WO2021232840A1 - 一种α-L-鼠李糖苷酶的截短突变体及其应用 - Google Patents

一种α-L-鼠李糖苷酶的截短突变体及其应用 Download PDF

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WO2021232840A1
WO2021232840A1 PCT/CN2021/073707 CN2021073707W WO2021232840A1 WO 2021232840 A1 WO2021232840 A1 WO 2021232840A1 CN 2021073707 W CN2021073707 W CN 2021073707W WO 2021232840 A1 WO2021232840 A1 WO 2021232840A1
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rhamnosidase
mutant
rhab1
enzyme
engineered bacteria
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王俊
游帅
朱林琳
李扬
郝卫东
耿丽恬
匡逸云
常雨潇
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江苏科技大学
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    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/0104—Alpha-L-rhamnosidase (3.2.1.40)

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  • the present invention belongs to the field of enzyme engineering and genetic engineering, and specifically relates to a mutant and its application, in particular to the transformation of an ⁇ -L-rhamnosidase gene by truncation mutation, and the use of the enzyme to transform rutin into a physiological Active isoquercitrin.
  • ⁇ -L-rhamnosidase (EC 3.2.1.40) is an important glycoside hydrolase, which can specifically hydrolyze and cleave the ⁇ -L-rhamnosyl group at the end of natural glycoside products, such as rutin and naringin , Hesperidin, stevioside, myricitin, quercetin, etc.
  • ⁇ -L-rhamnosidase has a wide range of applications in the pharmaceutical and food industries, and can be used for debittering and turbidity removal of citrus juices (Process Biochemistry, 2010, 45(8):1226-1235). Increase the aroma of wine by hydrolyzing the terpene-based glycosides in grape skins (Applied and Environmental Microbiology, 2011, 5317).
  • ⁇ -L-rhamnosidase can be used as a precursor of many drugs, and its hydrolyzed rutin, quercetin, and ginsenoside products have antioxidant, anti-cancer, and anti-inflammatory effects. (Process Biochemistry, 2004, 39(7):861-867).
  • ⁇ -L-rhamnosidase Different sources of ⁇ -L-rhamnosidase have different properties. Most of the characterized bacterial RHA are mainly derived from Bacillus, Lactobacillus, Staphylococcus, Clostridium, Streptomyces and Thermomicrobium, while fungal RHA is mainly derived from Aspergillus and Penicillium. Most fungal sources of RHA are acidic (pH 4.0-5.0), while bacterial sources tend to be neutral and alkaline, and are suitable for industrial production of hydrolyzed flavonoids to obtain L-rhamnoside to prepare pharmaceutical monomers.
  • the current ⁇ -L-rhamnosidase derived from microorganisms has low activity, high industrial production cost, low purity and high price of commercially available commercial enzymes; the research on its property modification is mainly on enzyme-producing strains and enzyme resources
  • the patent CN104312996 B expresses the ⁇ -L-rhamnosidase Rha1 derived from Aspergillus niger and applies it to the hydrolysis of rutin. There are few molecular modifications on functional modules.
  • the patent CN 106318957 B focuses on site-directed mutagenesis and addition. Starting with the stabilizer, it improved the thermal stability of the Aspergillus terreus CCF 3059 ⁇ -L-rhamnosidase mutant.
  • the present invention provides a truncated mutant of ⁇ -L-rhamnosidase and its application, a vector and strain containing the gene, and using the strain to transform Rutin is transformed into isoquercitrin with multiple biological activities, and its transformed product has high purity and easy separation, which expands the scope and field of application.
  • the gene sequence of the truncated mutant of ⁇ -L-rhamnosidase is shown in SEQ ID NO.1.
  • a recombinant vector in which the gene sequence of the above-mentioned mutant is inserted is inserted.
  • An engineered bacterium containing a truncated mutant gene of ⁇ -L-rhamnosidase which contains the gene sequence of the above-mentioned mutant.
  • the plasmid vector of the above-mentioned engineered bacteria is pET28a.
  • the host bacteria of the above-mentioned engineered bacteria are Escherichia coli.
