CN110283809A - 一种β-葡糖苷酶固定化方法及应用 - Google Patents
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Abstract
本发明公开了一种β‑葡糖苷酶固定化方法及应用,利用化学反应将β‑葡糖苷酶固定到聚甲基丙烯酸甲酯树脂上,装入层析柱中,作为大豆异黄酮降解生物反应器,用于连续生产大豆异黄酮苷元。该方法利用具有耐压性能好、孔径大、比表面积大的聚甲基丙烯酸甲酯树脂键合β‑葡糖苷酶,并装柱使用,具有操作方便、酶利用度高、酶活性好、酶寿命长、树脂耐污染、后续分离方便等优点,更重要的是可以进行连续生产,产率高,适合规模化生产大豆异黄酮苷元。
Description
技术领域
本发明属于生物工程技术领域,具体涉及一种β-葡糖苷酶固定化方法及应用。
背景技术
大豆异黄酮是大豆中的一种具有生理功能的活性物质。近年来,随着其保健作用研究开发的不断深入,大豆异黄酮的存在形式及生理功能的机理正逐渐被人们认识。研究表明,大豆中的异黄酮存在形式主要是结合型的糖苷,约占大豆中异黄酮含量的95%以上,虽然游离的苷元含量不足5%,但它们表现出来的活性要比结合型的糖苷高得多,为了提高大豆异黄酮的生物活性,提高在人体内的吸收率,将大豆异黄酮进行水解制成大豆异黄酮苷元,这成为近年来大豆异黄酮研究的一个热点。水解原理:大豆异黄酮苷属于氧苷类,是酚羟基与糖缩合而成的β-D葡萄糖苷。通过水解反应使苷键裂解得到大豆异黄酮苷元和葡萄糖配基。水解方法主要有以下几种:
酸水解:大豆异黄酮苷能被酸催化水解,反应在稀醇中进行,用醋酸、硫酸等均可发生水解,从成本和实际应用角度,最常使用盐酸。β-葡萄糖苷键首先发生质子化,然后苷键断裂生成苷元和糖的阳碳离子中间体,再水解,阳碳离子经溶剂化,再脱去氢离子而形成糖分子。酸催化水解的关键是O-苷基的苷质子化程度。但是酸性条件下大豆异黄酮苷元的稳定性值得怀疑。另外,强酸条件下不易进行工业化生产。
碱水解:糖苷键具有缩醛结构,对碱较稳定,但异黄酮中糖苷键具有酯苷性质,可用碱进行水解。分解为大豆异黄酮苷元和葡萄糖,但碱性条件下水解所得大豆异黄酮苷元很不稳定,容易降解。
Smith降解法:该法是一种氧化开裂法,比酸水解温和,能完整地保持苷元的结构,先用NaIO4将糖的二羟基氧化开裂为二元醛结构,第二步用NaBH4将二元醛结构还原成二元醇结构,第三步在室温条件下与稀酸作用使其水解。但由于此反应对葡萄糖进行开环,会有部分基团残留在苷元上,造成结构的复杂性以及不均一性。
酶催化水解:使用最多的大豆异黄酮糖苷水解酶是β-葡萄糖苷酶,大豆自身含有的内源β-葡萄糖苷酶水解活性不强,水解效率只有22%~29%。添加足量的高活性酶,如乳酸菌中提取的β-葡萄糖苷酶可使水解达到80%以上。能够水解大豆异黄酮糖苷的酶还有葡萄糖酸酶、α-半乳糖苷酶、生物乳糖酶、真菌乳糖酶和乳糖酶F等,它们都有很强的水解大豆异黄酮糖苷的能力。优势:酶具有高效率的催化能力,同时具有很高的专属性,能够对某一类特定物质或结构有专一性;作为生物催化剂,反应的前后不会发生性质和数量的改变,只有活性会有轻微的降低。且作用条件温和,与大豆异黄酮苷元在弱酸性的缓冲溶液中不易发生结构的变化。劣势:每次对大豆异黄酮进行酶解,得到活性较高的苷元部分时,需要对酶进行去除。普通的除酶手段,如进行煮沸离心,会有较大的蛋白质残留。同时也极大浪费了所用酶剩余的活性,造成生产成本的提高。
不同的固定化酶方式
因此,有必要研发一种高效降解大豆异黄酮制备大豆异黄酮苷元的方法,来解决现在大豆异黄酮苷元生产工艺复杂、效率低的难题。
发明内容
本发明目的是提供一种β-葡糖苷酶固定化方法,解决上述问题。
本发明的技术方案是:
一种β-葡糖苷酶固定化方法,包括步骤:
