WO2020186768A1 - 一种gdsl脂肪酶、基因工程菌及其应用 - Google Patents

一种gdsl脂肪酶、基因工程菌及其应用 Download PDF

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WO2020186768A1
WO2020186768A1 PCT/CN2019/114789 CN2019114789W WO2020186768A1 WO 2020186768 A1 WO2020186768 A1 WO 2020186768A1 CN 2019114789 W CN2019114789 W CN 2019114789W WO 2020186768 A1 WO2020186768 A1 WO 2020186768A1
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engineered bacteria
vitamin
lipase
olive oil
gdsl
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French (fr)
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王立梅
徐田甜
季亚美
齐斌
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常熟理工学院
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Priority to SG11202103103TA priority Critical patent/SG11202103103TA/en
Priority to JP2021521961A priority patent/JP7162374B2/ja
Priority to US17/279,733 priority patent/US20220033789A1/en
Publication of WO2020186768A1 publication Critical patent/WO2020186768A1/zh
Priority to US18/341,792 priority patent/US20240011001A1/en

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    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
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    • C12R2001/185—Escherichia
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  • the invention relates to the fields of genetic engineering and protein engineering, relates to a method for gene cloning and expression of a novel GDSL lipase and the application of enzymatic production of vitamin A palmitate, and belongs to the technical field of industrial microorganisms.
  • Lipase also known as acylglycerol hydrolase
  • acylglycerol hydrolase is a class of enzymes that can degrade natural oils into glycerol and free fatty acids. It is widely present in animals, plants and microorganisms. Microorganisms are an important source of lipase, including Rhizopus, Aspergillus and Candida, etc.
  • lipases can be divided into 8 families, of which the second family, also known as the GDSL family.
  • GDSL type lipase (lipase, EC 3.1.1.3) is a type of hydrolase that can hydrolyze various substrates such as thioesters, aryl esters, phospholipids and amino acids. Since GDSL lipase is a new type of lipase, there is little research on its expression and function. Due to the ester hydrolysis activity of GDSL lipase, people have been studying it more and more deeply.
  • Vitamin A palmitate can help maintain normal visual function and participate in various metabolic activities to maintain the health of the organism. It is currently the most commonly used vitamin A derivative and is widely used in food, cosmetics, medicine and other industries. At present, the synthesis of vitamin A palmitate mainly includes chemical methods and enzymatic methods. The chemical method of synthesizing vitamin A palmitate has environmental pollution and cost problems, while the enzymatic method has less pollution, high space-time yield and low cost. The enzyme producing vitamin A palmitate is a lipase with ester hydrolysis activity.
  • the purpose of the present invention is to provide a GDSL lipase, engineered bacteria and applications thereof.
  • the lipase has high activity and can be applied to the production of vitamin A palmitate.
  • a GDSL lipase whose amino acid sequence is shown in SEQ ID NO.2.
  • the present invention also provides a gene encoding the above-mentioned GDSL lipase.
  • nucleotide sequence of the gene encoding GDSL lipase is shown in SEQ ID NO.1.
  • GDSL lipase is derived from Streptomyces diastaticus CS1801, which has been disclosed in the applicant's prior application CN109337843A.
  • the present invention also provides a recombinant vector comprising a gene encoding GDSL lipase and an expression vector, the nucleotide sequence of the gene is shown in SEQ ID NO.1.
  • the expression vector is pET-32a(+).
  • the above-mentioned genes are inserted between the multiple cloning sites of the expression vector pET-32a(+).
  • the invention also provides a genetically engineered strain containing the above-mentioned recombinant vector
  • the host cell of the engineered bacteria is E. coli BL21 (DE3).
  • the present invention also provides the application of the above-mentioned engineered bacteria in the enzymatic production of vitamin A palmitate, including:
  • the method includes: inserting engineered bacteria cultured in LB medium at 37° C. and 200 r for 8-12 hours into the fermentation medium with an inoculum of 5%. After 8-12 hours of fermentation, IPTG was added to a final concentration of 0.4-1mmol/L, and fermentation was carried out at 37°C and 200r for 18-24 hours. 4000r, 10min, centrifuge to get the supernatant of the fermentation broth. The enzyme protein was precipitated with 50% ammonium sulfate solution, and the enzyme powder was obtained after freeze-drying for 48 hours.
  • fermentation medium components are:
  • the olive oil emulsifier configuration method is as follows: the olive oil emulsifier PVA is mixed with olive oil in a volume ratio of 3:1, and ultrasonic emulsification is used.
