CN112175926A - 一种牛胰凝乳蛋白酶及其在药物中间体异色满制备过程中的应用 - Google Patents

一种牛胰凝乳蛋白酶及其在药物中间体异色满制备过程中的应用 Download PDF

Info

Publication number
CN112175926A
CN112175926A CN202011089476.9A CN202011089476A CN112175926A CN 112175926 A CN112175926 A CN 112175926A CN 202011089476 A CN202011089476 A CN 202011089476A CN 112175926 A CN112175926 A CN 112175926A
Authority
CN
China
Prior art keywords
isochroman
bovine chymotrypsin
reaction
petroleum ether
ser
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202011089476.9A
Other languages
English (en)
Inventor
傅荣昭
谢志军
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangxi Bontac Green Biocatalysis Ecoindustrial Park Co ltd
Original Assignee
Jiangxi Bontac Green Biocatalysis Ecoindustrial Park Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangxi Bontac Green Biocatalysis Ecoindustrial Park Co ltd filed Critical Jiangxi Bontac Green Biocatalysis Ecoindustrial Park Co ltd
Priority to CN202011089476.9A priority Critical patent/CN112175926A/zh
Publication of CN112175926A publication Critical patent/CN112175926A/zh
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6427—Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/76—Benzo[c]pyrans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21001—Chymotrypsin (3.4.21.1)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

本发明涉及一种牛胰凝乳蛋白酶及其在药物中间体异色满制备过程中的应用，属于生物化学合成领域，采用Morita‑Baylis‑Hillman(MBH)反应，开发全新的酶合成工艺。以苯乙醇为原料，在牛胰凝乳蛋白酶催化下，与多聚甲醛和脱水剂如DABCO、DMAP、DBU及三聚氰氯等作用下生成异色满。与常规化学法相比与纯化学合成法相比更加绿色环保，三废排放少，合成的异色满产率和纯度高于化学合成法。

