WO2019011192A1 - 一种催化合成二肽的氨肽酶及其制备方法 - Google Patents

一种催化合成二肽的氨肽酶及其制备方法 Download PDF

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WO2019011192A1
WO2019011192A1 PCT/CN2018/094838 CN2018094838W WO2019011192A1 WO 2019011192 A1 WO2019011192 A1 WO 2019011192A1 CN 2018094838 W CN2018094838 W CN 2018094838W WO 2019011192 A1 WO2019011192 A1 WO 2019011192A1
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aminopeptidase
dipeptide
synthesis
reaction
amino acid
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French (fr)
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黄科学
祝俊
吴锋
王峰峰
张超
华俊国
余玉奎
徐飞
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江苏诚信药业有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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)
    • C12N9/485—Exopeptidases (3.4.11-3.4.19)
    • 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
    • C12P17/10—Nitrogen as only ring hetero atom

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  • the invention relates to a method for synthesizing a dipeptide, in particular to a dipeptide synthesis method using a highly efficient aminopeptidase catalysis and a modern separation technique, in particular to an aminopeptidase which catalyzes the synthesis of a dipeptide and a preparation method thereof.
  • L-carnosine was first discovered in beef in 1900. It is widely found in mammalian brain, muscle and other tissues. Its structure is mainly composed of ⁇ -alanine and L-histidine. It is a natural activity. The dipeptide is easily soluble in water, 0.1 mol/L hydrochloric acid and 0.1 mol/L sodium hydroxide, and is almost insoluble in methanol, acetonitrile, absolute ethanol, chloroform and acetone.
  • L-carnosine has anti-oxidation effect, anti-aging function, and has therapeutic effects on hypertension, heart disease, senile cataract, ulcer, etc., as well as anti-tumor and other biological activities, which can delay skin aging.
  • L-carnosine has the advantages of strong anti-oxidation ability, no toxic and side effects, and has a variety of physiological activities, and has broad application prospects in the fields of medicine, health care, hygiene and beauty. At present, the exploration of new physiological activities and mechanisms of L-carnosine is still a hot topic.
  • L-carnosine There are many chemical synthesis methods for L-carnosine reported in the literature. There are two main categories: (1) reactions involving ⁇ -alanine. The general method is that ⁇ -alanine is protected by an amino group, activated by a carboxyl group, and reacted with a protected L-histidine, and then the protective group is removed to obtain a product carnosine. (2) No direct involvement of ⁇ -alanine. The general method is that L-histidine is first reacted with a different ⁇ -alanine precursor to form a peptide bond, which is further converted into carnosine by further reaction.
  • aminopeptidase is used to catalyze the synthesis of carnosine from L-histidine and ⁇ -alanine, and the enzyme production cost is low, the synthesis time is short, the conversion rate is high, the environment is friendly, the process operation is simple, safe, etc., and has a large-scale industry. The broad prospects for application.
  • a first object of the present invention is to provide an aminopeptidase which catalyzes the synthesis of a dipeptide.
  • a second object of the present invention is to provide a nucleotide sequence encoding the aminopeptidase which catalyzes the synthesis of a dipeptide.
  • a third object of the present invention is to provide a process for the preparation of an aminopeptidase which catalyzes the synthesis of a dipeptide.
  • the present invention provides a method for synthesizing a dipeptide aminopeptide using an aminopeptidase method to catalyze a one-step synthesis of a dipeptide;
  • the present invention synthesizes a carnosine of a dipeptide.
  • the present invention employs an enzyme to efficiently catalyze the formation of carnosine by histidine and ⁇ -alanine, and adopts modern advanced separation technology to greatly reduce the industrial production cost of carnosine.
  • aminopeptidase which catalyzes the biosynthesis of dipeptide which is the amino acid sequence shown in SEQ ID NO: 2.
  • an aminopeptidase which catalyzes the biosynthesis of dipeptides which is derived from a genetically engineered strain recombinantly constructed in vitro.
  • the aminopeptidase which catalyzes the biosynthesis of a dipeptide the genetically engineered strain is Escherichia coli, Pichia pastoris, Bacillus subtilis.
