Highly Expression Of Agarase AgaD In E.coli And Its Preliminary Study

Agar, which is extracted mainly from marine red algae (including Gelidium and Gracilaria sp.), is a mixture of heterogeneous galactans composed mainly of 3,6-anhydro-L-galactose and D-galactose units alternately linked by α-(1,3) and β-(1,4) linkages. It is a major cell-wall component in red algae and has been used in various laboratory and industrial applications, owing to its gelation properties. Agarases are the enzymes which catalyze the hydrolysis of agar and they are classified into a-agarase and (3-agarase according to the cleavage pattern. So far, most known agarases are β-agarases and have been classified into four Glycoside Hydrolase (GH) families, GH-16, GH-50, GH-86 and GH-118. There are only four α-agarases, which belong to GH-96.3-agarases cleave the β-(1,4)-galactosidic bond of the polymer to release neoagaro-oligosaccharides (NAOS, various units of the neoagarobiose,3,6-AG-a-(1,3)-Gal). On the contrary, a-agarases cleave the α-(1,3) bond to release agaro-oligosaccharides (AOS,various units of the agarobiose, Gal-(3-(1,4)-3,6-AG). The neutral agar oligosaccharides exhibit important physiological and biological activities beneficial to the health of human being and are potential prebiotics oligosaccharides.Previously, a possible a-agarase producing strain was screened in our laboratory, which was named as Thalassomonas sp.LD5 according to the phylogenetic tree. An agarasse gene agaD was cloned from Thalassomonas sp.LD5 genomic. The full length of agaD consists of a 4401 bp open reading frame, encoding 1466 amino acid residues. Sequence analysis revealed that agarase AgaD shared 89% and 77% similarity with another two a-agarases of GH-96. Unfortunately, the low yield of both wild type strain and recombinant strain have hindered the further analysis of the enzyme.The aim of this study was to construct highly efficient expression systems for agarase AgaD and optimized its fermentation conditions. First, the codon usage of AgaD was optimized and synthesized to make it suitable for expression in E.coli. Then, the gene expression vectors were transformed into different E.coli hosts. Among them AD494(DE3) showed the highest enzyme activity,460U/L fermentation medium, which indicated that the correct formation of disulfide bond play an important role in the maturation of protein. According to the "N-end rule", which relates the in vivo half-life of a protein, the N-terminal second amino acid phenylalanine was replaced by alanine and the mutant gene was named as optagaDx. It turned out that this mutation significantly improved the culture agarase activity and shortened the fermentation period by half. In the following experiment we optimized the culture conditions and the exogenous additives. The best conditions were as follows:100mL fermentation broth in 500mL shake flask,0.0004mol/L 1PTG,25℃,0.005mol/L CaCl2 and 0.1mol/L Gly. After optimization, the extracellular enzyme production raised from 20U/L to 11300U/L, which was increased by more than 500 folds and laid a good foundation on the study of AgaD.Exploring the purification method indicated that the enzyme could be precipitated by 60% (NH4)2SO4, which showed strong inhibition of enzyme activity. We then tried to subsitute (NH4)2SO4 with NaCl. Strain LD5 was isolated from marine environment but with the presence of 2mol/L of NaCl the activity of agarase AgaD dropped by 50%. While the inhibition can be restored by 0.05mol/L CaCl2. SDS-PAGE analysis indicated there are multibands on the gel and this prompt us to analyze the matured enzyme. Western-blot analysis by antibody of Flag tag and His tag indicated that recombinant enzyme was fragmented, which was further verified by in-situ gel renaturation.The final degradation product of AgaD was analyzed by partially purified recombinant enzyme and the result revealed that the final product shared the same mobility with agarotriose in TLC(Thin-layer chromatography) and FACE (Application of Fluorophore-assisted Carbohydrate Electrophoresis). Time-course hydrolysis pattern indicated that AgaD displayed an endo-type degration pattern by giving high polymer of polysaccharide at the early stage and accumulating final products as the late stage. When using neoagaro-oligosaccharides as the substrate, neoagarooctaose and neoagarodecaose can be thoroughly degraded, while neoagarohexaose was partly digested. We deduced that the catalytic cavity of AgaD can accommodate at least neoagarohexaose.In a follow-up study, we will explore the cleavage mechanism of recombinant AgaD. Furthermore, the p-galactosidase activity was found combined in the whole purification procedure and was difficult to clean up. This activity might further degrade the final products of AgaD and causes a confusion in the analysis of the reducing end. Given this, we plan to chose a expresssion host without or knockingout of P-galactosidase activity or try other purification strategies. Furthermore. we will identify the property of AgaD by nuclear magnetic resonance or mass spectrometry.