Description

一种牛胰凝乳蛋白酶及其在药物中间体异色满制备过程中的 应用
技术领域
本发明属于生物化学合成领域，具体涉及一种牛胰凝乳蛋白酶及其在药物中间体异色满制备过程中的应用。
背景技术
异色满(CAS:493-05-0)是一种重要的药物中间体，也可以作为香料香精，香味佳，价格廉、稳定性好、无毒，广泛用作化妆品、皂用香精，其透发力及扩散性极佳，留香持久，用于调制调合香料及香精。同时也可用于合成多环麝香和佳乐麝香等。
异色满的化学合成方式很多，有一步法、两步法和三步法，普遍采用三氯化铝、三氯化磷等原料，反应条件毕竟苛刻，容易产生大量的污染物，对环境不太友好，因此，寻找简单高效、绿色环保的合成工艺是解决当前问题的关键。
发明内容
本发明结合酶催化Morita-Baylis-Hillman(MBH)反应，开发了全新的酶合成工艺。即以苯乙醇为原料，在牛胰凝乳蛋白酶催化下，与多聚甲醛和脱水剂如DABCO、DMAP、DBU及三聚氰氯等作用下生成异色满。
本发明是通过以下技术方案实现的：
一种牛胰凝乳蛋白酶，具有SEQIDNO：1所示的牛胰凝乳蛋白酶序列。
所述的牛胰凝乳蛋白酶序列，在药物中间体异色满制备过程中的应用。药物中间体异色满制备过程，包括以下步骤：
(1)将苯乙醇、多聚甲醛和脱水剂加入反应瓶，然后加入无水甲醇，搅拌溶解后，加入酶活80ug/g牛胰凝乳蛋白酶，并控温25-30℃；
(2)保温25-30℃反应1h，再升温至36℃反应2-3h，TLC监控反应进程；
(3)用6mol/L氢氧化钠溶液控温在40℃以下调PH＝7.0，在低温下蒸出甲醇，再用300ml二氯甲烷萃取3次，减压浓缩至干，加入石油醚稀释，过快速柱，石油醚洗脱，浓缩石油醚至干，得油状异色满。所述脱水剂为DABCO、DMAP、DBU中的一种。
步骤(1)中所述牛胰凝乳蛋白酶的加入量占苯乙醇质量的1/20-1/50。
本发明的有益效果
(1)与纯化学合成法相比更加绿色环保，三废排放少。
(2)合成的异色满产率和纯度高于化学合成法。
具体实施方式
以下实施例用于进一步阐述本发明，但不应理解为对本发明权利要求书请求保护范围的限制。
实施例1
牛胰凝乳蛋白酶的制备：
1.1提取：取新鲜、剥去脂肪及结缔组织的牛胰脏5.0kg，用绞肉机绞碎，将绞过的牛胰脏放入高速组织捣碎机中，加入适量配制好的0.125M硫酸，捣碎后倒进提取容器中，再加至4倍体积0.125M硫酸溶液用搅拌器开始搅拌、计时提取24h。
1.2离心：搅拌提取后的样品冷库放置24h后离心。将搅拌均匀的溶液加入500ml离心瓶中，用架盘天平平衡好后对称装进Beckman离心机中，(离心4500转，10min,4℃,JA-10转子)离心后将上清用四层纱布过滤至20L不锈钢桶中，累计离心收集上清，沉淀弃去。
1.3 40％盐析：离心后的样品，按242g/L加硫酸铵达到40％饱和度。搅拌溶解后，继续搅拌30min，放置24h后，(离心9500转，10min,4℃,JA-10转子)，离心后将上清用四层纱布过滤至20L不锈钢桶中，累计离心收集上清，沉淀弃去。
1.4 70％盐析：离心后的40％盐析上清，按205g/L加硫酸铵达到70％饱和度。搅拌溶解后，继续搅拌30min，放置24h后，(离心9500转，10min,4℃,JA-10转子)，收集沉淀，上清弃去，沉淀即为胰蛋白酶粗品。
1.5超滤：用10K超滤膜浓缩样品体积至1-2L，加10mM柠檬酸buffer至样品体积3倍左右，继续超滤重复此操作步骤三次(从超滤开始随时监测透过液A280值)，最后浓缩样晶体积至1L左右，除去进口压力用10mM柠檬酸buffer将膜中样品冲出，直至回流液A280值小于0.5,合并浓缩液和冲洗液至适当容器中，得牛凝乳蛋白酶精品。
实施例2
牛胰凝乳蛋白酶活力的测定：
将牛胰凝乳蛋白酶调pH8.10,加称量好的二水氯化钙，搅拌溶解后，用2mol氯氧化钠调pH值8.10士0.02,然后计时，测A280值随时检测活力至活力升至最高，速度缓慢时，用盐酸调pH值2.80,终止活化，检测trypsin活力及chymotrypsin活力，得到实施例1中牛胰凝乳蛋白酶活力为68.3U/mL。
实施例3
(1)室温下，将80g苯乙醇(MW:122.16)，21.6g多聚甲醛和3g DABCO加入反应瓶，然后加入200ml无水甲醇，搅拌溶解后，加入2g(酶活80ug/g)牛胰凝乳蛋白酶，并控温25-30℃；
(2)保温25-30℃反应1h，再升温至36℃反应2-3h，TLC监控反应进程；
(3)反应完，用6mol/L氢氧化钠溶液控温在40℃以下调PH＝7.0，先在低温下蒸出甲醇，再用300ml二氯甲烷萃取3次，减压浓缩至干，加入石油醚稀释，过快速柱，石油醚洗脱，浓缩石油醚至干，得油状异色满79.2g，收率88.3％。
对比例1
采用普通化学法制备异色满：
本实施例与实施例3的不同在于将牛胰凝乳蛋白酶替换为金属铜催化剂，金属铜催化剂的加入总质量为苯乙醇质量的1/500，其余同实施例3。得油状异色满68.3g，收率76.1％。
序列表
<110> 江西邦泰绿色生物合成生态产业园发展有限公司
<120> 一种牛胰凝乳蛋白酶及其在药物中间体异色满制备过程中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 381
<212> PRT
<213> 牛胰凝乳蛋白酶(chymotrypsinogen)
<400> 2
Met Arg Cys Leu Val Val Leu Leu Ala Val Phe Ala Leu Ser Gln Gly
1 5 10 15
Ala Glu Ile Thr Arg Ile Pro Leu Tyr Lys Gly Lys Ser Leu Arg Lys
20 25 30
Ala Leu Lys Glu His Gly Leu Leu Glu Asp Phe Leu Gln Lys Gln Gln
35 40 45
Tyr Gly Ile Ser Ser Lys Tyr Ser Gly Phe Gly Glu Val Ala Ser Val
50 55 60
Pro Leu Thr Asn Tyr Leu Asp Ser Gln Tyr Phe Gly Lys Ile Tyr Leu
65 70 75 80
Gly Thr Pro Pro Gln Glu Phe Thr Val Leu Phe Asp Thr Gly Ser Ser
85 90 95
Asp Phe Trp Val Pro Ser Ile Tyr Cys Lys Ser Asn Ala Cys Lys Asn
100 105 110
His Gln Arg Phe Asp Pro Arg Lys Ser Ser Thr Phe Gln Asn Leu Gly
115 120 125
Lys Pro Leu Ser Ile His Tyr Gly Thr Gly Ser Met Gln Gly Ile Leu
130 135 140
Gly Tyr Asp Thr Val Thr Val Ser Asn Ile Val Asp Ile Gln Gln Thr
145 150 155 160
Val Gly Leu Ser Thr Gln Glu Pro Gly Asp Val Phe Thr Tyr Ala Glu
165 170 175
Phe Asp Gly Ile Leu Gly Met Ala Tyr Pro Ser Leu Ala Ser Glu Tyr
180 185 190
Ser Ile Pro Val Phe Asp Asn Met Met Asn Arg His Leu Val Ala Gln
195 200 205
Asp Leu Phe Ser Val Tyr Met Asp Arg Asn Gly Gln Glu Ser Met Leu
210 215 220
Thr Leu Gly Ala Ile Asp Pro Ser Tyr Tyr Thr Gly Ser Leu His Trp
225 230 235 240
Val Pro Val Thr Val Gln Gln Tyr Trp Gln Phe Thr Val Asp Ser Val
245 250 255
Thr Ile Ser Gly Val Val Val Ala Cys Glu Gly Gly Cys Gln Ala Ile
260 265 270
Leu Asp Thr Gly Thr Ser Lys Leu Val Gly Pro Ser Ser Asp Ile Leu
275 280 285
Asn Ile Gln Gln Ala Ile Gly Ala Thr Gln Asn Gln Tyr Gly Glu Phe
290 295 300
Asp Ile Asp Cys Asp Asn Leu Ser Tyr Met Pro Thr Val Val Phe Glu
305 310 315 320
Ile Asn Gly Lys Met Tyr Pro Leu Thr Pro Ser Ala Tyr Thr Ser Gln
325 330 335
Asp Gln Gly Phe Cys Thr Ser Gly Phe Gln Ser Glu Asn His Ser Gln
340 345 350
Lys Trp Ile Leu Gly Asp Val Phe Ile Arg Glu Tyr Tyr Ser Val Phe
355 360 365
Asp Arg Ala Asn Asn Leu Val Gly Leu Ala Lys Ala Ile
370 375 380