  • an aminopeptidase which catalyzes the biosynthesis of a dipeptide, which is a myopeptide.
  • the present invention also provides a nucleotide sequence encoding the aminopeptidase, which is represented by SEQ ID NO: 1.
  • the invention also provides a preparation method of the aminopeptidase genetic engineering bacteria, comprising the following steps:
  • the inoculum amount is 5%
  • the method for synthesizing a dipeptide by the above aminopeptidase comprises the following steps:
  • the dipeptide is carnosine
  • the amino acid having an ⁇ -hydroxy group and the amino acid having an ⁇ -amino group are ⁇ -alanine methyl ester hydrochloride and L-histidine, respectively, and the method for synthesizing carnosine as follows:
  • the amount of the aminopeptidase is 1% to 3% of the volume of the reaction solution, stirring reaction at 25 to 45 ° C, the pH of the reaction system is controlled by an alkaline solution during the reaction is 7.0 to 9.0;
  • the nanofiltration concentrate is applied to the ionic resin column, the diameter ratio of the resin column is 0.05 to 0.2, and the loading pH is 5.0 to 7.0;
  • the crystallization solvent is one of methanol, ethanol, isopropanol or acetone.
  • the ultrafiltration machine has a membrane pore size of from 8,000 to 10,000 Daltons.
  • the nanofiltration membrane has a membrane pore size of from 200 to 300 Daltons.
  • aminopeptidase is used to catalyze the synthesis of a dipeptide, in particular, to catalyze the synthesis of carnosine from L-histidine and ⁇ -alanine, which has less pollution and a shorter reaction cycle than conventional chemical methods.
  • Low consumption, high enzyme reaction rate can reach more than 95%, which is more conducive to industrialization.
  • the invention adopts an ultrafiltration machine with a membrane pore diameter of 8000-10000 Dalton, which can effectively remove the macromolecular enzyme remaining in the reaction liquid after the enzyme reaction, thereby reducing impurities of the finished product.
  • the invention adopts a nanofiltration filter with a membrane pore size of 200-300 Dalton, which can effectively remove small molecular inorganic salts in the reaction liquid, reduce impurities of the finished product, has small pollution and low cost, and is suitable for enzymatic synthesis of dipeptide process. Industrialization.
  • the present application provides an aminopeptidase which catalyzes the synthesis of a dipeptide, a method for preparing the aminopeptidase, and a method for synthesizing a dipeptide by using the aminopeptidase.
  • the present application describes a synthetic carnosine as an example. In other embodiments, It can also be used to synthesize other dipeptides.
  • the whole gene-synthesized biological enzyme fragment (sequence as shown in SEQ ID NO: 1, synthesized by Changzhou Keyu Biotechnology Co., Ltd.), via restriction enzymes EcoR I and Not I (purchased from New England Biolabs, according to The instructions were manipulated and recombined into the yeast expression vector pPIC9k (invitrogen), transformed into E. coli Top10 competent state (purchased from Beijing Quanjin Biotechnology Co., Ltd.), and E. coli Top10 was placed in LB liquid medium. The recombinant plasmid was extracted by shaking at 37 ° C and 160 rpm overnight. The recombinant plasmid was linearized using restriction endonuclease Sal I (purchased from New England Biolabs, Inc., operating according to the instructions).
  • Pichia pastoris GS115 competent cells invitrogen: Pichia pastoris GS115 single colony was picked into YPD medium for activation, and activated Pichia GS115 was added to 50 ml of YPD medium at 30% inoculation at 0.5% inoculum. In several stages, the cells obtained by centrifugation were washed twice with 20 ml of sterile water, washed twice with 20 ml of sterile 1 M sorbitol, and the cells were resuspended by adding 1 ml of 1 M sorbitol solution to obtain P. pastoris GS115 competent cells.
