柑桔属生物类黄酮酶法结构修饰的研究
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摘要
对柑桔深加工及综合利用和类黄酮研究的现状进行较详细的综述；同时对橙皮苷及其衍生产品的性质、制备、分离、结构改性方面国内外的研究现状作了介绍。
通过初步建立筛选模型，对20来份土样和实验室保藏的菌种进行大量筛选，选育出了一株有较高选择性的橙皮苷酶活性黑曲霉菌株D_7(1)，初始酶活力为544.9u／ml。其最佳发酵培养基：麸皮8.0％、豆饼粉2.0％、KH_2PO_40.1％，每30mL培养基中加5mL0.1％的橙皮苷；最佳产酶条件：250mL的三角瓶中装量30mL，初始pH为6.0，以4层纱布包扎，在250r／min，30℃的摇床中培养4天；水相最佳酶反应条件：最佳pH4.0、反应温度为55℃。对该菌株进行了初步的紫外诱变处理，得到一株变株D_7(1)-1，橙皮苷酶活提高了19％，达到1146U／ml。
对黑曲霉菌株D_7(1)-1的产酶性质、酶学的特性及酶反应工艺作了一定的研究。通过硫酸铵分步盐析、反相柱层析、离子交换对酶进行纯化分离，证明橙皮苷酶包含两种酶的活性：鼠李糖苷酶和葡萄糖苷酶，其中鼠李糖酶为葡萄糖酶活性的9.5倍。经过对酶初步纯化后，酶的最适宜的反应条件为：最适作用pH为4.0，其pH值作用范围比较宽，pH稳定性在pH3.6—6.0之间较稳定；最适温度作用范围为30-55℃，在55℃时表现出最高酶活；在30℃下具有较好的稳定性，温度高于60℃热稳定性较差；粗酶可在4℃时保存4天酶活基本不变。酶的动力学参数为：K_m=0.54mmol／ml，V_m=0.8umol／l*min~(-1)。酶的作用方式是：该酶首先作用于鼠李糖—葡萄糖之间的糖苷键，对芸香糖苷水解活性较高，使得体系中的芸香糖含量很低。
通过在反应体系中添加DMF解决了橙皮苷溶解度、反应浓度很小的问题，少量的DMF几乎不影响酶活，当DMF加至40％，酶活还可保持在90％以上。酶的最适宜的反应条件为：在橙皮苷浓度为5％，加入酶量(酶活819.1u／ml)为1／4总反应液体积，酶反应的最佳pH 4.5，最佳酶反应温度为60℃，DMF／水=5：15，反应时间为0.5小时。将酵母和橙皮苷酶的共固定化，解决酶的重复利用、葡萄糖的反馈抑制和鼠李糖与葡萄糖分离的目的。装柱量约为3／4体积，水解温度为
浙江工业大学硕士学位论文
中文摘要
600C，循环速度5川1/，;lin 11寸，最千1毛反应jl寸j、ITJ为60分钟，循环10个尸刁
期酶活保持在85%左右，半衰期为1200分钟。
比较了产物的不同的分离方法，最后确定的分离方法为:反应混
合物用2倍乙醚提取4一5次，蒸发得橙皮素粗品。提取余液一再川31%
的乙酸乙醋萃取得橙皮素单糖昔粗品，将滤液经浓缩，结晶可得到较
纯的鼠李糖。
对产物的精制:将萃取得到的单糖营配成2cy0的乙醇溶液，上D.〔，【
柱，吸附30分钟后，用25%的乙醇洗去杂质，一再用40%的乙醇将单糖
二首洗下，纯度达98.02%。对橙皮素的精制为:配成2%的乙醇溶液，
上D，t，，柱，吸附30分钟后，先用10倍体积的水洗去杂质，然后一丙)}」
25%的乙醇洗脱，纯度达95%以_}_几。
最后，对橙皮什和水解户:物抗氧化性、。一糖二阵酶的抑制、氧和
烃基自山基的清除进行了初步的侧!究。结果表明:(l)在汕脂的J亢氧
化性方面:橙皮营及其水解产物均有一定的抗氧化效果，其「I，:橙皮素
的效果甚至优于V，:和BHT+BI认的混合物，但它们在汕脂的抗氧化方
面均表现为:前期效果较好后期效果较差。(2)在对一氧自山基和经基
自山基的清除方面:三者均有一定的自山基清除能力。对氧自山基清
除上，三者的强弱顺序为橙皮素>橙皮素单糖二阵>橙皮二首。对径基自山
基的清除上，低浓度时，三者的作用相当，在浓度较高时强弱顺序为
橙皮素>单糖二汗>橙皮二首。单糖二营和橙皮汁对轻基自山基的清除低于对
氧自山的清除，而橙皮素在高浓度时对氧自山基和烃基自山基清除相
当。(3)对。一糖有酶的抑制方面:三者都有抑制作用，一单糖二什的J一[1J
制效果较好，水溶液达82%而DMF液为8吐.5%。
In this paper, it was summarized that using of citrus by-products and studies of bioflavonoid in home and abroad.At the same time ,new progresses in present about structure-modification of hespeidin and it's derivate was introduced.
