CN110878288B - 多肽、核酸及其在合成橙花叔醇糖苷上的应用 - Google Patents
多肽、核酸及其在合成橙花叔醇糖苷上的应用 Download PDFInfo
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- CN110878288B CN110878288B CN201911253751.3A CN201911253751A CN110878288B CN 110878288 B CN110878288 B CN 110878288B CN 201911253751 A CN201911253751 A CN 201911253751A CN 110878288 B CN110878288 B CN 110878288B
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- Prior art keywords
- nerolidol
- glucoside
- nucleic acid
- polypeptide
- leu
- Prior art date
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Abstract
本发明公开了一种多肽、核酸及其在合成橙花叔醇糖苷上的应用,所述多肽的氨基酸序列如SEQ ID No.1所示。本发明通过分子克隆,得到了UGT91Q2基因及其表达蛋白,该蛋白能特异的催化橙花叔醇合成橙花叔醇葡萄糖苷。从而为生物合成橙化叔醇葡糖糖苷提供了新的途径。
Description
技术领域
本发明属于基因工程领域,具体涉及一种多肽、核酸及其在催化橙化叔醇合成橙化叔醇糖苷上的应用。
背景技术
Nerolidol(3,7,11-三甲基-1,6,10-12三萜-3-醇),是一种天然存在的倍半萜醇,存在于各种植物的EO中,具有花香。研究发现,Nerolidol是上述植物EOs所证实的具有生物活性的化合物之一。统计数据显示,nerolidol每年的全球使用量为10至100公吨。例如,nerolidol经常用于化妆品(如洗发水和香水)和非化妆品(如洗涤剂和清洁剂)。此外,自从nerolidol作为一种安全的食品调味剂被美国食品和药物管理局批准以来,它也被广泛应用于食品工业中作为许多食品中的增味剂。近年来国内外研究发现的nerolidol具有抗氧化、抗微生物、抗生物膜、抗寄生虫、杀虫、抗溃疡、皮肤渗透增强剂、抗肿瘤、抗伤害性和抗炎等多种药理和生物活性。
糖苷态香气物质是一类不具挥发性,与糖类物质通过糖苷键结合后以糖苷态形式存在的一类香气前体,具有重要的生物和药理功能。随着植物糖苷态香气前体研究的日益深入,大量糖苷态香气物质被分离并鉴定,有些还被证实具有重要的生物活性,如抗菌、抗炎和对神经的保护作用。糖苷态香气提高了游离香气物质的水溶性和稳定性,在特定的条件下,释放出人们需要的香气物质,在化妆品、食品和药物开发领域具有广泛的商业价值(如目前市场上每克香叶醇糖苷的价格为20万人民币),成为天然产物领域研究的热门课题。在自然界中,糖苷化是植物次生代谢最为重要的修饰反应之一,糖苷物质是由UDP-糖基转移酶催化完成,糖基转移酶可将糖基从活化的供体转移到小分子香气(苷元)上,从而调节受体分子在基体的生物活性、可溶性、亚细胞定位和稳定性。植物醇系香气糖基转移酶研究进展缓慢,限制了香气糖苷在体外的高效合成,制约了香气糖苷物质在食品、化妆品及医药行业的应用。
橙花叔醇,苯甲醇,苯乙醇,香叶醇,芳樟醇,芳樟醇氧化物,4-羟基香豆素,香叶醇,及顺/反-3-己烯醇,丁香酚等香气物质存在许多植物和水果中,具有怡人的芳香和花果香,但其本身稳定性差,在空气中容易变形变质,影响了其在食品,医药方面的应用,而这些物质的糖基化产物可以显著提高其稳定性,水溶性,降低其毒性,极大地拓宽这些香气物质应用到新的领域,具有很大的商业前景。
考虑到橙花叔醇在生产及生活中具有重要作用,以及糖苷态的化合物能有效的避免游离态苷元的存在的弊端,橙花叔醇糖苷化合物的化学合成效率低下,对环境污染大,且对产物没有选择性,因而这种环境友好型酶系方法合成香气糖苷化合物合成方法具有很大的应用价值。
目前现有技术中急需一种合成倍半萜香气物质橙花叔醇糖苷高效生物酶及其制备方法。
发明内容
本发明所要解决的技术问题为:如何提供一种橙花叔醇糖苷合成相关的酶及制备方法。
本发明的技术方案为:分离的多肽,其氨基酸序列如SEQ ID No.1所示。
编码上述多肽的核酸。
进一步地,所述核酸的核苷酸序列如SEQ ID No.2所示。
重组载体,包含有上述的核酸。
重组菌株,包含有上述的核酸或重组载体。
上述所述的分离的多肽或重组菌株在催化橙花叔醇合成橙花叔醇葡萄糖苷上的应用。
一种合成橙花叔醇葡糖糖苷的方法,以橙化叔醇为底物,以上述所述的分离的多肽为催化剂合成橙花叔醇葡糖糖苷。
进一步地,催化的温度为30℃、pH为8.5。
