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Glucose oxidase gene GOD, protein coded by GOD, pichia pastoris transformed by GOD and preparation method of pichia pastoris

A technology of glucose oxidase and oxidase, which is applied in the field of preparation of new genes, can solve the problems of low enzyme activity, cumbersome purification, and many experiments, and achieve the effects of improving fermentation enzyme activity, increasing enzyme production, and improving economic benefits

Inactive Publication Date: 2016-03-23
河北省微生物研究所有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the technical level of glucose oxidase in my country is still in the primary stage of research and development, and there are problems such as low yield, low enzyme activity, cumbersome purification, and complicated operation of enzyme activity detection methods, so that domestic glucose oxidase is still mainly imported
Moreover, the traditional method of improving glucose oxidation yield and enzyme activity by optimizing fermentation conditions has technical defects such as low accuracy, many experiments, long cycle, and inability to quickly and effectively improve the yield and enzyme activity of glucose oxidase.

Method used

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  • Glucose oxidase gene GOD, protein coded by GOD, pichia pastoris transformed by GOD and preparation method of pichia pastoris
  • Glucose oxidase gene GOD, protein coded by GOD, pichia pastoris transformed by GOD and preparation method of pichia pastoris
  • Glucose oxidase gene GOD, protein coded by GOD, pichia pastoris transformed by GOD and preparation method of pichia pastoris

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Experimental program
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Effect test

Embodiment 1

[0038] Construction of Pichia pastoris

[0039] Glucose oxidase gene GOD sequence and encoded protein sequence in this embodiment Such as figure 1 As shown, its specific preparation and functional identification of glucose oxidase gene GOD include:

[0040] A, the full sequence amplification and cloning of the glucose oxidase gene GOD;

[0041] B. Transformation of Pichia pastoris with the glucose oxidase gene GOD.

[0042] Step A The preparation of the glucose oxidase gene GOD, the preparation method is to use the 5' end ATGAAGTCCACTATTATCACCTCCA and the 3' end CTAGGCACTTTTGGCATAGTCTTCA as specific primers, and use Penicillium pointis DNA as a template, and pre-denature at 98°C for 5 minutes on the amplification instrument; 30 One PCR cycle: 98°C denaturation for 30 seconds, 65°C annealing for 30 seconds, 72°C extension for 1 minute; 72°C extension for 10 minutes for PCR amplification; PCR reactions were carried out according to the following components and dosage: The r...

Embodiment 2

[0048] Verification of enzyme activity of Pichia pastoris in test tube

[0049] The Pichia pastoris Pichia pastoris obtained in Example 1 is used as a production bacterium. After activation, the temperature is 30 ° C and the rotation speed is 200 rpm, and the seed culture solution with an OD600 of about 1.2 is transferred to the YPCS culture with an inoculum size of 2%. Base, cultured at a temperature of 30°C and a rotational speed of 200rpm; when cultured in YPCS medium until the OD600 value was about 1.2, the yeast cells were transferred to YPCS induction medium, and 1% methanol was added every day to induce expression for 3 days.

[0050] Medium: YPD medium for seeds and slant medium: 2% tryptone, 1% yeast extract, 2% glucose; 2% agar was added to the slant medium. The YPCS medium containing 100 μg / mL G418 contains the following raw materials in mass percentage: tryptone 2%, yeast extract 1%, casein hydrolyzate 2%, sorbitol 0.5%.

[0051] Obtained by protein electrophoresi...

Embodiment 3

[0053] Verification of enzyme activity of Pichia pastoris in 10L fermenter

[0054] Inoculate the activated recombinant strain in the YPD medium, inoculate it in the BMGY medium with a 3% inoculation amount at a temperature of 30°C and a rotation speed of 200-250rpm until the OD600≥2. Cultivate at 250rpm until OD600 reaches about 10. The cultured BMGY fermentation broth was put into a 10L fully automatic fermenter with a 10% inoculation amount. The initial liquid volume is 6L, and 4‰PTM1 is added, 6mL of sterile VC is added every 24h by aseptic operation, and 212ml of sterile VB is added by aseptic operation after induction for 48h.

[0055] Bacteria culture stage: 18-24h with aeration and stirring at 30°C, the glycerol in the medium is gradually consumed, and when the glycerol is exhausted, the bacteria will no longer grow, and DO will suddenly rise at this time and remain stable. During the cultivation process, the pH value was maintained at about 5.0 with ammonia water, a...

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Abstract

The invention belongs to the preparation of a new gene, and in particular discloses a glucose oxidase gene GOD, protein coded by the GOD and pichia pastoris transformed by the GOD. The glucose oxidase gene GOD is prepared by taking 5'-ATGAAGTCCACTATTATCACCTCCA and 3'-CTAGGCACTTTTGGCATAGTCTTCA as specific primers and penicillium notatum as a template through polymerase chain reaction (PCR) amplification. According to the invention, the problem of an existing recombinant glucose oxidase gene in heterologous expression is solved; the total-length GOD gene and a shuttle expression vector constructed by the gene are obtained and the gene and the shuttle expression vector thereof are further transformed to the pichia pastoris, and upon screening and identification, a strain, which is higher in secretion expression of the glucose oxidase than an original strain, is obtained. The invention can greatly save cost and expense for further industrialized production and can improve the economic benefits of the glucose oxidase.

Description

Technical field [0001] The present invention is prepared for new genes, especially refers to a glucose oxidase gene God, the protein encoded by it, and the transformed Pattihmaster PichiaPastoris. Background technique [0002] Glucose oxidase (EC1.1.3.4) is a special catalytic β-D-glucose oxidation to Δ-D-glucose acid, and uses molecular oxygen as a hydrogenidase to produce hydrogen peroxide.Glucose oxidase is one of the enzyme preparations allowed by the state. It has non -toxic and side use of the human body. It has the effects of removing glucose, dehydrogenation, sterilization, etc. It has been widely used in foods, textiles, chemicals, feed, medicine and other industriesmiddle. [0003] Glucose oxidase is widely distributed in animals and plants and microorganisms. The main strains currently used to study and produce glucose oxidase are Abperrillusniger and Penicilliumnotum.Given; but in the process of production of black mold and green mold, the presence of a large amount o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/04C12N1/19C12N15/10C12N15/81C12R1/84
CPCC12N9/0006C12N15/815C12Y101/03004
Inventor 高庆华胡美荣吴芳彤陶勇秦艳梅马清河王云鹏
Owner 河北省微生物研究所有限公司
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