  • the specific application steps are: adding the engineered bacteria to a buffer solution containing flavonoids, pH 5.0-7.0, shaking reaction; the buffer solution is a citric acid-disodium hydrogen phosphate buffer solution; the reaction temperature The temperature is 30-50°C; the reaction time is 1-24h.
  • the aforementioned flavonoid compound is rutin or naringin.
  • the optimal temperature of the mutant enzyme rhaB1- ⁇ N is 5°C lower than the original enzyme, and the optimal pH is the same, but compared with the original enzyme, the mutant enzyme is treated at 35°C for 60 minutes, and the remaining relative activity is as high as 95%.
  • the temperature stability and pH stability of the enzyme are greatly improved compared to the original enzyme.
  • the K m value and the maximum reaction rate V max of the mutant enzyme catalyzed hydrolysis of pNPR are 1.31 g/L and 61842.15 [ ⁇ mol/(min ⁇ mg)], respectively, which is 2.13 g/L compared to the original enzyme K m , which is different from the bottom It has higher affinity and can significantly improve the efficiency of catalyzing the hydrolysis of rutin to produce isoquercitrin.
  • Figure 1 is the electrophoresis diagram of rhaB1- ⁇ N verification, in which (A) PCR amplification rhaB1- ⁇ N M: DNA Marker (5000bp), 1: rhaB1- ⁇ N; (B) SDS-PAGE of RhaB1- ⁇ N and purified protein; M protein Marker; 1RhaB1- ⁇ N protein; 2 purified RhaB1-N protein; 3 recombinant strain BL21-pET28a;
  • Figure 2 is the optimum temperature analysis diagram of RhaB1- ⁇ N
  • Figure 3 is the thermal stability analysis diagram of RhaB1- ⁇ N
  • Figure 4 is a schematic diagram of the optimum pH of RhaB1- ⁇ N;
  • Figure 5 is a schematic diagram of RhaB1- ⁇ N pH stability.
  • the source of the ⁇ -L-rhamnosidase gene rhaB1 of the present invention is based on metagenomic technology and obtained from elephant excrement (Journal of Biotechnology, 2014, 191:38-45).
  • Use BLASTp to compare the amino acid sequence of rhaB1 with Genbank. According to its catalytic domain, design the initial upstream primer at the N-terminus to obtain the truncated mutant rhaB1- ⁇ N sequence, and obtain a new gene sequence such as SEQ ID NO.1 Shown.
  • the recombinant expression vector pET21a-rhaB1 is used as a template, and the primers in the following table are used for PCR amplification.
  • the PCR reaction system is: 2 ⁇ Taq Mix 25 ⁇ L, upstream primer rhaB1- ⁇ N-F, downstream primer rhaB1- ⁇ N-R each 2 ⁇ L, template 1 ⁇ L, add sterile water to a final volume of 50 ⁇ L.
  • PCR reaction conditions are: 94°C pre-denaturation for 5 minutes, 94°C denaturation for 30 seconds, 60°C annealing for 30 seconds, 72°C extension for 90 seconds, reaction for 30 cycles, 72°C extension for 5 minutes, and sequencing verification after PCR. After verification, a new one is obtained.
  • the rhaB1- ⁇ N mutant gene ( Figure 1), its sequence is shown in SEQ ID NO.1.
  • the obtained target gene rhaB1- ⁇ N was double-enzyme digested with Nhe I and Hind III at 37°C for 15 minutes, and the target fragment and pET28a vector were recovered at a molar ratio of 1:5. Ligated with T4 DNA ligase and placed at room temperature for 2 hours to obtain a recombinant plasmid. The ligation product was transformed into E.
  • coli DH5 ⁇ competent cells and spread evenly on LB plates with kanamycin resistance (50 ⁇ g/mL), cultured overnight at 37°C, and picked a single clone to inoculate with kanamycin In the LB liquid medium of 50 ⁇ g/mL (50 ⁇ g/mL), shake bacteria (4mL) to an OD600 value of 0.6-0.8. Perform PCR verification of bacterial liquid, verification of plasmid extraction and verification of restriction enzyme digestion, and obtain the recombinant expression vector pET28a-rhaB1- ⁇ N .
