【作者】 颜雅雯；

【导师】 陈清西；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 以锯缘青蟹（Scylla serrata）内表皮为材料,经过含0.2 mol/L NaCl的10 mmol/LTris-HCl（pH 7.5）缓冲液匀浆抽提处理,硫酸铵分级盐析纯化,获得粗酶。粗酶再经过SephadexG-100凝胶层析,DEAE-Cellulose离子交换柱层析,Phenyl Sepharose柱层析,及ConA Sepharose柱层析,获得比活为668.90 U/mg的聚丙烯酰胺凝胶电泳单一纯N-乙酰-β-D-氨基葡萄糖苷酶（N-Acetyl-β-D-glucosaminidase或NAGase,EC 3.2.1.52）制剂。通过Sephacryl-S 200凝胶过滤柱层析法测定该酶的分子量约为74.59 kD。该酶为糖蛋白。进一步对锯缘青蟹内表皮NAGase进行研究。化学修饰法研究表明:组氨酸的咪唑基、色氨酸的吲哚基和酸性氨基酸的侧链羧基是该酶的活性功能基团,而二硫键、赖氨酸的ε-氨基和精氨酸胍基不是该酶所必需的。分别选用水体污染物（金属离子和有机溶剂）、饲料成分（氨基酸和糖）及抗菌类药物为效应物,探讨这些物质对内表皮NAGase活力的影响。1.金属离子对酶活力的影响:结果表明Mg²⁺对酶活力几乎没有影响;Co²⁺和Mn²⁺分别在1.0-10.0 mmol/L和0.5-20.0 mmol/L浓度范围对酶有轻微的激活作用;Al³⁺对酶有一定的抑制作用,当其浓度为0.54 mmol/L时,可抑制44.4%的活力;而Pb⁺、Cu²⁺和Zn²⁺对酶具有强烈的抑制作用,其半抑制浓度(IC₅₀)分别为:0.4、0.05和0.04mmol/L。2.有机溶剂对酶活力的影响:结果表明甲醇、乙醇、丙醇、乙二醇、丙二醇、丙三醇、丙酮、甲醛、二甲亚砜、二氧六环及二甲基甲酰胺等对酶均有较强的抑制作用,其半抑制浓度(IC₅₀)分别为:3.7、2.3、1.15、2.4、2、2.3、0.80、1.15、0.68、0.88和0.8mol/L。3.氨基酸对酶活力的影响:结果表明除L-Lys具有明显的抑制作用,L-His和L-Asp具有轻微的抑制作用外,其余的几种氨基酸对该酶均无明显的影响。导致该酶活力丧失一半的L-Lys浓度(IC₅₀)为11 mmol/L。4.糖对酶活力的影响:结果表明当鼠李糖（rha）的浓度达0.2 mol/L,可激活酶活力约15%;0.8 mol/L的果糖（fru）、蔗糖（suc）、葡萄糖（glvu）和甘露糖（man）分别使酶的活力下降47.61%、57.54%、85.70%和90.50%;0.6 mol/L的阿拉伯糖（ara）可抑制酶41.62%的活力;0.09 mol/L的葡萄糖醛酸（glcUA）可使酶活力基本丧失;0.3 mol/L的半乳糖（gal）可抑制酶活力37.71%;而80 mmol/L的NAG可使酶丧失83%的活力。5.抗菌类药物对酶活力的影响:结果表明青霉素钾、卡那霉素和链霉素对酶活力基本没有影响;1.0-10.0 mg/ml的庆大霉素对酶有轻微的激活作用;而恩诺沙星对酶有强烈的抑制作用,使酶活力丧失一半的浓度(IC₅₀)为2.9 mg/ml。分别以锯缘青蟹的内表皮和肝胰脏为材料,通过匀浆、离心,获chtitnase和NAGase粗酶液。通过对不同生长期锯缘青蟹chitinase和NAGase的比较研究,结果显示不同生长期锯缘青蟹内表皮的chitinase和NAGase的比活力变化趋势基本一致:比活力在第1期最高,然后依次递减,第5期达到最低,此后维持在一稳定的水平。而肝胰脏chitinase和NAGase的比活力变化趋势虽然基本一致,但并没有一定的变化规律。内表皮和肝胰脏的NAGase最适pH都在5.6-5.8,最适温度分别为42℃和40±2℃,pH稳定性分别是5.0-10.0和5.0-9.0。温度稳定性方面的研究表明内表皮NAGase在大于45℃的条件下活力逐渐丧失,肝胰腺NAGase在大于42℃。的条件下活力开始降低。通过对不同季节锯缘青蟹chitinase和NAGase的比较研究,结果表明:锯缘青蟹内表皮chitinase和NAGase活力表现一致的季节性变化趋势,活力较高的月份集中在10-12月;一年当中,3月的活力较低,此后活力逐渐升高,直至11月,活力达到最高,然后又逐渐减小。不同季节肝胰脏chitinase和NAGase的活力变化规律与与内表皮的相似。不同季节NAGase的基本酶学性质较稳定,内表皮和肝胰腺的NAGase最适pH都在5.6-5.8,最适温度分别为42℃和40±2℃,pH稳定性分别是5.0-10.0和5.0-9.0。温度稳定性方面的研究表明内表皮NAGase在大于45℃的条件下活力逐渐丧失,肝胰腺NAGase在大于42℃。的条件下活力开始降低。更多还原

【Abstract】 N-Acetyl-β-D-glucosaminidase（NAGase,EC 3.2.1.52） was purified from epidermis of mud crab（Scylla serrata） by extraction with 0.01 mol/L Tris-HCl buffer（pH 7.5） containing 0.2 mol/L NaCl and ammonium sulfate fraction,the chromatography on Sephadex G-100, DEAE-Cellulose,Phenyl Sepharose and ConA Sepharose.The purified enzyme was a single band on polyacrylamide gel electrophoresis and the specific activity was determined to be 668.90 U/mg.The enzyme was determined to be 74.59 kD by Sephacryl S-200 gel filtration. It was glycoprotein.The enzyme（NAGase-E） was modified respectively by several chemical modification reagents,such as DL-Dithiothreitol（DTT）,bromoacetic acid（BrAc）,N-bromosuccinimide （NBS）,Azodicarboxylate（EDC）,acetic anhydride and acetyl acetone（AA） at certain condition,and the residue activity was assayed in normal reaction media.The results showed that the residues of histidine,tryptophan and carboxyl of acidic amino acids were necessary for the enzyme activity while the residues of disulfide bond,lysine and arginine were not necessary for the enzyme activity.The effects of water contamination（metal ions and organic solvent）,Feed Component （amino acid and saccharide） and antibacterial medicaments on the enzyme（NAGase-E） activity had been studied.1.The effects of several metal ions on the enzyme activity had been studied.The results show that:Mg²⁺ had no effects on the activity of the