(1)通过化学键合将β-葡糖苷酶固定到聚甲基丙烯酸树脂上,形成酶固定化树脂;
(2)将所述酶固定化树脂装入层析柱中制成生物反应器。
进一步的,步骤(1)中所述化学键合方式为环氧、邻二醇、乙胺基键合方式,所述化学键合的温度为10到60℃。
进一步的,步骤(1)中所述β-葡糖苷酶的来源为脑膜脓毒性黄杆菌、约氏黄杆菌、清酒酵母、黄孢原毛平革菌、蜜蜂、猪肝、猪小肠中的任意一种。
进一步的,步骤(1)中所述聚甲基丙烯酸树脂的粒径为50um~1000um、孔径为20nm~100nm。
进一步的,步骤(1)中所述通过化学键合将β-葡糖苷酶固定到聚甲基丙烯酸树脂上,形成酶固定化树脂具体为:
使用PBS溶液将50cm3带有环氧基的聚甲基丙烯酸树脂溶解,获得树脂溶液;
取1份含有β-葡糖苷酶的酶液稀释2~5倍,用滴加装置将所述酶液慢慢滴入所述树脂溶液中,并用玻璃棒不断搅拌,在20~40℃下反应3-6h,过滤后,用无菌PBS溶液洗涤树脂2-3次,形成酶固定化树脂,放入冰箱4℃保存。
进一步的,步骤(2)中所述层析柱的材质为玻璃、有机玻璃、聚合物或不锈钢中的任意一种
本发明的另一技术方案是:一种β-葡糖苷酶固定化方法在大豆异黄酮水解中的应用。
进一步的,利用输液泵输送大豆异黄酮溶液到生物反应器中,在β-葡糖苷酶的作用下,大豆异黄酮被降解成苷元。
进一步的,所述输液泵为计量泵、蠕动泵、隔膜泵、柱塞泵、无阀泵或电磁泵中的任意一种。
本发明提供了一种β-葡糖苷酶固定化方法及应用,优点为:投资少、转化效率高、适合大生产的特点。
附图说明
图1.酶固定化技术反应原理图;
图2.生物反应器降解大豆异黄酮效果图。
具体实施方式
实施例1
本发明一种β-葡糖苷酶固定化方法的技术方案如下:
1.酶的富集
酵母经28℃发酵48h后,4000×g离心30min收集菌体,用醋酸缓冲液(0.05mol/L,pH5.0)清洗2次。置–70℃中预冻4h,在冻干机上冻干菌体,用高速气流破碎仪将菌体破壁。将破壁粉悬浮于醋酸缓冲液(0.05mol/L,pH5.0)中搅拌1h,使蛋白质充分溶出,4000×g离心30min去除沉淀,得到酶液。
2.酶的固定化
使用PBS溶液将50cm3带有环氧基的聚甲基丙烯酸树脂溶解。取2mL的酶液稀释2倍,用滴加装置将其慢慢滴入树脂溶液中,并用玻璃棒不断搅拌,在20℃下反应3-6h。过滤后,用无菌PBS溶液洗涤树脂2-3次。放入冰箱4℃保存。
上述酶固定化技术反应原理见图1。
3.装柱与活化
将酶的固定化树脂使用蠕动泵进入玻璃柱(h=800mm,d=50mm),倒入pH7.0的PBS溶液冲洗,并进行活化。
4.洗涤
将大豆异黄酮10g溶解于2000mL pH7.0的PBS缓冲液,5mL/min流过玻璃柱,收集流出液。浓缩为500mL后,乙酸乙酯萃取三次,萃取液回收后液相监测。
5.HPLC条件
流动相为20%乙腈:80%水,流速为1mL/min,进样量20微升,检测波长280nm,色谱柱ODS C18色谱柱(4.6mm×150mm,5μm),柱温25℃。分析结果见图2。
实施例2
研究流速对大豆异黄酮苷元产率的影响
实验方法:将大豆异黄酮粗品上样至填装了键合固定化酶树脂的中压玻璃柱当中。装填压力为3MPa,采用PBS洗脱。实施步骤:
(1)采用PBS作为流动相。
(2)将10mL提取的大豆异黄酮糖苷上样到中压玻璃柱,然后采用平衡液平衡柱子至基线稳定。中压玻璃柱内装填键合100mgβ-葡糖苷酶的50cm3树脂。
(3)采用不同的流速:5ml/min;10ml/min;20ml/min;50ml/min。
(4)收集3倍柱体积溶液。
试验结果见表1
表1不同流速对大豆异黄酮苷元产率的影响
表1
实验结果表明,系统流速越小,其酶解后的大豆异黄酮苷元纯度和收率越高。从表1中可以看出,流速5ml/min对大豆异黄酮苷元的获得效果最好,且苷元收率也相对最高。因此,优选5ml/min的流速进行对大豆异黄酮苷元的制备。