  • the recombinant vector prepared above was inoculated into LB liquid medium, and kanamycin antibiotics at a concentration of 50 ⁇ g/mL were added, and cultured with shaking in a constant temperature air bath shaker to an OD600 of 0.6-0.8, and then IPTG (0.1% ) Perform low-temperature induction and expression for 16-22h. Bacteria were collected and ultrasonically disrupted using a cell disruptor, and then the supernatant was collected and stored as RhaB1- ⁇ N crude enzyme solution. A Ni-TED 2000 packed column was used to separate and purify the crude RhaB1- ⁇ N enzyme solution.
  • Enzyme activity unit (U) is defined as: under the measurement conditions, the amount of enzyme used to produce 1 ⁇ mol p-nitrophenol per minute is 1 enzyme activity unit.
  • the enzyme activity was measured every 5°C.
  • the buffer was 100mmol/L sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 6.5), and the optimal reaction temperature of rhaB1- ⁇ N was found to be 35°C ( Figure 2). Treated at different temperatures of 30-40°C for 5, 10, 15, 30, 45 and 60 minutes. The enzyme activity was measured separately, and the results ( Figure 3) showed that the relative enzyme activity of RhaB1- ⁇ N remained above 95% after being treated at the optimum temperature of 35°C for 60 minutes.
  • the present invention modified the ⁇ -L-rhamnosidase RhaB1 (Genbank:13557.1) of the GH78 family, truncated the N-terminal 336 amino acid residues, obtained mutant strains, and used the enzyme to catalyze flavonoids
  • the compound is hydrolyzed.
  • the mutant has a small molecular weight and an increase in enzyme activity by 12%. It is treated at the optimum temperature of 35°C for 60 minutes and kept at pH 6.5-7.0 for 1 hour.
  • the relative enzyme activity is basically unchanged, and it has stronger thermal stability and pH stability.
  • the m value and V max are 1.31 g/L and 61842.15 [ ⁇ mol/(min ⁇ mg)], respectively, which have higher affinity with the substrate and improve the catalytic hydrolysis efficiency of flavonoids.
