CN114457124A - 一种酶催化甲基腰果酚的制备方法 - Google Patents
一种酶催化甲基腰果酚的制备方法 Download PDFInfo
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- CN114457124A CN114457124A CN202210176560.7A CN202210176560A CN114457124A CN 114457124 A CN114457124 A CN 114457124A CN 202210176560 A CN202210176560 A CN 202210176560A CN 114457124 A CN114457124 A CN 114457124A
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- Prior art keywords
- cardanol
- preparation
- methyl
- strain
- enzymatic
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- FAYVLNWNMNHXGA-UHFFFAOYSA-N Cardanoldiene Natural products CCCC=CCC=CCCCCCCCC1=CC=CC(O)=C1 FAYVLNWNMNHXGA-UHFFFAOYSA-N 0.000 title claims abstract description 78
- JOLVYUIAMRUBRK-UHFFFAOYSA-N 11',12',14',15'-Tetradehydro(Z,Z-)-3-(8-Pentadecenyl)phenol Natural products OC1=CC=CC(CCCCCCCC=CCC=CCC=C)=C1 JOLVYUIAMRUBRK-UHFFFAOYSA-N 0.000 title claims abstract description 77
- YLKVIMNNMLKUGJ-UHFFFAOYSA-N 3-Delta8-pentadecenylphenol Natural products CCCCCCC=CCCCCCCCC1=CC=CC(O)=C1 YLKVIMNNMLKUGJ-UHFFFAOYSA-N 0.000 title claims abstract description 77
- PTFIPECGHSYQNR-UHFFFAOYSA-N cardanol Natural products CCCCCCCCCCCCCCCC1=CC=CC(O)=C1 PTFIPECGHSYQNR-UHFFFAOYSA-N 0.000 title claims abstract description 77
- -1 methyl cardanol Chemical compound 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 230000002255 enzymatic effect Effects 0.000 title claims abstract description 13
- JOLVYUIAMRUBRK-UTOQUPLUSA-N Cardanol Chemical compound OC1=CC=CC(CCCCCCC\C=C/C\C=C/CC=C)=C1 JOLVYUIAMRUBRK-UTOQUPLUSA-N 0.000 claims abstract description 38
- 238000006243 chemical reaction Methods 0.000 claims abstract description 26
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims abstract description 25
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- 239000007788 liquid Substances 0.000 claims abstract description 17
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 17
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- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims abstract 3