  • the linearized fragment was added to 80 ⁇ l of Pichia pastoris GS115 competent cells for 5 minutes in an ice bath, and after electrotransformation, 800 ⁇ l of sorbitol was added to wash the cells into a 1.5 ml sterile centrifuge tube, and after incubation at 25 ° C for 2 hours, The MD plate was centrifuged and cultured at 30 ° C until the bacteria grew out, and single colonies were separated by scribing. A single colony was picked into sterile water and added with appropriate amount of Lyticase (purchased from sigma company). The cell wall was digested at 37 ° C for 1 hour, and a part of the digestion product was added to the PCR system to detect positive clones.
  • the positive clones were picked into YPD liquid medium and transferred to BMGY liquid medium. When cultured until the OD was 1.0, 1% methanol was induced and induced for 72 hours. The methanol was added once every 24 hours to express the organism of the present invention. Enzyme.
  • the original strains were streaked in YPD, and cultured at 30 ° C overnight.
  • the cells were inoculated into a shake flask containing 300 ml of YPD liquid medium (1 L flask) at a 10% inoculation amount, and cultured at 30 ° C, shaking at 200 rpm, and the OD600 was increased to about 12 after about 24 hours.
  • the fermentation medium was transferred to a fermenter (30 L), sterilized at 121 ° C for 30 min; the temperature was lowered to 30 ° C, and the pH was adjusted to 5.0 using ammonia water.
  • the cultured bacterial solution was inoculated to the fermenter, and the inoculum amount was 5%. According to the dissolved oxygen regulation speed and ventilation, the dissolved oxygen is controlled to be more than 30%. After culturing for about 24 hours, the dissolved oxygen mutation increased. At this time, the wet weight was about 140g/L, and the 50% (w/v) glycerin aqueous solution was started to be fed. The feeding rate was about 15ml/L fermentation broth/hour.
  • the wet weight of the cells was induced to be about 340 g/L in 96 hours. After centrifugation, the supernatant was collected, concentrated by ultrafiltration, and lyophilized to obtain a bio-enzymatic lyophilized powder for synthesizing dipeptide.
  • the nanofiltration concentrate is applied to the ionic resin column, the diameter ratio of the resin column is 0.05 to 0.2, and the loading pH is 5.0 to 7.0;
  • the crystallization solvent is one of methanol, ethanol, isopropanol or acetone.
  • the ultrafiltration machine of the present invention has a membrane pore size of 8,000 to 10,000 Daltons; and the nanofiltration membrane has a membrane pore size of 200 to 300 Daltons.
  • aminopeptidase is used to catalyze the synthesis of carnosine from L-histidine and ⁇ -alanine, which has less pollution, shorter reaction cycle, lower energy consumption, and higher enzymatic reaction rate than conventional chemical methods, and can reach 95. More than %, more conducive to industrialization.
  • the invention adopts an ultrafiltration machine with a membrane pore diameter of 8000-10000 Dalton, which can effectively remove the macromolecular enzyme remaining in the reaction liquid after the enzyme reaction, thereby reducing impurities of the finished product.
  • the invention adopts a nanofiltration filter with a membrane pore size of 200-300 Dalton, which can effectively remove small molecular inorganic salts in the reaction liquid, reduce impurities of the finished product, has small pollution and low cost, and is suitable for enzymatic synthesis of dipeptide process. Industrialization.