一种合成橙化叔醇葡萄糖苷的方法,将上述所述的重组菌株接种到橙化叔醇原料中发酵,从而得到橙化叔醇葡萄糖苷。
与现有技术相比,本发明具有以下有益效果:
本发明通过分子克隆,得到了UGT91Q2基因及其表达蛋白,该蛋白能特异的催化橙花叔醇合成橙花叔醇葡萄糖苷,且转化效率高达90%。从而为生物合成橙化叔醇葡糖糖苷提供了新的途径。
附图说明
图1为不同底物的相对酶活;横坐标为筛选的38个底物,纵坐标为相对酶活数值。以酶活数值最高的底物做为100,按照占100的百分比排序。100%的认为该酶对该底物有最大的催化效率。
图2为CsUGT91Q2催化橙花叔醇合成橙花叔醇糖苷LC-MS色谱图,箭头所指的为合成的橙花叔醇糖苷的峰图,说明该酶催化橙花叔醇有糖苷产生;下方对应的为对照的峰图。
图3为CsUGT91Q2催化橙花叔醇合成橙花叔醇糖苷LC-MS质谱图,385为橙花叔醇加葡萄糖加氢条件下的M值(正离子模式),进一步验证橙花叔醇葡萄糖苷的存在。
图4为不同pH值的酶活数据;横坐标为不同条件的pH,2-5为柠檬酸缓冲液、5-8为磷酸缓冲液、8-10为Tris-HCl缓冲液。纵坐标为OD595条件下酶活相对应的吸光度值A。
图5为不同温度的酶活数据;横坐标为不同条件的温度,25-45℃。纵坐标为OD595条件下酶活相对应的吸光度值A。
图6为不同时间的酶活数据;横坐标为不同反应时间,10-50min。纵坐标为OD595条件下酶活相对应的吸光度值A。
图7为不同糖供体的酶活数据;横坐标为不同的糖供体,GA为葡萄糖醛酸,Gal为半乳糖,Glu为葡萄糖;纵坐标为OD595条件下酶活相对应的吸光度值A。相同的反应条件下加入不同的糖供体,葡萄糖具有最高的催化效率。
图8为酶动力学曲线。该酶催化橙花叔醇的动力学常数Km值为19.71uM,最大反应速率Vmax为0.37nKat.mg-1,其中Km值越小,说明酶与底物之间的亲和力越强。
具体实施方式
实施例1UGT91Q2的克隆
1、cDNA模板的获取:将茶叶的鲜叶液氮处理磨碎后,用商业化提取植物RNA试剂盒提取,之后用商业化反转录试剂盒进行反转录,获得cDNA。
PCR引物序列:
UGT91Q2F:GGTTCCGCGTGGATCCATGAACGGTGACTCCCAACAACA(SEQ ID No.3)UGT91Q2R:GGCCGCTCGAGTCGACTTAAGGGTGCTTACAAGCTTTGATTACCTCCAAC(SEQ ID No.4)
反应体系:
反应程序:
2、重组质粒pGEX4T1-UGT91Q2的构建
将完整的pGEX4T1载体根据需要进行双酶切,从而获得线性载体,之后通过商业化的试剂盒胶回收载体,获得纯化后的线性载体。用连接酶将单一的目的基因与线性载体进行连接,构建重组质粒pGEX4T1-UGT91Q2,之后转化到Trans1-T1感受态细胞进行过夜培养,选取阳性菌斑转入LB培养基中,经过菌落PCR验证后,将菌液送给通用生物有限公司完成测序工作。其核苷酸序列如SEQ ID No.2所示。
3、UGT91Q2基因的原核表达与纯化
将构建成功的表达载体pGEX4T1-UGT91Q2转化到BL21感受态细胞进行过夜培养,选取阳性菌斑转入LB培养基中37℃过夜培养,在37℃条件下进行扩大培养,直至OD600=0.6-0.8为止。冷却至16-18℃后,加入1M的IPTG在16℃培养室内诱导过夜。第二天离心收集菌落,超声破碎,按照文献中的优化的糖基转移酶的方法纯化蛋白(Song C,Hong X,ZhaoS,et al.Glucosylation of 4-Hydroxy-2,5-Dimethyl-3(2H)-Furanone,the KeyStrawberry Flavor Compound in Strawberry Fruit[J].Plant Physiol.2016,171(1):139-151;Song C,Ring L,Hoffmann T,et al.Acylphloroglucinol Biosynthesis inStrawberry Fruit[J].Plant Physiol.2015,169(3):1656-1670;Song C,Gu L,Liu J,etal.Functional Characterization and Substrate Promiscuity of UGT71Glycosyltransferases from Strawberry(Fragaria×ananassa)[J].Plantand CellPhysiology.2015,56(12)),并进行SDS-PAGE检测。
4、香气底物筛选
将纯化成功的蛋白进行香气底物筛选,活性测定参考本实验室优化的方法(SongC,Ring L,Hoffmann T,et al.Acylphloroglucinol Biosynthesis in Strawberry Fruit[J].Plant Physiol.2015,169(3):1656-1670;Song C,Gu L,Liu J,et al.FunctionalCharacterization and Substrate Promiscuity of UGT71 Glycosyltransferases fromStrawberry(Fragaria×ananassa)[J].Plant and Cell Physiology.2015,56(12)),测定UDP的生成量,从而确定测定糖苷的生成量。
