Purification Of β-agarases And Structure Study Of Neoagarobiose Hydrolase AgWH117A

Agarases, which could hydrolysis agar producing oligosaccharides, are consisting of a-agarases (EC 3.2.1.158) and β-agarases (EC 3.2.1.81). The applications of agarases were extensive, such as, a tool to separate protoplasts from seaweed; recovery the DNA from agarose gel and anylasis the structure of oligosaccharides. Up to now, many researches on biological activities of oligosaccharides from agar were reported, such as inhibitory effect on the growth of bacteria, whitening effect on melanoma cells, antioxidation, prebiotic effectiveness, antivirus activity and anti-inflammation et al. In this article, the marine red alga was used to screen the microorganism which can hydrolyze agar. After optimization of fermentation, two different β-agarases were purified. As a key enzyme in agarolytic bacteria, the neoagarobiose hydrolase AgWH117A was crystallized and characterized. The main context was as follows:(1) Six strains of bacteria were selected from marine red alga. After 16S rRNA sequence test, six strains were identified as different genus, including: Pseudoalteromonas sp., Agarivorans albus., Vibrio lentus, Microbulbifer sp. The strain with the highest agarase activity was Agarivorans albus, the strain was named as Agarivorans albus OAY2. As to improve the products of agarase, the fermentation was optimized. The optimal conditions were:tryptone 0.49 g/L, yeast extract 1.08 g/L, pH 9.85. The actual enzyme activity was improved to 2.65 U/ml.(2) Two extracellular β-agarases were purified from Agarivorans albus OAY2, Agarases were purified by ammonium sulfate fractionation and ion-exchange chromatography. Purification resulted in 107-fold of agarase a and 52-fold of agaras b with high specific activity of 2715 and 1338 U/mg, respectively. Molecular mass of the agarases were estimated to be 50 kDa and 107 kDa, respectively. The optimum temperatures of agarase-a and agarase-b were 40℃ and 50℃, respectively. Agarase a was stable at 30℃, while agarase-b was stable at 50℃. The optimum pH values for agarase-a and agarase-b were all 9.0. Cu2+, Co2+, Mn2+ showed strong inhibitory effect on the purified agarase-a and agarase-b, while DTT had strong stimulation on these two agarases with 596% and 600% of initial activity. The results of 13C-NMR and TLC showed that hydrolysis of agarose by agarase-a produced neoagarobiose (NA2), neoagarotase (NA4) and neoagarohexaose (NA6). Agarase-b hydrolyzed agarose to yield neoagarobiose (NA2) and neoagarotetraose (NA4). According to the results of matrix assisted laser desorption ionization/time of flight MS (MALD1-TOF-TOF/MS), the agarase-a belonged to glycoside hydrolase (GH) 118, while agarase-b belong to GH-50.(3) After X-ray diffraction, the amino acid sequence was blasted in the database NCB1. The highest identify was 74%from a-NABH (Saccharophagus Degradans 2-40). Compared to a-NABH(Saccharophagus Degradans 2-40), neoagarobiose hydrolase AgWH 117A has the same active site, such as, Asp52-Asp207, and Glu207. In the asymmetric unit, there are two molecules and the space grond was p21."TK-SA" model was used to study which amino acid affected the AgWH117A stability. The "TK-SA" model could calculate the interaction of amino acids to evaluate the effects of amino acids on the enzyme stability. With the exception of amino acid on the active sites and loop, eleven amino acids were selected to mutant. The thermostability of mutants was better compared to the nature enzyme.(4) In this article, Agarase-a and agarase-b with a high specific activity, thermostable properties and pH stability, could meet the requirements of industrial applications. The hydrolysis products of agarose resulting from agarase-a and agarase-b included diverse NAOS:NA2, NA4 and NA6. Both of these agarases and the bacteria are worthwhile for further research.Through crystallization, the structure of neoagarobiose hydrolase AgWH117A was obtained. With the structure, mechanism could be elaborated and benefit the enzyme modify.