Construction、Expression Of Deoxyriboaldolases Gene Engineering Bacteria And Preliminary Application Research Of Deoxyriboaldolases

Deoxyriboaldolases(DERA) is an aldol condensation lyase, which was existed inanimals, high plant and microorganism extensively. DERA plays an important role inC-C addition reactions. Especially, it can use three molecular acetaldehyde or otheraldehyde ketone structure compouds as substrate, through two step aldol condensationreaction, synthetize the chiral side-chain of statins. Statins can reduces the cholesterollevels in the body, which occupy an important place in the cholesterol-lowering drugs.DERA, as a biological catalyst, which can avoid the chiral selection problemencountered in the synthesis of statins by chemistry method, therefore, it has potentialapplication value in chiral synthesis reaction. In this paper, an eukaryotic engineeringstrain and a prokaryote engineering strain were constructed, and used the two strainsto express DERA. Also, the characteristics and application of DERA was researchedin this peper. The main research results are as follows:Firstly, Construction and expression of DERA eukaryotic engineering strain: TheDERA gene was amplified from E.coli BL21(DE3) plysS by PCR, then thepPIC9K-DERA expression plasmid was constructed by linked the DERA gene andpPIC9K plasmid. The expression plasmid pPIC9K-DERA was cut by Sal I enzyme,the linear plasmid was transformation into Pichia pastoris competence cell by clectricshock method, a multiple-copies of DERA recombinant Pichia pastoris(GS115-pPIC9K-DERA) was constructed by G418resistance screening. DERA wasexpressed after using1%methanol to induce the recombinant Pichia pastorisGS115,the molecular weight of DERA is28.6kDa which was evaluated bySDS-PAGE electrophoresis analysis. This enzyme was named GS-DERA in the paper.Secondly, Construction and expression of DERA prokaryote engineering strain:The DERA gene was amplified from BL21by PCR, then the expression plasmidpET-DsbA-DERA was constructed by linked the DERA gene and pET-DsbA plasmid.A DERA expressed engineering strain BL21(pET-DsbA-DERA) was constructed bytransforming pET-DsbA-DERA into BL21. The recombinant strains ofBL21(pET-DsbA-DERA) was induced cultivation by IPTG, and the cultivation cells were collected by centrifugal, then brokened with ultrasonic. Through SDS-PAGEelectrophoresis analysis, it was found that DERA and DsbA were fusion expressed,and the molecular weight of the fusion protein was about50kDa (DERA：28.6kDa，DsbA：21kDa). This fusion protein was named BL-DERA in the paper.Thirdly, The separation and purification of DERA and the study of Enzymaticcharacteristics of DERA: A single GS-DERA protein was isolated from thefermentation liquor of GS115-pPIC9K–DERA strains through freeze centrifuge. Thecrude enzyme of DERA was obtained from the broken cultivation cells, and thecultivation cells were collected from the fermentation liquor of BL21(pET-DsbA-DERA) which induced by IPTG. Target protein with purity more than90%wasobtained from the DERA crude enzyme which purified by nickel column purificationmaterials. The results of enzyme activity analysis showed: the specific activity ofGS-DERA was highest(2.4U/mg) when GS115-pPIC9K–DERA strains was inducedby1%methanol for72h, and the specific activity of purified BL-DERA was2.2U/mg,which was lower than GS-DERA. The research of enzymology characteristics showed:the optimum reaction pH value of GS-DERA and BL-DERA were6-8, and theoptimum reaction system was imidazole-HCl buffer (pH7.5).The BL-DERA canretain90%activity after heating in20-60℃for30min, while, the thermal stabilityrange of GS-DERA was20-40℃.The pH stability range of GS-DERA and BL-DERA,are3-10and5-10, respectively, which showed GS-DERA has better stability thanBL-DERA in lower pH.Finally, The application research of GS-DERA and BL-DERA: The results ofacetaldehyde resistant experiments showed GS-DERA and BL-DERA both had lowerresistant to acetaldehyde. The DERAs activity decreased sharply after the DERAsexposure to300mM acetaldehyde for30min. The GS-DERA and BL-DERA retained40%and24%activity, respectively, after exposure to300mM acetaldehyde for1h.Both of the DERAs are completely inactivated after exposure to the same conditionsfor2h. At the same time, it can be also found that the GS-DERA had stronger resistantto acetaldehyde than BL-DERA within30-120min. No condensation products wereobserved after the sequential aldol condensation with acetaldehyde catalyzed by the two DERAs, which showed the GS-DERA and BL-DERA both had low catalyticefficiency for sequential condensation reaction with acetaldehyde. But the twoDERAs can catalyze glyceraldehyde-3-phosphate reacting with acetaldehyde andgenerated2-deoxyribose-5-phosphate (DRP), and the catalytic efficiency was higherwhen the concentration of acetaldehyde was100-150mM.