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Coding beta-galactosidase gene and expression and application thereof

A galactosidase and gene technology, applied in the field of enzyme genetic engineering, can solve problems such as unclear mechanism

Inactive Publication Date: 2009-12-09
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using ONPG (o-nitrophenol-β-D galactoside) as a substrate, react at 40°C for 3 minutes, Km is 2.78mmol / L, and the maximum reaction speed is 0.1umol / min. It has a strong inhibitory effect. It is worth noting that the inhibitory effect of ribose is also extremely strong, but its mechanism is still unclear (Shen Weiqun et al., Acta Biological Engineering, 1993, 9(4): 348-354; Dickson R C et al., Journal of Bacteriology, 1980, 142(3): 777~785)

Method used

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  • Coding beta-galactosidase gene and expression and application thereof
  • Coding beta-galactosidase gene and expression and application thereof
  • Coding beta-galactosidase gene and expression and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Screening, isolation and identification of Cellulomonas producing β-galactosidase

[0052] 1.1 Materials

[0053] 1.1.1 Sample

[0054] A total of 12 kinds of soil samples were collected from Xinjiang.

[0055] 1.1.2 Tool enzymes and reagents

[0056] Pfu high-fidelity DNA polymerase was purchased from Qihuasheng Biological Company; restriction endonuclease and T4 DNA ligase were purchased from TaKaRa and NEB Company; RNase was purchased from Beijing Huamei Company; DNA purification kit was purchased from Shanghai Shenneng Gaming Company; The substance o-nitrophenol-β-D-galactoside (ONPG) was purchased from Pierce; 5-bromo-4-chloro-3-indole-D-galactoside (X-gal) was purchased from TaKaRa; dNTP was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; yeast extract and peptone were purchased from Oxford; dimethylformamide (DMF) was purchased from Shanghai Bioengineering Co., Ltd.; other reagents were of domestic analytical grade.

[0057] 1.1.3...

Embodiment 2

[0076] Isolation and cloning of embodiment 2β-galactosidase gene

[0077] 2.1 Materials

[0078] 2.1.1 Strains and plasmids

[0079] Cellulomonas Dm (Cellulomonas sp.) is obtained by screening in Example 1;

[0080] The pEASY-T1 cloning vector was purchased from Beijing Quanshijin Biological Company;

[0081] Top 10 Escherichia coli competent cells were purchased from Beijing Quanshijin Biological Company.

[0082] 2.1.2 Tool enzymes and reagents

[0083] LA TAq DNA polymerase was purchased from Takara Company;

[0084] Phusion ultra-fidelity PCR kit was purchased from NEB Company;

[0085] All other chemical reagents were of domestic analytical grade.

[0086] 2.1.3 Culture medium and related solution preparation

[0087] Lactose separation medium: 2.0% lactose, 0.5% yeast extract, 1.0% peptone, 0.5% NaCI, sterilized at 115°C for 15 minutes;

[0088] TAE (50×): 242g Tris base, 57.1ml glacial acetic acid, 100ml 0.5mol / L EDTA (pH8.0), dilute to 1L with sterile water.

[...

Embodiment 3

[0256] Expression of embodiment 3β-galactosidase gene galc

[0257] 3.1 Materials

[0258] 3.1.1 Strains and plasmids

[0259] Escherichia coli TOP10 and Escherichia coli BL21 (DE3) were purchased from Beijing Quanshijin Biological Company;

[0260] The pET-30a(+) vector was purchased from Novagen.

[0261] 3.1.2 Tool enzymes and reagents

[0262] IPTG was purchased from Promega;

[0263] Protein molecular weight standard DM101 and DR101 were purchased from Beijing Quanshijin Biological Company;

[0264] Acrylamide, N,N′-methylene acrylamide, and agarose were purchased from Sigma;

[0265] Affinity chromatography resin Ni-NTA Agarose was purchased from Qiagen;

[0266] Others are domestic analytical reagents.

[0267] 3.1.3 Configuration of medium and related solutions

[0268] Protein loading buffer (2×): 100mmol / L Tris-HCl (pH6.8), 200mmol / L dithiothreitol (DTT), 4% SDS, 0.2% bromophenol blue, 10% glycerol.

[0269] 30% acrylamide solution: 29g of acrylamide, 1g of ...

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Abstract

The invention discloses a coding beta-galactosidase gene and an expression and an application thereof. The invention sieves and clones a beta-galactosidase gene (SEQ ID NO:1) coming from Cellulononas sp. from Sinkiang soil. The DNA overall length of the beta-galactosidase gene group is 2076bp, and (G+C) percent content is 72.9% and the beta-galactosidase gene group is deduced to encode 629 amino acid. The beta-galactosidase encoded by genes has the highest similarity with the beta-galactosidase from Arthrobacter sp., and the similarity is 59%. Enzymology property measurements indicate that the beta-galactosidase of the invention is neutral normal-temperature enzyme. Most of the beta-galactosidase of other Arthrobacter sp. reported by documents belongs to low-temperature psychrophilic enzyme which is obvious different from the beta-galactosidase of the invention in terms of enzymology properties.

Description

technical field [0001] The invention relates to a gene encoding β-galactosidase, in particular to the cloning, expression and enzymatic property research of a gene encoding β-galactosidase, and belongs to the field of genetic engineering of enzymes. Background technique [0002] Milk and dairy products are rich in high-quality protein, fat, carbohydrates, and almost all known vitamins and various minerals, especially its high calcium content and appropriate calcium-phosphorus ratio, which are easily digested and absorbed by the human body, and also contain immunoglobulin It is an ideal food for human beings to enhance their physical fitness. [0003] Lactose is the main component in milk and whey. Lactose accounts for about 30% of the dry matter of milk, but its solubility is low. Its solubility in water at 20°C is only 20%, and its sweetness is only 16% of that of sucrose. Long-term storage of milk Due to the precipitation of lactose, the product has a sandy feeling and af...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/38C12N15/63C12N1/21C12N1/19
Inventor 张伟姚斌伍宁丰范云六郭军张宇宏
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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