- 239000000243 solution Substances 0.000 claims description 30
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- 239000008367 deionised water Substances 0.000 claims description 14
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- 241000894006 Bacteria Species 0.000 description 6
- 238000006911 enzymatic reaction Methods 0.000 description 6
- 238000001228 spectrum Methods 0.000 description 6
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 5
- 241001052560 Thallis Species 0.000 description 4
- KAOMOVYHGLSFHQ-UTOQUPLUSA-N anacardic acid Chemical compound CCC\C=C/C\C=C/CCCCCCCC1=CC=CC(O)=C1C(O)=O KAOMOVYHGLSFHQ-UTOQUPLUSA-N 0.000 description 4
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- ADFWQBGTDJIESE-UHFFFAOYSA-N anacardic acid 15:0 Natural products CCCCCCCCCCCCCCCC1=CC=CC(O)=C1C(O)=O ADFWQBGTDJIESE-UHFFFAOYSA-N 0.000 description 4
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- 238000004458 analytical method Methods 0.000 description 3
- 108010038633 aspartylglutamate Proteins 0.000 description 3
- KVVSCMOUFCNCGX-UHFFFAOYSA-N cardol Chemical compound CCCCCCCCCCCCCCCC1=CC(O)=CC(O)=C1 KVVSCMOUFCNCGX-UHFFFAOYSA-N 0.000 description 3
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- 238000006257 total synthesis reaction Methods 0.000 description 3
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- 108020004705 Codon Proteins 0.000 description 2
- SBANPBVRHYIMRR-UHFFFAOYSA-N Leu-Ser-Pro Natural products CC(C)CC(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O SBANPBVRHYIMRR-UHFFFAOYSA-N 0.000 description 2
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- 230000009286 beneficial effect Effects 0.000 description 2
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- 238000001816 cooling Methods 0.000 description 2
- 150000001993 dienes Chemical class 0.000 description 2
- 108010050848 glycylleucine Proteins 0.000 description 2
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- 238000005457 optimization Methods 0.000 description 2
