CN114214376B - 一种利用全细胞转化合成l-果糖的方法 - Google Patents
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Abstract
本发明涉及生物技术领域,尤其涉及一种利用全细胞转化合成L‑果糖的方法,方法中反应体系包括底物甘油、L‑甘油醛、焦磷酸,经过诱导后的重组大肠杆菌的湿菌体,所述反应体系经过催化反应后即可得到L‑果糖。利用该方法可提高L‑果糖的产率。
Description
技术领域
本发明涉及生物技术领域,尤其涉及一种利用全细胞转化合成L-果糖的方法。
背景技术
稀有己酮糖是一类在自然界中存在但含量很低、同时具有重要生理功能的一类单糖及其衍生物。L-构型包括L-山梨糖、L-果糖和L-塔格糖等,具有低热量、天然甜味等优势,可作为功能性甜味剂,同时具有抗癌、抗肥胖、神经保护、清除自由基等生理活性,在生命医药、化妆品、膳食等领域拥有广泛的应用前景。因此,完善稀有糖合成体系具有重要意义。
生产L型稀有己酮糖的方法有化学合成法和生物转化法两种。传统的化学合成法步骤繁琐,副反应较多,且很难得到单一构型的产物。相对于化学合成法,酶法因其反应条件温和,高效和立体选择性好而逐渐成为该领域的主流。迄今为止,许多酶促反应依赖于两种或多种糖之间通过异构化或差向异构化来获得各种稀有己酮糖(如Izumoring方法)。但是由于涉及热力学平衡,转化率较低,同时产品难以分离与纯化。因此,亟待开发一种低成本,低污染,高产率的适合规模化生产稀有己酮糖的新方法。
发明内容
本发明所要解决的技术问题是:针对现有技术存在的不足,提供一种利用全细胞转化合成L-果糖的方法,利用该方法可提高L-果糖的产率。
为解决上述技术问题,本发明的技术方案是:
一种利用全细胞转化合成L-果糖的方法,所述方法中反应体系包括底物甘油、L-甘油醛、焦磷酸,经过诱导后的重组大肠杆菌的湿菌体,所述反应体系经过催化反应后即可得到L-果糖。
作为一种改进的技术方案,催化反应时整个反应体系的甘油浓度为300-700mM,L-甘油醛浓度为10-50mM,焦磷酸浓度为10-100mM,所述反应体系的pH为4.5-8,反应温度为25-45℃,反应时间6-20h,所述重组大肠杆菌湿菌体的终浓度为50-250g/L。
作为一种优选的技术方案,催化反应时整个反应体系的甘油浓度为500mM,L-甘油醛浓度为40mM,焦磷酸浓度为80mM,pH为7.0,反应温度为30℃,反应时间为12h,所述重组大肠杆菌湿菌体终浓度为100g/L。
作为一种改进的技术方案,所述重组大肠杆菌为同时表达酸性磷酸酶PhoN、磷酸甘油氧化酶GPO和L-鼠李树胶糖-1-磷酸醛缩酶RhaD的重组大肠杆菌。
作为一种改进的技术方案,所述重组大肠杆菌的构建方法为:将编码L-鼠李树胶糖-1-磷酸醛缩酶RhaD的基因rhaD(来源于大肠杆菌MG1655)、磷酸甘油氧化酶GPO的基因glpO(来源于Streptococcus pneumoniae)和酸性磷酸酶PhoN的基因phoN(来源于Shigellaflexneri),依次***到pET28a质粒,得到重组质粒pET28a-rhaD-glpO-phoN,再将重组质粒pET28a-rhaD-glpO-phoN转化到大肠杆菌BL21(DE3)中,得到重组大肠杆菌。
作为一种改进的技术方案,所述重组大肠杆菌在LB培养基中37℃培养,OD600为0.8时,加入终浓度为0.1mM IPTG,16℃诱导20h,收集得到湿菌体。
采用了上述技术方案后,本发明的有益效果是:
(1)本发明直接采用全细胞作为催化剂不需要对酶进行纯化,原料价格低廉,且不用纯化酶和外加辅因子,有利于工业化生产;
(2)本发明制备方法不用过量表达甘油激酶,不需要外加ATP进行底物磷酸化生产甘油3-磷酸,大大降低生产L-果糖的成本;
(3)本发明以甘油为底物,加入L-甘油醛、焦磷酸以及重组大肠杆菌(可同时表达酸性磷酸酶PhoN、磷酸甘油氧化酶GPO和L-鼠李树胶糖-1-磷酸醛缩酶RhaD)经过诱导后的湿菌体,进行催化反应,通过该工艺可以大大提高L-果糖的转化率。
附图说明
附图1为实施例1条件下反应产物L-果糖的HPLC图;
附图2为L-鼠李树胶糖-1-磷酸醛缩酶RhaD、甘油磷酸氧化酶GPO、酸性磷酸酶PhoN的表达的SDS-PAGE凝胶电泳图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
实施例1