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Abstract

本发明提供一种催化合成二肽的氨肽酶及其制备方法和应用。所述氨肽酶为SEQ ID NO：2所示的氨基酸序列；生物酶的制备，首先构建生物酶的基因工程菌株，将全基因合成的生物酶基因片段，利用限制性内切酶SalⅠ线性化重组质粒。本发明构建的基因工程菌可催化组氨酸和β丙氨酸高效合成肌肽。

Description

一种催化合成二肽的氨肽酶及其制备方法 【技术领域】
本发明涉及一种合成二肽的方法，尤其是采用高效的氨肽酶催化和现代化的分离技术的二肽合成方法，具体是一种催化合成二肽的氨肽酶及其制备方法。
【背景技术】
L-肌肽1900年首次在牛肉中发现，在哺乳动物的大脑、肌肉和其他组织中广泛存在，其结构主要由β-丙氨酸和L-组氨酸两部分组成，是一种天然的活性二肽，在水、0.1mol/L盐酸和0.1mol/L氢氧化钠中易溶，在甲醇、乙腈、无水乙醇、三氯甲烷和丙酮中几乎不溶。
研究证明L-肌肽有抗氧化作用，有抗衰老功能，对高血压、心脏病、老年性白内障、溃疡等都有治疗效果，还有抗肿瘤等生物活性，可延缓皮肤衰老。与其他抗氧化剂相比，L-肌肽具有抗氧化能力强、无毒副作用的优点，并有多种生理活性，在医药、保健、卫生、美容等领域有广泛的应用前景。目前，L-肌肽新的生理活性与机制的探索仍是热点。
文献报道的L-肌肽的化学合成方法较多，主要有两大类：(1)有β-丙氨酸参与的反应。大致的方法是β-丙氨酸经氨基保护、羧基活化后与保护的L-组氨酸反应，然后脱掉保护基团得到产品肌肽。(2)无β-丙氨酸直接参与的反应。大致方法为L-组氨酸先与不同的β-丙氨酸前体反应生成肽键，再经进一步反应转换为肌肽。
在本发明中使用氨肽酶催化L-组氨酸与β-丙氨酸合成肌肽，制 酶成本低，合成时间短、转化率高、环境友好，工艺操作简单、安全等，具有大规模工业应用的广泛前景。
【发明内容】
为了解决以上问题，本发明的第一个目的在于提供一种催化合成二肽的氨肽酶。
本发明的第二个目的是提供一种编码所述催化合成二肽的氨肽酶的核苷酸序列。
本发明的第三个目的是提供一种催化合成二肽的氨肽酶的制备方法。
本发明提供二肽的氨肽合成方法采用氨肽酶法催化一步合成二肽；
其中，本发明合成二肽的一种肌肽，具体地，本发明采用酶高效催化组氨酸和β丙氨酸生成肌肽，并采用现代化的先进分离技术，使肌肽的工业化生产成本大大降低。
本发明的技术方案如下：
一种催化二肽生物合成的氨肽酶，所述氨肽酶为SEQ ID NO：2所示的氨基酸序列。
根据本发明所述催化二肽生物合成的氨肽酶，所述氨肽酶来源于体外重组构建的基因工程菌株。
根据本发明所述催化二肽的生物合成的氨肽酶，所述基因工程菌株为大肠杆菌、毕氏酵母、枯草芽孢杆菌。
根据本发明所述催化二肽的生物合成的氨肽酶，所述二肽为肌 肽。
本发明还提供一种编码所述的氨肽酶的核苷酸序列，所述核苷酸序列如SEQ ID NO：1所示。
本发明还提供一种所述的氨肽酶基因工程菌的制备方法，包括如下步骤：
(1)首先构建氨肽酶的基因工程菌株，将上述SEQ ID NO：1所示的核苷酸序列经酶切重组到表达载体，转化到宿主细胞中；
(2)取原种菌种在YPD平板上划线，30℃，倒置培养40h；
(3)在平板上挑取单菌落转移至液体培养基中，30℃，振荡培养24小时，OD600增长至4～5；
(4)以10％的接种量接种至液体培养基的摇瓶中，30℃，200rpm振荡培养，约24小时后OD600生长至12左右；
(5)将发酵培养基配制好后，转移至发酵罐中，121℃灭菌30min；降温至30±2℃，使用氨水调节pH值至5.0；
(6)将培养好的菌种液接种至发酵罐，接种量5％；
(7)根据溶氧调节转速和通气，保持溶氧在30％以上，诱导发酵96小时后，放罐；
(8)离心，收集清液；
