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Chymosin gene, amino acid sequence for coding chymosin gene and application of chymosin gene

A rennet, amino acid technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problem of lack of clear gene sequence, and achieve the effect of accelerating yak milk coagulation and broad application prospects.

Active Publication Date: 2013-08-14
成双林
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no literature or patent has reported microbial-derived chymosin that is specific for yak milk, thermostable, and maintains high activity at pH 6.0-8.0, let alone such a protein or the gene sequence encoding it

Method used

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  • Chymosin gene, amino acid sequence for coding chymosin gene and application of chymosin gene
  • Chymosin gene, amino acid sequence for coding chymosin gene and application of chymosin gene
  • Chymosin gene, amino acid sequence for coding chymosin gene and application of chymosin gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Cultivation of Bacillus subtilis YB-3 and preparation of fermentation broth.

[0034] Shake flask fermentation of Bacillus subtilis YB-3 was carried out in a 100ml Erlenmeyer flask containing 30ml of fermentation medium. The fermentation medium consists of 10g / L casein, 3g / L beef extract, 5g / LNaCl, 5g / LNa 2 HPO 4 12H 2 O composition, initial pH 7.0. The above medium was inoculated with 1 ml of logarithmic phase bacterial solution, and cultured with shaking at 37° C. at 150 rpm. After the fermentation is finished, the fermentation liquid is centrifuged for 5 minutes to remove the bacteria, the centrifugal force is 3020g, and the supernatant is the crude enzyme liquid.

Embodiment 2

[0036] Proteolytic activity assay of chymosin and determination of milk-clotting activity.

[0037] The determination method of chymosin refers to the method 157A:1997 of IDF.

[0038] The proteolytic activity assay method refers to the modified Lorry method.

Embodiment 3

[0040] Purification procedure for chymosin produced by strain Bacillus subtilis YB-3.

[0041] First, after fractional precipitation of the crude enzyme solution by 30-60% ethanol, the protein precipitate obtained by centrifugation at 12,000 g for 30 min was redissolved in 50 mM phosphate buffer solution of pH 6.0.

[0042] The above-mentioned reconstituted enzyme solution was further separated and purified by Waters protein pak i-125 molecular sieve chromatography column (7.5x300mm), and the above-mentioned chromatography column was pre-washed with 50mM pH6.0 phosphate buffer (containing 0.15M Na 2 SO 4 )balance. During the separation process, the sample detection wavelength is OD 280 , the mobile phase uses the same buffer solution, the flow rate is 1.0ml / min, and each 1.0ml eluate is regarded as a component, which is collected in sections.

[0043] Active fractions were collected and concentrated and desalted by ultrafiltration through Amicon Ultra (Milipore). The final...

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Abstract

The invention provides chymosin, wherein the amino acid sequence of the chymosin is as shown by SEQ ID NO:2. The chymosin has the specificity of a yak milk substrate, and can be used for preparing yak milk cheese. The invention also provides a gene for coding the chymosin described in the claim 1, wherein the nucleotide sequence of the gene is as shown by the SEQ ID NO:1. The invention has the advantages that the chymosin ensures that the yak milk can be quickly solidified, the problem that the conventional chymosin is not suitable for producing the yak milk cheese is solved, and the chymosin can be used as a substitute of calf abomasum chymosin and has wide application prospects in the field of deep processing of yak milk.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a gene sequence of chymosin secreted by a mutant strain of Bacillus subtilis and its protein sequence and application. Background technique [0002] In the field of food processing, especially cheese processing, coagulation enzyme is an essential additive. Rennet not only coagulates milk, but also plays a non-negligible role in cheese maturation (Kumar et al., 2005). Calf rennet was first discovered by chance. When people use animal skins to preserve milk, the milk sometimes coagulates. After that, calf rennet has been widely used in the field of cheese processing. Since 1961, due to the rapid development of the world's cheese industry, the supply of calf rennet has continued to be in short supply, and scientists have been looking for proteases that can replace calf rennet. [0003] Later, some proteases were found to have a similar curdling effect. These rennets are w...

Claims

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Application Information

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IPC IPC(8): C12N9/56C12N15/57A23C9/12A23C19/02
Inventor 不公告发明人
Owner 成双林
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