| L-aspartate α-decarboxylase(EC4.1.1.11),a key enzyme in the biosynthetic pathway of microbial pantothenate(include pantoate and beta-alanine),encoded by the panD gene and the pyruvoyl group as the active catalytical center,it has a stringent substrate specificity and can catalyze the removal of the a-carboxyl group from L-aspartic acid to generate P-alanine.The synthesis pathway of P-alanine exists only in the cell of microorganisms and plants,so the antifungal drugs,antibacterial drugs and herbicides with L-aspartate a-decarboxylase as the target are not detrimental to animals or humans.Beta-alanine has a wide range of uses,which could be used for the synthesis of medical products such as carnosine,calcium pantothenate,balsalazide and sodium pamidronate.At present,domestic β-alanine mainly produced by chemical synthesis method,which has disadvantages such as high cost and unfriendly production conditions to the environment.The development of a green and safe biological production method will have very notable economic and social benefits.However,the currently produced enzyme-producing strains have low enzyme production and low enzyme activity,and they are difficult for mass production and extended application.In this study,eighty strains with L-aspartic acid a-decarboxylase activity were isolated from the subtropical marine soil sediment samples by traditional pure culture techniques.After the second screening by paper chromatography,4 strains with relatively high enzyme activities were selected for the identification.Based on the morphological,physiological and biochemical characteristics,the 16S rRNA sequence alignment and phylogenetic analysis of the strains,two of them were identified as Acietobacter spp.,one as Providencia spp.,and one as Photobacterium spp..The Providence strain was selected as the study object,primers were designed based on the sequence of Aspartic acid decarboxylase gene(panD)from Providence bacteria in the GenBank database,and the L-aspartate a-decarboxylase(panDP)was cloned by PCR.Using pET-3 0a(+)as vector and Escherichia coli DH5a as host,recombinant bacteria were constructed.The bioinformatics analysis shows that the panDP gene contains a 381 bp long ORF encoding a polypeptide consisting of 126 amino acids with a predicted molecular mass of 13.92 kDa.The panDP gene has a 81%identity at nucleotide level with the aspartate 1-decarboxylase precursor gene(GenBank accession number:FM162591.1)from Photorhadus asymitica ATCC43949;and has a 77%identity with the aspartate decarboxylase gene(GenBank accession number:CP011104)from Photorhabdus temperata subsp.thracensis strain DSM 15199;at the amino acid level,PanDP is 98%identical to the L-aspartate a-decarboxylase(GenBank accession number:WP036958294.1)from Providencia rettgeri,but no relative report has been found on the functional characteristics.The constructed recombinant plasmid pET-30a-panDP was transformed into the expression host E.coli(DE3)plysS to construct a recombinant expression strain.The optimal conditions for the inducible expression are:a-lactose with a final concentration of 10 g/L as an inducer,30℃ induction for 10 h.The nickel column was applied for protein purification,the enzyme properties analysis shows:when the L-aspartic acid is the substrate,the optimal reaction temperature of PanDP is at 60℃,the optimum pH is 8.0,under the optimum reaction conditions,Ca2+,Al3+,and Fe3+ promoted the enzyme when their final concentration is 5 mM.Among them,the promotion effect of Al3+ is the highest,reaching 140%.However,Na+,Ba2+,Mn2+,Mg2+,Zn2+,and Cu2+ all have different degrees of inhibition.The inhibitory effect of Na+ is weak,and the inhibitory effects of Mn2+,Mg2+,and Cu2+ are distinct.The enzyme is stable at low temperature,but when exceeding 55 ℃,the stability drops along with time.However,the enzyme still has 40%of enzyme activity when stored at 80 ℃ for 1 hour.When the pH value is at 8.0 the enzyme has the best stability.In the range of pH 5.0 to 9.5,it still maintains the enzyme activity of 40%or more.In the pH range of 7.5 to 8.5,it maintains about 60%of the enzyme activity;Km is 0.01602 mmol/L,Vmax is 1.6861 μmol/min,kcat value is 0.28 s-1.Site-directed mutation of the panDP gene resulted in four mutants,K9A,K9D,S25E,and S25K;these four mutant proteins were purified and studied for their enzymatic properties.Compared to the wild recombinant PanDP,all of the enzymatic activities were lost,and thus it was found that thesites of the 9th and 25th of the amino acid sequence are related to the activity of the enzyme.This study completed the identification of the wild-type strains Providencia sp.GZl and Acinetobacter sp.GZ8 and the high yield expression and functional analysis of the panDP gene;the optimal reaction temperature of the PanDP enzyme was high,reaching 60℃,and retains high enzyme activity at 80 ℃.The property of high enzyme reaction temperature endows it with certain application and reference value in industrial production. |
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