CN106282272A - 一种利用脂肪酶催化合成c‑6’‑月桂酰基京尼平苷的方法 - Google Patents
一种利用脂肪酶催化合成c‑6’‑月桂酰基京尼平苷的方法 Download PDFInfo
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Abstract
本发明公开了一种利用脂肪酶催化合成C‑6’‑月桂酰基京尼平苷的方法,它将京尼平苷和桂酸乙烯酯溶于溶剂中,以固定化脂肪酶为催化剂进行反应。与现有技术相比,本发明具有如下优势:本发明首次利用脂肪酶催化合成天然产物京尼平苷的衍生物C‑6’‑月桂酰基京尼平苷,相对于化学修饰法,本发明具有反应条件温和,催化位点选择性强,工艺步骤简单,环境友好等特点。同时,本发明使用的固定化脂肪酶,易与底物及产物分离,寿命长可重复使用进行连续化生产,降低了生产成本。本发明制备得到的产品C‑6’‑月桂酰基京尼平苷较京尼平苷脂溶性得到了较大的改善,应用前景更为广阔。
Description
技术领域
本发明属于生物催化领域,具体涉及一种利用脂肪酶催化合成C-6’-月桂酰基京尼平苷的方法。
背景技术
京尼平苷是一种环烯醚萜葡萄糖苷,是栀子的主要药效成分。京尼平苷对消化***、心血管***和中枢神经***疾病均有显著疗效,此外,京尼平苷还有一定的抗炎、抗氧化、抗肿瘤和治疗软组织损伤的作用。京尼平苷除了药用以外,在其它领域也得到广泛的应用,如可用作植物增产剂、生物检测剂等。
作为一种环烯醚萜苷类化合物,京尼平苷拥有的极性官能团使其亲水性较强,更加容易溶解在水、甲醇中,可以溶解在乙醇、丙酮与正丁醇之中,但是难溶于氯仿、***、苯等亲脂性有机溶剂,脂溶性差,这很大程度上地限制了京尼平苷的应用。在应用中常将京尼平苷以某种形式进行修饰,得到更为稳定、脂溶性更好的京尼平苷衍生物。
目前,京尼平苷的修饰主要通过化学法进行。CN102875617A中汤文建等人以京尼平苷为原料,经过一系列乙酰化,脱乙酰基,缩合等一系列复杂反应制得了多种京尼平苷衍生物。然而化学修饰法存在工艺步骤复杂、反应条件苛刻、反应位点选择性差和环境污染较大等问题。
发明内容
本发明要解决的技术问题是提供一种利用脂肪酶催化合成C-6’-月桂酰基京尼平苷的方法,以解决现有技术存在的工艺步骤复杂、反应条件苛刻、反应位点选择性差和环境污染较大等问题
为解决上述技术问题,本发明采用的技术方案如下:
一种利用脂肪酶催化合成C-6’-月桂酰基京尼平苷的方法,它包括如下步骤:
将京尼平苷和桂酸乙烯酯溶于溶剂中,以固定化脂肪酶为催化剂进行反应,反应液经离心、溶解和柱纯化后,即得产物。
其中,京尼平苷和桂酸乙烯酯的摩尔比为1:1~10。
其中,所述的固定化脂肪酶由疏棉状嗜热丝孢菌脂肪酶、米黑根毛霉菌脂肪酶、南极假丝酵母脂肪酶B或南极假丝酵母脂肪酶A中的任意一种固定在载体上得到。
其中,所述的载体为硅胶、纺织制品或树脂。
其中,
所述硅胶优选为粒径5~20μm的硅胶颗粒;
所述树脂优选为粒径0.1~1mm的颗粒状树脂;
所述纺织制品优选为100目的尼龙86。
所述的固定化脂肪酶可以通过将脂肪酶通过吸附或化学键合固定在载体上制备得到,例如采用水相固定化法将脂肪酶吸附固定在载体上,具体操作为:将脂肪酶溶解在缓冲液中,加入载体,震荡或搅拌一定时间后,经抽滤、洗涤和干燥,获得固定化脂肪酶。
其中,所述的脂肪酶的酶活为250~10000U/g,脂肪酶酶活的定义为:在40℃下,在pH8.0的磷酸缓冲溶液中水解橄榄油,每分钟释放出1mmol脂肪酸所需的酶量为一个酶活单位。其中,脂肪酶和京尼平苷的质量比为0.5~5:1。