  • the invention obtains mutants with improved catalytic activity through truncated mutations, which lays a foundation for subsequent applications.

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Abstract

提供一种α-L-鼠李糖苷酶的截短突变体及其应用，该突变体是RhaB1酶截去了N端336个氨基酸残基构建而得。该突变体分子量小，相比野生型，酶活提高12％，具有更强热稳定性及pH稳定性，与底物亲和力更高，提高黄酮类化合物催化水解效率。

Description

一种α-L-鼠李糖苷酶的截短突变体及其应用 技术领域
本发明属于酶工程和基因工程领域，具体涉及一种突变体及其应用，尤其涉及利用截短突变改造一种α-L-鼠李糖苷酶基因，并利用该酶将芦丁转化为具有生理活性的异槲皮苷。
背景技术
α-L-鼠李糖苷酶(EC 3.2.1.40)是一种重要的糖苷水解酶，它能够特异性水解切割天然糖苷产物末端的α-L-鼠李糖基，如芦丁、柚皮苷、橘皮苷、甜菊苷、杨梅苷、槲皮苷等。α-L-鼠李糖苷酶在制药及食品工业具有广泛的应用价值，可用于柑橘类果汁的脱苦及去浊(Process Biochemistry,2010，45(8):1226-1235)。通过水解葡萄皮中的萜烯基糖苷来增加酒的香气(Applied and Environmental Microbiology，2011，5317)。此外，α-L-鼠李糖苷酶可作为许多药物的前体，其水解芦丁、槲皮素、人参皂苷的产物具有抗氧化、抗癌、抗炎等作用。(Process Biochemistry,2004，39(7):861-867)。
不同来源的α-L-鼠李糖苷酶性质不同，大多已表征的细菌RHA主要来自Bacillus、Lactobacillus、Staphylococcus、Clostridium、Streptomyces和Thermomicrobium，而真菌RHA则主要来源于曲霉菌和青霉菌。大多真菌来源的RHA最适pH为酸性(pH值4.0-5.0)，而细菌来源则偏向于中性与碱性，适合于工业生产水解黄酮化合物获得L-鼠李糖苷以制备药物单体。然而，目前微生物来源的α-L-鼠李糖苷酶活力较低，工业生产成本较高，市售的商品酶纯度低，价格高；对于其性质改造的研究主要在产酶菌株及酶的资源挖掘等，例如专利CN104312996 B表达了黑曲霉来源的α-L-鼠李糖苷酶Rha1并将其应用于水解芦丁，关于功能模块的分子改造则较少，专利CN 106318957 B从定点突变和添加稳定剂两方面入手，提高了土曲霉CCF 3059α-L-鼠李糖苷酶突变体热稳定性。然而，目前还未有通过结构域的截短来改造α-L-鼠李糖苷酶性质，提高其催化活力和稳定性的报道，通过设计截短突变，可以认识新酶功能域的功能，判断哪些区域是必须结构，以及该结构对整个酶的活性和稳定性的影响，从而为酶的结构和功能研究提供重要的依据。
发明内容
解决的技术问题：针对现有技术中所述的不足，本发明提供一种α-L-鼠李糖苷酶的截短突变体及其应用，包含该基因的载体和菌株，同时利用该菌株将芦丁转化为具有多种生物活性的异槲皮苷，其转化产物纯度高，易分离，扩大应用范围和领域。
技术方案：一种α-L-鼠李糖苷酶的截短突变体，其是RhaB1酶截去了N端336个氨基酸残基构建而得。
上述α-L-鼠李糖苷酶的截短突变体，基因序列如SEQ ID NO.1所示。
一种重组载体，载体中插入了上述突变体的基因序列。
一种含有α-L-鼠李糖苷酶的截短突变体基因的工程菌，含有上述突变体的基因序列。
上述工程菌的质粒载体为pET28a。
上述工程菌的宿主菌为大肠杆菌。
上述突变体在制备水解黄酮类化合物工程菌中的应用。
上述工程菌在水解黄酮类化合物中的应用。
应用具体步骤为，将所述工程菌加入到含有黄酮类化合物的缓冲液中，pH 5.0-7.0，振荡反应；所述的缓冲液为柠檬酸-磷酸氢二钠缓冲液；所述的反应温度为30-50℃；所述的反应时间为1-24h。
上述黄酮类化合物为芦丁或柚皮苷。
