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Construction method of kluyveromyces lactis engineering strains for producing ferment rennet

A technology of Kluyveromyces cerevisiae and engineering strains, applied in the directions of microorganism-based methods, biochemical equipment and methods, hydrolase, etc., can solve the problems of inconsistent product properties, bitterness, restrictions, etc.

Inactive Publication Date: 2011-09-07
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Plant rennet has a wide range of sources, such as ficin and papain, but it is limited by time, region, growth cycle and other conditions, and the production of cheese with plant-derived rennet will result in soft curd texture and low cheese yield And prone to adverse phenomena such as bitterness
The use of microbial rennet to produce cheese will lead to inconsistent product properties, and the microbial source of rennet has the characteristics of poor proteolytic specificity, strong heat resistance and high proteolytic activity, which not only reduces the cheese yield but also produces bitter taste after ripening , so its application is limited

Method used

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  • Construction method of kluyveromyces lactis engineering strains for producing ferment rennet
  • Construction method of kluyveromyces lactis engineering strains for producing ferment rennet
  • Construction method of kluyveromyces lactis engineering strains for producing ferment rennet

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specific Embodiment approach 1

[0016] Embodiment 1: The construction method of the rennet-producing Kluyveromyces lactis engineering strain of this embodiment is carried out according to the following steps:

[0017] 1. Design upstream primers and downstream primers according to the gene sequence of K. lactis PMR1 (AJ001018) published in Genebank, and then perform PCR amplification using K. lactis GG799 genomic DNA as a template to obtain K. lactis GG799 the PMR1 gene;

[0018] 2. The PMR1 gene of Kluyveromyces lactis GG799 and the plasmid pBluescript-II were simultaneously digested with Sac I and Xho I, and the desired target fragment was recovered and ligated to obtain the ligated product pBluescript-II-PMR1;

[0019] 3. The connection product pBluescript-II-PMR1 was transformed into Escherichia coli JM109 competent cells, and then spread on ampicillin-resistant LB solid medium for culture, and the positive clones were picked for culture in liquid shake flasks, and then the recombinant plasmid pBluescript...

specific Embodiment approach 2

[0031] Specific embodiment two: the difference between this embodiment and specific embodiment one is that the reaction system for PCR amplification in step one is a 50 μL reaction system, which consists of the following components:

[0032]

[0033] PCR amplification conditions were: 95°C pre-denaturation for 10 min, 95°C denaturation for 45 s, 55°C annealing for 30 s, 72°C extension for 2 min, a total of 30 cycles, 72°C extension for 10 min, and 4°C incubation. Other steps and parameters are the same as those in Embodiment 1.

specific Embodiment approach 3

[0034] Specific embodiment three: the difference between this embodiment and specific embodiment one is that the double enzyme digestion system of the PMR1 gene of Kluyveromyces lactis GG799 in step 2 is as follows:

[0035]

[0036] Enzyme digestion conditions: 37 ° C, 1.5 ~ 2h. Other steps and parameters are the same as those in Embodiment 1.

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Abstract

The invention discloses a construction method of kluyveromyces lactis engineering strains for producing ferment rennet, and relates to a construction method of engineering strains for producing ferment rennet. The method comprises the following steps of: inserting amplified PMR1 genes of GG799 into plasmids pBluescript-II to obtain recombinant plasmids pBluescript-II-PMR1, then inserting Zeocin resistance coding gene into the plasmids to obtain recombinant plasmids pBluesk-Z-PMR1, performing linearization, transforming kluyveromyces lactis GG799 competent cells, and constructing GG799 PMR1 gene defect strains; and optimizing calf ferment rennet genes, inserting the optimized calf ferment rennet genes into GG799 expression vectors pKLAC1 to obtain recombinant plasmids pKLAC1-S-chymosin, performing linearization, and transforming PMR1 gene defect GG799 competent cells to finish the construction. The obtained recombinant kluyveromyces lactis can be used for producing the recombinant ferment rennet for cheese.

Description

technical field [0001] The invention relates to a method for constructing a rennet-producing engineering bacterial strain. Background technique [0002] In the cheese production process, it is necessary to add rennet to the milk to coagulate the casein into a solid colloid, and at the same time add lactic acid bacteria starter, press molding and post-maturation to form cheese products of various shapes, structures and flavors. In recent years, the world's cheese production has been on the rise, so the demand for rennet is also increasing year by year. At present, the annual demand for chymosin preparations in the world is 2.5×10 6 L, the output accounts for 15% of the total enzyme preparations in the world, becoming the second largest enzyme preparation, and the demand is still increasing. The traditional rennet is calf rennet, which is a crude enzyme extracted from the salt extract of the fourth stomach (abomasum) of unweaned calves. The enzyme is the enzyme of choice fo...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N9/64C12R1/645
Inventor 冯镇张兰威
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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