其中,所述的溶剂的油水分配系数LogP为-0.5~+4.0,优选正己烷,叔戊醇,四氢呋喃或丙酮。
其中,所述反应的反应温度为35~55℃,反应时间为20~60h。
其中,反应时,反应体系置于摇床上,摇床转速为150~200r/min。
其中,
离心方法为以2000~5000r/min的转速离心5~10min;
溶解方法为取离心所得上层液体,吹干溶剂后,所得油状物溶解于乙腈中;
柱纯化所用色谱柱为SSepax Bio-C18 10μm,150mm x 21.2mm。色谱条件为:流动相:乙腈-水(80:20)柱温:25℃进样量:1.5mL流速:5mL/min检测波长:210nm。
本发明的反应式如下:
有益效果:
与现有技术相比,本发明具有如下优势:本发明首次利用脂肪酶催化合成天然产物京尼平苷的衍生物C-6’-月桂酰基京尼平苷,相对于化学修饰法,本发明具有反应条件温和,催化位点选择性强,工艺步骤简单,环境友好等特点。同时,本发明使用的固定化脂肪酶,易与底物及产物分离,寿命长可重复使用进行连续化生产,降低了生产成本。本发明制备得到的产品C-6’-月桂酰基京尼平苷较京尼平苷脂溶性得到了较大的改善,应用前景更为广阔。
附图说明
图1为实施例3中制备得到的C-6’-月桂酰基京尼平苷的正离子质谱图;
图2为实施例3中制备得到的C-6’-月桂酰基京尼平苷的核磁氢谱图;
图3为实施例3中制备得到的C-6’-月桂酰基京尼平苷的核磁碳谱图。
具体实施方式
以下通过具体实施例对本发明技术进一步说明。应当理解,此处所描述的实施例仅用于说明和解释本发明,并不用于限定本发明。
下述实施例中,柱纯化所用色谱柱为SSepax Bio-C18 10μm,150mm x21.2mm。色谱条件为:流动相:乙腈-水(80:20)柱温:25℃进样量:1.5mL流速:5mL/min检测波长:210nm。
实施例1
将两种底物0.04g京尼平苷和0.1g月桂酸乙烯酯(摩尔比约1:4)加入到反应媒体10mL的四氢呋喃中,反应以0.1g固定在平均粒径为10μm的硅胶颗粒上的疏棉状嗜热丝孢菌脂肪酶为催化剂,酶活为250U/g。在温度40℃条件下,摇床震荡(200r/min),反应60小时。反应结束后将反应液离心分离出固定化酶,取上层液体吹干溶剂,得到的油状物用乙腈溶解,经反相制备柱纯化获得产物。产物转化率达61.3%。
将制得的C-6'-月桂酰基京尼平苷与京尼平苷进行脂溶性实验,两者在水/正辛醇相中的分配比见表1。
表1
实施例2
将两种底物0.04g京尼平苷和0.1g月桂酸乙烯酯(摩尔比约1:4)加入到反应媒体10mL的叔戊醇中,反应以0.1g固定化在平均粒径为500μm的大孔树脂上的南极假丝酵母脂肪酶B为催化剂,酶活为1000U/g。在温度40℃条件下,摇床震荡(200r/min),反应60小时。反应结束后将反应液离心分离出固定化酶,取上层液体吹干溶剂,得到的油状物用乙腈溶解,经反相制备柱纯化获得产物。反应转化率达53.2%。
实施例3
将两种底物0.04g京尼平苷和0.15g月桂酸乙烯酯(摩尔比约1:6)加入到反应媒体10mL的四氢呋喃中,反应以0.1g固定在平均粒径为5μm的硅胶颗粒上的疏棉状嗜热丝孢菌脂肪酶为催化剂,酶活为250U/g。在温度45℃条件下,摇床震荡(200r/min),反应60小时。反应结束后将反应液离心分离出固定化酶,取上层液体吹干溶剂,得到的油状物用乙腈溶解,经反相制备柱纯化获得产物。反应转化率达78.7%。
实施例4
将两种底物0.04g京尼平苷和0.1g月桂酸乙烯酯(摩尔比约1:4)加入到反应媒体10mL的正己烷中,反应以0.1g固定在平均粒径为10μm的硅胶颗粒上的疏棉状嗜热丝孢菌脂肪酶为催化剂,酶活为250U/g。在温度40℃条件下,摇床震荡(200r/min),反应30小时。反应结束后将反应液离心分离出固定化酶,取上层液体吹干溶剂,得到的油状物用乙腈溶解,经反相制备柱纯化获得产物。反应转化率达33.5%。
