| Echinocandin antifungal drugs is an important kind of semisynthetic antifungal antibiotics. To date, micafungin, anidulafungin and caspofungin have been available for clinical use. Echinocandin B deacylase from Actinoplanes utahensis NRRL12052can catalyze the cleavage of the linoleoyl group of FR901379, a key step in the process of manufacturing micafungin. Echinocandin B deacylase in industry have been relying on fermentation of original producer, Actinoplanes utahensis, which have low yield and activity. The maximum of substrate concentration that can use was only1-2g/L. It can not meet the needs of large-scale industrial production. So it is urgent to construct recombinant strain of ECB deacylase to establish a basis for industrial production of micafungin.The aim of our study is to build a genetic engineering strain that can express the ECB deacylase efficiently. We get the gene of the echinocandin B deacylase from Actinoplanes utahensis and connect with expression vector PNW-S1. And the ECB deacylase was systematically overexpressed in the heterologous hosts Streptomyces lividans TK24. Results show that the engineering strain can produce10-fold enzyme more than Actinoplanes utahensis under the best conditions for industrialized production and the enzyme activity can reach125U/L. In contrast to the biotransformation of A. utahensis, recombinant deacylase is secreted into the culture broth, more suitable for a large scale production of FR901379nucleus. We also carry out mid-scale fermentation. The highest enzyme activity by300L fermentation tank can reach80U/L. With optimization of the reaction conditions with15g/liter FR901379, the molar conversion can achieve100%. And with ammonium sulfate and immobilized study, obviously simplify the process of biocatalysis and improve the utilization efficiency of the enzyme. The construction of recombinant echinocandin B deacylase producing strain will be must to improve the current production process of echinocandin antifungal drugs... |
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