一种利用全细胞转化合成L-果糖的方法,以甘油为底物(甘油的浓度为500mM),加入L-甘油醛(加入量为40mM)、焦磷酸(加入量为80mM)以及重组大肠杆菌经过诱导后的湿菌体(湿菌体反应体系的终浓度为100g/L),调pH至7.0,控制反应温度为30℃,经过12h的催化反应(其中上述整个反应体系为10mL),反应结束后离心10min收集上清,即得到含有L-果糖的溶液。
其中重组大肠杆菌为同时表达酸性磷酸酶PhoN、磷酸甘油氧化酶GPO和L-鼠李树胶糖-1-磷酸醛缩酶RhaD的重组大肠杆菌。
湿菌体的制备:
1)重组质粒pET28a-rhaD的构建:以大肠杆菌MG1655的基因组为模板,PCR扩增得到rhaD基因片段,将其与pET28a载体采用BamHI和EcoRI双酶切,通过割胶回收、连接、转化、测序等步骤获得pET28a-rhaD;
2)重组质粒pET28a-rhaD-glpO的构建:基因glpO由天霖生物科技无锡有限公司合成,以上述合成的glpO为模板,PCR扩增得到含有RBS序列的glpO基因片段将其与pET28a-rhaD载体采用SacI和HindIII双酶切,通过割胶回收、连接、转化、测序等步骤获得pET28a-rhaD-glpO;
3)重组质粒pET28a-rhaD-glpO-phoN的构建:基因phoN由天霖生物科技无锡有限公司合成,以上述合成的phoN为模板,PCR扩增得到含有RBS序列的phoN基因片段将其与pET28a-rhaD-glpO载体采用HindIII和XhoI双酶切,通过割胶回收、连接、转化、测序等步骤获得pET28a-rhaD-glpO-phoN;
4)重组质粒pET28a-rhaD-glpO-phoN的转化:从-80℃取BL21(DE3)感受态细胞置于冰上融化,将重组质粒与感受态细胞充分混合后冰浴25min,然后于42℃热激90s,加入1mL LB培养基于37℃预培养1h,涂布于LB卡那霉素的平板上。
5)重组蛋白酸性磷酸酶PhoN、磷酸甘油氧化酶GPO和L-鼠李树胶糖-1-磷酸醛缩酶RhaD的诱导表达:将重组大肠杆菌在LB培养基中37℃培养,至OD600为0.8时,加入终浓度为0.1mM IPTG,16℃诱导20h,收集得到湿菌体。
本发明反应体系的整个反应过程:
通过高效液相色谱(HPLC)检测底物甘油的消耗和产物L-果糖的产生,并计算转化率。
HPLC检测条件如下:Waters 2695(USA),检测器:示差折光检测器(RI),色谱柱:Bio-Rad有机酸柱(Aminex HPX-87H,300×7.8mm),流动相:5mM H2SO4溶液,流速:5mL/min,柱温:60℃,进样量:20μL。其中甘油标准品的保留时间为15.6min,L-果糖标准品的保留时间为11.8min(图1)。
实施例2
与实施例1的不同之处在于:反应的温度为25℃。
实施例3
与实施例1的不同之处在于:反应的温度为45℃。
实施例4
与实施例1不同之处在于:反应体系的pH为4.5。
实施例5
与实施例1不同之处在于:反应体系的pH为8.0。
实施例6
一种利用全细胞转化合成L-果糖的方法,以甘油为底物(甘油的浓度为300mM),加入L-甘油醛(加入量为10mM)、焦磷酸(加入量为10mM)以及重组大肠杆菌经过诱导后的湿菌体(湿菌体的终浓度为50g/L),调pH至7.0,控制反应温度为30℃,经过12h的催化反应(其中上述整个反应体系为10mL),反应结束后离心10min收集上清,即得到含有L-果糖的溶液。
实施例7
一种利用全细胞转化合成L-果糖的方法,以甘油为底物(甘油的浓度为700mM),加入L-甘油醛(加入量为50mM)、焦磷酸(加入量为100mM)以及重组大肠杆菌经过诱导后的湿菌体(湿菌体的终浓度为250g/L),调pH至7,控制反应温度为30℃,经过12h的催化反应(其中上述整个反应体系为10mL),反应结束后离心10min收集上清,即得到含有L-果糖的溶液。
为了更好的证明利用本发明的方法可以提高L-果糖的转化率,本发明同时做了6个对比例,其中对比例1、对比例2、对比例3、对比例4和对比例5分别以实施例1为参照进行对比。
对比例1
与实施例1不同的时,反应体系中不添加焦磷酸。
对比例2
与实施例1操作不同的是,反应体系的温度控制在35℃。
对比例3
与实施例1操作不同的是,反应体系的温度控制在40℃
对比例4
与实施例1操作不同的是,反应体系的pH控制在5.0。
对比例5
与实施例1操作不同的是,反应体系的pH控制在6.0。
对比例6
