Expression Of Penicillium Citrinum Nuclease P1 In Aspergillus Niger System

Penicillium citrinum nuclease P1?EC3.1.30.1,Nuc P1?is a nuclease specific enzyme come from nuclease S1/P1 family for cleaving single-stranded RNA and DNA into nucleotides.The producing 5'-nucleotides has been widely used in the fields of pharmaceutics,food industry and in the research related with nucleotides.In biopharmaceutics,it can be used to manufacture various biochemical drugs.In dietary supplement field,adding nuclease P1 to the yeast autolysis system increases the efficiency from RNA to 5'-GMP and 5'-AMP,which are widely used in the manufacture of various food flavoring agents.In the research of nucleic acids,it focuses on the structure of nucleic acid and its base composition.At present,the production of nuclease P1 in China falls short of demand.The purity of nuclease P1 produced by P.citrinum have high-background secreted proteins and toxin in the host affected the purifying process.The heterologous expression of nuclease P1 is mainly produced in Escherichia coli and yeast,and the production of nuclease P1 is not high enough.In this study,P.citrinum nuclease P1 was recombinantly expressed in Aspergillus niger and was used to producing nucleotides.Nuc P1 was cloned from P.citrinum and expressed in A.niger Bdel4?LB3ang02D4?with the low-background extracellular protein.We designed several expression plasmids,and selected the most efficient transformant.After purification using Ni-chelate purification,the characteristics of nuclease P1 was analysed.The main contents of this study are as follows:?1?According to NP1 protein sequence?P24289?in the Uniprot database,codon optimized Nuc P1 was synthesized after codon optimization.In order to purify nuclease P1through Ni-chelate purification,6×His tag was added to the C terminal of Nuc P1.We designed several expression cassettes using Kexlinker,2A peptide and fusion protein,to improve the enzyme activity of nuclease P1.Based on the principle of homogenous recombination,transformants were obtained using the glaA?A.niger?,18S rDNA and 28S rDNA sequence as integration sites.Recombinant transformants was screened based on the activity of nuclease P1.Finally,the transformant Bpnu-18S-2 with highest nuclease P1activity was obtained,and the activity of nuclease P1 reached 89.2 U/mL after 144 h fermentation.?2?we purified nuclease P1 from the fermentation broth.The purified protein owns a molecular mass of 38 kDa,and a protein band of approximately 30 kDa was produced after deglycosylation by PNGase F,suggesting that the molecular mass of Nuc P1 is consistent with the data in the PDB database.As for the characteristic of nuclease P1,the optimum pH and temperature of the recombinant nuclease P1 was 5.3 and 65°C.Zn²⁺,Cu²⁺and Mg²⁺with the concentration of 2 mmol/L,increased the activity and thermal stability of nuclease P1.Ni⁺decreased the activity of nuclease P1 by 20%,but increased its thermal stability.EDTA and K⁺significantly decreased activity of nuclease P1 by approximately 40%.The broth supernatant of transformant was used to improve the production of nucleotides.75 U nuclease P1 could produce 14.13 mg/mL nucleotides,which was 4.51 mg/mL higher than that without addition of nuclease P1.