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Dual-enhanced acidic lactase Pichia pastoris expression strain, and construction method and fermentation technology thereof

A technology of acid lactase and Pichia pastoris, applied in the directions of fermentation, glycosylase, botanical equipment and methods, etc., can solve the problems of high cost, complicated production process, low unit yield of lactase, etc., and improve industrial production. The effect of improving the amount and expression

Inactive Publication Date: 2020-03-24
NINGBO XINUOYA MARINE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is still a big gap between domestic and foreign countries. Lactase has not been widely used in China's food and dairy industry. The main reason is that the unit output of lactase is low, the production process is complicated, and the cost is high.

Method used

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  • Dual-enhanced acidic lactase Pichia pastoris expression strain, and construction method and fermentation technology thereof
  • Dual-enhanced acidic lactase Pichia pastoris expression strain, and construction method and fermentation technology thereof
  • Dual-enhanced acidic lactase Pichia pastoris expression strain, and construction method and fermentation technology thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Construction and screening of inducible acid lactase Pichia pastoris expression strain

[0038]Synthesize the acid lactase integrated gene (ie Lac gene, refer to the gene with the sequence number XM_025613095 in the genebank of NCBI), and clone it into the PMD-18T vector. Inoculate a single colony of PMD-18T-Lac into 50ml LB medium for culture, and extract the plasmid. Inoculate a single colony of pPIC9K-DH5α into LB medium for culture, and extract the plasmid. According to the method steps of the plasmid extraction kit, the plasmids PMD-18T-Lac and pPIC9K were extracted. Plasmids pMD-18T-Lac and pPIC9K were cut with restriction endonucleases. The Lac fragment and the pPIC9K vector were recovered by tapping the rubber. Lac and pPIC9K were ligated with T4 DNA ligase. The ligation product was transformed into top10 k to be competent, spread on the LB Amp antibiotic screening plate, cultured overnight at 37°C, picked a single clone, and carried out colony PCR...

Embodiment 2

[0045] Example 2 Construction and screening of composition and induction double enhanced acid lactase Pichia pastoris expression strain

[0046] Synthetic ARS_OPT_BglII gene (the gene sequence refers to Liachko I, Dunham M J.Anautonomously replicating sequence for use in a wide range of budding yeasts [J]. FEMS Yeast Research, 2014, 14 (2), 364-367, page 365 2 paragraphs), cloned into the puc57 vector. Inoculate a single colony of puc57-ARS-top10 into 50mL LB medium for expansion, and extract the plasmid. ARS and pGAPZαA were ligated using a similar scheme as in Example 1, and transformed to obtain clones. Cultivate overnight at 37°C, pick a single colony, and perform colony PCR verification. Positive clones were inoculated with LLB (including ZEOCIN), and the extracted plasmids were sent to the sequencing company for sequencing verification. After the verification is correct, save Glycerol bacterium parsGAPzαA and store it at -70°C.

[0047] Then the plasmids parsGAPzαA a...

Embodiment 3

[0054] The fermentation process of the fermentor of the expression strain of embodiment 3 acid lactase recombinant Pichia pastoris

[0055] The glycerol bacterium of the recombinant yeast strain with the highest expression of enzyme activity screened in Example 1 and Example 2 was activated and cultured on YPD for 2-3 days until a single colony grew. Use an inoculation loop to pick a single colony into 50ml of YPD liquid medium for overnight culture on a shaking table (28°C, 245rpm), OD to 0.5-1; as a fermentation seed solution, use the flame inoculation method to inoculate. The fermentation parameters of the fermenter are set to PH4.0-6.5, the temperature is 25-32°C, and the speed, ventilation and feeding speed are constantly adjusted to maintain the dissolved oxygen at 0%-40%. Samples were taken every 12 hours after inoculation to determine lactase activity. Continue to culture until OD reaches 170-250. At this time, stop supplementing 50% glycerol, and start supplementing...

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Abstract

The invention provides a composition and induction dual-enhanced acidic lactase recombinant Pichia pastoris expression strain constructed by combining a lactase gene-containing integrative plasmid with a free plasmid. The recombinant Pichia pastoris is sued to express lactase at a high density. The lactase gene-containing integrative plasmid is combined with the free plasmid to construct the expression strain to make the lactase expression quantity of the expression strain greatly improved, and the secretion expression quantity of the expression strain can reach 8000 ALU / mL or above through high-density fermentation, so that the expression strain can be industrially applied, and the industrial production quantity of lactase is improved.

Description

technical field [0001] The invention relates to a lactase biological fermentation technology, in particular to the construction of a lactase Pichia expressing strain and the fermentation process of the Pichia expressing strain. Background technique [0002] Lactase, scientific name β-galactoside galactohydrolase, also known as β-galactosidase, is a white powder, tasteless and odorless. Lactase hydrolyzes galactosidic bonds under specific conditions, and hydrolyzes lactose into glucose and galactose, which are easily absorbed by the intestinal tract. A large number of studies have shown that the lactase activity of mammals has a typical physiological decrease with age, and the irreversible decline of lactase in adults is controlled by genes. The incidence of lactase deficiency in the world is over 50%, while about 90% of adults in my country lack lactase. If a person with lactase deficiency ingests a lot of lactose at one time, the lactose cannot be digested and absorbed in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/66C12N9/24C12N15/56C12R1/84
CPCC12N9/2402C12N15/66C12N15/815C12Y302/01108
Inventor 曹阳田健张东风诸辉
Owner NINGBO XINUOYA MARINE BIOTECH CO LTD
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