- 238000010587 phase diagram Methods 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- JBVSSSZFNTXJDX-YTLHQDLWSA-N Ala-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](C)N JBVSSSZFNTXJDX-YTLHQDLWSA-N 0.000 description 1
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Abstract
本发明属于酶催化技术领域，具体涉及一种酶催化甲基腰果酚的制备方法，包括以下步骤：首先合成出甲基转移酶的原始基因，对其进行优化、重组，将重组的表达载体转移到菌株中进行表达，对表达菌株进行培养，然后破碎，得到含重组甲基转移酶菌体破碎液。向反应器中加入腰果酚、甜菜碱、重组甲基转移酶菌体破碎液、磷酸盐缓冲液，调节pH值、温度，反应后得到甲基腰果酚；本发明提供的酶催化甲基腰果酚的制备方法，制备出了一定转化率的甲基腰果酚，不使用易燃易爆物质硫酸二甲酯和碘甲烷，环境友好，对于安全生产具有十分重要的意义。
Description
技术领域
本发明涉及酶催化技术领域，尤其涉及一种酶催化甲基腰果酚的制备方法。
背景技术
天然腰果壳液(油)中含有腰果酸、腰果酚、强心酚和二甲基强心酚4种主要成分，其中腰果酸占90％，腰果酸在高温条件下会进行脱酸，使腰果酸转变为腰果酚，关于腰果酚的提取，目前已有许多报道的方法用于提取高纯度的腰果酚。腰果酚，是一种非常重要的工业烷基酚醛化合物，虽然其芳香环结构具有有益的化学耐性，长链脂肪酸碳链使得腰果酚具有脂肪族化合物的耐水性和耐温性，但是腰果酚因其苯酚基团的存在，仍具有酚类物质的危险性特征，并且具有皮肤腐蚀性。
腰果酚的结构式：
腰果酚经过衍生化之后，可以降低其腐蚀性，有研究表明甲基腰果酚具有部分抗原虫活性。现有技术中，实验室里用化学法进行甲基转移，如以碘甲烷为甲基供体；或以硫酸二甲酯为甲基供体，但是这些化学法安全系数低、成本高、环境污染大且碘甲烷和硫酸二甲酯存在易燃易爆等问题。
发明内容
基于现有技术中存在的上述缺点和不足，本发明的目的之一是至少解决现有技术中存在的上述问题之一或多个，换言之，本发明的目的之一是提供满足前述需求之一或多个的一种酶催化甲基腰果酚的制备方法。
为了达到上述发明目的，本发明采用以下技术方案：
甲基转移酶催化制备甲基腰果酚的反应路线为（腰果酚三烯为例）：
一种酶催化甲基腰果酚的制备方法，具体如下：
向反应器中加入腰果酚、甜菜碱、重组甲基转移酶、磷酸盐缓冲液，调节pH值、温度，反应后得到甲基腰果酚。
作为优选方案，所述甜菜碱浓度为20~50g/L。
作为优选方案，所述腰果酚与混合溶液的固液比为50~125g/L。
作为优选方案，所述pH值利用pH自动控制系统和1 M Na2CO3溶液控制酶反应的pH值在6.8~8.9之间。
作为优选方案，所述温度在28-35 ℃，反应时间不低于7小时。
作为优选方案，所述磷酸盐缓冲液浓度为0.08~0.12 M，pH为7。
作为优选方案，所述重组甲基转移酶是由菌株用离心机离心，用磷酸盐缓冲液重悬菌体，超声破碎后所得。
作为优选方案，所述菌株是经过优化甲基转移酶OMT3原始基因序列，得到OMT3基因序列，对其进行目的基因全合成后，连接到pET28a(+)上，得到载体pET28a-OMT3，将载体pET28a-OMT3转化到菌株BL21(DE3)中，挑取单菌落接入到卡那霉素的LB培养基中，振荡培养，得到种子液，种子液加入发酵培养基中后，添加IPTG，培养所得。
作为优选方案，所述LB培养基包括蛋白胨、酵母提取物、NaCl、去离子水。
作为优选方案，所述发酵培养基包括蛋白胨、酵母提取物、甘油、KH2PO4、K2HPO4、去离子水。
优化后的OMT3基因序列为：
ATGGTCTTGATCTCTGAGGATTCTAGGGAATTGTTGCAAGCTCATGTTGAGTTGTGGAACCAGACTTACTCTTTCATGAAGTCTGTCGCTTTGGCTGTTGCTTTGGATTTGCATATTGCTGACGCCATTCATAGAAGAGGTGGTGCTGCTACTTTGTCTCAAATTTTGGGCGAGATTGGTGTTAGACCATGTAAATTGCCAGGTTTGCATAGGATTATGAGGGTCTTGACTGTTTCTGGCACTTTTACTATTGTCCAGCCATCTGCTGAAACTATGTCTTCTGAGTCTGATGGTAGAGAACCAGTCTACAAATTGACTACTGCTTCCTCTTTGTTGGTTTCTTCTGAGTCTTCTGCTACTGCTTCTTTGTCTCCAATGTTGAATCACGTCTTGTCTCCATTTAGGGATTCTCCATTGTCTATGGGTTTGACTGCTTGGTTTAGGCATGATGAAGATGAACAAGCTCCAGGTATGTGTCCATTTACTTTGATGTACGGTACTACTTTGTGGGAAGTTTGCAGAAGGGACGATGCTATTAACGCTTTGTTCAACAACGCTATGGCTGCTGATTCTAATTTCTTGATGCAGATCTTGTTGAAGGAGTTCTCTGAGGTTTTCTTGGGTATTGACTCCTTGGTTGATGTTGCTGGTGGTGTTGGTGGTGCTACTATGGCTATTGCTGCTGCTTTTCCATGTTTGAAGTGCACTGTCTTGGATTTGCCACACGTTGTTGCTAAAGCTCCCTCTTCTTCTATTGGTAACGTCCAATTTGTTGGTGGTGACATGTTTGAATCTATTCCCCCAGCTAATGTCGTTTTGTTGAAGTGGATTTTGCACGACTGGTCTAATGATGAGTGCATTAAGATCTTGAAGAACTGCAAGCAAGCTATTCCATCTAGAGATGCTGGTGGTAAGATCATTATTATCGACGTCGTCGTTGGTTCTGATTCTTCTGACACCAAGTTGTTGGAAACCCAGGTCATTTACGACTTGCACTTGATGAAGATTGGTGGTGTCGAGAGAGATGAACAGGAGTGGAAGAAGATTTTCTTGGAGGCTGGTTTCAAAGACTACAAGATCATGCCAATCTTGGGCTTGAGGTCTATTATTGAGTTGTACCCATGA。
优化后的OMT3编码的氨基酸序列为：
MVLISEDSRELLQAHVELWNQTYSFMKSVALAVALDLHIADAIHRRGGAATLSQILGEIGVRPCKLPGLHRIMRVLTVSGTFTIVQPSAETMSSESDGREPVYKLTTASSLLVSSESSATASLSPMLNHVLSPFRDSPLSMGLTAWFRHDEDEQAPGMCPFTLMYGTTLWEVCRRDDAINALFNNAMAADSNFLMQILLKEFSEVFLGIDSLVDVAGGVGGATMAIAAAFPCLKCTVLDLPHVVAKAPSSSIGNVQFVGGDMFESIPPANVVLLKWILHDWSNDECIKILKNCKQAIPSRDAGGKIIIIDVVVGSDSSDTKLLETQVIYDLHLMKIGGVERDEQEWKKIFLEAGFKDYKIMPILGLRSIIELYP。
与现有技术相比，本发明的有益效果是：
（1）经过优化重组的表达载体在菌株中表达出的酶对催化制备甲基腰果酚的反应具有相当高的催化活性。
（2）本发明提供的酶法制备甲基腰果酚的方法，对环境友好，不使用易燃易爆物质硫酸二甲酯和碘甲烷，对于安全生产具有十分重要的意义。
（3）本发明制备出了一定转化率的甲基腰果酚。
附图说明
图1是腰果酚液相图谱；
图2是甲基腰果酚液相图谱；
图3是实施例1的产物液相图谱；
图4是实施例3的产物液相图谱；
图5是本发明方法的反应路线图。
具体实施方式
为了更清楚地说明本发明实施例，下面描述中的是本发明的一些实施例。显而易见地，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些实施例替换获得其他的实施方式。
本发明采用一种生物法酶催化方法通过腰果酚制备出了一定转化率的甲基腰果酚，腰果酚具有三种结构分别是腰果酚三烯、腰果酚二烯、腰果酚单烯。
在进行反应前，先制备出了重组甲基转移酶。制备时所述离心机转速为3000~4500rpm/min，离心时间为8~12 min；用浓度为0.05~0.15 M、pH为6.8~7.2的磷酸盐缓冲液重悬菌体，菌体与磷酸盐缓冲液的固液比为1~3 g/mL。
所述菌株是经过优化甲基转移酶OMT3原始基因序列，得到OMT3基因序列，对其进行目的基因全合成后，连接到pET28a(+)上，得到载体pET28a-OMT3，将载体pET28a-OMT3转化到菌株BL21(DE3)中，挑取单菌落，将单菌落接入到3~7 mL浓度为49~50 mg/L卡那霉素的LB培养基中，在36~38 ℃条件下震荡培养6~12 h，得种子液；取1 mL种子液加入到含浓度为49~50 mg/L卡那霉素的50 mL 发酵培养基中， 36~38 ℃振荡培养至OD600至3.2~3.5，然后添加IPTG至IPTG终浓度为0.05~0.15 mM，20~30 ℃下诱导培养6~12 h。
