CN114763566A - 一种生物催化合成3-羟基-3-甲基丁酸的方法 - Google Patents
一种生物催化合成3-羟基-3-甲基丁酸的方法 Download PDFInfo
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Abstract
本发明提供一种生物催化合成3‑羟基‑3‑甲基丁酸的方法,以亮氨酸为底物,由多酶或含有多酶的细胞催化合成,所述多酶由L‑氨基酸脱氨酶和4‑羟基苯丙酮酸双加氧酶组成。本发明在大肠杆菌中过表达L‑AAD和4‑HPPD从而构建一种应用于HMB生产全细胞催化***,实现了以低成本亮氨酸为底物催化HMB的合成。本发明对获得的重组大肠杆菌进行高密度培养后,获得酶的最大表达,收集大肠杆菌菌体进行全细胞高效催化,反应结束后直接离心即可结束反应。该工艺有效地节约了生产HMB的成本,提高了催化体系的转换效率,操作简单,有很好的应用前景。
Description
技术领域
本发明涉及生物工程技术领域,尤其涉及生物催化合成3-羟基-3-甲基丁酸的方法。
背景技术
3-羟基-3-甲基丁酸(β-羟基-β-甲基丁酸,Beta-hydroxy-beta-methylbutyrate,HMB)是一种必需氨基酸亮氨酸的代谢产物,目前已广泛应用于动物饲料添加剂(JAnim.Sci,1994,72(9):2331–2337.)、人体运动营养剂(Nutr.Biochem.1997,8(6),300–311.)以及减肥食品中(Sports Med.2000,30,105–116.),并且早在2002年有文献报道,HMB有望在***等恶病中发挥良好的功效(Am J Surg,2002,183(4):471–479.)。2011年1月18日,***批准β-羟基-β-甲基丁酸钙可作为新资源食品(批准翅果油和β-羟基-β-甲基丁酸钙为新资源食品的公告.食品与发酵工业,2011,37(1):93–93.)。
HMB传统的生产方法一般采用以次氯酸钠(液氯和氢氧化钠)、二丙酮醇、盐酸、乙酸乙酯、乙醇为主要原料,经氧化合成、酸化、萃取、中和等步骤进行化学合成,该方法在工业生产中得到了广泛的应用(Lima D A,Mccoy L W,Miller M E.US 1996.)。然而,有机合成过程中含有较多挥发性有毒有机物,反应步骤较为复杂。相比化学合成方法,生物合成法更环保,对人体的危害更小。早在1997年,Lee等人尝试以β-甲基丁酸(MBA)为底物,Galactomyces reessii为细胞工厂生产HMB,通过G.reessii将β-甲基丁酸转化为β-羟基β甲基丁酸。136h后HMB的最大浓度为38g/L,发酵过程中摩尔转化率大于0.50mol HMB/molof MBA(J Ferment.Bioeng.1996.81:79–82.)。2002年,Ye等在Lee工作的基础上优化了G.reessii培养条件。在最佳条件下,102h时HMB的产率达到29.0g/L,平均产率为0.098g/(L·h),MBA摩尔转化率为57.3%,为当时报道的最高转换率和生产速率(华东理工大学学报,2002.28(6):601–605)。然而,微生物发酵法仍存在副反应、反应途径长、产物成分复杂、不易纯化等问题,使得最终产率和转化率较低。同时,底物MBA不仅价格昂贵,而且气味很难闻、挥发性强,易残留在环境中难以去除。因此,有必要开发一种经济、高效的HMB生产方法。
目前,酶和全细胞生物催化在绿色生物制造中更为常用,具有产率较高、底物特异性好以及易于纯化产物等优点。而与酶催化法相比,全细胞转化法具有屏蔽外界环境变化影响、提供辅酶再生体系、催化剂成本低等优点,并更有利于提高酶的稳定性和催化活性。因此,构建一个步骤短、副反应少、底物成本低的全细胞转化体系是本发明要实现的目标。
发明内容
本发明的第一个目的在于,提供一种以亮氨酸为底物经由α-酮异己酸生产HMB,底物亮氨酸价格低廉且反应过程均无有害物质的产生的生物催化合成3-羟基-3-甲基丁酸的方法。
本发明的第二个目的在于,提供一种合成3-羟基-3-甲基丁酸的重组大肠杆菌。
本发明的第三个目的在于,提供利用重组大肠杆菌合成3-羟基-3-甲基丁酸的方法。
为了实现上述第一个目的,本发明提供了一种生物催化合成3-羟基-3-甲基丁酸的方法,以亮氨酸为底物,由多酶或含有多酶的细胞催化合成,所述多酶由L-氨基酸脱氨酶和4-羟基苯丙酮酸双加氧酶组成。
