Expression, Purification And Characteristics Analysis Of Serine Racemase(SR) From Zea Mays

Serine racemase （SR） is a bifunctional enzyme which catalyzes not only serine（L-serine, D-serine） racemization but also dehydration of L-serine and D-serine topyruvate.The enzymatic properties of SRs have been studied in the Arabidopsis, barley andrice, while the recombinant proteins contain C-terminal histidine tag.Serine racemase coulddegrade D-serine. Zea mays had a higher tolerance on the D-serine than other plants. Nomechanism for the toxicity has yet been proposed.In this study, different labels on a varietyof recombinant maize activity of SR were analyzed. Enzymatic characterizations ofrecombinant protein were assayed. We try to use SR as a selection marker in tobacco. Themain results are as follows:1. In this work, the SR genes were cloned with different restriction sites from Zeamays. The sequencing results showed99%identity with that of a putative Zea mays serineracemase in data banks（NM₀01143028.1）, and87%,91%,94%and69%identity with aserine racemase from barley（BAF63026.1）, as well as rice （NP₀01053521.1）, sorghum（AAT42169.1），A. thaliana （XP₀02872605.1）, respectively. It has high homology withmonocot serine racemase and low homology with Arabidopsis.2. The genes with different restriction sites were directly subcloned into differentexpression veetors and were transformed into Escherchia coli BL21（DE3）. The resultsshowed that the N-terminal His-tag of His-SR，SR subcloned into the pET28-TEV, could beremoved well. The molecular weight was estimated38kDa and36kDa, respectively. Theirexpression levels were2.5mg/ml and1.93mg/ml, respectively.3. The activities of serine racemase with or without fusion tags were discussed. Theresults showed that the SR without His-tag serine racemase and dehydratase activities werehighest. His-tag at C-terminus effected the dehydratase activities markedly, while effectedthe racemase activities slightly. His-tag at N-terminus had no significantly effect on bothracemase and dehydratase activities.4. In addition, the serine dehydratase activity of SR whose His-tag was cleaved wasassayed. The optimal reaction pH and temperature of SR were7.5and37℃, respectiviely.Mn²⁺, Ca²⁺and Mg²⁺activated SR obviously. Zn²⁺inhibited SR obviously. The Kmand Vmaxvalues are12.469mM and8.425nmol/min/mg, respectively.5. The SR gene was subcloned into pCAMBIA1301, the resulting recombinantplasmid was named as p1301-SR. The recombinant plasmid was introduced into tobaccovia Agrobacterium tumefaciens mediated method. The D-serine resistant tobaceo plants were obtained and all of these plants were alive. PCR detection showed that the serineracemase gene has been integrated into their genome.In summary, the SR from Zea mays was inserted into different expression vectors forprotein expression. The effects of the His-tag label on different proteins were studied. Apreliminary analysis of SR serine dehydratase activity was conducted. We obtainedtransgenic tobacco plants by selecting with10mM D-serine.