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Kluyveromyces lactis mutant strain as well as glycosidase and application thereof

A Kluyveromyces and glycosidase technology, applied in the field of Kluyveromyces lactis mutant strains, can solve the problems of catalytic efficiency, substrate range high-temperature activity and stability, unsatisfactory yield, glycosidase safety and poor activity, etc. problems, to achieve the effects of reduced bacterial contamination, high safety, and reduced energy consumption

Active Publication Date: 2018-06-01
董颖军
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the safety and poor activity of most glycosidases at present, they are far from meeting the needs of modern industrial applications in terms of catalytic efficiency, substrate range, high temperature activity and stability, and yield. It is necessary to further find a new, safe source Glycosidase with high thermal stability and activity to meet the needs of industrial production, especially food industry production

Method used

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  • Kluyveromyces lactis mutant strain as well as glycosidase and application thereof
  • Kluyveromyces lactis mutant strain as well as glycosidase and application thereof
  • Kluyveromyces lactis mutant strain as well as glycosidase and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0066] Compound mutagenesis of embodiment 1 Kluyveromyces lactis strain ATCC 8585

[0067] 1.1 Preparation of Kluyveromyces lactis ATCC 8585 single colony

[0068] The preserved Kluyveromyces lactis ATCC 8585 was inoculated into YPD liquid medium, and cultured continuously at 28° C. and 250 rpm for 2 to 3 days. Dilute the fermentation broth 10 with sterile water -3 , 10 -4 , 10 -5 After doubling, spread them on the YPD solid medium plate, culture statically at 28°C for 2 to 3 days, after a single colony grows, pick a single colony and transfer it to the YPD liquid medium, continuously culture at 28°C, 250rpm for 48 Hour.

[0069] 1.2 UV treatment

[0070] After the cultivation, the bacterial solution was diluted with sterile physiological saline so that the yeast cell concentration was about 10 7 pieces / ml. Take 25ml of bacterial solution and place it in a sterile empty plate, and carry out medium-speed magnetic stirring. Irradiate for 1min, 2min, 3min, 4min and 5min r...

Embodiment 2

[0077] Example 2 Screening of Kluyveromyces lactis Mutants

[0078] 2.1 Cultivation of mutant strains

[0079] Pick the clones on the plate in 1.5 and transfer them to a 96-deep well plate containing 1ml YPD liquid medium. Continuous culture was carried out at 30°C and 250 rpm for 5 days. After the incubation, the deep-well plate was centrifuged at 5000 rpm for 5 min. Take 100 μl of fermentation broth into a 96 shallow-well plate and store it at 4°C until use.

[0080] 2.2 Determination of glycosidase activity of mutant strains

[0081] The fermentation broth obtained in 2.1 was incubated at 95°C for 30 minutes, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected. The assay was carried out with p-nitrophenol β-D-glucoside (pNPG) as the substrate. The system for measuring activity is: sodium phosphate buffer 0.05ml (100mM, pH7.0) + 0.05ml enzyme solution to be tested + 15μl pNPG (100mM, dissolved in DMSO), with no enzyme solution as the blank contro...

Embodiment 3

[0084] Example 3 Obtaining of Kluyveromyces lactis XT1412 glycosidase

[0085] 3.1 Fermentation production

[0086] Kluyveromyces lactis XT1412 preserved in 2.3 was activated with 100 ml of liquid YPD medium, and cultured continuously at 28° C. and 250 rpm for 2 days. After the culture was completed, the microscopic observation showed that there was no bacterial contamination, and then transferred to a 5L fermenter of liquid YPD medium, and fermented continuously for 5 days at 28° C. and 300 rpm. After the fermentation, the cells were collected by centrifugation at 5000 rpm for 10 min. Suspend cells in 500ml sterile 50mM pH7.0Na 2 HPO 4 / NaH 2 PO 4 In the buffer solution, use a high-pressure homogenizer to break the cells at 0°C and 1000MPa. After the cells were broken, they were centrifuged at 5000rpm for 20min, the supernatant was collected, and incubated at 95°C for 20min. Centrifuge again at 5000rpm for 10min, collect the supernatant, and store at 4°C.

[0087] 3.2 A...

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Abstract

The invention relates to a kluyveromyces lactis mutant strain as well as glycosidase and an application thereof. With kluyveromyces lactis ATCC 8585 as an initial strain, the kluyveromyces lactis mutant strain is obtained through composite mutagenesis; the glycosidase, which is produced by the mutant strain, is good in stability and reserves high-temperature activity, and meanwhile, the glycosidase, which conforms to natural standard specifications and source safety, is applicable to industrial production. The invention also provides polynucleotide, a recombinant vector containing the polynucleotide, a host cell containing the recombinant vector and a composition containing the glycosidase. In addition, the invention also provides a method for producing the glycosidase and hydrolyzing a material containing a glucosidic bond.

Description

technical field [0001] The invention relates to Kluyveromyces lactis, in particular to a Kluyveromyces lactis mutant strain producing thermophilic glycosidase. Background technique [0002] Glycosidase, also known as glycoside hydrolase (Glycoside hydrolase, EC3.2.1), is a general term for enzymes that act on various glycosides or oligosaccharides to hydrolyze glycosidic bonds. They can convert carbohydrates (cellulose, hemicellulose, starch, pectin, alginic acid, chitin, etc.), which account for 70% of the world's total biomass, into monosaccharides, and can also convert non-polysaccharide glycoside compounds into aglycones . Therefore, in the field of biocatalysis and transformation, glycosidase has a very wide range of applications in many industries such as medicine, chemical industry, materials, energy, textiles, papermaking, food, feed, and environment. [0003] The essence of an enzyme is a protein, and its suitable reaction temperature is usually around 40°C. Howev...

Claims

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Application Information

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IPC IPC(8): C12N1/16C12N9/24C12N15/56C12N15/81C12N1/19C12R1/645
CPCC12N9/2402C12N15/815
Inventor 董颖军
Owner 董颖军
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