According to screening model, Aspergillus D7 (1) was selected from over 20 different soils and strains reserved by our lab. The optimized formula of fermentation medium with orthogonal experiment comprised: bran 8.0%, soybean powder 2.0%, KH2PO4 0.1% and 5ml 0.1% hesperidin per 30ml culturing base. The highest output of hesperidase could be obtained when the optimized fermentation medium with 30ml in 250ml flask, kept fermenting for 4 days, at the starting pH6 and the optimum temperature 50℃, after incubated 2% seed. After the treatment of UV mutation, Aspergillus D7 (1)-1 was obtained with hesperidinase activity more 19% than strain D_(7) (1).
After separation and purification of cultured broth by means of salting-out, hydrophobic interaction chromatograph (Hiprep 16/10) and cationg-exchange (Hitrap), it showed that hesperidinase was made of rhamnosidase and glucosidase.In the hesperidinase; the activity of rhamnosidase was 8.5 times higher than that of glucosidase. The studies showed that it's optimal pH for activity was 4.0 ,stability range was 3.6-6.0,the optimum temperature was at 55 ℃ ,the enzyme was stable below 55℃ and unstable above 60℃ The activity of raw hesperidinase was remained in 4 days at 4℃ . Enzymatic parameters (Km=0.54mmol/ml, Vm=0.8ummol/l.min ) had been obtained by Lincweaver-Burk with hesperidin as substrate.Merchanic of hesperidinase reaction was analysised: the bond between rhamnoside and glucose was cut down firstly and glucopyranoside was hydrolyzed rapidly by hesperidinase, so the content of glucopyranoside was very low during the reaction.
The low solubility of hesperidin in water was solved by adding DMF (N,N-dimethylformamide),DMF have a little effect on the activity of hesperidinase, hesperidinase has remained 90% activity when DMF took up 40% in reaction system, the optimal reaction condition:5% hesperidn DMF/
buffer ,1/4 enzyme of total volume, pH4.5,reaction temperature 60℃,DMF: buffer =5:15,reaction time 30 minute. Through immobilizing hesperidinase and 2% yeast together with 4% PVC, 2%alginate, 0.15%tannin and 10% hesperidinase, the feedback restrain of glucose and the separation of rhamnoside and glucose was settled down. The experimental results show that the highest outputs of monoglucosyhesperetin in the column reaction can be attained at 60 ℃ ,velocity of flow 5ml/min, reacting time 60min. After 10 times recycle, immobilized hesperidase had remained about 85% of activity.it's halflife is 1200 minutes.
Comparing many isolating methods of different products, the final method was determined as follow: Through extraction by aether and ethyl acetate in turns, raw hesperetin and monoglucosyhesperetin was gained. After column chromatography with D_(101) macroporous resin as adsorbent, eluted by grads aqueous-alcohol ,the products in which the purity of monoglucosyhesperetin was 98.02% was obtained.
Antioxidation, scavenging hydroxy radical and superoxide radical and inhabiting a-glucosidase enzyme of hesperidin and its hydrolysate was studied. The results showed as follow: 1) hesperidin and it's hydrolysate have strong antioxidation to plant oil, hesperetin excelled VE and mixture of BHA and BHT.But early stage was better than later stage to the effect. 2) Hesperidin and it's hydrolyte showed good ability to scavenge hydroxyl radical and superoxide radical. About the effect to scavenge hydroxyl radical and superoxide radical, hesperetin was better than monoglucosyhesperetin and hesperidin. 3) hesperidin and it's hydrolysate both have strong inhibitive to a-glucosidase. The inhibitive activity of monoglucosyhesperetin was 82% of glucobay in water and 84.5% in DMF solvent.The enzymologic property of hesperidin and it's hydrolysate had strong affinity to a-glucosidase.
