Study Of Degradases For Polysaccharides From Enteromorpha Prolifera Produced By Alteromonas Sp. A321

Polysaccharides from Enteromorpha prolifera (PE) are recognized as one kind ofpolyanion polysaccharides and composed of rhamnose, xylose, glucuronic acid, andsome sulfate. Recently, PE are becoming increasingly popular due to their uniquestructural features, bioactivity, and abundant source. Degradase for polysaccharidesfrom E. prolifera (DPE) is one kinds of enzyme to degrade PE for micromoleculeoligosaccharides. The research of DPE enriches and perfects the types of seaweed toolenzymes. Thus, this thesis studies the screening of the DPE producing strains, theidentification of the strain, enzyme production characteristics of the strain,purification, and characterization of DPE.1. In this research, we used tablet screening method and rescreening method toscreen a high-yield DPE strain A321from surfaces of decayed E. prolifera. The strainwas identified as Alteromonas sp. according to the morphological characteristics and16S rDNA sequence analysis.2. The Fermentation conditions for DPE were optimized using single factorexperiments and response surface methodology design. As a result, the optimumcultivation conditions: initial pH value, medium volume, inoculum size, andtemperature were7.3,75ml/250ml,8%, and28oC, respectively. The optimizedmedia: PE0.93%, NaNO30.41%, NaCl1.03%, MgSO40.10%, and K2HPO40.40%.The maximum yield of DPE was0.744U/ml after48h under these conditions.3. The DPE produced by Alteromornas sp. A321was successively purified. Theresults showed that DPE was purified as two type of enzyme named as degradase L1and degradase L2, respectively. The degradase L1and degradase L2were purified to homogeneity level with35.78-fold and42.35-fold, respectively. The specific activityof purified degradase L1and degradase L2were19.32U/mg and22.86U/mg,respectively. The molecular weight of degradase L1and degradase L2wasapproximately76.4kDa and102.5kDa, respectively, by SDS-PAGE. The N-terminalamino acid sequence of degradase L1was determined. And the N-terminal amino acidsequence of degradase L1was A-N-P-A-A-P-G-E.4. The characteristics of the degradase L1and degradase L2were studied. Theresults showed that the purified L1exhibited the maxium degradase activity at theoptimal temperature35oC and pH7.0. The purified L2exhibited the maxiumdegradase activity at the range of temperature30-35oC and optimal pH6.0. The Zn2+,Hg2+, Cu2+, and Co2+had a strong inhibitory effect on the activity of degradase L1anddegradase L2. The Al3+had inhibitory effect on the activity of degradase L1anddegradase L2. The low concentration of SDS activated the activity of degradase L1and degradase L2, while the high concentration inhibited the acticity of both enzymes.Phenanthroline activated the activity of degradase L1but inhibited the acticity ofdegradase L2. The results of mass analysis showed that degradase L1could degradesulfate PE. Degradase L2could degrade neutral PE.The Vmaxand Kmvalues ofdegradase L1and degradase L2were8.87μmol/min/ml,46.69mg/ml and2.99μmol/min/ml,3.37mg/ml, respectively when PE was used as substrate.The enzyme has a broad application prospects in many aspects such asresearching the function and structure of PE. Besides, it is also important technologiesfor comprehensive application of green macroalga biomass. Meanwhile, the enzymeis also used for the preparation of low-molecular-weight sulfate oligosaccharides.Compared to the currently used methords such as acid hydrolysis and physicalhydrolysis, the enzymatic production for low molecular weight sulfateoligosaccharides does not require advanced laboratory equipments, and will not pollute the environment. Therefore, screening a strain of bacteria producing DPEwith high activity has important theoretical and practical significance.