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Application of N-acetylglucosamine isomerase in production of N-acetylmannosamine

A technology of acetylglucosamine and acetylmannosamine, which is applied in the field of the invention, can solve the problems of no PmAGE and other problems, and achieve the effect of reducing production costs

Active Publication Date: 2014-06-04
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no report of PmAGE being used for ManNAc synthesis has been found

Method used

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  • Application of N-acetylglucosamine isomerase in production of N-acetylmannosamine
  • Application of N-acetylglucosamine isomerase in production of N-acetylmannosamine
  • Application of N-acetylglucosamine isomerase in production of N-acetylmannosamine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 实施例1:重组大肠杆菌E.coli Rosseta(pET28a-PmAGE)的构建。

[0034] 1、N-乙酰葡萄糖胺异构酶基因的获取:

[0035] 海洋来源的蓝藻Prochlorococcus marinus str.MIT9215(来自于Massachusetts Institute of Technology的chisholm教授)细胞在液氮中冷冻1分钟后再转移到100℃沸水中温浴10分钟。将如此处理过的海藻作为PCR的模板。

[0036] 构建表达载体所用的引物加设酶切位点,引物序列如下:

[0037] 上游引物(PmAGE-sense含EcoR I)为:

[0038] 5′CCG GAATTC ATGCAAAAATATATAAACGAATATCTAAG

[0039] 下游引物(PmAGE-anti含Not I)为:

[0040] ATTT GCGGCCGC TTATTTAAACAATGTTGTATTTTTT

[0041] 所有引物均由南京金斯瑞公司合成。

[0042] 基因的PCR条件:

[0043] 94℃变性5min,按如下参数循环30次:94℃变性1min,60℃退火50s,72℃延伸1.5min。 Finally, extend at 72°C for 10 min.

[0044] 2、重组大肠杆菌E.coli Rosseta转化:

[0045] 用EcoR I及Not I分别酶切pET-28a(pET-28a购于Novagen(默克中国))及所扩增含有两个酶切位点的目的基因,分别胶回收已双酶切的目的片段和表达载体,将已双酶切的表达载体pET-28a与目的基因用T4连接酶进行连接过夜,将10uL的连接产物pET-28a-PmAGE加入100uL的Rosetta(DE3)感受态细胞中,冰上放置30min,42℃热激90sec。冰上放置2min。加入预热的0.45mL培养基。220rpm37℃1h。将200uL菌液加入分别含有100μg / mL的卡那霉素和氯霉素的LB平板上,37℃过夜培养12~16h。构建图谱见 figure 1 .

Embodiment 2

[0046] 实施例2:异构酶PmAGE的获取极其性质研究。

[0047] 1、N-乙酰葡萄糖胺异构酶PmAGE的表达。

[0048] 挑取重组菌E.coli Rosseta(pET-28a-PmAGE)至含抗生素的LB液体培养基中,37℃振荡培养过夜。然后按2%接种量分别接种到新鲜培养液中,37℃培养至OD 600 约为0.6时,加入IPTG至终浓度0.5mmol·L -1 ,15℃,220rpm,诱导表达12h后,离心(4℃,10000rpm,10min)。

[0049] 2、N-乙酰葡萄糖胺异构酶PmAGE的纯化。

[0050] 将收集的菌泥用含300mM NaCl和30%甘油的磷酸缓冲(50mM,pH7.5)重悬,超声破碎细胞(功率300W,超声5s,间歇5s,共5min),离心(4℃,12000rpm,15min)取上清。

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Abstract

The invention discloses application of N-acetylglucosamine isomerase of which the amino acid sequence is shown as SEQ ID NO:2 in catalytic synthesis of N-acetylmannosamine. The N-acetylmannosamine is prepared by taking the N-acetylglucosamine isomerase of which the amino acid sequence is shown as SEQ ID NO:2 as a catalyst and taking N-acetylglucosamine as a substrate. According to the invention, the N-acetylglucosamine isomerase of which the amino acid sequence is shown as SEQ ID NO:2 is used in the preparation of the N-acetylmannosamine for the first time, and favorable effect is achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an application of N-acetylglucosamine isomerase (N-acetylglucosamine 2-epimerase, AGE), in particular to a kind of N-acetylglucosamine isomerase from seaweed Prochlorococcus marinus str.MIT9215. Application of PmAGE in the production of N-acetyl-D-mannosamine (ManNAc) with N-acetyl-D-glucosamine (GlcNAc) as substrate. Background technique [0002] N-acetyl-mannosamine (N-acetyl-D-mannosamine, ManNAc) is an important intermediate, which can be used as anti-influenza drug precursor and infant milk powder additive N-acetylneuraminic acid (N-acetyl-D- neuraminic acid, Neu5Ac) synthesis. [0003] Using N-acetyl-D-glucosamine (GlcNAc) as a substrate for the synthesis of ManNAc, and then using ManNAc and pyruvate as substrates through N-acetylneuraminic acid lyase (N-acetylneuraminic acid lyase, Nal) catalyzed synthesis of N-acetylneuraminic acid is currently the most important way to synth...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/26C12P19/24C12P19/02C12R1/19
Inventor 应汉杰季雯燕谢婧婧孙梧进陈勇陈晓春吴菁岚
Owner NANJING TECH UNIV