Claims (5)

1.一种牛胰凝乳蛋白酶，其特征在于具有SEQ ID NO：1所示的牛胰凝乳蛋白酶序列。
2.根据权利要求1所述的牛胰凝乳蛋白酶序列，其特征在于：在药物中间体异色满制备过程中的应用。
3.根据权利要求2所述药物中间体异色满制备过程，其特征在于：包括以下步骤：
(1)将苯乙醇、多聚甲醛和脱水剂加入反应瓶，然后加入无水甲醇，搅拌溶解后，加入酶活80ug/g牛胰凝乳蛋白酶，并控温25-30℃；
(2)保温25-30℃反应1h，再升温至36℃反应2-3h，TLC监控反应进程；
(3)用6mol/L氢氧化钠溶液控温在40℃以下调PH＝7.0，在低温下蒸出甲醇，再用300ml二氯甲烷萃取3次，减压浓缩至干，加入石油醚稀释，过快速柱，石油醚洗脱，浓缩石油醚至干，得油状异色满。
4.根据权利要求3所述的制备方法，其特征在于：所述脱水剂为DABCO、DMAP、DBU中的一种。
5.根据权利要求3所述的制备方法，其特征在于：步骤(1)中所述牛胰凝乳蛋白酶的加入量占苯乙醇质量的1/20-1/50。
CN202011089476.9A 2020-10-13 2020-10-13 一种牛胰凝乳蛋白酶及其在药物中间体异色满制备过程中的应用 Pending CN112175926A (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202011089476.9A CN112175926A (zh) 2020-10-13 2020-10-13 一种牛胰凝乳蛋白酶及其在药物中间体异色满制备过程中的应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202011089476.9A CN112175926A (zh) 2020-10-13 2020-10-13 一种牛胰凝乳蛋白酶及其在药物中间体异色满制备过程中的应用

