Cloning And Expression Of The Agarase Gene From Marine Microorganisms

Agarase is a kind of glycoside hydrolase,which can catalyse the hydrolysis of agar into agar oligosaccharides.It has been recognized that agar oligosaccharides have potentially wide applications in food,drugs,cosmetics and other fields because of their activities.Agarase strain BY and BN were isolated from Marine Microorganisms by transparent circle method.Strain BY was identified as Vibrio sp.and strain BN was identified as Microbulbifer sp.based on its morphological observation and phylogenetic analysis of 16S rRNA sequence.Research on the enzymatic properties of agarase,we learned that the optimum temperature and pH of strain BY were 50?and 6.0;the optimum temperature and pH of strain BN were 40? and 6.0.Touch down PCR and chromosome walking techniques were used to obtain agarase Vibrio sp.BY full-length gene sequence,the full-length gene was 2232 bp,encoding 744 amino acids,theory molecular was 85.4 kD,recombinant vector pET22a-Vibrio sp.BY was succeed expression in E.coli BL21(DE3).After induced with IPTG,the recombinant agarase reached 71.73 U/mL.The optimum pH and temperature of recombinant agarase were 7.0 and 50?,respectively.Furthermore,this agarase exhibited remarkable stability at pH between 5.0-7.0 and temperature between 30-50 ?.Na+?K+?Mg2+?Ca2+ had a slightly positive effect on the activity of the recombinant agarase.But some heavy metal ions,such as Zn2+?Fre2+?Mn2+ had an apparent negative effect on the recombinant agarase activity.In addition,SDS?EDTA inhibited the recombinant agarase activity.Agarase gene N3 was cloned from strain BN by PCR amplified,the full-length of gene N3 was 924 bp,encoding 308 amino acids,theory molecular was 34.3 kD,engineering strain pET28a-N3 was built,and was succeed expression in E.coli BL21(DE3),the recombinant agarase reached 228.3 U/mL which was 3.04 folds of the original strain BN.The optimal induction conditions were as follows:sonication 15 min,inducing time 16 h,induction at 30?,IPTG to a final concentration of 1 mM,which was 1.35 times before the optimal.Then we used affinity chromatography to purified recombinant protein,the purified recombinant agarase was analyzed by SDS-PAGE and the result showed a single band,the specific activity was 700.59 U/mg.The optimum pH and temperature of recombinant agarase were 6.0 and 50?,respectively.And it has a good heat stability and pH stability.The recombinant agarase was stimulated by Na+?K+?Mg2+,while Ca2+,Zn2+,Fe2+,Mn2+,SDS and EDTA inhibited the enzyme activity.Determined from Lineweaver-Burk plots,the kinetic parameters Km and Vmax for the enzyme activity on agar were 13.41 mg/mL and 10.82 umoL/min/mL.Finally,the enzyme hydrolyzed agar and asparagus dish to generate disaccharides and tetrasaccharides as the main products.