岩藻聚糖硫酸酯酶产生菌的筛选、酶学性质研究及酶解产物抗氧化活性预测系统的建立
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摘要
海带是我国重要的经济藻类，因其营养价值高，具有多种生理活性，受到人们的喜爱。目前，我国海带产量居世界第一位，但海带的工业利用率仅有30%，并且整体加工水平较低，导致海带产业始终无法发展壮大。因此，对海带的生理活性成分进行深入研究，进而实现海带的高值化利用，是发展海带产业的重要途径。本文围绕海带中的活性物质岩藻聚糖硫酸酯（HDFuc）及其降解产物展开研究，鉴于低分子量HDFuc在活性应用以及结构研究方面的优势，对HDFuc进行降解进而获取低分子量的HDFuc成为重要的课题。酶法降解是最理想的方式，但是由于相应商品酶制剂的缺乏，目前无法实现对HDFuc的酶法降解。
本文首先进行产酶微生物的筛选。以HDFuc为唯一碳源，从荣成海域海带中筛选出一株产酶细菌RC2，经过生理生化和16s rDNA鉴定，判断RC2为黄杆菌科的一个新成员。RC2能够稳定产生岩藻聚糖硫酸酯酶（FUCenzyme），该酶属于胞内酶。经过高效凝胶排阻色谱及薄层色谱的方法确认了RC2的产酶能力，并证实该酶能够将HDFuc降解为低分子量产物。
为了提高RC2的产酶能力，对其发酵培养基及产酶条件进行优化。确定RC2产酶最优培养基配方为0.2%HDFuc、0.4%牛肉膏、用过膜海水配制；最优培养条件为25℃、pH值自然、摇床转速150rpm、250mL三角瓶装液量20%、接种量10%。在上述条件下，菌株RC2培养72h后产酶活力可达178U/mL，该酶活力高于所有已报道的有可比性的细菌。并在此基础上，对RC2进行了5L发酵罐扩大发酵试验，在25℃，搅拌速度200rpm，通气量2.0L/min的条件下，经过96h的培养后收集菌体并进行细胞破碎，可获得总酶活力110530U。
随后，对FUCenzyme进行了分离纯化与酶学性质研究。胞内酶经过硫酸铵沉淀和Q-Sepharose Fast Flow离子交换层析纯化，可得到单一酶活性组分，对该组分进行SDS-PAGE检测，结果为单一条带，证明得到了纯化酶。通过分子量标准曲线计算其分子量为41.0KDa。纯化过程总的酶活力回收率为11.3%，纯化倍数为11.8倍。经酶学性质分析表明，该酶最适反应温度为50℃；热稳定性较差，仅在20℃和30℃稳定，但在4℃稳定性很好，酶活力保持一个月无显著性下降。酶的最适反应pH值为8.0；在pH8.0最稳定，其次为pH7.0。该酶必须在NaCl存在下才能够显示酶活力，最适NaCl浓度为0.4mol/L或0.6mol/L。Zn~(2+)、K~+、 Ca~(2+)对该酶有激活作用，其中Zn~(2+)激活作用最强；Cu~(2+)、Hg~(2+)、Ag+对该酶有较大抑制作用，其中Cu~(2+)的抑制作用最强。另外，NaF和EDTA-2Na对该酶有较强的抑制作用。另外，还监测了该酶催化过程还原糖含量的变化，发现反应12h内还原糖明显上升，12h后变化较小，说明酶催化反应速率在12h以后减慢。
利用纯化酶对HDFuc进行酶解，首次建立了酶解产物抗氧化活性可视化预测系统。利用三个BP网络模型分别实现了对酶解过程产物的DPPH˙清除率、˙OH清除率和O2˙-清除率的预测。经验证，三个BP网络模型的最大误差均小于10%，完全能够满足应用要求，并在Matlab平台建立了抗氧化活性可视化预测系统。进一步应用遗传算法对BP网络模型进行寻优，确定最佳酶解条件为25.2℃，酶解6.4h，加酶量22.0U/(mg HDFuc)，此时酶解产物的DPPH˙、˙OH和O2˙-清除率分别为39.85±1.00%、82.08±5.74%、27.67±3.45%，还原能力为0.2348±0.0044，金属离子螯合能力达到35.64±3.01%。
对HDFuc及其酶解产物进行体内抗氧化活性研究。在最优酶解条件下对HDFuc进行酶解，获得酶解产物。通过建立D-半乳糖诱导的小鼠亚急性衰老模型，评估了二者的体内抗氧化活性。结果表明二者均具有显著的抗氧化活性，酶解产物在保护小鼠血清抗氧化酶活性方面的作用要优于HDFuc。
综上所述，本文成功筛选到FUCenzyme产酶细菌，并对该酶进行了分离纯化。利用该酶获得低分子量的酶解产物，首次建立了HDFuc酶解产物抗氧化活性可视化预测系统，并实现了联合遗传算法进行寻优，获得了具有最高抗氧化活性的产物。经动物实验证实，该酶解产物具有显著的抗氧化活性。本文的结果为海带岩藻聚糖硫酸酯的开发应用奠定了基础，有望促进海带的高值化利用及海带产业的发展。
Kelp is a kind of important economic alga in China, and it is popular for its highnutritional value and various biological activities. At present, the output of kelp inChina ranks first in the world. However, the kelp industry is unable to grow vigorousdue to the current low-level processing and low utilization rate, which is just30%.Therefore, in order to realize development of kelp industry, an important way is torealize value-added utilization through further research on biological activities of kelp.This paper conducts research on the HDFuc in kelp, and its degrading products.Because of the great advantages of lower molecular weight HDFuc in activityapplication and structure study, it is essential to degrade HDFuc and to obtain lowermolecular weight components. Enzymic hydrolysis is the best way for fucoidandegrading, but it cannot be achieved due to the lack of Fucoidan degrading enzyme(FUCenzyme) in the market.