Publications (1)

Publication Number Publication Date
CN112175926A true CN112175926A (zh) 2021-01-05

Family

ID=73949499

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202011089476.9A Pending CN112175926A (zh) 2020-10-13 2020-10-13 一种牛胰凝乳蛋白酶及其在药物中间体异色满制备过程中的应用

Country Status (1)

Country Link
CN (1) CN112175926A (zh)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3978090A (en) * 1974-05-01 1976-08-31 International Flavors & Fragrances Inc. Process for production of isochromans
US5607939A (en) * 1994-04-28 1997-03-04 Takeda Chemical Industries, Ltd. Condensed heterocyclic compounds, their production and use

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3978090A (en) * 1974-05-01 1976-08-31 International Flavors & Fragrances Inc. Process for production of isochromans
US5607939A (en) * 1994-04-28 1997-03-04 Takeda Chemical Industries, Ltd. Condensed heterocyclic compounds, their production and use

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
PRALHAD N. JOSHI 等: ""Protein self-assembly induces promiscuous nucleophilic biocatalysis in Morita-Baylis-Hillman(MBH) reaction"" *
SARAVANAN GOWRISANKAR 等: ""Synthesis of isochroman and tetrahydroisoquinoline derivatives from Baylis-Hillman adducts by radical cyclization"" *
SUN, D. AND JIANG, Y.: ""preprochymosin [Bos taurus]，ABU41411.1"" *
杜利利: ""2-芳基异喹啉类化合物合成及体外细胞毒活性研究"" *
郭雪飞; 刘向阳: "制备异色满的方法" *

Similar Documents

Publication Publication Date Title
KR101285259B1 (ko) Ｗｎｔ 계열 유래 펩타이드 및 이의 용도
US10080775B2 (en) Peptide having skin whitening activity, and use thereof
JP5230152B2 (ja) プロフィラグリン産生促進剤及びフィラグリン産生促進剤
US20040266699A1 (en) Flavonoid compounds capable of modifying the dynamic and/or physical state of biological membranes and to stimulate the endogenous synthesis of stress proteins in eukaryotic cells, relative synthesis and their use
AU655087B2 (en) New oligodeoxyribonucleotides having anti-ischemic activity and methods of preparations thereof
CN101812496A (zh) 一种高纯度鱼鳞胶原蛋白的制备方法
CN109619233A (zh) 一种抗氧化功能速溶茶粉的制备方法
EP2985275A1 (en) Beta-hydroxy-beta-methylbutyric acid purification method
EP0980363B9 (en) Preparation of isoflavones from legumes
CN101269090A (zh) 一种低分子量阿胶水解物的制备方法
CN102732548A (zh) 一种表达蛇毒激肽原酶的高效麦胚无细胞蛋白合成系统的建立及应用
JP5925414B2 (ja) 高圧酵素分解技法を利用した植物抽出物の製造方法
CN112174922A (zh) 一种5-甲基色酮及其制备方法和应用
CN112175926A (zh) 一种牛胰凝乳蛋白酶及其在药物中间体异色满制备过程中的应用
CN110642753A (zh) 一种氨基酸衍生物
US10695279B2 (en) Peptide having skin-whitening activity and use thereof
CN101829196A (zh) 一种高含量荞麦总黄酮的制备方法
Hocquemiller et al. Alcaloides du peripterygia marginata (baill.) lues.(célastracées)—I: Révision de la structure de la périphylline
KR101209117B1 (ko) 변형된 ｗｎｔ１０?유래 펩타이드 및 그의 용도
JP2008280308A (ja) 毛乳頭細胞増殖促進剤、テストステロン５α−リダクターゼ阻害剤、アンドロゲン受容体結合阻害剤、血管内皮増殖因子産生促進剤、骨形成タンパク質−２産生促進剤、インスリン様増殖因子−１産生促進剤、育毛剤及び頭髪化粧料
CN101759803A (zh) 水蛭素的生理盐水-聚乙二醇双水相提取法
CN113880916B (zh) 一种牦牛皮抗氧化多肽及其制备方法和应用
CN106478516A (zh) 一种5‑氯哒嗪酮的合成方法
CN105037356A (zh) 3,6-二溴咪唑并[1,2-a]吡啶的合成方法
US3893995A (en) Method for obtaining polyfructosans

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20210105

WD01 Invention patent application deemed withdrawn after publication