(9)将清液超滤浓缩后冻干，得到合成二肽用氨肽酶的冻干粉。
采用上述的氨肽酶催化合成二肽的方法，包括如下步骤：
提供具有α-羟基的氨基酸和具有α-氨基的氨基酸；
将所述α-羟基氨基酸、α-氨基氨基酸和去离子水进行混合得到第 一混合液；
向所述第一混合液中加入上述的氨肽酶，在预定条件下进行反应；
反应结束，对反应液进行处理，得到二肽。
具体地，所述二肽为肌肽，所述具有α-羟基的氨基酸和具有α-氨基的氨基酸分别为β-丙氨酸甲酯盐酸盐和L-组氨酸，所述合成肌肽的方法如下：
1)β-丙氨酸甲酯盐酸盐合成：
1.1)向反应釜中加入甲醇，控温0～20℃将氯化亚砜滴入其中，滴加完毕，加入β-丙氨酸，30～60℃保温搅拌；
1.2)30～50℃减压，真空度≤-0.09MPa，浓缩，浓缩至无馏分流出，得β-丙氨酸甲酯盐酸盐；
2)酶催化反应：
2.1向反应釜中加入L-组氨酸、去离子水、上述反应制备的β-丙氨酸甲酯盐酸盐；
2.2在15～45℃温度下，用碱性溶液调节pH值至7.0～9.0；
2.3加入上述的氨肽酶，该氨肽酶的用量为反应液体积的1％～3％，25～45℃搅拌反应，反应过程中用碱性溶液控制反应体系pH值在7.0～9.0；
3)肌肽粗品制备：
3.1反应完毕，放料，料液离心除去不溶物，收集滤液；
3.2将得到的滤液通过超滤机进行超滤，收集超滤透析液；
3.3将得到的超滤透析液通过纳滤机进行纳滤，收集纳滤浓缩液；
3.4将纳滤浓缩液上离子树脂柱，树脂柱的径高比为0.05～0.2，上样pH值为5.0～7.0；
3.5用纯化水冲洗树脂柱；
3.6用1％～5％的氨水洗脱解吸附树脂柱，收集茚三酮显色部分洗脱液；
3.7将收集的洗脱液50～80℃减压，真空度≤-0.09MPa，浓缩肌肽含量至20～40％；
3.8加入析晶溶剂，0～25℃，搅拌析晶10～24h；
3.9析晶完毕，放料离心，30～50℃，减压，真空度≤-0.09MPa，干燥6～8h，得肌肽粗品。
作为优选，所述的析晶溶剂为甲醇、乙醇、异丙醇或丙酮中的一种。
作为优选，所述的超滤机的滤膜孔径为8000～10000道尔顿。
作为优选，所述的纳滤机的滤膜孔径为200～300道尔顿。
有益的效果：在本发明中使用氨肽酶催化合成二肽，特别地，催化L-组氨酸与β-丙氨酸合成肌肽，相比传统化学法，其污染小、反应周期短，能耗低，酶反应生成率高，可到达95％以上，更利于产业化。本发明采用滤膜孔径为8000-10000道尔顿的超滤机，可在酶反应后有效去除反应液中残留的大分子酶，减少成品的杂质。本发明采用滤膜孔径为200-300道尔顿的纳滤机，可有效去除反应液中的小分子无机盐，减少成品的杂质，且污染小，成本低，适于酶催化合成二 肽工艺的产业化。
【具体实施方式】
以下结合具体实施例，对本发明作进一步说明。应理解，以下实施例仅由于说明本发明而非用于限定本发明的范围。
本申请提供一种催化合成二肽的氨肽酶、制备该氨肽酶的方法，以及用该氨肽酶催化合成二肽的方法，本申请以合成肌肽为例进行说明，在其他实施方式中，也可以用于合成其他二肽。
实施例1：氨肽酶的基因工程菌株构建
将全基因合成的生物酶片段(序列如SEQ ID NO：1所示，由常州基宇生物科技有限公司合成)，经限制性内切酶EcoR Ⅰ和Not Ⅰ(购自New England Biolabs公司，根据说明书进行操作)酶切后重组到酵母表达载体pPIC9k(invitrogen公司)，转化到E.coli Top10感受态(购自北京全式金生物技术有限公司)，将E.coli Top10置于LB液体培养基中，37℃、160rpm转振荡培养过夜，提取重组质粒。利用限制性内切酶Sal Ⅰ(购自New England Biolabs公司，根据说明书进行操作)线性化重组质粒。
