Dysoxia-resistant lactase yeast strain and construction method thereof

A technology of yeast strain and lactase, which is applied in the biological field, can solve problems such as the lack of reports on lactase strains, and achieve the effects of increasing yield, improving utilization rate, and promoting growth

Inactive Publication Date: 2016-11-09
SHANDONG ACADEMY OF PHARMACEUTICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the construction and application of lactase-producing yeast genetically engineered strains have been reported, the lactase strains capable of anoxic tolerance have not been reported.

Method used

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  • Dysoxia-resistant lactase yeast strain and construction method thereof
  • Dysoxia-resistant lactase yeast strain and construction method thereof
  • Dysoxia-resistant lactase yeast strain and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Construction of anaerobic-tolerant lactase strains

[0027] 1) Construction of pPIC9-vgb recombinant vector and yeast transformation

[0028] According to the published vgb sequence, design upstream amplification primer vgb-up: 5'-CGGGATCCAAACGATGTTAGACCAGCAAACCAT-3' and downstream amplification primer vgb-do: 5'-CGGAATTCTTATTCAACCGCTTGAGC-3', respectively containing BamHI and EcoRI restriction enzymes For the recognition site, use the recombinant plasmid pSET152-vgb containing the vgb gene sequence in the laboratory as a template, and amplify the vgb gene sequence according to the following system: 10*PCR buffer with Mg 2+5µl, 10mM dNTP mix 1µl, pSET152-vgb template 0.5µl, 50mM vgb-up primer 1µl, 50mM vgb-do primer 1µl, PFUDNA Polymerase 0.5µl, dd water 41µl, the total volume is 50µl. The PCR reaction conditions were: denaturation at 94°C for 30s, annealing at 58°C for 30s, extension at 72°C for 40s, 30 cycles, and extension at 72°C for 10 minutes.

[0029]...

Embodiment 2

[0037] Embodiment 2 Determination of the hypoxia tolerance of shake flask level engineering strains

[0038] The engineering strain GS115-vgb-lac was compared with the lactase-lactase recombinant yeast strain GS115-lac without vgb constructed in the laboratory to compare the hypoxia tolerance at the shake flask level. Two groups of shake flasks were set up for the two kinds of bacteria, one group was the normal control group, which was sealed with 6 layers of gauze; the other group was the hypoxia group, which was sealed with PARAFILM film. The expression process is as follows: Streak the recombinant bacteria stored at -80°C on the YPD plate, culture it upside down at 30°C for 2 days, pick a single colony and culture it in 10 ml BMGY medium at 30°C, 300 rpm to OD 600 =2-6, centrifuge, collect the bacteria, insert into 20ml BMMY medium, induce expression at 30°C, 300 rpm, add methanol to the initial concentration every 24 hours. After 96 hours of induction, the cells were remo...

Embodiment 3

[0039] Example 3 Determination of Lactase Activity

[0040] 1) Standard curve drawing

[0041] Prepare 0.2M NaAc-HAc buffer pH5.2, 0.2M NaAc-HAc buffer pH5.2, 40umol / ml o-nitrophenyl β-D-galactoside (o-NPG), and 5% Na 2 CO 3 solution, fill the tubes as indicated in the table below.

[0042] .

[0043] Make 3 parallel tubes for each gradient, draw the standard curve with o-NP content (umol) as the abscissa and A420 as the ordinate.

[0044] 2) Enzyme activity assay

[0045] Sample tube: Substrate preheated for 5 minutes; 50ul appropriately diluted enzyme solution + 450ul preheated substrate, reacted at 60°C for 10 minutes; immediately added 500ul 5% Na2CO3 to stop the reaction and develop color; add 2ml of experimental buffer to a final volume of 3ml; measure A420 .

[0046] Control tube: No enzyme solution is added during the reaction, just add enzyme solution before measuring the absorbance.

[0047] Enzyme activity unit definition: at 60°C and pH 5.2, the hydrolysis...

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Abstract

The invention discloses a dysoxia-resistant lactase yeast strain with a preservation number of the dysoxia-resistant lactase yeast strain is CCTCC NO:M2016252. The invention further discloses a construction method of the dysoxia-resistant lactase yeast strain, wherein the construction method comprises the following steps: sequentially transferring expression vectors in vitreoscilla hemoglobin cells and lactase secretory expression vectors into pichia pastoris; screening a strain with high lactase activity to obtain the dysoxia-resistant lactase yeast strain. The strain disclosed by the invention can be used for preparing recombinant lactase protein by high-density fermentation; a utilization rate, on oxygen gas, of cells is increased by synthesizing hemoglobin (VHB) in cells, and growth, under an oxygen-lean condition, of the cells is promoted, so that the expression yield of extracellular recombinant lactase is increased.

Description

technical field [0001] The invention relates to an anoxic-resistant lactase yeast strain and a construction method thereof, in particular to transferring the hemoglobin gene and the lactase gene of Vitella hyaline into a Pichia pastoris strain at the same time, so as to improve the anoxic-resistant ability of the strain and the production of lactase. Belongs to the field of biotechnology. Background technique [0002] The scientific name of lactase (Lactose) is β-D-galactoside galactohydrolase (β-D-galactosido-galatohydrolase, EC3.2.1.23), which can not only hydrolyze lactose to generate galactose and glucose, but also have transglycosidic effect . It can be used to treat "lactose intolerance" in the medical field, and can be used in the preparation of low-lactose milk and other low-lactose dairy products in the food field, and has extremely high application value. [0003] Lactase in nature is mostly derived from microorganisms, such as bacteria, yeast, mold and actinomyc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12R1/84
CPCC07K14/195C12N9/2471C12N15/815C12N2800/102C12Y302/01023
Inventor 刘飞钊倩倩张秀华朱希强侯重文袁超
Owner SHANDONG ACADEMY OF PHARMACEUTICAL SCIENCES
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