In this thesis, the first step of research is to screen FUCenzyme-producing microbe.Using HDFuc as the sole carbon source, a FUCenzyme-producing bacterium namedRC2is obtained from Laminaria japonica in the sea area of Rongcheng. It isidentified to be a new member of Flavobacteriacea by its physiology andbiochemistry characteristic and its16s rDNA analysis. FUCenzyme, a kind ofendoenzyme, can be produced stably by RC2. According to tests of HPSEC and TLC,RC2is confirmed to produce FUCenzyme and generate lower molecular weighthydrolyzate.
To increase the FUCenzyme-producing ability of RC2, fermentation medium andenzyme-producing conditions are optimized. The optimal medium is0.2%Fucoidanand0.4%beef extract, prepared with filtered seawater. FUCenzyme activity reachesto178U/mL after72h-cultivating by RC2under the optimal fermentation conditions: 25℃, natural pH,150rpm of shaking speed,20%of the liquid loading size in250mLflask,10%of inoculation size. On this base, the expanding fermentation test in5Lfermentation tank is done and110530U enzyme activity in total is obtained after96h-cultivating by RC2under the conditions:25℃,200rpm of stirring speed and1.0L/min of air flow.
Thereafter, the FUCenzyme is purified and its characteristics are analyzed. CrudeFUCenzyme is separated by ammonium sulfate precipitation and QFF ion exchange,and then a single component with enzymic activity is obtained. The single componentis proved to be a purified FUCenzyme by the evidence of a single band in SDS-PAGE.Its molecular weight is41.0KDa according to standard molecular weight marker. Inthe whole process of purification, FUCenzyme is purified up to11.8times and therecovery of enzymic activity is11.3%. Enzymic property research shows that theoptimal conditions are50℃, pH8.0. The FUCenzyme, stable only at20℃and30℃,is with poor thermal stability. But it is with good stability when stored at4℃. It isstable at pH8.0, then at pH7.0. And the enzyme can show its activity only in thesituation with NaCl, and the optimal NaCl concertration is0.4mol/L or0.6mol/L.Zn~(2+), K~+and Ca~(2+)have function of activation to the enzyme, and among them, thefunction of Zn~(2+)is greatest. While, Cu~(2+), Hg~(2+)and Ag+have function of inhibition,and among them, the function of Cu~(2+)is greatest. In addition, NaF and EDTA-2Nainhibit the FUCenzyme strongly. Change of reducing sugars content is monitoredduring the catalyzing process, and the results show that the content of reducing sugarsincreases markedly in the early12hours, and is with little change after12hours. It isproved that the rate of catalyzing declines clearly after12hours.
