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Recombinant pichia pastoris capable of expressing keratinase and application of recombinant pichia pastoris

A technology of keratinase and Pichia pastoris, which is applied in the field of genetic engineering, can solve the problems of product enzyme activity reduction, keratinase toxicity to host bacteria, and inability to industrialize production, etc.

Active Publication Date: 2015-11-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the original strains derived from keratinase usually have very low expression of keratinase and cannot be used for industrial production, so it is very important to construct a high-yielding keratinase strain using biotechnology
[0004] To solve the problem of low enzyme production efficiency of wild bacteria, the target gene is usually cloned into a commonly used expression vector, and then the target product is expressed heterologously using a known high-efficiency expression system
However, due to the self-modification of the host bacteria, the enzyme activity of the expressed product is reduced, and keratinase has a strong toxic effect on the host bacteria itself. Therefore, the yield has been very low and cannot be used for industrial production.

Method used

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  • Recombinant pichia pastoris capable of expressing keratinase and application of recombinant pichia pastoris
  • Recombinant pichia pastoris capable of expressing keratinase and application of recombinant pichia pastoris
  • Recombinant pichia pastoris capable of expressing keratinase and application of recombinant pichia pastoris

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Embodiment 1

[0014] Construction of embodiment 1 recombinant strain

[0015] The gene encoding keratinase was cloned from Stenotrophomonas maltophilia BBE11-1, and after optimization, the gene encoding keratinase with nucleotide sequence as shown in SEQ ID NO.1 was obtained, and then cloned into expression plasmid pPIC9k to obtain recombinant plasmid pPIC9k -kerD. Afterwards, the recombinant plasmid was linearized and integrated into Pichia pastoris SMD1168 competent cells.

[0016] details as follows:

[0017] According to the gene sequence of kerD, primers EcoRI-F and NotI-R as shown in SEQ ID NO.2 and SEQ ID NO.3 were designed, and EcoRI and NotI restriction sites were added to both ends of the gene by PCR. The PCR product and the vector pPIC9k were digested with EcoRI and NotI respectively, and the digested products were purified and then ligated. The ligation product was transformed into Escherichia coli GM109 competent cells, positive clones were screened by LB plates containing k...

Embodiment 2

[0021] Induced expression and detection of embodiment 2keratinase

[0022] The recombinant strain was induced by methanol in a 250ml shake flask, and the keratinase activity of the supernatant of the fermentation broth was detected at different times, and the supernatant of the fermentation broth was analyzed by SDS-PAGE.

[0023] The induced expression of the recombinant strain is as follows:

[0024] 1) Transfer the recombinant strain to the YPD plate, then pick a single colony and inoculate it into a 250ml shake flask containing 25mL of BMGY medium, and culture it to OD at 28-30°C / 250-300rpm 600 =2-6(16-18h);

[0025] 2) Centrifuge at 1500-3000g for 5min at room temperature, collect the bacteria, and resuspend the bacteria with 50mL of BMMY medium to make the OD 600 = around 1.0;

[0026] 3) Put the bacterial liquid obtained in step 2 (about 100-200mL) into a 500mL shaker flask, seal it with double-layer gauze or cheesecloth, and place it on a shaker at 28-30°C / 250-300rp...

Embodiment 3

[0030] Verification on the enzyme production performance of embodiment 3 recombinant strains on the 3L fermenter

[0031] The recombinant strain was transferred to the YPD plate, and a single colony was picked and inoculated into a 1L shake flask containing 100 mL of YPD medium, and placed in a shaker at 30°C and 220 rpm for 24 hours as a seed solution. It was then inoculated into a 3L fully automatic fermenter (LiFlus GM BioTRON, Korea) containing 800 mL of BSM medium. Use 50% ammonia water and phosphoric acid solution to control pH5.5, temperature at 30°C, adjust the stirring speed to 500rpm, and maintain the ventilation at 2vvm; Add 350mL of 50% (W / V) glycerol (containing 12mL / L PTM1) to maintain the specific growth rate at 0.18h -1Left and right, and gradually adjust the stirring speed to 1000rpm, and the ventilation volume to 4vvm. After the glycerol is exhausted again, continue to maintain the matrix-deficient state in the system for about 1 hour and DO>60%, start feed...

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Abstract

The invention discloses recombinant pichia pastoris capable of expressing keratinase and application of the recombinant pichia pastoris and belongs to the field of gene engineering. The recombinant pichia pastoris expresses the keratinase by taking pichia pastoris SMD1168 as a host and pPIC9k as a vector. The recombinant pichia pastoris capable of expressing the keratinase has the advantages that the constructed recombinant pichia pastoris is a recombinant strain obtained by means of fermentation culture, and when fermentation culture reaches 168 hours, dry cell weight is up to 174.1g / L, and enzyme activity of extracellular keratinase is up to 1156U / mL, thus, the recombinant pichia pastoris is more suitable for industrial production of the keratinase.

Description

technical field [0001] The invention relates to a recombinant Pichia pastoris expressing keratinase and an application thereof, belonging to the field of genetic engineering. Background technique [0002] Hundreds of thousands of tons of waste such as feathers, wool and animal cutin are produced every year in the production process of aquaculture, animal husbandry and textile industry in my country. The main component of these wastes is keratin, which is difficult to be fully recovered due to its compact structure. Most of them become garbage, which not only pollutes the environment, but also is a waste of resources. [0003] Keratinase can specifically degrade keratin, and the degradation products contain essential amino acids needed for animal growth, so keratin can be used to degrade keratin waste such as feathers, wool and animal cutin, to produce animal feed and organic fertilizers. However, the original strains derived from keratinase usually have a very low expression...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N9/60C12R1/84
Inventor 张娟陈坚李光磊堵国成方真
Owner JIANGNAN UNIV
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