香气底物筛选的反应体系:
香气底物筛选的对照体系:
Tris-HCl pH=7.5(50mM) 4.6ul
DTT(50mM) 0.2ul
UDPG(2.5mM) 0.2ul
所用的香气底物有:橙花叔醇、芳樟醇、香叶醇、丁子香酚、香草酸、水杨酸、氧化芳香醇、1-萘酚、金合欢醇、橙花醇、山梨酸等。
发现UGT91Q2编码的蛋白可以特异性催化橙花叔醇合成橙花叔醇葡萄糖苷。相对活性见图1所示。
5、产物鉴定
把反应体系中反应产物用乙酸乙酯进行萃取,上清液通过浓缩后用甲醇进行溶解,溶解液过滤后直接用LC-MS/MS进行分析。采用全波长扫描,进样量体积为2ul,流速为1.0mL/min,流动相为水和甲醇,鉴定方法参考实验室优化的方法(Song C,Hong X,Zhao S,et al.Glucosylation of 4-Hydroxy-2,5-Dimethyl-3(2H)-Furanone,the KeyStrawberry Flavor Compound in Strawberry Fruit[J].Plant Physiol.2016,171(1):139-151)。图2为CsUGT91Q2催化橙花叔醇合成橙花叔醇糖苷LC-MS色谱图,箭头所指的为合成的橙花叔醇糖苷的峰图,说明该酶催化橙花叔醇有糖苷产生;下方对应的为对照的峰图。图3为CsUGT91Q2催化橙花叔醇合成橙花叔醇糖苷LC-MS质谱图,385为橙花叔醇加葡萄糖加氢条件下的M值(正离子模式),进一步验证橙花叔醇葡萄糖苷的存在。
6、UGT91Q2体外酶动力学分析
研究UGT91Q2基因编码的蛋白跟橙花叔醇体外反应在不同反应时间、温度、pH下,酶活的差异,优化出最反应的最适条件。在最适条件下,改变底物浓度,做酶动力学实验,从而得到酶的Km和Vm。优化不同反应时间、温度、pH的反应体系为:
不同不同pH、不同温度、反应时间的酶活数据分别见图4、图5和图6。
图7为不同糖供体的酶活数据;横坐标为不同的糖供体,GA为葡萄糖醛酸,Gal为半乳糖,Glu为葡萄糖;纵坐标为OD595条件下酶活相对应的吸光度值A。相同的反应条件下加入不同的糖供体,葡萄糖具有最高的催化效率。
根据最适温度(30℃)、最适pH值(8.5),反应时间10min,求得酶动力学参数。见图8,其Km:19.71±2.76μM,Vmax:0.37±0.01nKat·mg-1。
7、橙花叔醇葡萄糖苷的合成
利用本发明的分离的多肽或重组菌株可催化橙花叔醇合成橙花叔醇葡萄糖苷。
其中,利用分离的多肽催化橙花叔醇合成橙花叔醇葡萄糖苷的具体方法为:以橙化叔醇为底物,以上述所述的分离的多肽为催化剂合成橙花叔醇葡糖糖苷。最优的催化条件为:催化的温度为30℃、pH为8.5。
其中,利用重组菌株催化橙花叔醇合成橙花叔醇葡萄糖苷的具体方法为:将含有SEQ ID No.2所示的核酸的重组菌株或可表达SEQ ID No.1所示氨基酸序列的蛋白的重组菌株接种到橙化叔醇原料中发酵,从而得到橙化叔醇葡萄糖苷。
8、利用整细胞转化技术
将构建成功的表达载体pGEX4T1-UGT91Q2转化到BL21感受态细胞进行过夜培养,选取阳性菌斑转入LB培养基中37℃过夜培养,在37℃条件下进行扩大培养,直至OD600=0.6-0.8为止。冷却至16-18℃后,加入1M的IPTG在16℃培养室内诱导过夜。第二天离心收集菌落,获得的菌体用生理盐水(或缓冲液)悬浮洗涤后,5000r/min离心10min,收集菌体并于-20℃储存备用,100mL带塞透气性封口膜的锥形瓶中装入50mL缓冲液(pH为7.5),加入干菌体10mg/mL,并同时加入底物20、40、60、80、100、120、140mmol/L,在20-45℃、200r/min的条件下反应,定时取样,用液质进行分析产物。该方法得到的产物转化率可达到90%。
序列表
<110> 安徽农业大学
<120> 多肽、核酸及其在合成橙花叔醇糖苷上的应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 452
<212> PRT
<213> 茶树UGT91Q2蛋白(Camellia sinensis)
<400> 1
Met Glu Gly Lys Glu Ile His Val Val Leu Leu Pro Trp Leu Ala Phe
1 5 10 15
Gly His Met Met Pro Phe His Glu Leu Ala Ile Ser Leu Ala Lys Ala
20 25 30
Gly Ile Lys Val Ser Tyr Ile Ser Thr Pro Asn Asn Leu Arg Arg Leu
