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Preparation method and application of small molecular weight hyaluronic acid, and hyaluronate lyase genetic vector and engineering bacteria

A technology of hyaluronic acid and gene carrier, applied in the field of hyaluronic acid production, can solve problems such as unfavorable industrial production, and achieve the effects of controllable molecular weight, mild reaction conditions and energy saving

Inactive Publication Date: 2016-01-27
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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Problems solved by technology

Chinese patent (CN101633931) uses the pBV220 expression vector to express the hyaluronan lyase gene and induces it at 42°C, but the obtained product is an inclusion body, which requires protein renaturation, which is not conducive to large-scale industrial production

Method used

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  • Preparation method and application of small molecular weight hyaluronic acid, and hyaluronate lyase genetic vector and engineering bacteria
  • Preparation method and application of small molecular weight hyaluronic acid, and hyaluronate lyase genetic vector and engineering bacteria
  • Preparation method and application of small molecular weight hyaluronic acid, and hyaluronate lyase genetic vector and engineering bacteria

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Embodiment Construction

[0041] The technical solutions of the present invention will be further described below through specific examples.

[0042]Using the Streptococcus zooepidemicus ATCC39920 genome as a template, the hylb gene encoding the hyaluronan lyase gene was cloned into the starting vector pET28a, and after verification by PCR, double enzyme digestion and sequencing, the hyaluronan lyase expression vector pET28a::hylb was successfully constructed . The vector was transferred to the host bacteria Escherichia coli BL21 (DE3), and the engineering strain producing hyaluronic acid lyase was successfully obtained. After the above-mentioned engineering bacteria fermentation analysis, enzyme liquid extraction, enzyme purification and separation, and enzyme activity determination, the preparation highly active hyaluronan lyase.

[0043] The hyaluronan lyase gene hylb is derived from Streptococcus zooepidemicus (Streptococcus zooepidemicus), the strain preservation number is ATCC39920, the gene fra...

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Abstract

The invention provides a preparation method and application of small molecular weight hyaluronic acid, and a hyaluronate lyase genetic vector and engineering bacteria. An expression vector successfully constructed is converted to Escherichia coli BL21 (DE3) by: cloning part of fragments encoding hyaluronate lyase gene (hylb) in genome of Streptococcus zooepidemicus ATCC 39920 and constructing the expression carrier pET 28a:hylb, and high-activity soluble hyaluronate lyase is obtained by the induced expression of Escherichia coli. Specific enzyme activity of purified liquid of the lyase is 4655 U / mg (equivalently 4.655*107 IU / mg) that is measured by N-acetyl-glucosamine (NAG) measurement. The small molecular weight hyaluronic acid is prepared by controlling the enzyme content and reaction conditions to degrade large molecular weight hyaluronic acid, the molecular weight of the hyaluronic acid is measured by means of an intrinsic viscosity method and agarose gel electrophoresis, and the obtained molecular weight is that of small molecular weight hyaluronic acid of 6000Da level, an infrared spectrogram of which is consistent with standard atlas in national pharmacopoeia.

Description

technical field [0001] The invention relates to the field of hyaluronic acid production, and more specifically relates to a preparation method and application of hyaluronic acid with a small molecular weight, a hyaluronan lyase gene carrier and engineering bacteria. Background technique [0002] Hyaluronic acid (HA), also known as hyaluronic acid, is a linear high-density compound composed of D-glucuronic acid and N-acetylglucosamine disaccharide units connected alternately by β-1,3 glycosidic bonds and β-1,4 glycosidic bonds. Molecule acid mucopolysaccharide. HA is widely distributed in the tissue interstitium of the body and exhibits different physiological effects depending on the location of distribution, such as better water retention in the skin, lubrication and anti-wear effects in the synovial fluid of the joints, and It plays an important role in the regeneration of tissues and blood vessels, so hyaluronic acid is widely used in the fields of cosmetics, food health...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12N9/88C12P19/26C12P19/14C12R1/19
Inventor 刘浩张海洋
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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