采用来源于Proteus vulgaris的L-氨基酸脱氨酶(L-amino acid deaminase,L-AAD)和来源于Rattus norvegicus的4-羟基苯丙酮酸双加氧酶(4-hydroxyphenylpyruvatedioxygenase,4-HPPD)首次实现了以亮氨酸为底物经由α-酮异己酸生产HMB,蛋白L-AAD、4-HPPD的基因序列如SEQ ID NO:1和SEQ ID NO:2所示。
作为一个优选方案,本发明提供了一种提高4-HPPD活性的突变体,是将SEQ IDNO:2所示DNA序列中第268–270位的GGC(对应氨基酸为甘氨酸)突变成CCG(对应氨基酸为丙氨酸),该突变体命名为4HPPD-1,其DNA序列如SEQ ID NO:3所示。
为了实现上述第二个目的,本发明提供了一种生成3-羟基-3-甲基丁酸的重组大肠杆菌,将L-AAD和4-HPPD这两个基因片段利用现有技术分别整合到质粒载体上,转化获得重组大肠杆菌。
作为一个优选方案,所述4-羟基苯丙酮酸双加氧酶经优化,优化后的基因序列如SEQ ID NO:3所示。将L-AAD和4HPPD-1这两个基因片段利用现有技术分别整合到质粒载体上,转化获得重组大肠杆菌。
本发明的一种实施方式中,将L-AAD、4-HPPD、4HPPD-1通过同源重组的方法分别连接到pET28a质粒中,并将基因过表达。
为了实现上述第三个目的,本发明提供了利用上述重组大肠杆菌合成3-羟基-3-甲基丁酸的方法,以亮氨酸为底物经过两步酶反应合成3-羟基-3-甲基丁酸。以亮氨酸为底物经过两步酶反应合成3-羟基-3-甲基丁酸,亮氨酸首先经L-AAD催化转化为α-酮异己酸,再经4-HPPD催化转化为3-羟基3-甲基丁酸。底物亮氨酸价格低廉且反应过程均无有害物质的产生,可以有效地解决HMB合成过程中的底物价格贵、环境污染、转化率低等问题。
作为一个优选方案,反应温度为30–45℃,pH为4.0–8.0。本发明的优选实施例中,催化反应的底物浓度为L-亮氨酸5–25mM、亚铁离子盐0.1–5mM、二硫苏糖醇0.1–5mM。亚铁离子盐可以为FeCl2、FeSO4等催化反应常用亚铁离子盐。
本发明的优点在于,本发明在大肠杆菌中通过质粒分别过表达L-AAD和4-HPPD从而构建一种应用于HMB生产全细胞催化***,实现了以低成本亮氨酸为底物催化HMB的产生,同时通过理性设计获得了活性提高的突变体4HPPD-1。本发明对获得的重组大肠杆菌进行高密度培养后,获得两个酶的最大表达,收集大肠杆菌菌体进行全细胞高效催化,反应结束后直接离心即可结束反应。该工艺有效地节约了生产HMB的成本,提高了催化体系的转换效率,操作简单,有很好的应用前景。
附图说明
图1为生物催化路径设计。
图2为大肠杆菌Rosetta(DE3)中分别过表达L-AAD、4-HPPD。
图3为L-AAD与4-HPPD在最优条件下生产HMB。
图4为理性设计后获得活性提高突变体4HPPD-1。
图5为L-AAD与突变体4HPPD-1在最优条件下生产HMB。
具体实施方式
下面结合具体的相关实施例,对本发明实施例中的技术方案进行清楚、完整地描述。但是所描述的实施例仅为本发明的一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域的普通技术人员在未做出创造性劳动的前提下所获得的所有其它实施例都属于本发明的保护范围。
下述实施例中所使用的实验方法,如无特殊说明均为常规方法,所用的材料、试剂等,如无特殊说明均可从商业途径得到。
实施例1.将L-AAD、4-HPPD分别构建重组表达质粒以及工程菌
选取NCBI网站筛选到的L-AAD和4-HPPD,(基因序列由上海生物工程有限公司合成;4HPPD-1序列以4-HPPD基因序列为模板,4HPPD1F、4HPPD1R为引物扩增得到),分别将基因序列通过同源重组的方式连接到pET28a载体上,将质粒分别转入大肠杆菌Rossetta(DE3),得到的阳性克隆培养至OD600为0.6–0.8,加入终浓度为0.2mM的IPTG,18℃,220rpm诱导培养16h。对诱导后的细胞进行破碎,并用SDS-PAGE分析,结果如图2所示.其中1号泳道为protein maker;2号泳道为Proteus vulgaris来源L-氨基酸脱氨酶;3号泳道为Rattusnorvegicus来源4-羟基苯丙酮酸双加氧酶。具体的引物序列见表1,序列送上海生物工程有限公司合成。
