Expression Of Agarase Genes From Marine Sediments And Theirs Application In The Preparation Of Neoagarobiose

Agarase,a glycoside hydrolase(GH),can hydrolyse agarose and produce agar oligosaccharides.Agarases have applications in the food,medical industries and cosmetic for the production of oligosaccharides from agar.Agarase is used to the preparation of a series of different degree of polymerization of oligosaccharides,which is considered to be the most ideal green production technology.It is of great practical significance to screen the strains with high efficiency of producing agarase and to mine agarase with excellent enzymatic properties and preparation of agar oligosaccharides.There are abundant microbial resources in marine sediments.agarase genes are extracted from the sediments of offshore and deep-sea sources,and the neoagarobiose is prepared from Gracilaria lemaneiformis.The main research contents and results are as follows:Our group predicted a agarase enzyme gene aga M1 with amino acid sequence similarity of 58.64% from mangrove sediment microbial metagenomics.The properties of recombinant AgaM1(rAgaM1)were studied using Bacillus subtilis for extracellular expression.The optimum temperature and p H were 50 °C and 7.0.The rAgaM1 exhibited a high adaptability to wide ranges of temperature.In addition,rAgaM1 can degrade Gracilaria lemaneiformis and generate neoagarotetraose and neoagarohexaose.Flammeovirga pacifica WPAGA1 was isolated from deep sea sediment of the West Pacific Ocean in our previous study,which could directly degrade Gracilaria lemaneiformis to produce oligosaccharides.The genome of Flammeovirga pacifica WPAGA1 was sequenced,agarose-related genes were identified and analyzed.This paper focuses on the analysis of the agarase gene aga2660,the only GH50 family in the strain WPAGA1.The agarase gene aga2660 was cloned and transformed into E.coli BL21(DE3)for expression and purification.The results showed that recombinant Aga2660(Recombiant Aga2660,r Aga2660),unlike other GH50 family agarases,can degrade the low degree of polymerization of the neoagaro-oligosaccharides into neoagarobiose,but not the macromolecule agar.The optimum temperature of r Aga2660 was 30 °C and exhibited high thermal stability.The above results indicated that the agarase had great potential in industrial production of neoagarobiose.The agarase rAgaM1 and r Aga2660 were applied to prepare neoagarobiose by using Gracilaria lemaneiformis as the substrate,and cytotoxicity test and whitening efficacy evaluation of neoagarobiose.The results showed that the safety of the neoagarobiose on the cells was much higher than that of the neoagarotetraose and neoagarohexaose,and the whitening effect of the neoagarobiose was better than that of the neoagarotetraose and neoagarohexaose.It shows that the technology of rAgaM1 and r Aga2660 has good application value.