Acid protease Candidapepsin as well as heterogeneous expression and purification method thereof

A technology of acid protease and heterologous expression, applied in biochemical equipment and methods, botany equipment and methods, microbial-based methods, etc., can solve the problem of low yield and achieve the effect of increasing yield

Inactive Publication Date: 2019-04-26
SOUTH CHINA UNIV OF TECH
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

At present, the yield of recombinant expression of Candida acid protease is low. For example, Zuzana Vinterová et al. expressed Candida parapsilosis SAPP1 in Saccharomyces cerevisiae with a yield of 3mg / L; E. Hochuli et al. expressed SAP2 in E. coli with an enzyme activity of 2.3U / mg; Jing Li et al expressed the enzyme activity of SAP6 in Escherichia coli as 19.6U / mg

Method used

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  • Acid protease Candidapepsin as well as heterogeneous expression and purification method thereof
  • Acid protease Candidapepsin as well as heterogeneous expression and purification method thereof
  • Acid protease Candidapepsin as well as heterogeneous expression and purification method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Example 1 The heterologous expression of Candida tropicalis Candidapepsin protease

[0050] (1) Cultivation of Candida tropicalis

[0051] Candida tropicalis GIM2.147 (purchased from Guangdong Microbial Culture Collection Center) was inoculated into YPD solid medium (glucose 2g, peptone 2g, yeast extract 1g, agar powder 1g) and cultured at 30°C for 2 days.

[0052] (2) Candida tropicalis total RNA extraction

[0053] (1) Ingest about 100 mg of Candida with tweezers after high-pressure steam sterilization, put it into a mortar pre-cooled with liquid nitrogen, add a small amount of liquid nitrogen, grind quickly with the mortar, then add a small amount of liquid nitrogen, continue grinding, repeat 3 times, until all Candida completely turns into white powder.

[0054] (2) Add 2mL RNAiso Plus (purchased from Dalian Bao Biological Engineering Co., Ltd.) to the mortar, try to cover the powder as completely as possible, then let it stand at room temperature until the RNAiso...

Embodiment 2

[0111] Fermentation induction, purification and enzyme activity assay of embodiment 2 Candidapepsin

[0112] Select the positive recombinant Pichia pastoris strain obtained in Example 1 to streak the MD plate, cultivate it for 2 days at 30° C., pick a single colony and inoculate it in 50 mL of BMGY culture fluid (yeast extract 10 g, tryptone 20 g, YNB 13.4 g, glycerol 10mL, 1M potassium phosphate (pH 6.0) 100mL, distilled water to 1000mL) Erlenmeyer flask, 30°C, 250rpm shaking culture to OD600≈5.0. Then the cells were collected by centrifugation, and the cell pellet was transferred in equal amounts to 100 mL of BMMY culture medium (yeast extract 10 g, tryptone 20 g, YNB 13.4 g, methanol 10 mL, 1M potassium phosphate (pH 6.0) 100 mL, distilled water to volume 1000 mL) in a Erlenmeyer flask at 28° C., shake culture at 250 rpm, add 1.5% methanol solution every 24 hours to induce expression, and induce for 7 days. The fermentation broth was centrifuged at 5000rpm at 4°C for 10min...

Embodiment 3

[0115] The SDS-PAGE detection of embodiment 3 recombinant protein

[0116] SDS-PAGE gel electrophoresis was used to confirm the expression, purity and molecular weight of the recombinant protease. The concentration of the stacking gel used was 12% and the concentration of the separating gel was 5%, the sample volume was 20 μL, and a standard protein with a standard molecular weight was used as a marker. For the operation process of SDS-PAGE gel electrophoresis, please refer to "Protein Electrophoresis Experimental Technique". For the preparation of fermentation broth samples, the amount of recombinant protease produced by induced expression is relatively high. The fermentation broth can be directly diluted 1-fold and mixed with loading buffer, boiled in boiling water for 10 minutes, centrifuged at 12,000 rpm for 1 minute, and electrophoresis after loading.

[0117] The SDS-PAGE electrophoresis of the crude enzyme liquid (referring to the fermentation liquid that has not been ...

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Abstract

The invention provides acid protease Candidapepsin as well as a heterogeneous expression and purification method thereof. The acid protease Candidapepsin is sourced from candida tropicalis, an amino acid sequence of the acid protease Candidapepsin is as shown in SEQ ID NO.1, the enzyme activity of the acid protease Candidapepsin can be up to 268.2U / mL, and the specific enzyme activity can be up to2086.7U / mL and is far higher than the enzyme activity of like reported acid protease; the hydrolysis effect of the acid protease Candidapepsin is superior to that of commercial papain and commercialacid protease. The invention also provides the heterogeneous expression and purification method of the acid protease Candidapepsin. According to the heterogeneous expression and purification method provided by the invention, the yield of Candidapepsin protease is increased to a large extent, the yield can be up to 384.3mg / L, and a wide popularization and application prospect is obtained.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to acid protease Candidapepsin and a heterologous expression and purification method thereof, in particular to a heterologous expression and purification method of Candida tropicalis acid protease Candidapepsin. Background technique [0002] Candida is the causative agent of 8% to 10% of hospital-acquired bloodstream infections worldwide, with Candida albicans being the most common, but other species (eg, Candida tropicalis) are also increasing in frequency . Candida tropicalis, second only to Candida albicans, is one of the most common pathogens in the tropics and has also been found to be associated with tropical schistosomiasis, urinary tract infections, and hematologic malignancies. It can be transmitted between medical staff and patients. In tropical areas, Candida tropicalis can survive outside the body for up to 24 hours, so it is easy to cross-infect. The probability o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/60C12N15/57C12N15/81C12N1/19C12R1/84
CPCC12N9/60C12N15/815
Inventor 罗晓春邓俊劲李志伟史丹茅和花梁爽
Owner SOUTH CHINA UNIV OF TECH
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