Study On Molecular Mechanism Of The Degradation Of ?-selenocarrageenan By Deep-sea Microbial Enzymes

Deep-sea microorganisms have evolved a variety of biological enzyme systems under multiple extreme living environments.The development and utilization of microbial resources in deep sea is conducive to the mining of new enzyme resources.In this paper,the bacteria which can degrade?-selenocarrageenan were isolated from cold spring sediments for the first time and the gene encoding?-selenocarrageenase was heterologously expressed by genetic engineering technology.Besides,the structure and function of?-selenocarrageenan oligosaccharides were studied.The above work provides technical support for large-scale green production of?-selenocarrageenan oligosaccharides by enzymatic method,and lays a foundation for the wide application of?-selenocarrageenan oligosaccharides.The specific results are as follows:(1)Three strains of bacteria capable of degrading?-selenocarrageenan were screened from cold spring sediments in the South China Sea.The extracellular enzyme activity was compared by DNS method,and Bacillus sp.N1-1 had the highest activity,which reached 30.12 U/mg.(2)The whole genome sequencing analysis showed that the circular genome size of Bacillus sp.N1-1was 4,497,340 bp,and 4,683 coding genes were predicted.The functional annotation of GO,KEGG,CAZy and other databases was completed.The results of the annotated Carbohydrate Active Enzymes(CAZy)database showed that the Bacillus sp.N1-1genome contained 138 genes encoding active carbohydrate enzymes.(3)Through screening coding genes and annotation results,candidate gene of?-selenocarrageenase was obtained,which belongs to glycoside hydrolases 16(GH16)family with an ORF length of 2133 bp.The gene sequence without the signal peptide was heterologously expressed and its function was verified.It was found that supernatant of the engineering strain which was successfully expressed had function of?-selenocarrageenan degradation.The homology between this gene and the currently known?-carrageenase genes is only 27.04%-28.12%,indicating that this sequence is novel.(4)After optimization,the induced expression conditions were as follows:0.7 m M IPTG was added at 20?for induction.The recombinant?-selenocarrageenase was purified through Ni-agarose affinity and its enzymatic properties were studied.The experiment showed that 40?was the best temperature for the recombinase reaction,and 7 was the best p H for the reaction in buffer.Besides,Cu²⁺could make the recombinant enzyme lose 80%of its activity.The kinetic constant of the enzyme was 0.2389 mg/m L,indicating that the enzyme had strong affinity with substrate.(5)It was found that?-selenocarrageenan oligosaccharides with polymerization degree of 2–4 were mainly produced after enzymatic hydrolysis of?-selenocarrageenan.The results of infrared spectrum showed that the infrared absorption curve of oligosaccharides obtained was basically consistent with that of original?-selenocarrageenan,indicating that the basic skeleton of?-selenocarrageenan was not greatly affected.(6)The purified?-selenocarrageenan oligosaccharides were tested in tumor cells,showing?-selenocarrageenan oligosaccharides can inhibit the proliferation of K562 cells and HUVEC cells to a certain extent.At the concentration of 12.5?g/m L,the inhibition rate of?-selenocarrageenan oligosaccharides on the proliferation of K562 cells was significantly higher than that of?-selenocarrageenan(P<0.001).At concentration of25?g/m L(P<0.01)and 50?g/m L(P<0.05),the inhibition rate of?-selenocarrageenan oligosaccharides on the proliferation of HUVEC cells was significantly higher than that of?-selenocarrageenan.(7)The application of?-selenocarrageenan in sea cucumber culture was studied.The sea cucumber diet was supplemented with?-selenocarrageenan at selenium(Se)levels of 2.0?g/g.Then the results of antioxidant test showed that the activity of GSH-Px(glutathione peroxidase)and T-AOC(total antioxidant capacity)in sea cucumbers was significantly increased(P<0.05),and the content of malondialdehyde(MDA)decreased significantly(P<0.001).According to the results of intestinal microbial diversity,dietary?-selenocarrageenan significantly increased the diversity of intestinal flora of Apostichopus japonicus,and the abundance of beneficial bacteria such as Pseudophaeobacter,Tropicibacter and Sulfobacter also increased.The above results showed that the addition of?-selenocarrageenan was beneficial to the healthy cultivation of sea cucumber,while the most suitable amount of?-selenocarrageenan should be further studied.In this study,a new type of?-selenocarrageenase was obtained from deep-sea microorganisms,which not only enriched the enzyme resources from deep-sea microorganisms,but also provided scientific basis for large-scale preparation of?-selenocarrageenan oligosaccharides and its application in medicine and aquaculture.