实施例5
将两种底物0.04g京尼平苷和0.1g月桂酸乙烯酯(摩尔比约1:4)加入到反应媒体10mL的四氢呋喃中,反应以0.2g固定化在平均粒径为1mm的大孔树脂上的南极假丝酵母脂肪酶A为催化剂,酶活为500U/g。在温度40℃条件下,摇床震荡(200r/min),反应60小时。反应结束后将反应液离心分离出固定化酶,取上层液体吹干溶剂,得到的油状物用乙腈溶解,经反相制备柱纯化获得产物。反应转化率达12.3%。
实施例6
将两种底物0.04g京尼平苷和0.1g月桂酸乙烯酯(摩尔比约1:4)加入到反应媒体10mL的丙酮中,反应以0.1g固定在平均粒径为5μm的硅胶颗粒上的疏棉状嗜热丝孢菌脂肪酶为催化剂,酶活为250U/g。在温度35℃条件下,摇床震荡(200r/min),反应60小时。反应结束后将反应液离心分离出固定化酶,取上层液体吹干溶剂,得到的油状物用乙腈溶解,经反相制备柱纯化获得产物。反应转化率达58.7%。
实施例7
将两种底物0.04g京尼平苷和0.1g月桂酸乙烯酯(摩尔比约1:4)加入到反应媒体10mL的四氢呋喃中,反应以0.1g固定在100目尼龙86上的米黑根毛霉菌脂肪酶为催化剂,酶活为3000U/g。在温度40℃条件下,摇床震荡(150r/min),反应60小时。反应结束后将反应液离心分离出固定化酶,取上层液体吹干溶剂,得到的油状物用乙腈溶解,经反相制备柱纯化获得产物。反应转化率达17.2%。
实施例8
脂肪酶的固定化:作为载体的D311离子交换树脂先用去离子水浸泡胀润,去杂,然后用4%NaOH和4%HCl交替在磁力搅拌器下搅拌浸泡2小时,用去离子水洗至中性。随后将预处理后的载体树脂真空抽滤后取1.0g放入三角烧瓶中,加入含有1.5mL南极假丝酵母脂肪酶B酶液的缓冲液(pH 10)5mL,恒温35℃,转速110rpm,在恒温振荡器中吸附固定10小时。固定完成后用去离子水洗涤,真空抽滤,干燥,得到固定化酶。
Claims (9)
1.一种利用脂肪酶催化合成C-6’-月桂酰基京尼平苷的方法,其特征在于,它包括如下步骤:
将京尼平苷和桂酸乙烯酯溶于溶剂中,以固定化脂肪酶为催化剂进行反应,反应液经离心、溶解和柱纯化后,即得产物。
2.根据权利要求1所述的合成方法,其特征在于,京尼平苷和桂酸乙烯酯的摩尔比为1:1~10。
3.根据权利要求1所述的合成方法,其特征在于,所述的固定化脂肪酶由疏棉状嗜热丝孢菌脂肪酶、米黑根毛霉菌脂肪酶、南极假丝酵母脂肪酶B或南极假丝酵母脂肪酶A中的任意一种固定在载体上得到。
4.根据权利要求3所述的合成方法,其特征在于,所述的载体为硅胶、纺织制品或树脂。
5.根据权利要求3所述的合成方法,其特征在于,所述的脂肪酶的酶活为250~10000U/g;其中,所述的脂肪酶酶活的定义为:在40℃下,在pH8.0的磷酸缓冲溶液中水解橄榄油,每分钟释放出1mmol脂肪酸所需的酶量为一个酶活单位。
6.根据权利要求1所述的合成方法,其特征在于,脂肪酶和京尼平苷的质量比为0.5~5:1。
7.根据权利要求1所述的合成方法,其特征在于,所述的溶剂的油水分配系数Log P为-0.5~+4.0。
8.根据权利要求1所述的合成方法,其特征在于,所述的溶剂为正己烷,叔戊醇,四氢呋喃或丙酮。
9.根据权利要求1所述的合成方法,其特征在于,所述反应的反应温度为35~55℃,反应时间为20~60h。
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