  • the genetically engineered bacteria constructed by the enzyme of the present invention is used for enzymatic production of vitamin A palmitate, and the maximum content of vitamin A palmitate obtained by the conversion is 16.35 mg/L. The highest conversion efficiency is 81.75%.
  • the lipase provides a new way for enzymatic synthesis of vitamin A palmitate and has important application prospects.
  • Figure 1 shows the target band amplified by GDSL lipase PCR
  • Figure 2 is an SDS-PAGE electrophoresis diagram of Escherichia coli
  • Figure 3 shows the conversion time of GDSL lipase to produce vitamin A palmitate.
  • This example illustrates the PCR amplification method of the GDSL lipase gene derived from Streptomyces amylase.
  • the primer design for GDSL lipase gene amplification is:
  • the specific amplification procedure is as follows, pre-denaturation at 95°C for 10 min, and Taq enzyme added. Denaturation at 95°C for 1 min, annealing temperature at 55°C, annealing time at 30s, extension temperature at 72°C, and extension time at 1 min. This process is carried out for 29 cycles, and finally at 72°C for 30 min. Take the product for agarose gel electrophoresis, cut the gel and recover the target band and store it. The gel recovery kit was purchased from Shenggong.
  • This example illustrates the PCR amplification method of GDSL lipase gene with restriction site.
  • the primers for GDSL lipase gene amplification with restriction sites are designed as:
  • Example 2 Using the gel recovery product in Example 1 as a template, the above primers and Shanghai Shenggong's high GC content PCR amplification kit were used for amplification.
  • the specific amplification procedure is as follows, pre-denaturation at 95°C for 2 min. Denaturation at 95°C for 1 min, annealing temperature at 55°C, annealing time at 30s, extension temperature at 72°C, and extension time at 1 min. This process is carried out for 29 cycles, and finally at 72°C for 30 min. Take the product and perform agarose gel electrophoresis.
  • the target band is shown in Figure 1. The target band is cut and recovered and sent to the laboratory for sequencing to obtain the sequence SEQ ID NO.1.
  • This example illustrates the construction method of the recombinant cloning vector of GDSL lipase.
  • the gel recovery product in Example 2 was connected with the T vector, and after transformation into DH-5 ⁇ , positive clones were picked for verification, and the plasmid was extracted and sequenced for verification.
  • This example illustrates the method for constructing a recombinant expression vector of GDSL lipase.
  • the plasmid in Example 3 was double-enzyme-cut with XhoI and EcoRI and the target band was recovered.
  • the pET32a(+) vector was double-enzyme-cut with XhoI and EcoRI to recover the larger fragments in the vector, and the recovered target gene fragments After ligation with the vector fragment, it was introduced into the host cell E. coli DH5 ⁇ , and after resistance screening, positive clones were selected for sequencing verification.
  • This example illustrates the construction method of GDSL lipase genetically engineered bacteria.
  • the plasmid of the positive clone with correct sequencing in Example 4 was extracted and directly transformed into the host cell Escherichia coli BL21 (DE3).
  • the GDSL lipase genetic engineering strain was successfully constructed. In the fermentation process, an inducer such as IPTG must be added to induce its high efficiency. Express GDSL lipase protein. The successful expression of the fusion protein was verified by SDS-PAGE. The SDS-PAGE electrophoresis chart is shown in Figure 2. Lane 1 is uninduced E. coli pET32a-GDSL, Lane 2, 3, and 4 are recombinant E. coli pET32a-induced by IPTG for 4, 8, and 16 hours, respectively.
  • GDSL Compared with other lanes, GDSL has a clear band at a molecular weight of 44kDa. The protein fusion expressed on the plasmid is removed, which is consistent with the predicted target protein size, indicating that the GDSL lipase was successfully expressed in the recombinant bacteria.
  • This example illustrates the application of GDSL lipase genetically engineered bacteria in the production of vitamin A palmitate.
  • the genetically engineered bacteria cultured in LB medium at 37°C, 200r, for 8-12 hours are connected to the fermentation medium with an inoculum of 5%.
  • IPTG was added to a final concentration of 0.4-1mmol/L, and the fermentation was cultured at 37°C and 200r for 18-24 hours.
  • the GDSL lipase enzyme activity was determined to be 1.53 U/mg.
  • the components of the fermentation medium are:
  • the preparation method of the olive oil emulsion is as follows: the olive oil emulsifier PVA is mixed with olive oil in a volume ratio of 3:1, and ultrasonic emulsification is used.
  • the described method for determination of vitamin A palmitate is: high performance liquid method, which adopts external standard method for quantification.
  • the chromatographic conditions are: chromatographic column: Alltech C18 (250x4.6mm, 4.5 ⁇ m); mobile phase: 100% methanol; detector: Shimadzu 10A UV detector; detection wavelength: 327nm; flow rate: 1mL/min.
  • the conversion rate calculation formula is:

