Research On Agar Degradation Enzymes And Preparation Of Neoagarooligosaccharides By Enzymatic Hydrolysis

The oceans are the Earth's largest ecosystem, covering 70% of our planet and providing goods and services for the majority of the world's population, particularly the algae. The extraction of agar is currently based on the procedure of alkali treatment, water washing, acid treatment, bleaching and water washing on raw algae materials, which not only cause serious environmental pollution, but also consumes vast quantities of water resources. On the other hand, the exported agar-relevant products are mainly low value-added agar, while processed agarose and agar oligosaccharides are imported with higher prices in China. Therefore, it is essential to improve the current process of extraction by applying the enzymatic treatment to degrade the sulfates of algae and produce the high value-added agar oligosaccharides, which will help to prevent environmental pollution and to gain the maximum economic benefits, thus promoting a faster and better development of the agar industry.In this study, we isolated a sulfatase-producing bacterium S9 from the mangrove soil samples.16S rRNA analysis indicated that it belonged to genus Pseudomonas. The genome analysis suggests that the genome of Pseudomonas sp. S9 is consisted of a circular DNA. The draft genome excluding the gaps has a total of 4,796,832 bases comprising 4,665 predicted open reading frames (ORFs), with a G+C content of 56.52%. There are two rRNA operons and 56 predicted tRNA genes. In addition, the genome of Pseudomonas sp. S9 encodes amount of ABC tansporter genes and heavy metal tolerance genes. These are likely to bear systems to cope with toxic mangrove environment. According to the KEGG and COGs analysis, Pseudomonas sp. S9 is predicted that 38.3% function genes are related to the metabolism function. Moreover, it possesses the complete pathway of carbohydrate metabolism, lipid metabolism and amino acid metabolism. Additional, there are amounts of ABC transporter genes and heavy metal tolerance genes. These suggest Pseudomonas sp. S9 can cope with toxic metals in mangrove environment. In S9 genome, seven predicted ORFs are annotated as arylsulfatases, six of whose sequences are complete. All these complete coding sequences have the CX[P/A]XRX motif, a characteristic of the cysteine-type sulfatases. Using SignalP to search for the possible signal peptides in cysteine-type sulfatases, we find four of them carrying a signal peptide which is accordant with that the arylsulfatases are exited in extracellular of Pseudomonas sp. S9. It is the first report of the extracellular cysteine-type sulfatases.Meanwhile, arylsulfatase genes were cloned from strain S9 and expressed in E. coli BL21, and the recombinant enzyme was characterized to degrade the sulfate of polysaccharide from Gracilaria Lemaneiformis. That would provide a foundation for enzymatic degradation instead alkaline degradation in producing agar. Moreover, a novel alkylsulfatase gene, sdsAP, was cloned from a newly isolated bacterium Pseudomonas sp. S9 and expressed in Escherichia coli. The noticeable thermal stability makes SdsAP an ideal candidate for the application in disposing SDS-containing waste.We described the isolation and identification of a new agarolytic bacterium Agarivorans sp. LQ48 from marine algae, and the cloning and expression of a novel endo-typeβ-agarase gene, agaA, in Escherichia coli. We also explored the characteristics of the purified recombinant agarase. On account of its noticeable pH stability, the enzyme can be utilized extensively in many kinds of industries. Then a series of neoagaroligosaccharides with different degree of polymerization were prepared by the crude agarases of strain LQ48. Neoagarobiose and neoagarotetraose were purified by Sephadex G-15 and identified by TLC. Furthermore, their antioxidant activity was investigated by various antioxidant assay in vitro systems, including DPPH radical scavenging, superoxide anion radical scavenging, hydrogen radical scavenging. The results indicated that neoagarobiose and neoagarotetraose producing by enzyme exhibited the antioxidant activities.This study is the first step of improving the technique of agar production and exploring the methods of producing the neoagaroligosaccharides by agarases.