卡那霉素是一种蛋白质生物合成抑制剂，被作为标记基因用于分子克隆中。本发明中卡那霉素作为表达载体的标记，在LB培养基和发酵培养基中添加了卡那霉素，培养出的菌即带有表达的目的菌。OD600指的是某种溶液在600 nm波长处的吸光值。吸光值正比于溶液中的吸光物质的浓度。测量细菌培养液在600 nm处的吸光值，得到的OD600的数值如果在0.6-0.8之间，表明细菌处于旺盛生长的对数生长期，OD600>3表明细菌已经饱和。将菌种液在发酵培养基中培养至饱和后再进行诱导。IPTG是一种作用极强的诱导剂，是半乳糖的结构类似物，能够实现诱导但是不会被降解，从而更方便的实现蛋白的诱导表达。
LB培养基含有单菌落所需的营养物质，包括蛋白胨、酵母提取物、NaCl、去离子水。每1 L LB培养基中含有蛋白胨5~15 g、酵母提取物2~8 g、NaCl 5~15 g，余量为去离子水。
发酵培养基中含有种子液扩大培养所需的营养物质和无机盐，包括蛋白胨、酵母提取物、甘油、KH2PO4、K2HPO4、去离子水。每1 L发酵培养基中含有蛋白胨10~15 g、酵母提取物20~28 g、甘油2~6 mL、KH2PO4 2~2.7 g、K2HPO4 12~13 g，余量为去离子水。所述培养基的制备方法为：将蛋白胨、酵母提取物、甘油加入900~950 mL去离子水中，制成溶液I；将KH2PO4、K2HPO4溶于50~100 mL去离子水中，制成溶液II；将溶液I和溶液II分别灭菌后冷却至20~60 ℃混合均匀。
一种酶催化甲基腰果酚的制备方法，其中重组甲基转移酶的制备包括以下步骤：
(1)甲基转移酶原始基因的合成：按照大肠杆菌密码子分析用表对甲基转移酶OMT3原始基因序列进行密码子优化，得到优化后的OMT3基因序列，对其进行目的基因的全合成，合成后的基因重组并连接到pET28a(+)上，得到重组表达载体pET28a-OMT3。
(2)甲基转移酶的表达：将重组表达载体pET28a-OMT3通过热激转化法转化到表达菌株BL21(DE3)中，挑取单菌落，将单菌落接入到5 mL含50 μg/mL卡那霉素的LB培养基中，在37 ℃条件下，振荡培养9 h，得到种子液。每1 mL种子液加入到100 mL含浓度为50 μg/mL卡那霉素的发酵培养基中，在37 ℃条件下培养至OD600为3.3，然后添加IPTG至IPTG的终浓度为0.5 mM，在25 ℃培养9 h。
(3)细胞破碎：将培养好的菌株用离心机离心，离心机的转速为4000 rpm/min，离心时间为10 min，收集菌体，用浓度为100 mM、pH=7.40的磷酸盐缓冲液重悬菌体，菌体与磷酸盐缓冲液的固液比为2 g/ mL，超声破碎后所得液体即为含重组甲级转移酶菌体破碎液。
其中，LB培养基中含有以下成分的物质：每1 L LB培养基中含有蛋白胨10 g、酵母提取物5 g、NaCl 10 g，余量为去离子水。
发酵培养基中含有以下成分的物质：每1 L 发酵培养基中含有蛋白胨12 g、酵母提取物24 g、甘油4 mL、KH2PO4 2.31 g、K2HPO4 12.54 g，余量为去离子水。其制备方法为：将蛋白胨、酵母提取物、甘油加入到900 mL去离子水中，制成溶液I；将KH2PO4、K2HPO4溶于100 mL去离子水中，制成溶液II；将溶液I和溶液II分别灭菌后冷却至40 ℃混和均匀。
本发明中提供甲基的物质，选用甜菜碱，酶可以把甜菜碱的甲基转移到腰果酚上，反应中甜菜碱的浓度在20~50g/L之间，甜菜碱也可以换成S-腺苷甲硫氨酸。
本发明的反应温度在28-35℃之间，反应的最佳温度为30℃。反应中的pH利用pH自动控制系统和1 M Na2CO3溶液控制酶反应的pH值，使其在6.8~8.9之间，实施例中控制pH值为7.0左右。磷酸盐缓冲液浓度为0.08~0.12 M，pH为7，实施例中采用0.1M，pH为7。下述实施例腰果酚与混合溶液的固液比为50~125g/L，浓度不同底物转化率及产物浓度会有所不同。
实施例1
将0.2 g底物腰果酚加入到50 mL反应器中。随后，将10 mL含有20 g/L甜菜碱、20g/L 含重组甲基转移酶菌体破碎液、0.1 M磷酸盐缓冲液(pH 7.0)加入该反应器中，腰果酚与混合溶液的体积比为20 g/L，利用pH自动控制系统和1 M Na2CO3溶液控制酶反应的pH值为7.0左右。30 ℃反应8 h。底物转化率大于30%，产物浓度为6 g/L。
实施例2
将1 g底物腰果酚加入到50 mL反应器中。随后，将15 mL含有40 g/L甜菜碱、20 g/L 含重组甲基转移酶菌体破碎液、0.1 M磷酸盐缓冲液(pH 7.0)加入该反应器中，腰果酚与混合溶液的固液比为66.7 g/L，利用pH自动控制系统和1 M Na2CO3溶液控制酶反应的pH值为7.0左右。30 ℃反应8 h。底物转化率大于33%，产物浓度为22 g/L。
实施例3