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Abstract

提供了一种GDSL脂肪酶、基因工程菌及其在生产维生素A棕榈酸酯中的应用，所述的GDSL脂肪酶来源于淀粉酶链霉菌(Streptomyces diastaticus)CS1801，其氨基酸序列如SEQ ID NO.2所示，编码该GDSL脂肪酶的核苷酸序列如SEQ ID NO.1所示。

Description

一种GDSL脂肪酶、基因工程菌及其应用 技术领域
本发明涉及基因工程及蛋白质工程领域,涉及到一种新型GDSL脂肪酶的基因克隆及表达的方法以及酶法生产维生素A棕榈酸酯的应用,属于工业微生物领域技术。
背景技术
脂肪酶，又称酰基甘油水解酶,是一类能将天然油脂降解为甘油和游离脂肪酸的酶，其广泛存在于动植物及微生物中。微生物是脂肪酶的重要来源,主要包括根霉、曲霉及假丝酵母等等。根据对不同脂肪酶的氨基酸序列及其基本生物学性质分析，可将脂肪酶分为8个家族,其中第2家族,又称GDSL家族。GDSL型脂肪酶(lipase,EC 3.1.1.3)是一类水解酶,能水解硫酯、芳基酯、磷脂和氨基酸等多种底物。由于GDSL脂肪酶是一种新型脂肪酶，目前人们对其表达及功能都研究较少。由于GDSL脂肪酶具有酯类水解活性,人们对其研究越来越深入。
维生素A棕榈酸酯能帮助维持正常视觉功能并参与各种代谢活动维持生物机体健康，是目前最常用的维生素A衍生物，广泛应用于食品、化妆品、医药等行业。目前合成维生素A棕榈酸酯主要有化学法和酶法。化学方法合成维生素A棕榈酸酯存在环境污染、成本等问题，而酶法则污染少，时空产率高，成本低。而生产维生素A棕榈酸酯的酶就是具有酯类水解活性的脂肪酶。
发明内容
本发明的目的在于提供一种GDSL脂肪酶、工程菌及其应用，该脂肪酶活性高，可应用于维生素A棕榈酸酯的生产。
为实现上述技术目的，本发明采用如下技术方案：
一种GDSL脂肪酶，其氨基酸序列如SEQ ID NO.2所示。
本发明还提供了编码上述GDSL脂肪酶的基因。
具体地，所述的编码GDSL脂肪酶的基因的核苷酸序列如SEQ ID NO.1所示。
上述GDSL脂肪酶来源于淀粉酶链霉菌(Streptomyces diastaticus)CS1801，该菌株已在申请人的在先申请CN109337843A中公开。
本发明还提供了一种重组载体，其包含编码GDSL脂肪酶的基因和表达载体，所述基因的核苷酸序列如SEQ ID NO.1所示。所述表达载体为pET-32a(+)。上述基因在表达载体pET-32a(+)的多克隆位点间插入。
本发明还提供了一种包含上述的重组载体的基因工程菌
进一步地，所述工程菌的宿主细胞为E.coli BL21(DE3)。
本发明还提供了上述的工程菌在酶法生产维生素A棕榈酸酯中的应用，包括：
(1)将工程菌接入LB培养基中进行种子培养；
(2)种子液转入发酵培养基中进行发酵培养，之后加入诱导剂诱导酶类的表达；
(3)对发酵液进行离心，获取上清液，通过硫酸铵沉淀、冷冻干燥获得酶粉；
(4)将酶粉添加至含有维生素A和棕榈酸的有机相体系，生产维生素A棕榈酸酯。
具体地，所述方法包括：将在37℃，200r条件下于LB培养基中培养8～12h的工程菌以5％的接种量接入发酵培养基。发酵8～12h后添加IPTG至终浓度0.4～1mmol/L，在37℃、200r条件下发酵培养18～24h。4000r，10min，离心取发酵液的上清液。用50％的硫酸铵溶液沉淀酶蛋白，冷冻干燥48h后获得酶粉。将酶粉按5％加入有机相体系(维生素A：棕榈酸＝10g：10g溶于1L正己烷)转化一定时间后测定维生素A棕榈酸酯的含量，并计算转化率。
进一步的，所述的发酵培养基成分为：
胰蛋白胨10g/L，酵母粉5g/L，NaCl 10g/L，MgSO 4·7H 2O 1g/L，KH 2PO 4 0.5g/L，K 2HPO 4 0.5g/L，橄榄油乳化液12mL/L。
进一步的，橄榄油乳化剂配置方法为：橄榄油乳化剂PVA与橄榄油以体积比3：1混合，利用超声波乳化。
利用本发明的酶构建的基因工程菌进行酶法生产维生素A棕榈酸酯，转化所得维生素A棕榈酸酯含量最高为16.35mg/L。转化效率最高为81.75％。该脂肪酶为酶法合成维生素A棕榈酸酯提供新途径，具有重要应用前景。
附图说明
图1为GDSL脂肪酶PCR扩增的目的条带；
图2为大肠杆菌SDS-PAGE电泳图；
图3为GDSL脂肪酶生产维生素A棕榈酸酯的转化时间。
具体实施方式
实施例1
本实施例说明淀粉酶链霉菌来源的GDSL脂肪酶基因的PCR扩增方法。
