone PCR strategy. The error-prone PCR products were recombinated in the expression vector p ET-28a(+)-pel B and then introducted into BL21(DE3) to construct mutant library. The high-throughput screening method for pullulanase in this study was used to screen the mutant library efficiently. The optimum mutant 4A4 was screened and the pullulanase activity in supernatant was improved by 1.46 folds. This method not only is also applicable to high-throughput screening of amylase and cellulase, but also provides a new way for high-throughput screening of strain library and directed evolution of proteins.5. The recombinant pullulanase was purified by using Beaver Beads? His-tag Protein Purification. The optimal temperature and p H activity of the purified recombinant pullulanase were 60 ?C and 5.0, in accordance with the wild type.The results of this study provides theoretical basis for heterologous expression and secretion of pullulanase. The high-throughput screening method for pullulanase of the mutant library presents guidance for library screening.