将5 g底物腰果酚加入到250 mL反应器中。随后，将50 mL含有40 g/L甜菜碱、30g/L 含重组甲基转移酶菌体破碎液、0.1 M磷酸盐缓冲液(pH 7.0)加入该反应器中，腰果酚与混合溶液的固液比为100 g/L，利用pH自动控制系统和1 M Na2CO3溶液控制酶反应的pH值为7.0左右。30 ℃反应8 h。底物转化率大于32%，产物浓度为32 g/L。
实施例4
将10 g底物腰果酚加入到250 mL反应器中。随后，将100 mL含有40 g/L甜菜碱、35g/L 含重组甲基转移酶菌体破碎液、0.1 M磷酸盐缓冲液(pH 7.0)加入该反应器中，腰果酚与混合溶液的固液比为100 g/L，利用pH自动控制系统和1 M Na2CO3溶液控制酶反应的pH值为7.0左右。30 ℃反应8 h。底物转化率大于31%，产物浓度为31 g/L。
实施例5
将25 g底物腰果酚加入到500 mL反应器中。随后，将200 mL含有40 g/L甜菜碱、30g/L含重组甲基转移酶菌体破碎液、0.1 M磷酸盐缓冲液(pH 7.0)加入该反应器中，腰果酚与混合溶液的固液比为125 g/L，利用pH自动控制系统和1 M Na2CO3溶液控制酶反应的pH值为7.0。30 ℃反应7 h。底物转化率大于28%，产物浓度为35 g/L。
为了进一步说明反应产生了一定转化率的甲基腰果酚，本实验通过液相图谱加以验证。
液相分析条件：分析色谱柱：Aglient-XDB-C18(4.6 × 250 mm, 5 μm)；流动相：纯乙腈；流速：1.5 mL/min；柱温：30 ℃。
首先配置了标样腰果酚，并通过液相得到图谱如图1所示，从腰果油中分离得到的腰果酚样品，从左至右分别为：1.腰果酚(三烯)；2.腰果酚(二烯)；3.腰果酚(单烯)。
为了探究甲基腰果酚的出峰情况，配制了甲基腰果酚标样并通过液相仪器得到了甲基腰果酚样品液相谱图如图2所示，从左至右分别为：4.甲基腰果酚(三烯)；5.甲基腰果酚(二烯)；6.甲基腰果酚(单烯)。
通过甲基转移酶经过如图5所示反应路线催化转化的腰果酚样品，得到的液相图谱如图3、4所示。从图中可以看出，组分4甲基腰果酚(三烯)保留时间介于组分2和3之间，从而说明分析方法合适，8小时后的转化率为30%左右。
上述实施例中制备得到甲基腰果酚的样品，通过液相LC确定产物浓度，转化率是用产物摩尔浓度/底物投料摩尔浓度得到。实施例1的产物浓度低是因为实施例1的产物浓度是根据底物投料量来确定的，底物投料是20 g/L，最后得到产物浓度6 g/L，实际转化率28.65%。通过上述实验数据可以看出，经过优化重组的表达载体在菌株中表达出的酶对催化制备甲基腰果酚的反应具有相当高的催化活性。本发明提供的酶催化制备甲基腰果酚的方法，对环境友好，不使用易燃易爆物质硫酸二甲酯和碘甲烷，对于安全生产具有十分重要的意义，并制备出了转化率在30%左右的甲基腰果酚，展现了本发明的优势所在。
以上所述仅是对本发明的优选实施例及原理进行了详细说明，但是本发明并不限于上述实施方式中的具体细节，对本领域的普通技术人员而言，依据本发明提供的技术构思，在技术方案具体实施方式上会有改变之处，而这些改变也应视为本发明的保护范围。
序列表
<110> 浙江工业大学
<120> 一种酶法制备甲基腰果酚的方法
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1125
<212> DNA
<213> 未知(Unknown)
<400> 1
atggtcttga tctctgagga ttctagggaa ttgttgcaag ctcatgttga gttgtggaac 60
cagacttact ctttcatgaa gtctgtcgct ttggctgttg ctttggattt gcatattgct 120
gacgccattc atagaagagg tggtgctgct actttgtctc aaattttggg cgagattggt 180
gttagaccat gtaaattgcc aggtttgcat aggattatga gggtcttgac tgtttctggc 240
acttttacta ttgtccagcc atctgctgaa actatgtctt ctgagtctga tggtagagaa 300
ccagtctaca aattgactac tgcttcctct ttgttggttt cttctgagtc ttctgctact 360
gcttctttgt ctccaatgtt gaatcacgtc ttgtctccat ttagggattc tccattgtct 420
atgggtttga ctgcttggtt taggcatgat gaagatgaac aagctccagg tatgtgtcca 480
tttactttga tgtacggtac tactttgtgg gaagtttgca gaagggacga tgctattaac 540