有益效果：1.突变酶rhaB1-ΔN的最适温度比原酶低5℃，最适pH相同，但与原酶相比，突变酶在35℃处理60min，剩余的相对活力高达95％，突变酶的温度稳定性及pH稳定性相对于原酶有较大提高。
2.突变酶催化水解pNPR的K m值和最大反应速率V max分别为1.31g/L和61842.15[μmol/(min×mg)]，相较于原酶K m为2.13g/L，与底物亲和力更高，能够显著提高催化水解芦丁生成异槲皮苷的效率。
附图说明
图1为rhaB1-ΔN验证电泳图，其中(A)PCR扩增rhaB1-ΔN M:DNA Marker(5000bp),1:rhaB1-ΔN；(B)RhaB1-ΔN及纯化蛋白的SDS-PAGE；M蛋白质Marker；1RhaB1-ΔN蛋白；2纯化的RhaB1-N蛋白；3重组菌株BL21-pET28a；
图2为RhaB1-ΔN的最适温度分析图；
图3为RhaB1-ΔN热稳定性分析图；
图4为RhaB1-ΔN最适pH示意图；
图5为RhaB1-ΔN pH稳定性示意图。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
本发明所述的α-L-鼠李糖苷酶基因rhaB1的来源基于宏基因组技术且从大象排泄物中获取(Journal of Biotechnology，2014,191:38-45)。使用BLASTp将rhaB1的氨基酸序列与Genbank进行比对，依据其催化结构域，在N端设计起始的上游引物来获得截短突变体rhaB1-ΔN序列，得到新的基因序列如SEQ ID NO.1所示。将rhaB1-ΔN基因插入载体pET28a中，构建了重组载体pET28a-rhaB1-ΔN，并将其导入大肠杆菌BL21(DE3)中，获得含有表达载体的菌株E.coli.pET28a-rhaB1-ΔN，上述菌株经诱导表达后，用于与底物芦丁充分接触，并将芦丁催化转化为单一活性产物异槲皮苷。
实施例1目的基因的获得
本发明分别以重组表达载体pET21a-rhaB1为模板，利用下表中的引物进行PCR扩增。
PCR反应体系为：2×Taq Mix 25μL，上游引物rhaB1-ΔN-F、下游引物rhaB1-ΔN-R各2μL，模板1μL，加无菌水至终体积为50μL。
PCR反应条件为：94℃预变性5min，94℃变性30s，60℃退火30s，72℃延伸90s，反应30个循环，72℃再延伸5min，PCR结束后进行测序验证，验证正确后获得新的rhaB1-ΔN突变体基因(图1)，其序列见SEQ ID NO.1。
引物序列
Figure PCTCN2021073707-appb-000001
实施例2构建pET28a-rhaB1-ΔN重组载体
将已获得的目的基因rhaB1-ΔN分别用Nhe I和Hind III在37℃下双酶切PCR产物基因rhaB1-ΔN和质粒pET28a 15min，回收后目的片段和pET28a载体按摩尔数1:5的比例，用T4DNA连接酶连接在室温放置2h以获得重组质粒。将连接产物转化大肠杆菌DH5α感受态细胞，并均匀涂布于带有卡那霉素抗性(50μg/mL)的LB平板上，37℃培养过夜，挑取单克隆接种到带有卡那霉素抗性(50μg/mL)的LB液体培养基中摇菌(4mL)至OD600值为0.6-0.8，进行菌液PCR验证，提取质粒验证和酶切验证，获得重组表达载体pET28a-rhaB1-ΔN。
实施例3重组酶的表达及纯化
将上述制备的重组载体接种于LB液体培养基中，并加入浓度为50μg/mL的卡那霉素抗生素，于恒温气浴摇床中振荡培养至OD600为0.6-0.8，然后加入IPTG(0.1％)进行低温诱导表达16-22h。收集菌体并使用细胞破碎仪进行超声破碎，然后收集上清，保存为RhaB1-ΔN粗酶液。利用Ni-TED 2000packed column色谱柱对RhaB1-ΔN粗酶液进行分离纯化。先用4mL1×LEW清洗色谱柱，取10mL粗酶液加入色谱柱，然后用4mL 1×LEW清洗两次，再用3mL1×Elution清洗三次，接取滤液，即rhaB1-ΔN纯酶液。用PSB(pH 7.4)缓冲液浸泡，在4℃下透析过夜，用4mL 1×LEW清洗色谱柱，于4℃保存并进行SDS-PAGE分析(图1)。
实施例4RhaB1-ΔN性质测定
1.α-L-鼠李糖苷酶的酶活测定
反应体系200μL，100μL酶液先在45℃孵育5min，再加入100μL 1mM对硝基苯α-L-鼠李糖苷酶(pNPR)45℃反应10min，显色后再加入1mol/L的碳酸钠溶液150μL终止反应。在405nm下测定吸光值。酶活力单位(U)定义为：在测定条件下，每分钟产生1μmol p-硝基苯酚所用的酶量为1个酶活力单位。