表1
线性化载体的准备:以pET28a为模板,分别以pET28a-F和pET28a-R为上下游引物,进行PCR扩增。反应体系为:模板2μl;Prime STAR Max 25μl;正反向引物各20mM;加无菌水至总体积为50μl。反应条件为:98℃预变性5min;98℃变性10s;60℃退火30s;72℃延伸4min,共30个循环;结束后,72℃终延伸10min。扩增得到的产物经核酸电泳跑胶验证后通过PCR Purification Kit试剂盒纯化,随后通过Dpn I消化模板质粒。
DNA重组片段的准备:分别以上海生工公司合成的质粒pTrc99a-LAAD、pTrc99a-4HPPD为模板,以Table 1中所对应的上下游引物进行扩增。反应体系同上,反应条件为:98℃预变性5min;98℃变性10s;60℃退火30s;72℃延伸5min,共30个循环;结束后,72℃终延伸10min。以上述相同方法对产物进行回收。
将线性化载体与目的片段按浓度比1:3混合,加水补齐至10μl,在向其中加入10μl的HB-infusion,于50℃连接30–40min,随后转化至Rossetta(DE3)感受态细胞,涂布与LB平板上(含50μg/ml的Kanamycin),筛选出阳性转化子,并分别提取出重组质粒,送上海杰李生物技术有限公司测序验证。
LB培养基:10g/L胰蛋白胨、5g/L酵母提取物、10g/L氯化钠,pH 7.2,121℃高温灭菌20min。(固体培养基另加20g/L琼脂粉)。
HMB检测方法:采用C18色谱柱(4.6×150mm,Wondasil,shimadu-gl)高效液相色谱法检测反应液中HMB的含量。流动相由磷酸氢二铵(0.01mM,pH2.50)和甲醇组成(90:10,V/V),流速0.6mL/min。柱温维持在35℃,检测波长设定在203nm。样品首先用2M盐酸稀释,然后过滤,柱上样量为10μL。使用Dionex Chromeleon软件对数据进行分析。
实施例2.L-AAD、4-HPPD以亮氨酸为底物生产HMB的验证
以亮氨酸为底物生产HMB时,亮氨酸首先转化为α-酮异己酸,再转化为HMB。我们将第一步反应的酶确定为来源于普通变形杆菌的L-AAD。4-HPPD是体内酪氨酸代谢过程中的重要酶,几乎存在于所有需氧生物中。N.P.Crouch等人首次提出,位于小鼠肝脏中的4-HPPD可能具有α-酮异己酸氧化酶(α-ketoisocaproate dioxygense,α-KICD)的功能(Tetrahedron,1997,53(20):6993–7010.)。随后于2000年,N.P.Crouch等人通过测序以及N端胰蛋白酶消化等方法确定了4-HPPD确实与α-KICD为同一种酶(Method Enzymol,2000,324:342–355.),这表示4-HPPD可以催化α-KIC转化为HMB,因此我们选择了来源小鼠的4-HPPD酶作为第二步反应的催化剂。反应路径如图2所示。
首先在含有Tris-Maleic acid(缓冲液浓度:20-200mM,pH 4–6.5)的1.2mL体系中,加入25mM L-亮氨酸、亚铁离子盐(0.3–1.6mM)、菌体液(即酶液,30–55OD600),在20–45℃、140–300rpm下反应12h,以验证反应的可行性。每3h取一次样,加入2M盐酸终止反应,然后12000rpm离心10min。离心取上清,使用HPLC检测HMB含量,反应结果如图3所示,证明该路径可行且能够连续反应。
实施例3.L-AAD和4HPPD-1以亮氨酸为底物生产HMB