取试管斜面保存的淀粉酶链霉菌CS1801进行平板活化，将单菌落接种到LB液体培养基中，30℃，培养2～3天。取培养液，于8000r离心2min，收集菌体，用细菌基因组提取试剂盒进行总的基因组提取，提取步骤参照上海生工的细菌基因组提取试剂盒的说明书。
GDSL脂肪酶基因扩增引物设计为：
GDSL2-上：5’-GTGGCCGGGCTCACGTCCTC-3’
GDSL2-下：5’-TCATTCCGGCAGGCTCCG-3’
以提取的淀粉酶链霉菌基因组为模板，采用上述引物及上海生工的高GC含量PCR扩增试剂盒进行扩增，但不加Taq酶。
具体的扩增程序如下，预变性95℃10min，加Taq酶。95℃变性1min，退火温度55℃，退火时间为30s，延伸温度为72℃，延伸时间为1min，此过程进行29个循环，最后72℃延伸30min。取产物进行琼脂糖凝胶电泳，将目的条带切胶回收后保存。凝胶回收试剂盒购自生工。
实施例2
本实施例说明带有酶切位点的GDSL脂肪酶基因的PCR扩增方法。
带有酶切位点的GDSL脂肪酶基因扩增引物设计为：
GDSL2-上：5’-CCGGAATTCGTGGCCGGGCTCACGTCCTC-3’
GDSL2-下：5’-CCGCTCGAGTCATTCCGGCAGGCTCCG-3’
以实施例1中的胶回收产物为模板，采用上述引物及上海生工的高GC含量PCR扩增试剂盒进行扩增。
具体的扩增程序如下，预变性95℃2min。95℃变性1min，退火温度55℃，退火时间为30s，延伸温度为72℃，延伸时间为1min，此过程进行29个循环，最后72℃延伸30min。取产物进行琼脂糖凝胶电泳，目的条带如图1，将目的条带切胶回收后送生工测序得到序列SEQ ID NO.1。
实施例3
本实施例说明GDSL脂肪酶的重组克隆载体的构建方法。
将实施例2中的胶回收产物与T载体连接，转化至DH-5α后，挑阳性克隆子验证，提取质粒后测序验证。
实施例4
本实施例说明GDSL脂肪酶的重组表达载体的构建方法。
将实施例3中的质粒用XhoI和EcoRI进行双酶切并回收目的条带，同时用XhoI和EcoRI对pET32a(+)载体双酶切，回收载体中较大的片段，将回收的目的基因片段与载体片段进行连接，导入宿主细胞大肠杆菌DH5α，抗性筛选后，挑取阳性克隆子测序验证。
实施例5
本实施例说明GDSL脂肪酶基因工程菌的构建方法。
提取实施例4中的测序正确的阳性克隆子的质粒，直接转化导入宿主细胞大肠杆菌BL21(DE3)中，GDSL脂肪酶基因工程菌构建成功，在其发酵过程需加入IPTG等诱导剂诱导其高效表达GDSL脂肪酶蛋白。通过SDS-PAGE验证融合蛋白表达成功。SDS-PAGE电泳图如图2所示，泳道1为未经诱导的E.coli pET32a-GDSL，泳道2、3、4为经过IPTG分别诱导4、8、16h的重组大肠杆菌E.coli pET32a-GDSL，与其它泳道相比，在分子量为44k Da处有明显条带，除去质粒上融合表达的蛋白，与预测目标蛋白大小相一致，表明GDSL脂肪酶在重组菌中成功表达。
实施例6
本实施例说明GDSL脂肪酶基因工程菌在维生素A棕榈酸酯生产中的应用。
(1)将37℃，200r，LB培养基中培养8～12h的基因工程菌以5％的接种量接入发酵培养基。
(2)发酵8～12h后添加IPTG至终浓度0.4～1mmol/L，在37℃、200r条件下发酵培养18～24h。
(3)4000r，10min，离心取发酵液的上清液。用50％的硫酸铵溶液沉淀酶蛋白，冷冻干燥48h后获得酶粉，按照国标GBT23535-2009测定GDSL脂肪酶酶活为1.53U/mg。
(4)将酶粉按5％的添加量(w/v)加入有机相体系(维生素A：棕榈酸＝10g：10g溶于1L正己烷)转化2、4、6、8、10h后测定维生素A棕榈酸酯的含量，并计算转化率。如图3所示，转化8h后，维生素A棕榈酸酯含量最高为16.35mg/L，转化率为81.75％。
所述的发酵培养基成分为：
胰蛋白胨10g/L，酵母粉5g/L，NaCl 10g/L，MgSO 4·7H 2O 1g/L，KH 2PO 4 0.5g/L，K 2HPO 40.5g/L，橄榄油乳化液12mL/L，蒸馏水定容至1L。
橄榄油乳化液配制方法为：将橄榄油乳化剂PVA与橄榄油以体积比3：1混合，利用超声波乳化。
所述的维生素A棕榈酸酯测定方法为：高效液相法，采用外标法定量。色谱条件为：色谱柱：Alltech C18(250x4.6mm，4.5μm)；流动相：100％甲醇；检测器：岛津10A紫外检测器；检测波长：327nm；流速：1mL/min。
转化率计算公式为：
Figure PCTCN2019114789-appb-000001