For the first time, a visual antioxidant activity prediction system is established,which realizes the prediction of antioxidant activity of the hydrolyzate catalyzed byFUCenzyme. Three BP network models are established to predict respectively thescavenging ratio of DPPH˙,˙OH and O2-. The three models are verified to be suitablefor use, with the maximum error lower than10%. On the basis of BP netwo rk models,a visual antioxidant activity prediction system is established under the platform ofMatlab, making the visual antioxidant activity prediction of hydrolyzate comes true. Further, GA is used for seeking optimal value of BP network models. The resultsshow that when adding FUCenzyme22.0U to1mg HDFuc and hydrolyzing at25.2℃for6.4hours, the hydrolyzate is generated with highest antioxidant activity (DPPH˙scavenging ratio39.85±1.00%,˙OH scavenging ratio82.08±5.74%, O2-scavengingratio27.67±3.45%, reducing power0.2348±0.0044, iron-chelation activity35.64±3.01%).
Then the in vivo antioxidant study of HDFuc and enzymic hydrolyzate is done.Hydrolyzate is obtained when catalyzing HDFuc under the optimal conditionsmentioned above. It shows significant antioxidant activity in mice of HDFuc and itshydrolyzate when using D-galactose induced subacute aging model. Furthermore, thehydrolyzate is better than HDFuc in the aspect of protecting the mice antioxidantenzyme activity.
In conclusion, a FUCenzyme producing bacterium is successfully obtained and thepurification of the FUCenzyme is conducted. Lower molecular weight enzymatichydrolyzate is obtained taking advantage of the enzyme, and a visual antioxidantactivity prediction system of hydrolyzate is established for the first time. Using GAfor optimizing, highest activity enzymatic hydrolyzate is got. And the enzymatichydrolyzate is proved to be with good antioxidant activity by animal experiments. Theachievement in this thesis lays a foundation for development and application ofHDFuc, which will promote the high-value utilization of kelp and development ofkelp industry.
本文首先进行产酶微生物的筛选。以HDFuc为唯一碳源，从荣成海域海带中筛选出一株产酶细菌RC2，经过生理生化和16s rDNA鉴定，判断RC2为黄杆菌科的一个新成员。RC2能够稳定产生岩藻聚糖硫酸酯酶（FUCenzyme），该酶属于胞内酶。经过高效凝胶排阻色谱及薄层色谱的方法确认了RC2的产酶能力，并证实该酶能够将HDFuc降解为低分子量产物。
为了提高RC2的产酶能力，对其发酵培养基及产酶条件进行优化。确定RC2产酶最优培养基配方为0.2%HDFuc、0.4%牛肉膏、用过膜海水配制；最优培养条件为25℃、pH值自然、摇床转速150rpm、250mL三角瓶装液量20%、接种量10%。在上述条件下，菌株RC2培养72h后产酶活力可达178U/mL，该酶活力高于所有已报道的有可比性的细菌。并在此基础上，对RC2进行了5L发酵罐扩大发酵试验，在25℃，搅拌速度200rpm，通气量2.0L/min的条件下，经过96h的培养后收集菌体并进行细胞破碎，可获得总酶活力110530U。
