Biological preparation method of Jinggang mycylamine

A technology of Jinggang mycoenamine and Jinggang mycoenone, which is applied in the field of biological preparation of Jinggang mycoenamine, can solve problems such as complicated production process, and achieve improved production efficiency, strong stereoselectivity, reduced operation and complicated control steps Effect

Active Publication Date: 2020-12-29
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since this process requires the fermentation synthesis of Jinggangmycin and product separation, as well as the preparation of Jinggangmycin enamine by secondary fermentation of microorganisms using Jinggangmycin as the main carbon source, the production process is complicated and the production efficiency needs to be improved urgently.

Method used

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  • Biological preparation method of Jinggang mycylamine
  • Biological preparation method of Jinggang mycylamine
  • Biological preparation method of Jinggang mycylamine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1, with WecE enzyme is prepared Jinggang mycoenamine by Jinggang mycoenone biotransformation

[0033] With 5mM jinggangmycetenone as substrate, 10mM glutamine as amino donor, 0.3mM pyridoxal phosphate (PLP) as cofactor, 1mg / mL WecE aminotransferase as biocatalyst, in 20mM PBS at pH 7.4 Buffer, react in a 37°C water bath for 3 hours.

[0034] Adopt 2% o-phthalaldehyde (OPA) to carry out precolumn derivatization to reaction product under room temperature condition, derivatization product is separated through Eclipse XDB-C18 (5 μ m, 4.6 * 150mm) chromatographic column, 0~8min acetonitrile-water (22: 78 volume ratio); 8-12min 100% acetonitrile; 12-17min acetonitrile: water (22:78 volume ratio) gradient elution; fluorescence absorption was detected when the excitation wavelength was 240nm and the emission wavelength was 450nm. OPA derivatization HPLC results such as figure 1 As shown, o-phthalaldehyde reacts with WecE catalyzed product to generate a single pro...

Embodiment 2

[0037] Embodiment 2, construct genetically engineered bacterium by WecE, ferment and produce Jinggang mycylamine

[0038] Jinggangmycin is an intermediate product in the biosynthetic pathway of Jinggangmycin. Jinggangmycin production strains such as Streptomyces hygroscopicus var. 5008 or Streptomyces hygroscopicus subsp. . In this example, Streptomyces hygroscopicus S5008 was selected as the host, and the phosphokinase ValC that recognized jinggang ketenone in its metabolic pathway was knocked out by PCR-Target method, and the downstream metabolic pathway of jinggang ketenone was blocked, and jinggang ketenone was constructed. Production strain; and the aminotransferase gene WecE is cloned to the downstream of the promoter PvalA of the Jinggangmycin biosynthetic gene valA, and the WecE gene is integrated into the Jinggangmycin enone-producing bacterium by the Streptomyces conjugative transfer operation method through the integration vector pPM927 On the chromosome of , the...

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Abstract

The invention discloses a biological preparation method of valienamine. The preparation method comprises a step of biologically converting valienone into valienamine by using amino transferase (WecE), or integrating WecE gene into bacterial strains that can generate the precursor compounds of valienone so that the bacterial strains integrated with WecE gene can directly produce valienamine through fermentation. The conventional chemical synthesis method has the disadvantages such as many reaction steps, low yield, strict reaction conditions, organic reagent pollution, and the like, and the provided biological preparation method overcomes the abovementioned problems. The biological preparation method is based on amino transferase catalyzed amino transferring reactions, the direct biological synthesis is convenient and high efficient, the stereo-selectivity is high, and the valienamine can be used as a synthetic intermediate for synthesis of voglibose for treating II type diabetes, synthesis of acarbose, and development of glycosidase inhibitors.

Description

technical field [0001] The invention relates to enzymology, genetic engineering and other life science technology means, and in particular relates to a biological preparation method of Jinggang mycylamine. Background technique [0002] Jinggang valienamine (valienamine) belongs to aminocyclic alcohol compounds and is the core structure of pseudoaminoglycohydrolase inhibitors. Jinggang valienamine and its derivatives can not only participate in the treatment of diseases related to glycosidase, such as diabetes, It can also be used as a treatment for cancer, AIDS and related syndromes (Kajimoto T, Node M. Inhibitors against glycosidases as medicines. Current Topics in Medicinal Chemistry, 2009, 9(1): 13-33.). The anti-type II diabetes drug voglibose (Bexin) containing the structure of Jinggang mycetin is a typical hypoglycemic drug of glycosidase inhibitors (Kataoka Y, Yasuda S, Miyamoto Y, Sase K, Kosuge M, Kimura K , YoshimasaY, Miyazaki S.Effects of voglibose and nateglini...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/00C12N15/76C12N15/54C12R1/55
CPCC12N9/1096C12N15/76C12P13/001C12Y206/01
Inventor 冯雁崔莉白林泉邓子新刘璋敏
Owner SHANGHAI JIAOTONG UNIV
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