DNA Shuffling Of Thermoascus Aurantiacus Endo-β-glucanase I Gene And The Study Of Enzymic Properties

Cellulose is the most abundant and widely distrbuted renewable biopolymers on earth. the transformation of cellulose has very important significance for the solution of environmental pollution and energy crisis problems. Cellulose can be hydrolyzed by Cellulase to produce glucose, and it is a pollution free and efficient use of cellulose approach. But cellulase had some drawbacks such as high cost, low activity and thermal instability. so we will improve and obtain new performance of cellulase by directed evolution.In this study, the Thermoascus aurantiacus Endo-β-glucanase I Gene was mutated to obtain a strain with high CMCase activity and stability via DNA shuffling. The main research results are listed below:1. The method is established by optimizing the time of DNaseⅠdigestion and the quantity of DNA template of primerless PCR and primer PCR, which adapted to DNA shuffling of Thermoascus aurantiacus Endo-β-glucanase I Gene.2. The wide-type gene of Endo-β-glucanaseⅠ(egⅠ) was amplified by PCR from plamid pMD18-T- egⅠ. About 400 clones were obtained by DNA shuffling . 20 clones were selected to sequence and analysis randomly. The mutation rate was 0.11%~0.43%.3. After digestion with SnaBⅠ/NotⅠ, the mutational egⅠwas inserted into the expression vector pPIC9K digested with the same restriction endonucleases to construct the recombinant plasmid pPIC9K-egⅠcontaining different mutation. Linearized pPIC9K-egⅠwas transformed into Pichia pastoris GS115 with the method of LiCl. Transformants were screened and identified by MD plates. By measuring enzyme activity of the fermentation supernatant, a strain named BY2 with high activity was elected. The activity was up to 23.75 U/mL , which is 10 fold higher activity than that of wild type .4. To obtained high-copy strain, the mutant genomic was extracted. The mutant gene was amplified with primers EG-S and EG-A . We founded that the mutant was the mutant gene which number is 13. Then the mutant gene and pPIC9K were Ligaged and transformed into P.pastoris GS115 with the method of electroporation. Through activity screen,a mutant named BY213 with improved activity was obtained. The highest activity of Endo-β-glucanaseⅠand the protein content were up to 78.63U/mL and 1.14 mg/mL at incubation time of 168 h in Basic Salt Medium. The molecular size of the expression product was estimated by SDS-PAGE to be 34.1 KDa.5. The enzyme properties of the strain BY213 was researched. The optimal temperature and pH value of the enzyme activity was 65℃and 3.5, respectively. The enzyme activity can remained over 90% when the pH was 2.5~4.0 and above 85% after preincubation at 55oC for 30 min. But The enzyme of recombinant protein was un stable, When the enzyme was preincubated over a pH range of 2.5 to 8.5 at normal temperature for 24 h, only 40%~65% activity was remained.