Claims (10)

  1. 一种GDSL脂肪酶，其特征在于，氨基酸序列如SEQ ID NO.2所示。
  2. 编码权利要求1所述GDSL脂肪酶的基因。
  3. 根据权利要求2所述的基因，其特征在于，核苷酸序列如SEQ ID NO.1所示。
  4. 一种包含权利要求3所述基因的重组载体。
  5. 根据权利要求4所述的重组载体，其特征在于，表达载体为pET 32a(+)。
  6. 一种包含权利要求5所述的重组载体的工程菌。
  7. 根据权利要求6所述的工程菌，其特征在于，宿主细胞为大肠杆菌E.coli BL21(DE3)。
  8. 权利要求6或7任一项所述的工程菌在生产维生素A棕榈酸酯中的应用，包括：
    (1)将工程菌接入LB培养基中进行种子培养；
    (2)种子液转入发酵培养基中进行发酵培养，之后加入诱导剂诱导酶类的表达；
    (3)对发酵液进行离心，获取上清液，通过硫酸铵沉淀、冷冻干燥获得酶粉；
    (4)将酶粉添加至含有维生素A和棕榈酸的有机相体系，生产维生素A棕榈酸酯。
  9. 根据权利要求8所述的应用，其特征在于，所述发酵培养基成分为：
    胰蛋白胨10g/L，酵母粉5g/L，NaCl 10g/L，MgSO 4·7H 2O 1g/L，KH 2PO 40.5g/L，K 2HPO 40.5g/L，橄榄油乳化液12mL/L，蒸馏水定容至1L。
  10. 根据权利要求9所述的应用，其特征在于，所述橄榄油乳化液配制方法为：将橄榄油乳化剂PVA与橄榄油以体积比3：1混合，利用超声波乳化。
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