Abstract:

【Objective】 In order to exploit novel disease-resistance gene and biological pesticide sources, antifungal protein secreted by Bacillus subtilis B111 was purified and identified, then its gene was cloned and expressed in Pichia pastoris. 【Method】Antifungal protein was purified by ultrafiltration, 90% ammonium sulfate precipitation and QAE Sephadex A50 ion exchange chromatogram column, then it was identified via CapLC-ESI-MS-MS. Its gene encoding antifungal protein was cloned. The recombinant expression vector pPIC9K-PT was constructed and transformed into P. astoris GS115. The transformants were identified by PCR analysis．The products of the transformants induced by methanol were identified by neutral protease and antifungal activity assay. 【Result】 An antifungal protein BS2 was purified and identified to be a neutral protease from B. subtilis. Antifungal gene BS2 with pro-sequence was cloned. The recombinant expression vector pPIC9K-PT was constructed and BS2 gene was expressed in P. pastoris. The recombinant BS2 protein exhibited a molecular mass of approximately 69 kD and presented neutral protease activity of 27 800 U?g-1 and antifungal activity. Its production reached up to as high as 29.77 mg?L-1. 【Conclusion】 In this study, an antifungal neutral protein BS2 was purified and identified, then its gene was cloned and successfully expressed in P. pastoris.

Key words: Bacillus subtilis, neutral protease, antifungal activity, Pichia pastoris expression