现有技术将假单胞菌来源的D-塔格糖-3-差向异构酶(D-TE)固定在chitopearlbeads上,采用固定化酶来催化L-阿洛酮糖转化得到L-果糖,转化率为65%(Journal ofFermentation and Bioengineering 1996,81,351-353),具体反应体系(100mL)如下:2%L-阿洛酮糖、D-TE固定化酶(32U,湿重为2g)反应pH为7.5,42℃反应7天。然而该方法需要纯化酶,反应时间高达7天且L-阿洛酮糖本身是稀有糖,成本较高。
本发明实施例1-7以及对比例1-5中L-果糖的转化率分别见下表1。
表1
实施例 | 底物浓度 | 湿菌体浓度 | L-甘油醛浓度 | 温度 | 焦磷酸 | pH | 转化率 |
实施例1 | 500mM | 100g/l | 40mM | 30℃ | 80mM | 7.0 | 93% |
实施例2 | 500mM | 100g/l | 40mM | 25℃ | 80mM | 7.0 | 90% |
实施例3 | 500mM | 100g/l | 40mM | 45℃ | 80mM | 7.0 | 85% |
实施例4 | 500mM | 100g/l | 40mM | 30℃ | 80mM | 4.5 | 80% |
实施例5 | 500mM | 100g/l | 40mM | 30℃ | 80mM | 8.0 | 90% |
实施例6 | 300mM | 50g/l | 10mM | 30℃ | 10mM | 7.0 | 82% |
实施例7 | 700mM | 250g/l | 50mM | 30℃ | 100mM | 7.0 | 90% |
对比例1 | 500mM | 100g/l | 40mM | 30℃ | 0 | 7.0 | 40% |
对比例2 | 500mM | 100g/l | 40mM | 35℃ | 80mM | 7.0 | 88% |
对比例3 | 500mM | 100g/l | 40mM | 40℃ | 80mM | 7.0 | 86% |
对比例4 | 500mM | 100g/l | 40mM | 30℃ | 80mM | 5.0 | 80% |
对比例5 | 500mM | 100g/l | 40mM | 30℃ | 80mM | 6.0 | 85% |
通过表1数据可以发现,本发明以甘油为底物,加入L-甘油醛、焦磷酸以及重组大肠杆菌(可同时表达酸性磷酸酶PhoN、磷酸甘油氧化酶GPO和L-鼠李树胶糖-1-磷酸醛缩酶RhaD)经过诱导后的湿菌体,进行催化反应,通过该工艺可以大大提高L-果糖的转化率。该工艺与现有技术(对比例6中的工艺方法)以及对比例1-5相比较,可以大大提高L-果糖的转化率,大大节约了成本。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (4)
1.一种利用全细胞转化合成L-果糖的方法,其特征在于:所述方法中反应体系包括底物甘油、L-甘油醛、焦磷酸,经过诱导后的重组大肠杆菌的湿菌体,所述反应体系经过催化反应后即可得到L-果糖;
催化反应时整个反应体系的甘油浓度为300-700mM,L-甘油醛浓度为10-50mM,焦磷酸浓度为10-100mM,所述反应体系的pH为4.5-8,反应温度为25-45℃,反应时间6-20h,所述重组大肠杆菌湿菌体的终浓度为50-250g/L;
所述重组大肠杆菌为同时表达酸性磷酸酶PhoN、磷酸甘油氧化酶GPO和L-鼠李树胶糖-1-磷酸醛缩酶RhaD的重组大肠杆菌。
2.根据权利要求1所述的一种利用全细胞转化合成L-果糖的方法,其特征在于:催化反应时整个反应体系的甘油浓度为500mM,L-甘油醛浓度为40mM,焦磷酸浓度为80mM,pH为7.0,反应温度为30℃,反应时间为12h,所述重组大肠杆菌湿菌体终浓度为100g/L。
3.根据权利要求1所述的一种利用全细胞转化合成L-果糖的方法,其特征在于:所述重组大肠杆菌的构建方法为:将编码L-鼠李树胶糖-1-磷酸醛缩酶RhaD的基因rhaD、磷酸甘油氧化酶GPO的基因glpO和酸性磷酸酶PhoN的基因phoN,依次***到pET28a质粒,得到重组质粒pET28a-rhaD-glpO-phoN,再将重组质粒pET28a-rhaD-glpO-phoN转化到大肠杆菌BL21(DE3)中,得到重组大肠杆菌。
4.根据权利要求3所述的一种利用全细胞转化合成L-果糖的方法,其特征在于:所述重组大肠杆菌在LB培养基中37℃培养,OD600为0.8时,加入终浓度为0.1mM IPTG,16℃诱导20h,收集得到湿菌体。
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