Research On Optimization Of Fermentation Conditions, Heterologous Expression And Enzymatic Properties Of Agarase

Agarases are the enzymes with the ability of catalyzing the hydrolysis of agar.Various agarases have been isolated from different genera of bacteria found in seawater,marine sediments and engineered microorganisms. According to the different cleavagepatterns, agarase are classified into α-agarase (E.C.3.2.1.158) and β-agarase (E.C.3.2.1.81). In food industry, cosmetics, as well as medical fields agarases have wideapplications because the oligosaccharides they produced have remarkable activities.Also, agarase are used as a tool enzyme for physiological, biological, and cytologicalstudies.Using agarose as the only carbon source, we isolated6marine bacterium with theefficient agar degradation ability from the fresh seaweed samples of Qingdao coast. Theextra-cellular enzymatic activity of them was determined by DNS method afterinclubated at the proper fermentation conditions. The16SrDNA was cloned formolecular identify and some of the physiological characteristics were also studied. Fromthe original results, the determination of genus of OAG04bacterium needs further study.The OAG07bacterium may belong to the same genus with the marine bacterium strainAgarivorans gilvus WH0801saved by our laboratry.The marine bacterium strain Agarivorans gilvus WH0801was isolated from thefresh seaweed samples of Weihai coast with the efficient agar degradation ability by Duet. This paper studied on the optimal fermentation condition of Agarivorans gilvusWH0801and the enzymatic properties of agarases from it. First, it was determined thatthe optimal time for the determination of the activity of agarase was48h. Then theeffects of medium composition and culture conditions on agarase production byAgarivorans gilvus WH0801were investigated in shake flasks.The most importantnutritional components and culture conditions influencing agarase production wereselected by Plackett-Burman design.Among all the factors studied, agar, yeast extractand seed age had significant effect on agarase production.The optimum levels of thesevariables were further determined using Box-Behnken design. The highest agaraseproduction was obtained in the medium consisting of2.50g/L agar and0.88g/L yeastextract when the seed age was25.64h. The levels of other factors are1g/L peptone,0.01g/L ironic citrate at initial pH7.0and the culture temperature28oC. The wholeoptimization strategy resulted in the activity of agarase reached1.158U/mL, which was about6.2-fold increase compared with that using the original medium and also had agreat advantage compared to reported. In addition, enzymatic properties of Agarasesfrom WH0801were also studied. Effects of temperature and pH on enzymatic reactionwere evaluated by one-factor-at-a-time design.45oC,1%concentration of substrateand pH7.6were found to be most suitable for agarase production.The recombinant E.coli BL21-pETAg2was supplied by East China University ofScience and Technology. It was obtined by transforming the sequence of agar gene ofAgarivorans gilvus WH0801into E. coli BL21. We studied on the optimal mediumcomposition and culture conditions of recombinant E.coli BL21-pETAg2byone-factor-at-a-time design in shake flasks. A clear Zone was observed around thecolony of the E. coli BL21-pETAg2which certificated that the pET24a expressionvectors were successfully constructed. The ultimate result was the efficient expressionof recombinant agarase.