随后，对FUCenzyme进行了分离纯化与酶学性质研究。胞内酶经过硫酸铵沉淀和Q-Sepharose Fast Flow离子交换层析纯化，可得到单一酶活性组分，对该组分进行SDS-PAGE检测，结果为单一条带，证明得到了纯化酶。通过分子量标准曲线计算其分子量为41.0KDa。纯化过程总的酶活力回收率为11.3%，纯化倍数为11.8倍。经酶学性质分析表明，该酶最适反应温度为50℃；热稳定性较差，仅在20℃和30℃稳定，但在4℃稳定性很好，酶活力保持一个月无显著性下降。酶的最适反应pH值为8.0；在pH8.0最稳定，其次为pH7.0。该酶必须在NaCl存在下才能够显示酶活力，最适NaCl浓度为0.4mol/L或0.6mol/L。Zn~(2+)、K~+、 Ca~(2+)对该酶有激活作用，其中Zn~(2+)激活作用最强；Cu~(2+)、Hg~(2+)、Ag+对该酶有较大抑制作用，其中Cu~(2+)的抑制作用最强。另外，NaF和EDTA-2Na对该酶有较强的抑制作用。另外，还监测了该酶催化过程还原糖含量的变化，发现反应12h内还原糖明显上升，12h后变化较小，说明酶催化反应速率在12h以后减慢。
利用纯化酶对HDFuc进行酶解，首次建立了酶解产物抗氧化活性可视化预测系统。利用三个BP网络模型分别实现了对酶解过程产物的DPPH˙清除率、˙OH清除率和O2˙-清除率的预测。经验证，三个BP网络模型的最大误差均小于10%，完全能够满足应用要求，并在Matlab平台建立了抗氧化活性可视化预测系统。进一步应用遗传算法对BP网络模型进行寻优，确定最佳酶解条件为25.2℃，酶解6.4h，加酶量22.0U/(mg HDFuc)，此时酶解产物的DPPH˙、˙OH和O2˙-清除率分别为39.85±1.00%、82.08±5.74%、27.67±3.45%，还原能力为0.2348±0.0044，金属离子螯合能力达到35.64±3.01%。
对HDFuc及其酶解产物进行体内抗氧化活性研究。在最优酶解条件下对HDFuc进行酶解，获得酶解产物。通过建立D-半乳糖诱导的小鼠亚急性衰老模型，评估了二者的体内抗氧化活性。结果表明二者均具有显著的抗氧化活性，酶解产物在保护小鼠血清抗氧化酶活性方面的作用要优于HDFuc。
综上所述，本文成功筛选到FUCenzyme产酶细菌，并对该酶进行了分离纯化。利用该酶获得低分子量的酶解产物，首次建立了HDFuc酶解产物抗氧化活性可视化预测系统，并实现了联合遗传算法进行寻优，获得了具有最高抗氧化活性的产物。经动物实验证实，该酶解产物具有显著的抗氧化活性。本文的结果为海带岩藻聚糖硫酸酯的开发应用奠定了基础，有望促进海带的高值化利用及海带产业的发展。
Kelp is a kind of important economic alga in China, and it is popular for its highnutritional value and various biological activities. At present, the output of kelp inChina ranks first in the world. However, the kelp industry is unable to grow vigorousdue to the current low-level processing and low utilization rate, which is just30%.Therefore, in order to realize development of kelp industry, an important way is torealize value-added utilization through further research on biological activities of kelp.This paper conducts research on the HDFuc in kelp, and its degrading products.Because of the great advantages of lower molecular weight HDFuc in activityapplication and structure study, it is essential to degrade HDFuc and to obtain lowermolecular weight components. Enzymic hydrolysis is the best way for fucoidandegrading, but it cannot be achieved due to the lack of Fucoidan degrading enzyme(FUCenzyme) in the market.
In this thesis, the first step of research is to screen FUCenzyme-producing microbe.Using HDFuc as the sole carbon source, a FUCenzyme-producing bacterium namedRC2is obtained from Laminaria japonica in the sea area of Rongcheng. It isidentified to be a new member of Flavobacteriacea by its physiology andbiochemistry characteristic and its16s rDNA analysis. FUCenzyme, a kind ofendoenzyme, can be produced stably by RC2. According to tests of HPSEC and TLC,RC2is confirmed to produce FUCenzyme and generate lower molecular weighthydrolyzate.
