Heterologous Expression,rational Modification And Immobilization Of Oscillatoria Acuminata Chlorophyllase

Chlorophyllase is a key enzyme in the chlorophyll metabolism pathway of photosynthetic organisms,such as plants,algae and cyanobacteria.It can hydrolyze chlorophylls and produce chlorophyllides and phytol.The catalytic activity of chlorophyllase has potential industrial and pharmaceutical applications.It can remove green pigments from edible oils to improve their oxidative stability,and can partly replace the expensive adsorptive bleaching technique currently used in the edible oil refining industry.in vitro,chlorophyllides and their derivatives have been shown to have anti-oxidant,anti-viral,anti-mutagenic,and anti-carcinogenic properties.So far,many studies have been conducted on the plant and algal chlorophyllases.However,the heterologous expression of plant chlorophyllases is generally extremely low,making its extensive use in industrial applications difficult.Microbial-derived chlorophyllases are likely to be more highly heterologously expressed than higher plant and algal chlorophyllases.In this paper,Oscillatoria acuminata chlorophyllase（OaCLH）was heterologously expressed in Escherichia coli and Pichia pastoris,respectively.The molecular structure and enzymatic characteristics of recombinant OaCLH were studied.The catalytic efficiency of OaCLH was enhanced by rational site-directed mutagenesis.In addition,the fermentation process of recombinant P.pastoris for the OaCLH production and the immobilization of OaCLH were also investigated.The recombinant OaCLH was successfully expressed in E.coli BL21（DE3）.The expression of the recombinant OaCLH reached 150 mg/L in growth medium,significantly higher than that of plant chlorophyllase.The putative N-terminal 28-amino-acid signal peptide sequence of OaCLH is essential for its activity,but may confer poor solubility on OaCLH.The C-terminal fusion of a 6×His tag caused a partial loss of activity in recombinant OaCLH,but an N-terminal 6×His tag did not destroy its activity.The optimal p H and temperature for recombinant OaCLH activity are 7.0 and 40°C,respectively.Recombinant OaCLH has hydrolysis activities against chlorophyll a,chlorophyll b,bacteriochlorophyll a,and pheophytin a,but prefers chlorophyll b and chlorophyll a as substrates.Catalytic efficiency(k_cat/K_m)of the recombinant OaCLH is comparable to those of chlorophyllase 1 and chlorophyllase 2 from Brassica oleracea.The results of site-directed mutagenesis experiments indicated that the catalytic triad of OaCLH consists of Ser159,Asp226,and His258.Three single-site mutant enzymes（W160R,V228C,and D224N）were constructed by site-directed mutagenesis to improve the catalytic efficiency of OaCLH using rational design based on multiple sequence alignment and homology modeling.Trp160 was found to be important in substrate access and binding,while Val228 was found to be involved in substrate recognition.In comparison with wild-type enzyme,the mutant enzyme W160R showed a13.39-fold,15.63-fold,and 19.28-fold improvement in the catalytic efficiency for substrate chlorophyll a,chlorophyll b,and bacteriochlorophyll a,respectively.In comparison with wild-type enzyme,mutant enzyme V228C increased the catalytic efficiency by 2.98-fold for chlorophyll a,6.86-fold for chlorophyll b,and 3.45-fold for bacteriochlorophyll a.Asp224played an important role in substrate specificity.In comparison with wild-type enzyme,the mutant enzyme D224N showed a 2.13-fold improvement for chlorophyll a,a 1.48-fold improvement for bacteriochlorophyll a,but a 11%reduction for chlorophyll b in the catalytic efficiency.Mutant D224N changed its preferred substrate from chlorophyll b to chlorophyll a.According to the circular dichroism spectra,the introduced mutations had no significant effect on the overall secondary structures of three mutant enzymes.When compared to mutant W160R,double mutant W160RV228C showed a slight improvement in enzyme affinity and catalytic activity.The double mutant W160RV228C was successfully expressed in P.pastoris GS115.The apparent molecular weight of P.pastoris-OaCLH is about 42 k Da,which is significantly larger than its theoretical molecular weight（34.78 k Da）.It may be due to the glycosylation of recombinant OaCLH in P.pastoris.The result of deglycosylation experiment showed that the glycosylation of P.pastoris-OaCLH did not significantly reduce its catalytic activity.The optimal temperature and p H for P.pastoris-OaCLH activity are 40°C and 6.0-6.5,respectively.When compared with E.coli-OaCLH,the thermal stability of P.pastoris-OaCLH was significantly improved at 70°C and 80°C.The optimal temperature and initial p H for enzyme production by recombinant P.pastoris are 28°C and 5.0-5.5,respectively.After 148 hours of high-density fermentation of recombinant P.pastoris,the final activity in the fermentation supernatant was 366.70 m U/m L.The enzyme activity expression level showed 3.76-fold improvement when compared with that of shake flask fermentation.The covalent binding of LX-1000EP did not cause significant conformational changes of the enzyme,so that the immobilized OaCLH could exhibit normal catalytic activity.When compared with the free OaCLH,the immobilized OaCLH showed significant improvement on its catalytic performance.Its thermal stability was significantly improved,while the optimal reaction temperature（40°C）did not change.Its optimal reaction p H shifted from 6.0-6.5 to7.0.The catalytic performance at p H 5.0,5.5,7.5,or 8.0 was also improved.The immobilized OaCLH exhibited a broader p H tolerance.The residual activity of immobilized OaCLH reached about 80%after 20 batches of continuous reactions,which can greatly reduce the enzyme cost.