Abstract:In order to explore the effects of N-terminal on acid stability of xylanase, this paper cloned and expressed a gene sequence screened from NCBI database, designed response surface test to obtain the best expression conditions, and constructed N-terminal displacement mutants from N-terminal by overlapping extension SORPCR technology, characterized its enzyme activity and related enzymatic properties, and finally applied it to apple juice to explore the clarification effect. The results showed that the optimal xylanase xynP expression condition was 1 884 U/mL by adding 1.5% seed solution into 100 mL TB medium, the induction time was 3.5 h, 200 μL of 500 mmol/L isopropyl β-D-thiogalactoside (IPTG) was added, and the enzyme activity was induced at 20 ℃ for 21.6 h. The optimum temperature of xylanase xynP and mutant xynP-1 was 40 ℃, and the optimum pH value was 4.5 and 5.0, respectively. After different pH value treatments, it was found that the stability of mutant enzyme xynP-1 was slightly weaker than that of wild type xylanase xynP under acid conditions (pH=4.0), but with the increase of pH (pH 4.5~5.5), its acid stability was all plateau enzyme xynP, indicating that N-terminal had an impact on the acid stability of xylanase. Metal ions Pb2+ and chemical reagents sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetic acid (EDTA) have a significant inhibitory effect on the activity of the two enzymes. When beechwood xylan was used as substrate, the Km values of the two enzymes were 1.72 mg/mL and 2.57 mg/mL, respectively. The vmax values were 0.93 nmol/(mg·min) and 1.15 nmol/(mg·min), respectively, indicating that the substrate affinity and catalytic efficiency of the mutant enzyme were lower than those of the original enzyme. Two enzymes were applied to apple juice. After the addition of the original enzyme xynP and the mutant enzyme xynP-1, the light transmittance of the juice increased from 24.33% to 90.03% and 95.70%, respectively, indicating that the addition of xylanase xynP had a significant clarifying effect on apple juice. The results provided a reference for further research on the application of xylanase in fruit juice processing.