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Eosinophilic lactase BGALA, gene and application thereof

A lactase and lactose technology, applied in the field of genetic engineering, can solve the problems of increasing product processing and cost, and achieve good protease resistance, high lactose hydrolysis rate, and high enzyme activity stability

Active Publication Date: 2013-03-20
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method of casing coating will increase the processing process and cost of the product

Method used

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  • Eosinophilic lactase BGALA, gene and application thereof
  • Eosinophilic lactase BGALA, gene and application thereof
  • Eosinophilic lactase BGALA, gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Acidophilic fungus Bispora sp.MEY-1 and its enzyme production characteristics

[0042] A sample of uranium mine wastewater from a mine in Jiangxi was cultured on potato juice medium, spread on an enzyme-producing medium plate, and incubated at 30°C for 5-6 days, and a transparent circle could be seen on the enzyme-producing medium plate. Proved to have lactase activity.

[0043] The acidophilic fungus Bispora sp.MEY-1 was preserved on May 19, 2008 in the General Microbiology Center of the China Committee for the Collection of Microorganisms (Datun Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101), and its preservation number For: CGMCC No.2500.

Embodiment 2

[0044] Example 2 Cloning of the acidophilic fungus Bispora sp.MEY-1 lactase-encoding gene bgalA

[0045] Extraction of acidophilic fungus Bispora sp.MEY-1 genomic DNA:

[0046] Filter the mycelium cultured in liquid for 3 days with sterile filter paper, put it into a mortar, add 2mL of extract, grind for 5min, then put the grinding solution in a 50mL centrifuge tube, lyse in a water bath at 65°C for 20min, and mix every 10min. Homogenize once and centrifuge at 10,000 rpm for 5 min at 4°C. The supernatant was extracted in phenol / chloroform to remove impurity proteins, and then an equal volume of isopropanol was added to the supernatant. After standing at room temperature for 5 minutes, centrifuge at 10,000 rpm for 10 minutes at 4°C. The supernatant was discarded, the precipitate was washed twice with 70% ethanol, dried in vacuum, dissolved by adding an appropriate amount of TE, and stored at -20°C for later use.

[0047] According to the conserved sequence of the lactase gene...

Embodiment 3

[0052] RT-PCR analysis of embodiment 3 lactase gene

[0053] Extract the total RNA of Bispora sp.MEY-1, the extraction method of total RNA is as follows: the bacterial cell centrifugation that will cultivate 54hr in the bran culture medium, get wet weight 100mg, use the phosphate buffer (PH7.0) of 1mmol / L Wash three times, grind into powder in liquid nitrogen, add ice-cooled 600 μL denaturing solution (26mM sodium acetate, pH4.0, 0.5% lauryl sarcosine, 0.125M β-mercaptoethanol, 4M guanidine thiocyanate ), then sequentially add 60 μL 2mol / L sodium acetate (PH4.0), mix well, add 600 μL phenol-chloroform-isoamyl alcohol (125:24:1, pH4.7), shake vigorously, and place on ice for 15 minutes Centrifuge at 10,000g for 20min at 4°C, suck out the supernatant, add an equal volume of isopropanol, place at -20°C for 30min, centrifuge at 10,000g for 10min at 4°C, discard the supernatant, add 1 mL of ice-cold 75% ethanol to the precipitate for washing Finally, the precipitated RNA was dried...

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Abstract

The invention relates to genetic engineering field, in particular to eosinophilic lactase BGALA, gene and application thereof. The invention provides a lactase BGALA of eosinophilic Bispora sp.; the amino acid sequence of the lactase BGALA is shown in SEQ ID NO.1; and the invention provides genome and cDNA coding gene bgalA for coding the lactase. The invention obtains an eosinophilic lactase with the most proper pH value of 1.5, which maintains high enzymatic activity in acid environment, has better pH stability and heat stability, and antiprotease hydrolysis. The eosinophilic lactase can effectively hydrolyze the lactose in milk in simulated digestive tract environment. The eosinophilic lactase BGALA with the properties can be used for treating lactose intolerance, diary industry no processing cow milk and whey.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to an acidophilic lactase BGALA and its gene, a recombinant vector containing the gene and application thereof. Background technique [0002] β-galactosidase (β-D-galactoside galactohydrolase, β-D-galactosido-galatohydrolase, EC3.2.1.23) is generally called lactase (Lactase). This enzyme can hydrolyze lactose into galactose and glucose, and also has the function of transferring galactoside (Zhang Shuzheng et al., Enzyme Industry, Science Press, 1984, p818-819). Lactase exists in plants (especially in apricots, peaches, apples), bacteria (lactic acid bacteria, Escherichia coli, etc.), fungi (Aspergillus oryzae, Aspergillus niger, Aspergillus sulfovus, brittle-walled yeast, milk-binding yeast, lactic acid yeast, Candida tropicalis Yeast) and actinomycetes (Streptomyces coelicolor), animal intestines (especially infants). Lactose is the main component in milk...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/38C12N15/56C12N15/63C12N1/21C12N1/19A23C9/12A61K38/47A61P1/00C12R1/225C12R1/07C12R1/645C12R1/19
Inventor 姚斌罗会颖王亚茹孟昆杨培龙袁铁铮柏映国史秀云石鹏君王慧
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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