2.最适反应温度及热稳定性的测定
在4-50℃范围内，每隔5℃，分别测定酶活。缓冲液为100mmol/L磷酸二氢钠-磷酸氢二钠缓冲液(pH 6.5)，发现rhaB1-ΔN的最适反应温度为35℃(图2)。在不同温度30-40℃下分别处理5、10、15、30、45和60min。分别测定酶活，结果(图3)表明RhaB1-ΔN在最适温度35℃下处理60min后相对酶活力仍保持在95％以上。
3.最适反应pH及pH稳定性的测定
用pH 5-8.5的柠檬酸-磷酸氢二钠缓冲液稀释，于35℃下与底物进行酶促反应，发现最适pH为6.5(图4)。将RhaB1-ΔN在不同pH的Buffer中于35℃下保温1h，再用最适Buffer稀释至适当倍数，结果(图5)表明在pH6.5-7.0之间相对酶活力保持在90％以上。
4.RhaB1-ΔN水解pNPR的反应动力学
将pNPR用pH 5.0的PBS缓冲液稀释成0.2-8mmol/L的梯度浓度，将其与纯化的RhaB1-ΔN酶液于最适条件下进行酶促反应。通过Lineweaver-Burk模型拟合，绘制反应动力学曲线，计算其反应动力学K m及V m。K m值和V max分别为1.31g/L和61842.15[μmol/(min×mg)]。
实施例5 RhaB1-ΔN催化水解芦丁合成异槲皮苷
将制备的RhaB1-ΔN催化剂加入到含有0.6g/L芦丁的缓冲液中，40℃，pH＝5，180r/min振荡培养进行转化反应，分别在反应时间的2-24h取样，进行HPLC检测，并计算产物异槲皮苷的含量，产物异槲皮苷的得率为83.9±3.7％。
实施例6 RhaB1-ΔN催化水解芦丁合成异槲皮苷
将上述制备的催化剂加入到含有0.6g/L芦丁的缓冲液中，35℃，pH＝6.5，180r/min振荡培养进行转化反应，分别在反应时间的2-24h取样，进行HPLC检测，并计算产物异槲皮苷的含量，产物异槲皮苷的得率为62.9±4.4％。
实施例7RhaB1-ΔN催化水解芦丁合成异槲皮苷
将上述制备的催化剂加入到含有0.6g/L芦丁的缓冲液中，40℃，pH＝6.5，180r/min振荡培养进行转化反应，分别在反应时间的2-24h取样，进行HPLC检测，并计算产物异槲皮苷的含量，产物异槲皮苷的得率为82.4±6.9％。
综上所述，本发明对GH78家族的α-L-鼠李糖苷酶RhaB1(Genbank:13557.1)进行改造，截去N端336个氨基酸残基，获得突变体菌株，并利用该酶催化黄酮类化合物水解。该突变体分子量小，酶活提高12％，在最适温度35℃下处理60min及在pH 6.5-7.0内保温1h，相对酶活力基本不变，具有更强热稳定性及pH稳定性，K m值和V max分别为1.31g/L和61842.15[μmol/(min×mg)]，与底物亲和力更高，提高黄酮类化合物催化水解效率。本发明通过截短突变获得催化活力提高的突变体，为后续应用奠定了基础。
Figure PCTCN2021073707-appb-000002
Figure PCTCN2021073707-appb-000003

Claims (10)

  1. 一种α-L-鼠李糖苷酶的截短突变体，其特征在于，其是RhaB1酶截去了N端336个氨基酸残基构建而得。
  2. 根据权利要求1所述α-L-鼠李糖苷酶的截短突变体，其特征在于基因序列如SEQ ID NO.1所示。
  3. 一种重组载体，其特征在于载体中插入了权利要求2所述突变体的基因序列。
  4. 一种含有α-L-鼠李糖苷酶的截短突变体基因的工程菌，其特征在于含有权利要求2所述突变体的基因序列。
  5. 根据权利要求4所述含有α-L-鼠李糖苷酶的截短突变体基因的工程菌，其特征在于所述工程菌的质粒载体为pET28a。
  6. 根据权利要求4所述含有α-L-鼠李糖苷酶的截短突变体基因的工程菌，其特征在于所述工程菌的宿主菌为大肠杆菌。
  7. 权利要求1或2所述突变体在制备水解黄酮类化合物工程菌中的应用。
  8. 权利要求4～6任一所述工程菌在水解黄酮类化合物中的应用。
  9. 根据权利要求8所述的应用，其特征在于将所述工程菌加入到含有黄酮类化合物的缓冲液中，pH 5.0-7.0，振荡反应；所述的缓冲液为柠檬酸-磷酸氢二钠缓冲液；所述的反应温度为30-50℃；所述的反应时间为1-24h。
  10. 根据权利要求9所述的应用，其特征在于将所述黄酮类化合物为芦丁或柚皮苷。
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