通过初步建立筛选模型，对20来份土样和实验室保藏的菌种进行大量筛选，选育出了一株有较高选择性的橙皮苷酶活性黑曲霉菌株D_7(1)，初始酶活力为544.9u／ml。其最佳发酵培养基：麸皮8.0％、豆饼粉2.0％、KH_2PO_40.1％，每30mL培养基中加5mL0.1％的橙皮苷；最佳产酶条件：250mL的三角瓶中装量30mL，初始pH为6.0，以4层纱布包扎，在250r／min，30℃的摇床中培养4天；水相最佳酶反应条件：最佳pH4.0、反应温度为55℃。对该菌株进行了初步的紫外诱变处理，得到一株变株D_7(1)-1，橙皮苷酶活提高了19％，达到1146U／ml。
对黑曲霉菌株D_7(1)-1的产酶性质、酶学的特性及酶反应工艺作了一定的研究。通过硫酸铵分步盐析、反相柱层析、离子交换对酶进行纯化分离，证明橙皮苷酶包含两种酶的活性：鼠李糖苷酶和葡萄糖苷酶，其中鼠李糖酶为葡萄糖酶活性的9.5倍。经过对酶初步纯化后，酶的最适宜的反应条件为：最适作用pH为4.0，其pH值作用范围比较宽，pH稳定性在pH3.6—6.0之间较稳定；最适温度作用范围为30-55℃，在55℃时表现出最高酶活；在30℃下具有较好的稳定性，温度高于60℃热稳定性较差；粗酶可在4℃时保存4天酶活基本不变。酶的动力学参数为：K_m=0.54mmol／ml，V_m=0.8umol／l*min~(-1)。酶的作用方式是：该酶首先作用于鼠李糖—葡萄糖之间的糖苷键，对芸香糖苷水解活性较高，使得体系中的芸香糖含量很低。
通过在反应体系中添加DMF解决了橙皮苷溶解度、反应浓度很小的问题，少量的DMF几乎不影响酶活，当DMF加至40％，酶活还可保持在90％以上。酶的最适宜的反应条件为：在橙皮苷浓度为5％，加入酶量(酶活819.1u／ml)为1／4总反应液体积，酶反应的最佳pH 4.5，最佳酶反应温度为60℃，DMF／水=5：15，反应时间为0.5小时。将酵母和橙皮苷酶的共固定化，解决酶的重复利用、葡萄糖的反馈抑制和鼠李糖与葡萄糖分离的目的。装柱量约为3／4体积，水解温度为
浙江工业大学硕士学位论文
中文摘要
600C，循环速度5川1/，;lin 11寸，最千1毛反应jl寸j、ITJ为60分钟，循环10个尸刁
期酶活保持在85%左右，半衰期为1200分钟。
比较了产物的不同的分离方法，最后确定的分离方法为:反应混
合物用2倍乙醚提取4一5次，蒸发得橙皮素粗品。提取余液一再川31%
的乙酸乙醋萃取得橙皮素单糖昔粗品，将滤液经浓缩，结晶可得到较
纯的鼠李糖。
对产物的精制:将萃取得到的单糖营配成2cy0的乙醇溶液，上D.〔，【
柱，吸附30分钟后，用25%的乙醇洗去杂质，一再用40%的乙醇将单糖
二首洗下，纯度达98.02%。对橙皮素的精制为:配成2%的乙醇溶液，
上D，t，，柱，吸附30分钟后，先用10倍体积的水洗去杂质，然后一丙)}」
25%的乙醇洗脱，纯度达95%以_}_几。
最后，对橙皮什和水解户:物抗氧化性、。一糖二阵酶的抑制、氧和
烃基自山基的清除进行了初步的侧!究。结果表明:(l)在汕脂的J亢氧
化性方面:橙皮营及其水解产物均有一定的抗氧化效果，其「I，:橙皮素
的效果甚至优于V，:和BHT+BI认的混合物，但它们在汕脂的抗氧化方
面均表现为:前期效果较好后期效果较差。(2)在对一氧自山基和经基
自山基的清除方面:三者均有一定的自山基清除能力。对氧自山基清
除上，三者的强弱顺序为橙皮素>橙皮素单糖二阵>橙皮二首。对径基自山
基的清除上，低浓度时，三者的作用相当，在浓度较高时强弱顺序为
橙皮素>单糖二汗>橙皮二首。单糖二营和橙皮汁对轻基自山基的清除低于对
氧自山的清除，而橙皮素在高浓度时对氧自山基和烃基自山基清除相
当。(3)对。一糖有酶的抑制方面:三者都有抑制作用，一单糖二什的J一[1J
制效果较好，水溶液达82%而DMF液为8吐.5%。
In this paper, it was summarized that using of citrus by-products and studies of bioflavonoid in home and abroad.At the same time ,new progresses in present about structure-modification of hespeidin and it's derivate was introduced.
According to screening model, Aspergillus D7 (1) was selected from over 20 different soils and strains reserved by our lab. The optimized formula of fermentation medium with orthogonal experiment comprised: bran 8.0%, soybean powder 2.0%, KH2PO4 0.1% and 5ml 0.1% hesperidin per 30ml culturing base. The highest output of hesperidase could be obtained when the optimized fermentation medium with 30ml in 250ml flask, kept fermenting for 4 days, at the starting pH6 and the optimum temperature 50℃, after incubated 2% seed. After the treatment of UV mutation, Aspergillus D7 (1)-1 was obtained with hesperidinase activity more 19% than strain D_(7) (1).
After separation and purification of cultured broth by means of salting-out, hydrophobic interaction chromatograph (Hiprep 16/10) and cationg-exchange (Hitrap), it showed that hesperidinase was made of rhamnosidase and glucosidase.In the hesperidinase; the activity of rhamnosidase was 8.5 times higher than that of glucosidase. The studies showed that it's optimal pH for activity was 4.0 ,stability range was 3.6-6.0,the optimum temperature was at 55 ℃ ,the enzyme was stable below 55℃ and unstable above 60℃ The activity of raw hesperidinase was remained in 4 days at 4℃ . Enzymatic parameters (Km=0.54mmol/ml, Vm=0.8ummol/l.min ) had been obtained by Lincweaver-Burk with hesperidin as substrate.Merchanic of hesperidinase reaction was analysised: the bond between rhamnoside and glucose was cut down firstly and glucopyranoside was hydrolyzed rapidly by hesperidinase, so the content of glucopyranoside was very low during the reaction.
The low solubility of hesperidin in water was solved by adding DMF (N,N-dimethylformamide),DMF have a little effect on the activity of hesperidinase, hesperidinase has remained 90% activity when DMF took up 40% in reaction system, the optimal reaction condition:5% hesperidn DMF/
buffer ,1/4 enzyme of total volume, pH4.5,reaction temperature 60℃,DMF: buffer =5:15,reaction time 30 minute. Through immobilizing hesperidinase and 2% yeast together with 4% PVC, 2%alginate, 0.15%tannin and 10% hesperidinase, the feedback restrain of glucose and the separation of rhamnoside and glucose was settled down. The experimental results show that the highest outputs of monoglucosyhesperetin in the column reaction can be attained at 60 ℃ ,velocity of flow 5ml/min, reacting time 60min. After 10 times recycle, immobilized hesperidase had remained about 85% of activity.it's halflife is 1200 minutes.
Comparing many isolating methods of different products, the final method was determined as follow: Through extraction by aether and ethyl acetate in turns, raw hesperetin and monoglucosyhesperetin was gained. After column chromatography with D_(101) macroporous resin as adsorbent, eluted by grads aqueous-alcohol ,the products in which the purity of monoglucosyhesperetin was 98.02% was obtained.
Antioxidation, scavenging hydroxy radical and superoxide radical and inhabiting a-glucosidase enzyme of hesperidin and its hydrolysate was studied. The results showed as follow: 1) hesperidin and it's hydrolysate have strong antioxidation to plant oil, hesperetin excelled VE and mixture of BHA and BHT.But early stage was better than later stage to the effect. 2) Hesperidin and it's hydrolyte showed good ability to scavenge hydroxyl radical and superoxide radical. About the effect to scavenge hydroxyl radical and superoxide radical, hesperetin was better than monoglucosyhesperetin and hesperidin. 3) hesperidin and it's hydrolysate both have strong inhibitive to a-glucosidase. The inhibitive activity of monoglucosyhesperetin was 82% of glucobay in water and 84.5% in DMF solvent.The enzymologic property of hesperidin and it's hydrolysate had strong affinity to a-glucosidase.
引文
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