Shahid Bahonar University of Kerman and Iranian Biotechnology Society Agricultural Biotechnology Journal 2228-6705 12 4 2021 01 20 Cloning and expression of recombinant xylanase enzyme (xynA) in E. coli. Cloning and expression of recombinant xylanase enzyme (xynA) in E. coli. 1 21 2774 10.22103/jab.2020.16372.1255 FA Amin Sadeghi Alikelayeh Department of Biotechnology, Faculty of Agriculture, University of Shahrekord, Chaharmahal and baktyari province, IRAN Neda Mirakhorli Assistant Professor, Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Shahrekord University, Shahrekord, Iran Mohammad Reza Akbari Assistant Professor, Department of Animal and Poultry Science, Faculty of Agriculture, Shahrekord University, Shahrekord, Iran Behzad Shareghi Brojeni Professor, Department of Biochemistry, Faculty of Basic Science, Shahrekord University, Shahrekord, Iran. Journal Article 2020 09 19 Objective Today, global increasing in corn prices has led to the replacement of wheat instead of corn in poultry diets. The NSPs in the cell wall prevents the uptake of wheat nutrients by poultry. Using xylanase is one of the methods to remove NSPs. Xylanases can digest xylan. The aim of current investigation was to produce xylanase (xynA) gene as a supplement of poultry diets in E. coli. Materials and methods In present study, the thermostable xylanase (xynA) gene in a bacterial secretory expression system along with: Shine-Dalgarno sequence, Usp45 signal peptide and T7 promoter was cloned in plasmid pET-22b (+) from E. coli strain BL21 (DE3) using The heat shock method. IPTG were used at different times to induce the expression of recombinant xylanase gene. In addition, catalytic indicators were done according to the DNS method. Results PCR colony confirmed the presence of 743 bp recombinant xylanase gene sequence on 1% agarose gel. SDS-PAGE showed protein with 27 kDa molecular weight. Also, the activity of recombinant protein was 189.47 (unit/ml) using DNS method at optimum temperature of 65 °C and pH=6. As well as, michaelis-menten curve determined Km and Vmax 4.1 (mg/ml) and 87 (unit/ml), respectively. Conclusions Xylanase are the most important complementary enzymes in poultry diets that are added to wheat-based diets. Results showed that produced recombinant xylanase is thermostable enzyme with acceptable activity and concentration. Furthermore, because of the secretory expression system, it is produced at a lower cost. Objective Today, global increasing in corn prices has led to the replacement of wheat instead of corn in poultry diets. The NSPs in the cell wall prevents the uptake of wheat nutrients by poultry. Using xylanase is one of the methods to remove NSPs. Xylanases can digest xylan. The aim of current investigation was to produce xylanase (xynA) gene as a supplement of poultry diets in E. coli. Materials and methods In present study, the thermostable xylanase (xynA) gene in a bacterial secretory expression system along with: Shine-Dalgarno sequence, Usp45 signal peptide and T7 promoter was cloned in plasmid pET-22b (+) from E. coli strain BL21 (DE3) using The heat shock method. IPTG were used at different times to induce the expression of recombinant xylanase gene. In addition, catalytic indicators were done according to the DNS method. Results PCR colony confirmed the presence of 743 bp recombinant xylanase gene sequence on 1% agarose gel. SDS-PAGE showed protein with 27 kDa molecular weight. Also, the activity of recombinant protein was 189.47 (unit/ml) using DNS method at optimum temperature of 65 °C and pH=6. As well as, michaelis-menten curve determined Km and Vmax 4.1 (mg/ml) and 87 (unit/ml), respectively. Conclusions Xylanase are the most important complementary enzymes in poultry diets that are added to wheat-based diets. Results showed that produced recombinant xylanase is thermostable enzyme with acceptable activity and concentration. Furthermore, because of the secretory expression system, it is produced at a lower cost.

https://jab.uk.ac.ir/article_2774_989bbdd1904e85e3aae1007ca01218e8.pdf