gctttgttca acaacgctat ggctgctgat tctaatttct tgatgcagat cttgttgaag 600
gagttctctg aggttttctt gggtattgac tccttggttg atgttgctgg tggtgttggt 660
ggtgctacta tggctattgc tgctgctttt ccatgtttga agtgcactgt cttggatttg 720
ccacacgttg ttgctaaagc tccctcttct tctattggta acgtccaatt tgttggtggt 780
gacatgtttg aatctattcc cccagctaat gtcgttttgt tgaagtggat tttgcacgac 840
tggtctaatg atgagtgcat taagatcttg aagaactgca agcaagctat tccatctaga 900
gatgctggtg gtaagatcat tattatcgac gtcgtcgttg gttctgattc ttctgacacc 960
aagttgttgg aaacccaggt catttacgac ttgcacttga tgaagattgg tggtgtcgag 1020
agagatgaac aggagtggaa gaagattttc ttggaggctg gtttcaaaga ctacaagatc 1080
atgccaatct tgggcttgag gtctattatt gagttgtacc catga 1125
<210> 2
<211> 374
<212> PRT
<213> 未知(Unknown)
<400> 2
Met Val Leu Ile Ser Glu Asp Ser Arg Glu Leu Leu Gln Ala His Val
1 5 10 15
Glu Leu Trp Asn Gln Thr Tyr Ser Phe Met Lys Ser Val Ala Leu Ala
20 25 30
Val Ala Leu Asp Leu His Ile Ala Asp Ala Ile His Arg Arg Gly Gly
35 40 45
Ala Ala Thr Leu Ser Gln Ile Leu Gly Glu Ile Gly Val Arg Pro Cys
50 55 60
Lys Leu Pro Gly Leu His Arg Ile Met Arg Val Leu Thr Val Ser Gly
65 70 75 80
Thr Phe Thr Ile Val Gln Pro Ser Ala Glu Thr Met Ser Ser Glu Ser
85 90 95
Asp Gly Arg Glu Pro Val Tyr Lys Leu Thr Thr Ala Ser Ser Leu Leu
100 105 110
Val Ser Ser Glu Ser Ser Ala Thr Ala Ser Leu Ser Pro Met Leu Asn
115 120 125
His Val Leu Ser Pro Phe Arg Asp Ser Pro Leu Ser Met Gly Leu Thr
130 135 140
Ala Trp Phe Arg His Asp Glu Asp Glu Gln Ala Pro Gly Met Cys Pro
145 150 155 160
Phe Thr Leu Met Tyr Gly Thr Thr Leu Trp Glu Val Cys Arg Arg Asp
165 170 175
Asp Ala Ile Asn Ala Leu Phe Asn Asn Ala Met Ala Ala Asp Ser Asn
180 185 190
Phe Leu Met Gln Ile Leu Leu Lys Glu Phe Ser Glu Val Phe Leu Gly
195 200 205
Ile Asp Ser Leu Val Asp Val Ala Gly Gly Val Gly Gly Ala Thr Met
210 215 220
Ala Ile Ala Ala Ala Phe Pro Cys Leu Lys Cys Thr Val Leu Asp Leu
225 230 235 240
Pro His Val Val Ala Lys Ala Pro Ser Ser Ser Ile Gly Asn Val Gln
245 250 255
Phe Val Gly Gly Asp Met Phe Glu Ser Ile Pro Pro Ala Asn Val Val
260 265 270