35 40 45
Pro Thr Pro Pro Pro Pro Leu Ala Ala Leu Ile Thr Pro Val Ala Leu
50 55 60
Pro Leu Pro Pro Leu Asp Leu Pro Glu Asn Ala Glu Ala Thr Val Asp
65 70 75 80
Ile Pro Met Glu Lys Leu Leu Ser Leu Thr Met Ala Phe Asp Leu Leu
85 90 95
His Gln Pro Phe Lys Gln Phe Val Ser Asp Phe Ser Pro Asp Trp Ile
100 105 110
Ile Ser Asp Phe Ile Pro His Trp Thr Ser Asp Val Ala Arg Asp Leu
115 120 125
Gly Val Pro Leu Leu Thr Phe Ser Ala Phe Ser Ala Ala Thr Asn Val
130 135 140
Phe Phe Gly Pro Pro Glu Phe Leu Ser Gly Glu Gly Gln Lys Arg Val
145 150 155 160
Arg Ser Ser Ile Glu Ser Leu Thr Ser Pro Pro Glu Trp Val Thr Phe
165 170 175
Pro Ser Ala Val Ala Tyr Arg Arg Phe Glu Ala Ala Gly Ala Leu Phe
180 185 190
Gly Phe Phe Gly Asp Asn Pro Thr Gly Ile Ser Ala Ala Gly Arg Val
195 200 205
Gly Lys Thr Leu Glu Gly Ser Arg Ala Val Ala Ile Arg Ser Cys Arg
210 215 220
Glu Ile Glu Gly Glu Tyr Leu Ser Leu Phe Glu Gln Ile Ile Gly Lys
225 230 235 240
Pro Val Ile Pro Val Gly Leu Leu Pro Pro Leu Lys Ser Asn Lys Ala
245 250 255
Gln Lys Gln Thr Arg Asp Glu Asn Trp Thr Gln Ile Phe Lys Trp Leu
260 265 270
Asp Tyr Gln Lys Pro Arg Ser Val Leu Phe Val Gly Phe Gly Ser Glu
275 280 285
Cys Lys Leu Asn Lys Glu Glu Ile His Glu Ile Ala His Gly Leu Glu
290 295 300
Lys Ser Glu Leu Pro Phe Leu Trp Ala Leu Arg Lys Pro Thr Trp Ala
305 310 315 320
Ile Glu Asp Leu Asp Ser Val Pro Ile Glu Phe Thr Asp Arg Thr Leu
325 330 335
Glu Arg Gly Arg Val Ser Phe Gly Trp Ala Pro Gln Arg Glu Ile Leu
340 345 350
Glu His Pro Ser Ile Gly Gly Ser Leu Phe His Ala Gly Trp Gly Ser
355 360 365
Val Ile Glu Thr Leu Gln Phe Gly His Ser Met Val Val Leu Pro Leu
370 375 380
Ile Ile Asp Gln Gly Leu Asn Ala Arg Leu Met Val Glu Lys Gly Leu
385 390 395 400
Ala Ile Glu Val Asp Arg Ser Glu Asp Gly Ser Phe Ser Arg Asp Asp
405 410 415
Ile Ala Lys Ala Leu Lys Leu Ala Met Val Ser Lys Glu Gly Asp Glu
420 425 430
Met Arg Ala Arg Leu Arg Glu Ala Ala Lys Met Ala Gly His Gln Lys
435 440 445
Leu His Asp Gly
450
<210> 2
<211> 1359
<212> DNA
<213> 茶树UGT91Q2基因(Camellia sinensis)
<400> 2
atggagggaa aagaaattca cgttgttctc cttccatggc tagcattcgg tcacatgatg 60
