CAJ | 학술논문

为了获得分子量集中，生物活性显著的岩藻低聚糖片断，该试验从九孔鲍肝胰腺中分离纯化出岩藻聚糖裂解酶裂解岩藻聚糖。通过等电点法、硫酸铵分布盐析法及Sephadex G-150柱分离纯化出2种底物酶（岩藻聚糖酶、α-L-岩藻糖苷酶），同时采用黏度和还原糖法作为酶活的评价指标评价酶的作用特点。结果表明：等电点法中pH值4.5、5.0时的沉淀物酶活最高，且pH值4.5的沉淀物在硫酸铵质量分数为30%时分离纯化出岩藻聚糖酶，而pH值5.0的沉淀物在硫酸铵质量分数40%时分离纯化出α-L-岩藻糖苷酶。此种工艺方法能得到2种岩藻聚糖裂解酶，底物降解率为8%～15%，获得硫酸基团破坏性小的低分子量岩藻聚糖。因此，九孔鲍岩藻聚糖裂解酶可作为酶法制备低分子量岩藻聚糖的工具酶，具有重要的开发应用价值。
위료획득분자량집중，생물활성현저적암조저취당편단，해시험종구공포간이선중분리순화출암조취당렬해매렬해암조취당。통과등전점법、류산안분포염석법급Sephadex G-150주분리순화출2충저물매（암조취당매、α-L-암조당감매），동시채용점도화환원당법작위매활적평개지표평개매적작용특점。결과표명：등전점법중pH치4.5、5.0시적침정물매활최고，차pH치4.5적침정물재류산안질량분수위30%시분리순화출암조취당매，이pH치5.0적침정물재류산안질량분수40%시분리순화출α-L-암조당감매。차충공예방법능득도2충암조취당렬해매，저물강해솔위8%～15%，획득류산기단파배성소적저분자량암조취당。인차，구공포암조취당렬해매가작위매법제비저분자량암조취당적공구매，구유중요적개발응용개치。
In order to obtain fuco-oligosaccharides which possessed centralized molecular weight and remarkable bioactivity, an enzymic degradation method, due to its mild reaction conditions and no destructive effect on the structure of fucoidan, was adopted in this work. So the fucoidan lyases, derived from hepatopancreas of abalone (Haliotis diversicolor supertexta), were isolated and partially purified to hydrolyze fucoidans which were derived from Laminaria japonica. According to the electric properties of protein, an organic solvent method, an ammonium sulfate precipitation method and an isoelectric precipitation method were employed to isolate and purify the fucoidan lyases, and their purification effects were compared. First, for the isoelectric precipitation, the enzymic activity of precipitates at pH value 3.0 to 9.0 were measured with the viscometric method, aiming to shrink the isoelectric range of fucoidan lyases. The results showed that the precipitate at pH value 5.0 condition exhibited observably (p<0.05) higher enzymic activity than the other ones. It was indicated that the isoelectric point of fucoidan lyases ranged from pH value 4.0 to 6.0. Then the enzyme activity of precipitates which were obtained by the methods of isoelectric precipitation at pH value 4.5, 5.0, and 5.5, ammonium sulfate precipitation, and organic solvent, were measured with the DNS method. Finally, the results indicated that the crude fucoidan lyases had the highest enzymic activity at pH value 4.5 and 5.0, and that the enzyme activity recovery rates of the fucoidan lyases were 44.4%and 42.5%, respectively. Meanwhile, the content of sulfate groups of the hydrolyzed products (fuco-oligosaccharides) at pH value 4.0 to 6.0 were measured. The results showed that there were no significantly (P>0.05) decrease of sulfate groups for the fuco-oligosaccharides compared with that of the original fucoidans, which implied that no sulfatase existed in the crude fucoidan layses at pH value 4.5 and 5.0. Due to the better enzymic activity of the crude fucoidan layses precipitated at pH value 4.5 and 5.0, their enzymology characteristics were further discussed. The results displayed that the optimum hydrolysis reaction conditions were 2 g/mL of substrate concentration for 60 min at 38℃. Then the crude fucoidan layses were further isolated and purified with an ammonium sulfate precipitation and gel filtration chromatography. The results showed that when the saturation of ammonium sulfate reached 30% and 40%, the crude fucoidanase and α-L-fucosidase were obtained, respectively. In the end, the results indicated that the enzyme activity of fucoidanase was 0.43 U andα-L-fucosidase was 0.27 U after gel filtation. The enzyme activity recovery rate of the two fucoidan lyases were both 16%, which manifested that the enzymic activity of fucoidan lyases had a loss in the process of isolation and purification. The experimental results showed that two kinds of fucoidan lyases could effectively degrade fucoidans whose degration rates were 8%-15%, and that the sulfate groups of fucoidans were not obviously destroyed. Consequently, low molecular weight oligosaccharides (Fuco-Oligosaccharides) were produced, and the relative binding rate of sulfate groups were maintained at over 85%. Therefore, the fucoidan lyases from hepatopancreas of abalone might be used as a tool enzyme to hydrolyze high molecular weight fucoidans, and can have an important application value in the market in the future.