实施例3
研究树脂不同粒径对大豆异黄酮苷元产率的影响
实验方法:将大豆异黄酮粗品上样至填装了键合固定化酶树脂的中压玻璃柱当中。装填压力为3MPa,采用PBS洗脱。实施步骤:
(1)采用PBS作为流动相。
(2)将10mL提取的大豆异黄酮糖苷上样到中压玻璃柱,然后采用平衡液平衡柱子至基线稳定。中压玻璃柱内装填键合100mgβ-葡糖苷酶的50cm3树脂。
(3)采用不同粒径的树脂:50μm;600μm;800μm;1000μm。
(4)收集3倍柱体积溶液。
试验结果见表2
表2不同树脂粒径对大豆异黄酮苷元产率的影响
实验结果表明,树脂的粒径越大,大豆异黄酮苷元的收率越低,在600μm粒径时,大豆异黄酮苷元的纯度达到最高,且此时苷元的收率与50μm时几乎一致,因此从生产成本与苷元纯度角度考虑,优选600μm的树脂粒径进行对大豆异黄酮苷元的制备。
综上所述,本发明所述的一种β-葡糖苷酶固定化方法及应用,利用具有耐压性能好、孔径大、比表面积大的聚甲基丙烯酸甲酯树脂键合β-葡糖苷酶,并装柱使用,具有操作方便、酶利用度高、酶活性好、酶寿命长、树脂耐污染、后续分离方便等优点,更重要的是可以进行连续生产,产率高,适合规模化生产大豆异黄酮苷元。
应说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的精神和范围,其均应涵盖在本发明的权利要求范围当中。
Claims (9)
1.一种β-葡糖苷酶固定化方法,其特征在于,包括步骤:
(1)通过化学键合将β-葡糖苷酶固定到聚甲基丙烯酸树脂上,形成酶固定化树脂;
(2)将所述酶固定化树脂装入层析柱中制成生物反应器;
(3)利用恒流泵输送反应液通过生物反应器,经过降解后,得到产物。
2.根据权利要求1所述的一种β-葡糖苷酶固定化方法,其特征在于:步骤(1)中所述化学键合方式为环氧、邻二醇、乙胺基键合方式,所述化学键合的温度为10到60℃。
3.根据权利要求1所述的一种β-葡糖苷酶固定化方法,其特征在于:步骤(1)中所述β-葡糖苷酶的来源为脑膜脓毒性黄杆菌、约氏黄杆菌、清酒酵母、黄孢原毛平革菌、蜜蜂、猪肝、猪小肠中的任意一种。
4.根据权利要求1所述的一种β-葡糖苷酶固定化方法,其特征在于:步骤(1)中所述聚甲基丙烯酸树脂的粒径为50um~1000um、孔径为20nm~100nm。
5.根据权利要求4所述的一种β-葡糖苷酶固定化方法,其特征在于,步骤(1)中所述通过化学键合将β-葡糖苷酶固定到聚甲基丙烯酸树脂上,形成酶固定化树脂具体为:
使用PBS溶液将带有环氧基的聚甲基丙烯酸树脂溶解,获得树脂溶液;
取1份含有β-葡糖苷酶的酶液稀释2~5倍,用滴加装置将所述酶液慢慢滴入所述树脂溶液中,并用玻璃棒不断搅拌,在20~40℃下反应3-6h,过滤后,用无菌PBS溶液洗涤树脂2-3次,形成酶固定化树脂,放入冰箱4℃保存。
6.根据权利要求1所述的一种β-葡糖苷酶固定化方法,其特征在于:步骤(2)中所述层析柱的材质为玻璃、有机玻璃、聚合物或不锈钢中的任意一种。
7.根据权利要求1-6中任意一项所述的一种β-葡糖苷酶固定化方法在大豆异黄酮水解中的应用。
8.根据权利要求7所述的一种β-葡糖苷酶固定化方法在大豆异黄酮水解中的应用,其特征在于,包括步骤:
利用输液泵输送大豆异黄酮溶液到生物反应器中,在β-葡糖苷酶的作用下,大豆异黄酮被降解成苷元。
9.根据权利要求8所述的一种β-葡糖苷酶固定化方法在大豆异黄酮水解中的应用,其特征在于:所述输液泵为计量泵、蠕动泵、隔膜泵、柱塞泵、无阀泵或电磁泵中的任意一种。
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