巴斯德毕赤酵母GS115感受态细胞(invitrogen公司)：将Pichia Pastoris GS115单菌落挑入YPD培养基中活化，活化的Pichia GS115按0.5％的接种量接入50mlYPD培养基中30℃培养至对数期，离心获得的菌体用20ml无菌水洗2次，再用20ml无菌1M山梨醇洗2次，加入1ml1M山梨醇溶液重悬菌体获得巴斯德毕赤酵母GS115感受态细胞。
将线性化片段加入到80μl巴斯德毕赤酵母GS115感受态细胞中冰浴5分钟，电转化后加入800μl山梨醇将细胞洗至1.5ml无菌离心管中，25℃温育2小时后，离心涂MD平板，30℃培养至长出菌落后，划线分离出单菌落。将单菌落挑入无菌水中加入适量Lyticase(购自sigma公司)后37℃温育1小时消化细胞壁，取部分消化产物加入PCR体系检测阳性克隆。
将阳性克隆挑入YPD液体培养基后转接入BMGY液体培养基中，培养至OD为1.0时接入1％甲醇诱导，诱导72小时，每24小时补加一次甲醇用于表达本发明的生物酶。
实施例2：氨肽酶的发酵制备
取原种菌种在YPD划线，30℃，倒置培养过夜。在平板上挑取单菌落(直径1mm)至50mlYPD液体培养基(酵母粉10g，蛋白胨10g，葡萄糖10g，加水定容至1L)中，30℃，200rpm振荡培养过夜(24h)，OD600增长至4-5。以10％的接种量接种至含300mlYPD液体培养基(1L三角瓶)摇瓶中，30℃、200rpm摇床振荡培养，约24小时后OD600长到12左右。将发酵培养基配制完成后，转入发酵罐(30L)，121℃灭菌30min；降温至30℃，使用氨水调节pH值至5.0。将培养好的菌种液接种至发酵罐，接种量5％。根据溶氧调节转速和通气，控制溶氧在30％以上。培养24小时左右，待溶氧突变上升，此时湿重约为140g/L，开始补料50％(w/v)甘油水溶液，补料速度约为15ml/L发酵液/小时，补料速度控制溶氧保持在30％以上；待菌种湿重长至180g/L左右，停止补加甘油，以7.2ml/L发酵液/小时流 速流加100％甲醇，诱导10小时后，pH值调节为6.0，诱导24小时，pH调节为7.0，补料速度保持不变，根据溶氧调节转速和通气，保持溶氧在30％以上。诱导96小时菌体湿重约为340g/L左右放罐。离心，收集清液，超滤浓缩后冻干，得到合成二肽用生物酶冻干粉。
实施例3氨肽酶催化合成肌肽
包括如下步骤：
1)β-丙氨酸甲酯盐酸盐合成：
1.1)向反应釜中加入甲醇，控温0～20℃将氯化亚砜滴入其中，滴加完毕，加入β-丙氨酸，30～60℃保温搅拌；
1.2)30～50℃减压，真空度≤-0.09MPa，浓缩，浓缩至无馏分流出，得β-丙氨酸甲酯盐酸盐；
2)酶催化反应：
2.1向反应釜中加入L-组氨酸、去离子水、上述反应制备的β-丙氨酸甲酯盐酸盐；
2.2在15～45℃温度下，用碱性溶液调节pH值至7.0～9.0；
2.3加入上述制备的氨肽酶，该氨肽酶的用量为反应液体积的1％～3％，25～45℃搅拌反应，反应过程中用碱性溶液控制反应体系pH值在7.0～9.0；
3)肌肽粗品制备：
3.1反应完毕，放料，料液离心除去不溶物，收集滤液；
3.2将得到的滤液通过超滤机进行超滤，收集超滤透析液；
3.3将得到的超滤透析液通过纳滤机进行纳滤，收集纳滤浓缩液；
3.4将纳滤浓缩液上离子树脂柱，树脂柱的径高比为0.05～0.2，上样pH值为5.0～7.0；
3.5用纯化水冲洗树脂柱；
3.6用1％～5％的氨水洗脱解吸附树脂柱，收集茚三酮显色部分洗脱液；
3.7将收集的洗脱液50～80℃减压，真空度≤-0.09MPa，浓缩肌肽含量至20～40％；
3.8加入析晶溶剂，0～25℃，搅拌析晶10～24h；
3.9析晶完毕，放料离心，30～50℃，减压，真空度≤-0.09MPa，干燥6～8h，得肌肽粗品。
本实施例中，所述的析晶溶剂为甲醇、乙醇、异丙醇或丙酮中的一种。
