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Updated: May 17, 2026
Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris
Published on: February 25, 2010
A thermolabile aspartic proteinase from Mucor mucedo DSM 809: gene identification, cloning, and functional expression
Sirma Yegin1, Marcelo Fernandez-Lahore
1Jacobs University Bremen, Bremen, Germany. s.yegin@jacobs-university.de
Abstract:
In this study, the cDNA encoding the aspartic proteinase of Mucor mucedo DSM 809 has been identified by RNA ligased-mediated and oligo-capping rapid amplification of cDNA ends (RACE) technique. The gene contained an open reading frame of 1,200 bp and encoded for a signal peptide of 21 amino acid residues. Two N-glycosylation sites were observed within the identified sequence. The proteinase gene was cloned into the vector pGAPZαA and expressed in Pichia pastoris X-33 for the first time. The protein has been secreted in functionally active form into the culture medium. The expression system does not require any acid activation process. The factors affecting the expression level were optimized in shaking flask cultures. Maximum enzyme production was observed with an initial medium pH of 3.5 at 20 °C and 220 rpm shaking speed utilizing 4 % glucose as a carbon and energy source. The enzyme was purified with cation exchange chromatography and further studies revealed that the enzyme was secreted in glycosylated form. The purified enzyme exhibited remarkable sensitivity to thermal treatment and became completely inactivated after incubation at 55 °C for 10 min. These results indicated that the recombinant proteinase could be considered as a potential rennet candidate for the cheese-making industry.
Insights
Researchers identified and expressed the Mucor mucedo aspartic proteinase gene in Pichia pastoris. This active, glycosylated enzyme shows potential as a rennet substitute for the cheese-making industry.
Area of Science:
- Biotechnology
- Enzyme Engineering
- Molecular Biology
Background:
- Aspartic proteinases are crucial enzymes in various industrial applications.
- Mucor mucedo DSM 809 is a fungal source of potential industrial enzymes.
- Efficient expression systems are needed for large-scale enzyme production.
Purpose of the Study:
- To identify and characterize the cDNA encoding the aspartic proteinase from Mucor mucedo DSM 809.
- To establish a recombinant expression system for this proteinase in Pichia pastoris.
- To evaluate its potential as a rennet substitute in the cheese-making industry.
Main Methods:
- cDNA identification using RNA ligase-mediated and oligo-capping rapid amplification of cDNA ends (RACE).
- Cloning the gene into pGAPZαA vector and expression in Pichia pastoris X-33.
- Optimization of expression conditions (pH, temperature, shaking speed, carbon source) and purification via cation exchange chromatography.
Main Results:
- The identified gene has a 1,200 bp open reading frame encoding a signal peptide and two N-glycosylation sites.
- Functional, active aspartic proteinase was secreted into the culture medium without acid activation.
- Optimal production conditions identified: pH 3.5, 20 °C, 220 rpm, 4% glucose.
- The purified enzyme is glycosylated and thermally sensitive, inactivated at 55 °C for 10 min.
Conclusions:
- The recombinant Mucor mucedo aspartic proteinase can be successfully expressed and secreted in Pichia pastoris.
- The enzyme's active secretion and glycosylated form are confirmed.
- Its characteristics suggest potential application as a rennet candidate in cheese production.