To increase the FUCenzyme-producing ability of RC2, fermentation medium andenzyme-producing conditions are optimized. The optimal medium is0.2%Fucoidanand0.4%beef extract, prepared with filtered seawater. FUCenzyme activity reachesto178U/mL after72h-cultivating by RC2under the optimal fermentation conditions: 25℃, natural pH,150rpm of shaking speed,20%of the liquid loading size in250mLflask,10%of inoculation size. On this base, the expanding fermentation test in5Lfermentation tank is done and110530U enzyme activity in total is obtained after96h-cultivating by RC2under the conditions:25℃,200rpm of stirring speed and1.0L/min of air flow.
Thereafter, the FUCenzyme is purified and its characteristics are analyzed. CrudeFUCenzyme is separated by ammonium sulfate precipitation and QFF ion exchange,and then a single component with enzymic activity is obtained. The single componentis proved to be a purified FUCenzyme by the evidence of a single band in SDS-PAGE.Its molecular weight is41.0KDa according to standard molecular weight marker. Inthe whole process of purification, FUCenzyme is purified up to11.8times and therecovery of enzymic activity is11.3%. Enzymic property research shows that theoptimal conditions are50℃, pH8.0. The FUCenzyme, stable only at20℃and30℃,is with poor thermal stability. But it is with good stability when stored at4℃. It isstable at pH8.0, then at pH7.0. And the enzyme can show its activity only in thesituation with NaCl, and the optimal NaCl concertration is0.4mol/L or0.6mol/L.Zn~(2+), K~+and Ca~(2+)have function of activation to the enzyme, and among them, thefunction of Zn~(2+)is greatest. While, Cu~(2+), Hg~(2+)and Ag+have function of inhibition,and among them, the function of Cu~(2+)is greatest. In addition, NaF and EDTA-2Nainhibit the FUCenzyme strongly. Change of reducing sugars content is monitoredduring the catalyzing process, and the results show that the content of reducing sugarsincreases markedly in the early12hours, and is with little change after12hours. It isproved that the rate of catalyzing declines clearly after12hours.
For the first time, a visual antioxidant activity prediction system is established,which realizes the prediction of antioxidant activity of the hydrolyzate catalyzed byFUCenzyme. Three BP network models are established to predict respectively thescavenging ratio of DPPH˙,˙OH and O2-. The three models are verified to be suitablefor use, with the maximum error lower than10%. On the basis of BP netwo rk models,a visual antioxidant activity prediction system is established under the platform ofMatlab, making the visual antioxidant activity prediction of hydrolyzate comes true. Further, GA is used for seeking optimal value of BP network models. The resultsshow that when adding FUCenzyme22.0U to1mg HDFuc and hydrolyzing at25.2℃for6.4hours, the hydrolyzate is generated with highest antioxidant activity (DPPH˙scavenging ratio39.85±1.00%,˙OH scavenging ratio82.08±5.74%, O2-scavengingratio27.67±3.45%, reducing power0.2348±0.0044, iron-chelation activity35.64±3.01%).
Then the in vivo antioxidant study of HDFuc and enzymic hydrolyzate is done.Hydrolyzate is obtained when catalyzing HDFuc under the optimal conditionsmentioned above. It shows significant antioxidant activity in mice of HDFuc and itshydrolyzate when using D-galactose induced subacute aging model. Furthermore, thehydrolyzate is better than HDFuc in the aspect of protecting the mice antioxidantenzyme activity.
In conclusion, a FUCenzyme producing bacterium is successfully obtained and thepurification of the FUCenzyme is conducted. Lower molecular weight enzymatichydrolyzate is obtained taking advantage of the enzyme, and a visual antioxidantactivity prediction system of hydrolyzate is established for the first time. Using GAfor optimizing, highest activity enzymatic hydrolyzate is got. And the enzymatichydrolyzate is proved to be with good antioxidant activity by animal experiments. Theachievement in this thesis lays a foundation for development and application ofHDFuc, which will promote the high-value utilization of kelp and development ofkelp industry.
引文
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