本发明所述的超滤机的滤膜孔径为8000～10000道尔顿；所述的纳滤机的滤膜孔径为200～300道尔顿。
在本发明中使用氨肽酶催化L-组氨酸与β-丙氨酸合成肌肽，相比传统化学法，其污染小、反应周期短，能耗低，酶反应生成率高，可到达95％以上，更利于产业化。本发明采用滤膜孔径为8000-10000道尔顿的超滤机，可在酶反应后有效去除反应液中残留的大分子酶，减少成品的杂质。本发明采用滤膜孔径为200-300道尔顿的纳滤机，可有效去除反应液中的小分子无机盐，减少成品的杂质，且污染小，成本低，适于酶催化合成二肽工艺的产业化。

Claims (15)

  1. 一种催化合成二肽的氨肽酶，其中，所述氨肽酶为SEQ ID NO：2所示的氨基酸序列。
  2. 根据权利要求1所述催化合成二肽的氨肽酶，其中，所示氨肽酶来源于体外重组构建的基因工程菌株；所述基因工程菌株为大肠杆菌、毕氏酵母、枯草芽孢杆菌。
  3. 根据权利要求1所述催化合成二肽的氨肽酶，其中，所述二肽为肌肽。
  4. 一种编码权利要求1所述的氨肽酶的核苷酸序列，所述核苷酸序列如SEQ ID NO：1所示。
  5. 一种权利要求1所述的氨肽酶的制备方法，包括如下步骤：
    (1)首先构建氨肽酶的基因工程菌株，将权利要求4所述的核苷酸序列经酶切重组到表达载体，转化到宿主细胞中；
    (2)取原种菌种在YPD平板上划线，30℃，倒置培养40h；
    (3)在平板上挑取单菌落转移至液体培养基中，30℃，振荡培养24小时，OD600增长至4～5；
    (4)以10％的接种量接种至液体培养基的摇瓶中，30℃，200rpm振荡培养，约24小时后OD600生长至12左右；
    (5)将发酵培养基配制好后，转移至发酵罐中，121℃灭菌30min；降温至30±2℃，使用氨水调节pH值至5.0；
    (6)将培养好的菌种液接种至发酵罐，接种量5％；
    (7)根据溶氧调节转速和通气，保持溶氧在30％以上，诱导发酵96小时后，放罐；
    (8)离心，收集清液；
    (9)将清液超滤浓缩后冻干，得到合成二肽用氨肽酶的冻干粉。
  6. 一种采用权利要求1所述的氨肽酶催化合成二肽的方法，其中，所述包括：
    提供具有α-羟基的氨基酸和具有α-氨基的氨基酸；
    将所述α-羟基氨基酸、α-氨基酸和去离子水进行混合得到第一混合液；
    向所述第一混合液中加入如权利要求1所述的氨肽酶，在预定条件下进行反应；
    反应结束，对反应液进行处理，得到二肽。
  7. 根据权利要求6所述的氨肽酶催化合成二肽的方法，其中，所述二肽为肌肽，所述提供具有α-羟基的氨基酸和具有α-氨基的氨基酸包括：提供β-丙氨酸甲酯盐酸盐和L-组氨酸。
  8. 根据权利要求7所述的氨肽酶催化合成二肽的方法，其中，所述提供β-丙氨酸甲酯盐酸盐包括：
    向反应釜中加入甲醇，控温0～20℃将氯化亚砜滴入其中，滴加完毕，加入β-丙氨酸，30～60℃保温搅拌；
    30～50℃减压，真空度≤-0.09MPa，浓缩，浓缩至无馏分流出，得β-丙氨酸甲酯盐酸盐。
  9. 根据权利要求7所述的氨肽酶催化合成二肽的方法，其中，所 述将α-羟基氨基酸、α-氨基氨基酸和去离子水进行混合得到第一混合液包括：
    向反应釜中加入所述L-组氨酸、去离子水和β-丙氨酸甲酯盐酸盐；
    在15～45℃温度下，用碱性溶液调节pH值至7.0～9.0。
  10. 根据权利要求7所述的氨肽酶催化合成二肽的方法，其中，所述向第一混合液中加入如权利要求1所述的氨肽酶，在预定条件下进行反应包括：
    向所述第一混合液中加入如权利要求1所述的氨肽酶，所述氨肽酶的用量为反应液体积的1％～3％，25～45℃搅拌反应，反应过程中用碱性溶液控制反应体系pH值在7.0～9.0；
  11. 根据权利要求7所述的氨肽酶催化合成二肽的方法，其中，所述对反应液进行处理，得到二肽包括：