enzyme,while Co²⁺ and Mn²⁺ had a little active effect on the enzyme.Al³⁺ could inhibit the enzyme activity by 44.4%when its concentration reached to 0.54 mmol/L.Pb⁺,Cu²⁺ and Zn²⁺ can inhibit the enzyme activity with the inhibitor concentration leading to 50%of enzyme activity lost(IC₅₀) were estimated to be 0.4 mmol/L,0.05 mmol/L and 0.04 mmol/L,respectively.2.The effects of several organic solvent on the enzyme activity had been studied.The results show that:methanol,ethanol,propanol,ethylene glycol,propanediol,glycerol,acetone, formaldehyde,dimethyl sulfoxide,1,4-dioxane and N,N-dimethylformamide inhibit the enzyme activity in different degree with the inhibitor concentration leading to 50%of enzyme activity lost(IC₅₀) were estimated to be 3.7 mol/L,2.3 mol/L,1.15 mol/L,2.4 mol/L,2 mol/L, 2.3 mol/L,0.80 mol/L,1.15 mol/L,0.68 mol/L,0.88 mol/L and 0.8 mol/L.3.The effects of several amino acid on the enzyme activity had been studied.The results show that:L-His and L-Asp had just a little effect on the enzyme activity,and L-Lys could inhibit the enzyme activity,obviously.4.The effects of several saccharides on the enzyme activity had been studied.The results show that:rha could active the enzyme and enhanced the enzyme activity by 15%when its concentration reached to 0.2 mol/L.0.8 mol/L concentration of fru、suc、glu and man could obtain inhibit rate 47.61%、57.54%、85.70%and 90.50%.0.6 mol/L and 0.3 mol/L concentration of ara and gal could obtain inhibit rate 41.62%and 37.71%,respectively.0.09 mol/L glcUA could make NAGase almost loose its activity.And NAG had obvious inhibit rate 83%,when its concentration reached to 80 nmol/L.5.The effects of several antibacterial medicaments on the enzyme activity had been studied.The results show that:benzylpenicillic,kanamycin,and streptomycin had no effects on the enzyme activity.The gentamicin had just a little active effects on the enzyme,while the inhibition effect of enroflxacin was most prominent.2.9 mg/ml enroflxacin could make the enzyme loose 50%activity.The changes of the activities of NAGase and chitinase from the mud crab during different growing stages were investigated in this contribution.The results exhibited that the activities of chitinase-E and NAGase-E showed a significant rhythm,namely decreasing gradually from stagel to stage5,and then keeping in a steady level.Contrarily,the activity changes of NAGase-H and chitinase-H were found to be random.But the change of NAGase and chitinase were synchronously.The optimum pH for NAGase-E and NAGase-H were 5.6-5.8, and the optimum temperatures for them were 42℃and 40±2℃,respectively.The pH stabilities for NAGase-E and NAGase-H were 5.0-10.0 and 5.0-9.0,respectively.Besides,the temperature which NAGase-E remained steady was below 45℃,while that for NAGase-H was below 42℃.The changes of the activities of NAGase and chitinase from the mud crab during different seasons had been studied.The results show that the activities of chitinase and NAGase from epidermis and hepatopanereas showed a synchronously significant rhythm.The lower activity was present in March,then decreasing gradually until November.During different season,the optimum pH for NAGase-E and NAGase-H were 5.6-5.8,and the optimum temperatures for them were 42℃and 40±2℃,respectively.The pH stabilities for NAGase-E and NAGase-H were 5.0-10.0 and 5.0-9.0,respectively.Besides,the temperature which NAGase-E remained steady was below 45℃,while that for NAGase-H was below 42℃.更多还原