Leu Leu Lys Trp Ile Leu His Asp Trp Ser Asn Asp Glu Cys Ile Lys
275 280 285
Ile Leu Lys Asn Cys Lys Gln Ala Ile Pro Ser Arg Asp Ala Gly Gly
290 295 300
Lys Ile Ile Ile Ile Asp Val Val Val Gly Ser Asp Ser Ser Asp Thr
305 310 315 320
Lys Leu Leu Glu Thr Gln Val Ile Tyr Asp Leu His Leu Met Lys Ile
325 330 335
Gly Gly Val Glu Arg Asp Glu Gln Glu Trp Lys Lys Ile Phe Leu Glu
340 345 350
Ala Gly Phe Lys Asp Tyr Lys Ile Met Pro Ile Leu Gly Leu Arg Ser
355 360 365
Ile Ile Glu Leu Tyr Pro
370
Claims (10)
1.一种酶催化甲基腰果酚的制备方法，其特征在于：
向反应器中加入腰果酚、甜菜碱、重组甲基转移酶、磷酸盐缓冲液，调节pH值、温度，反应后得到甲基腰果酚。
2.根据权利要求1所述的一种酶催化甲基腰果酚的制备方法，其特征在于，所述甜菜碱浓度为20~50g/L。
3.根据权利要求1所述的一种酶催化甲基腰果酚的制备方法，其特征在于，所述腰果酚与混合溶液的固液比为50~125g/L。
4.根据权利要求1所述的一种酶催化甲基腰果酚的制备方法，其特征在于，所述pH值在6.8~8.9之间。
5.根据权利要求1所述的一种酶催化甲基腰果酚的制备方法，其特征在于，所述反应温度在28-35 ℃，反应时间不低于7小时。
6.根据权利要求1所述的一种酶催化甲基腰果酚的制备方法，其特征在于，所述磷酸盐缓冲液浓度为0.08~0.12 M，pH为7。
7.根据权利要求1所述的一种酶催化甲基腰果酚的制备方法，其特征在于，所述重组甲基转移酶是把菌株用离心机离心，用磷酸盐缓冲液重悬菌体，超声破碎后所得。
8.根据权利要求7所述的一种酶催化甲基腰果酚的制备方法，其特征在于，所述菌株是经过优化甲基转移酶OMT3原始基因序列，得到OMT3基因序列，对其进行目的基因全合成后，连接到pET28a(+)上，得到载体pET28a-OMT3，将载体pET28a-OMT3转化到菌株BL21(DE3)中，挑取单菌落接入到卡那霉素的LB培养基中，振荡培养，得到种子液，种子液加入发酵培养基中后，添加IPTG，培养所得。
9.根据权利要求8所述的一种酶催化甲基腰果酚的制备方法，其特征在于，所述LB培养基包括蛋白胨、酵母提取物、NaCl、去离子水。
10.根据权利要求8所述的一种酶催化甲基腰果酚的制备方法，其特征在于，所述发酵培养基包括蛋白胨、酵母提取物、甘油、KH2PO4、K2HPO4、去离子水。
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|---|---|---|---|---|
| JP2006000023A (ja) * | 2004-06-16 | 2006-01-05 | Kissei Pharmaceut Co Ltd | ヒトカテコールｏ−メチルトランスフェラーゼの結晶 |
| WO2010100831A1 (ja) * | 2009-03-02 | 2010-09-10 | アサヒビール株式会社 | メチルトランスフェラーゼ酵素 |
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| CN110699399A (zh) * | 2019-10-22 | 2020-01-17 | 浙江大学 | 柑橘氧甲基转移酶CitOMT2的体外酶活应用 |
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| JP2006000023A (ja) * | 2004-06-16 | 2006-01-05 | Kissei Pharmaceut Co Ltd | ヒトカテコールｏ−メチルトランスフェラーゼの結晶 |
| WO2010100831A1 (ja) * | 2009-03-02 | 2010-09-10 | アサヒビール株式会社 | メチルトランスフェラーゼ酵素 |
| CN107893065A (zh) * | 2017-11-24 | 2018-04-10 | 宁夏乙征生物工程有限公司 | 一种固定化酶的制备方法 |
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