High expression and characterization of N-acyl-homoserine lactonase-xylanase recombinant protein

Abstract:

Abstract: ［Objective］ The fusion protein SL2B with both xylanase and N-acyl-homoserine lactonase activities was expressed in Pichia pastori． Characterization of the purified xylanase and N-acyl-homoserine lactonase fusion protein SL2B was investigated．［Methods］ The fusion gene sl2b was amplified from the N-acyl-homoserine lactonase gene aiiA-B546 and the xylanase gene xynAS27cd via overlap PCR technique． After the recombinant vector pPIC9/sl2b was transformed into P． pastoris，transformants with both xylanase and N-acyl-homoserine lactonase activity were screened． The purified SL2B was obtained with ammonium sulfate precipitation and molecular sieve． Both N-acyl-homoserine lactonase and xylanase activities of SL2B were characterized． ［Results］ The purified SL2B showed that the xylanase had optimal pH and temperature at pH 6. 5 and 60℃，respectively． The enzyme was stable between pH 6. 0 and 8.0，retained over 80% enzyme activity between 50 and 65℃．It resisted various neutral proteases and chemical reagents． With oat spelt xylan as substrate，the Km value of SL2B was 2. 9 mg /L．The N-acyl-homoserine lactonase had optimal pH and temperature at pH 8.0 and 30℃，respectively．The enzyme was stable between pH 4. 0 and 10.0，retained over 80% enzyme activity between 0 and 50℃．It resisted various neutral proteases and chemical reagents． The fusion protein can hydrolyze many Nacyl homoserine lactones substrates． With N-(3-oxo-octanoyl)-L-homoserine lactone as substrate，the Km value of SL2B was 0.050 mmol/L．Conclusion］ High level expression is achieved by fusing N-acyl-homoserine lactonase to the xylanase．