为进一步提高反应速率,我们对4-HPPD的底物进出通道进行理性设计,即将SEQID NO:2所示DNA序列中第268–270位的GGC(对应氨基酸为甘氨酸)突变成CCG(对应氨基酸为丙氨酸),并将该突变体命名为4HPPD-1,序列如SEQ ID NO:3所示。在下述反应体系中对其进行催化活力测定。即在含有Tris-Maleic acid(缓冲液浓度:60mM,pH 5.0)的1.2mL体系中,加入25mM L-亮氨酸、亚铁离子盐(1.0mM)、菌体液(即酶液,45OD600),在35℃、220rpm下反应45min,加入2M盐酸终止反应,然后12000rpm离心10min。离心取上清,使用HPLC检测HMB含量,反应结果显示突变体活性提高了20%,如图4所示。使用突变体在最优条件下生产HMB,即在含有Tris-Maleic acid(缓冲液浓度:60mM,pH 5.0)的5mL体系中,加入25mM L-亮氨酸、亚铁离子盐(1.0mM)、菌体液(即酶液L-AAD=35OD600,4HPPD-1=45OD600),在35℃、220rpm下反应12h,每3h取一次样,加入2M盐酸终止反应,然后12000rpm离心10min。离心取上清,使用HPLC检测HMB含量,最终转化率约为86%(图5)。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 华东理工大学
<120> 一种生物催化合成3-羟基-3-甲基丁酸的方法
<130> /
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Claims (6)
1.一种生物催化合成3-羟基-3-甲基丁酸的方法,其特征在于,以亮氨酸为底物,由多酶或含有多酶的细胞催化合成,所述多酶由L-氨基酸脱氨酶和4-羟基苯丙酮酸双加氧酶组成。
2.根据权利要求1所述的一种生物催化合成3-羟基-3-甲基丁酸的方法,其特征在于,所述4-羟基苯丙酮酸双加氧酶经优化,优化后的基因序列如SEQ ID NO:3所示。
3.一种合成3-羟基-3-甲基丁酸的重组大肠杆菌,其特征在于,将L-氨基酸脱氨酶和4-羟基苯丙酮酸双加氧酶的基因片段分别整合到质粒载体上,转化获得重组大肠杆菌。
4.根据权利要求3所述的一种合成3-羟基-3-甲基丁酸的重组大肠杆菌,其特征在于,所述4-羟基苯丙酮酸双加氧酶经优化,优化后的基因序列如SEQ ID NO:3所示。
5.利用权利要求3或4所述重组大肠杆菌合成3-羟基-3-甲基丁酸的方法,其特征在于,以亮氨酸为底物经过两步酶反应合成3-羟基-3-甲基丁酸。
6.根据权利要求5所述的利用重组大肠杆菌合成3-羟基-3-甲基丁酸的方法,其特征在于,反应温度为30–45℃,pH为4.0–8.0。
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102348802A (zh) * | 2009-01-22 | 2012-02-08 | 先正达参股股份有限公司 | 突变体羟基苯丙酮酸双加氧酶多肽及其使用方法 |
CN109504645A (zh) * | 2018-12-27 | 2019-03-22 | 华东理工大学 | 异亮氨酸双加氧酶、突变体及在合成4-羟基异亮氨酸中的应用 |
-
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102348802A (zh) * | 2009-01-22 | 2012-02-08 | 先正达参股股份有限公司 | 突变体羟基苯丙酮酸双加氧酶多肽及其使用方法 |
CN109504645A (zh) * | 2018-12-27 | 2019-03-22 | 华东理工大学 | 异亮氨酸双加氧酶、突变体及在合成4-羟基异亮氨酸中的应用 |
Non-Patent Citations (3)
Title |
---|
NICHOLAS P. CROUCH ET AL: "Cloning, Expression, and Purification of Mammalian 4-Hydroxyphenylpyruvate Dioxygenase /a-Ketoisocaproate Dioxygenase", 《METHODS IN ENZYMOLOGY》, vol. 324, pages 343 * |
RUICHEN GAO ET AL: "Biosynthesis of 3‑Hydroxy-3-Methylbutyrate from L‑Leucine by Whole-Cell Catalysis", 《J. AGRIC. FOOD CHEM.》, vol. 69 * |
宋阳等: "L‐氨基酸脱氨酶全细胞转化亮氨酸生产α-酮异己酸", 《中国生物工程学会2014年学术年会暨全国生物技术大会》, pages 3 - 1 * |
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