ccatttcacg agctcgccat atccctagcc aaagccggca tcaaagtctc ctacatctca 120
accccaaaca atctccgccg cctccccacc cctccgccgc ctctcgccgc cctcatcact 180
ccggtggcgc ttccacttcc gccgctcgac ttgccggaaa acgccgaagc caccgtcgac 240
attcccatgg agaaactcct ctccttaacc atggccttcg atctcctcca ccaacccttc 300
aagcaattcg tctccgattt ctcgccggac tggatcatct ccgatttcat cccccattgg 360
acctccgacg tagctcgaga cttgggtgtt cctctattga ccttctctgc tttctcggcg 420
gcgaccaatg tgttcttcgg cccgccggag tttctctccg gcgagggtca gaaaagagtc 480
cgatcatcca tcgagagcct gacttcgccg ccggagtggg tcacgtttcc ttcggcggtg 540
gcgtaccgga gattcgaagc cgccggagct cttttcgggt tttttgggga taatccgacc 600
gggatctccg cggcgggaag agtcgggaaa actctagaag gttctagagc tgttgcgatt 660
cggagttgta gagagatcga gggtgagtat ttgagcttgt tcgagcagat cattgggaag 720
cctgtgattc cagtgggttt gcttccgcca ttgaaatcga ataaagctca aaaacaaacc 780
agagatgaga attggactca aatcttcaaa tggcttgatt atcagaaacc cagatcggtt 840
ctttttgttg ggtttgggag tgagtgtaaa ctcaacaaag aagaaattca cgagatcgct 900
catgggcttg agaaatcgga gcttccattt ttgtgggctc tgagaaaacc cacttgggca 960
attgaagatc tcgattctgt gccgattgaa ttcactgatc ggacattgga gagagggaga 1020
gtgagcttcg gatgggcacc gcagagagag attctggaac acccatcaat cggagggtct 1080
ctgtttcacg caggttgggg atcggtgatt gaaacactgc aatttggaca ctcgatggtg 1140
gttcttcctt tgataatcga tcagggtttg aatgcgaggt tgatggttga aaagggtttg 1200
gcgattgaag tggacagaag tgaagatggg tcgtttagca gagacgacat agctaaggct 1260
ctgaaactag ctatggtgtc caaggaagga gatgaaatga gagctcggtt gagagaagct 1320
gcgaagatgg ctggacatca gaaactgcat gatggttag 1359
<210> 3
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ggttccgcgt ggatccatga acggtgactc ccaacaaca 39
<210> 4
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ggccgctcga gtcgacttaa gggtgcttac aagctttgat tacctccaac 50
Claims (9)
1.分离的多肽,其氨基酸序列如SEQ ID No.1所示。
2.编码权利要求1所述的多肽的核酸。
3.根据权利要求2所述的核酸,其特征在于,所述核酸的核苷酸序列如SEQ ID No.2所示。
4.重组载体,包含有权利要求2或3所述的核酸。
5.重组菌株,包含有权利要求2所述的核酸或权利要求4所述的重组载体。
6.权利要求1所述的分离的多肽在催化橙花叔醇合成橙花叔醇葡萄糖苷上的应用。
7.根据权利要求6所述的应用,其特征在于,以橙化叔醇为底物,以权利要求1所述的分离的多肽为催化剂合成橙花叔醇葡糖糖苷。
8.根据权利要求7所述的应用,其特征在于,催化的温度为30℃、pH为8.5。
9.一种合成橙化叔醇葡萄糖苷的方法,将权利要求5所述的重组菌株接种到橙化叔醇原料中发酵,从而得到橙化叔醇葡萄糖苷。
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