    反应完毕，放料，料液离心除去不溶物，收集滤液；
    将得到的滤液通过超滤机进行超滤，收集超滤透析液；
    将得到的超滤透析液通过纳滤机进行纳滤，收集纳滤浓缩液；
    将纳滤浓缩液上离子树脂柱，树脂柱的径高比为0.05～0.2，上样pH值为5.0～7.0；
    用纯化水冲洗树脂柱；
    用1％～5％的氨水洗脱解吸附树脂柱，收集茚三酮显色部分洗脱液；
    将收集的洗脱液50～80℃减压，真空度≤-0.09MPa，浓缩肌肽含 量至20～40％；
    加入析晶溶剂，0～25℃，搅拌析晶10～24h；
    析晶完毕，放料离心，30～50℃，减压，真空度≤-0.09MPa，干燥6～8h，得肌肽粗品。
  12. 根据权利要求11所述的氨肽酶催化合成二肽的方法，其中，所述的析晶溶剂为甲醇、乙醇、异丙醇或丙酮中的一种。
  13. 根据权利要求11所述的氨肽酶催化合成二肽的方法，其中，所述的超滤机的滤膜孔径为8000～10000道尔顿。
  14. 根据权利要求11所述的氨肽酶催化合成二肽的方法，其中，所述的纳滤机的滤膜孔径为200～300道尔顿。
  15. 根据权利要求6所述的氨肽酶催化合成二肽的方法，其中，所述如权利要求1所述的氨肽酶利用如权利要求5所述的方法制得。
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011139667A (ja) * 2010-01-07 2011-07-21 Tottori Univ プロリンおよびβ−アラニンをＮ末端に有するジペプチド、及びその環化ジペプチドの酵素合成法
CN107217048A (zh) * 2017-07-10 2017-09-29 江苏诚信药业有限公司 一种催化合成肌肽的氨肽酶及其制备方法和应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011139667A (ja) * 2010-01-07 2011-07-21 Tottori Univ プロリンおよびβ−アラニンをＮ末端に有するジペプチド、及びその環化ジペプチドの酵素合成法
CN107217048A (zh) * 2017-07-10 2017-09-29 江苏诚信药业有限公司 一种催化合成肌肽的氨肽酶及其制备方法和应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE NCBI [O] 14 September 2015 (2015-09-14), XP055567874, Database accession no. XP_013961656 .1 *
HECK, T.: "Kinetic Analysis of L-Camosine Formation by beta-Aminopeptidases", ADVANCED SYNTHESIS & CATALYSIS, vol. 352, 9 February 2010 (2010-02-09), pages 407, XP055567858 *
HEYLAND, J.: "Simple Enzymatic Procedure for L-carnosine Synthesis: Whole- cell Biocatalysis and Efficient Biocatalyst Recycling", MICROBIAL BIOTECHNOLOGY, vol. 3, no. 1, 31 December 2010 (2010-12-31), pages 74, XP055567859 *

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