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Beta-mannase codon optimization sequence and method for improving expression level of pichia pastoris engineering bacteria

A technology of mannanase and codon optimization, which is applied in the field of β-mannanase codon optimization sequence and improving the expression level of Pichia pastoris engineering bacteria, which can solve the problem of low expression level

Inactive Publication Date: 2020-01-10
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for some genes derived from bacteria, the expression level is often low, even after codon optimization, it may not be able to achieve the ideal expression level

Method used

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  • Beta-mannase codon optimization sequence and method for improving expression level of pichia pastoris engineering bacteria
  • Beta-mannase codon optimization sequence and method for improving expression level of pichia pastoris engineering bacteria
  • Beta-mannase codon optimization sequence and method for improving expression level of pichia pastoris engineering bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Man5AS11R and VHb co-expressed recombinant vector construction

[0042] The man5AS11R gene sequence was amplified by PCR, with EcoRI and NotⅠ restriction sites at both ends. After double digestion and purification, it was ligated with the same double digestion pPIC9k vector to construct the cloning vector pPIC9k-man5AS11R and transform it into Escherichiacoli DH5α competent cells. , Pick a single clone for PCR identification and enzyme digestion identification, and obtain a successfully constructed fusion expression plasmid after the gene sequencing is correct. VHb gene GenBank accession number is AY278220.1, synthesized by BGI. Both ends of the synthesized VHb gene contain restriction sites of BstBI and NotⅠ. After double-enzyme digestion and purification, the VHb fragment was connected to the same double-enzyme-digested pPICZ vector to construct the cloning vector pPICZ-VHb. Transform Escherichia coli DH5α competent cells, pick a single clone for PCR, enzy...

Embodiment 2

[0043] The 10L fermenter of embodiment 2 GS115 / man5AS11R-opt produces enzyme

[0044] GS115 / man5AS11R and GS115 / man5AS11R-opt were subjected to 10L fermenter induced enzyme fermentation, the results are as follows figure 2 As shown, the maximum mannanase activity of GS115 / man5AS11R-opt fermented for 120h reached 14641U / ml, which was 38% higher than that of GS115 / man5AS11R fermented for 120h. It can be seen that codon optimization can significantly improve the activity of Man5AS11R in Pichia pastoris. The expression level.

Embodiment 3

[0045] Example 3 Enzyme production in 10L fermenter of GS115 / man5AS11R-VHb

[0046]GS115 / man5AS11R and GS115 / man5AS11R-VHb were subjected to 10L fermenters to induce enzyme production fermentation, and the results were as follows: image 3 As shown, the mannanase activity of GS115 / man5AS11R-VHb fermented for 120 hours reached a maximum of 15641U / ml, which was 48% higher than that of GS115 / man5AS11R fermented for 120 hours. It can be seen that co-expression of VHb can significantly improve the activity of Man5AS11R in Pichia pastoris. The expression level.

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Abstract

The invention, which relates to the technical field of biology, particularly discloses a beta-mannase codon optimization sequence and a method for improving the expression level of the pichia pastorisengineering bacteria, thereby improving the induced expression quantity of heat-resistant acidic beta-mannase Man5AS11R in the pichia pastoris engineering bacteria. Codon bias optimization is carriedout on a nucleotide sequence of a man5AS11R gene by using Gene Designer (DNA2.0, Menlo Park, CA and USA) software; and a pichia pastoris engineering bacteria GS115 / man5AS11R-opt is successfully constructed by a pPIC9k-man5AS11R-opt recombinant vector. And co-expression of hemoglobin VHb is completed in a bacterial strain GS115 / man5AS11R-opt by a recombinant vector pPICZ-alpha A-vgb, so that the oxygen dissolving level of the pichia pastoris engineering bacteria fermentation process is obviously prompted and the fermentation density and yield of the engineering bacteria are increased. The fermentation enzyme production of a 10L fermentation tank reaches 20000 U / mL or above and is doubled by being compard with the yield of the original strain GS115 / man5AS11R. Therefore, the method has the broad industrial production prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a codon-optimized sequence of β-mannanase and a method for improving the expression level of Pichia pastoris engineering bacteria. Background technique [0002] In recent years, with the development and utilization of natural hemicellulose resources, the elimination of mannan anti-nutritional factors in diets and the discovery of the medicinal value of mannan oligosaccharides, the demand for β-mannanase has increased. bigger. At present, the commercial β-mannanase enzyme preparations on the market include Hemei Enzyme produced by Chengem Company of the United States, as well as Huafenzyme produced by the domestic Bosco Group and β-mannanase produced by the Challenge Group, but because of the small output, The relative price of the product is high, which greatly reduces the scope of application of the enzyme. [0003] The Pichia pastoris expression system has developed into a mature e...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/2491C12N15/815C12N2800/22C12Y302/01025
Inventor 郑宏臣宋诙徐健勇甄杰付晓平
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI