Homologous Expression Of Monascus Acid Protease Gene And Optimization Of Liquid Fermentation Conditions

Monascus as one kind of important filamentous fungi which is widely used in the industry. Currently, productions of Monascus pigment, Monacolin K and ?-aminobutyric acid have been widely investigated. As for genetic transformation, the filamentous fungi such as Aspergillus niger and Aspergillus oryzae are deeply investigated rather than Monacus. Acid protease is one kind of important products producted by Monascus.It's also known as aspartic proteases which is a commonly used proteolytic enzymes. The hydrolysis of proteinscan be enhanced to increase the contents ofamino acids by rising the acid protease content in production,thus,the generation of esters in the process of making wine can be facilitated. However, the production cost of acid protease was too high to be used in industry.In this work, acid proteases of Monascus was investigated. First, the gene sequence of the Asp was obtained by PCR.Then the bioinformation characteristics was analyzed. The result reveals that the whole sequence of Asp contented 1188 bp and molecular weight of protease predition was 41.4Ku. The protein was hydrophilic with a coiled-coil region and without ligand. The protein without transmembrane helix had signal peptide and the splice site likely was between 20 and 21 points. The secreted protein dould not cause signal transduction of Monascus. The protein had 9 phosphorylation sites of serine kinase,one phosphorylation site of threonine kinase and 4 phosphorylation sites of tyrosine kinase. The Asp sequence with two function sites matched with that of acid protease family pattern and kept the same evolution rate with Aspergillus ruber. Meanwhile environment conditions plays an important role in expression of Asp and the optimal conditions included 80% water, 23?,respectively,the maximal activity of Asp was obtained at pH6-7.In this work, Asp recombinant vector was constructed and transformed into Monascus strain using Agrobacterium tumefaciens-mediated transformation. The results show that the constructed plasmid pBc-Hygro could realize the homologous expression of Monascus gene and could keep stability. At the same time, high expression of the target gene was achieved with mediation of the promoter Neurospora crassa cpc-1. The expression level of Asp in transformants was 3.30 times that of the wild Monascus.Thus, the genetic of combination of Monascus by grobacterium tumefaciens-mediated was carried out using Monascus spore as recipient.Furthermore,the culture medium was optimized by single factor testing.The testing result shows that the enzyme activity of acid protease was up to 68U/mL at malt sugar 13 g, yeast extract 10.4g,CuCl2·2H2O 0.04g?KCl 0.018g?CaCl2 0.022 g in 100 mL water.The result of response surface analysis reveals that the highest enzyme activity, 72.345U/mL,was obtained at the culture temperature of 30.9?,pH6, strring rate of 244 rpm under liquid fermentation condition.With the optimized culture medium and fermentation conditions the growth of Monascuss biomass became stable after the 8th day of inoculation, while the enzyme activity of acid protease trended to decrease 7th day of inoculation.