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A novel alkaline protease from wild edible mushroom Termitomyces albuminosus
Suyue Zheng1, Hexiang Wang, Guoqing Zhang
1Hebei Engineering University, Handan, China.
Abstract:
A protease with a molecular mass of 30 kDa and the N-terminal sequence of GLQTNAPWGLARSS, was isolated from fresh fruiting bodies of the wild edible mushroom Termitomyces albuminosus. The purification protocol included ion exchange chromatography on DEAE-cellulose, Q-Sepharose, SP-Sepharose and FPLC-gel filtration on Superdex 75. The protein was unadsorbed on DEAE-cellulose and Q-Sepharose, but adsorbed on SP-Sepharose. The optimal pH and temperature of the purified enzyme were 10.6 and 60 °C, respectively. The enzyme was stable in the presence of 2 % (v/v) Tween 80 and 4 M urea. More than 80 % of the enzyme activity was retained in 2 % (v/v) Triton X 100, 54 % in 10 mM EDTA and 31 % in 2 % (w/v) SDS. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), but not inhibited by dithiothreitol (DTT), pepstatin or lima bean trypsin inhibitor suggesting that it was a serine protease but not a trypsin-like one. The protease was inhibited by Hg(2+), Cu(2+), and Fe(3+) ions. The K(m) and V(max) values of the purified enzyme for casein were 8.26 mg ∙ ml(-1) and 0.668 mg ∙ ml(-1) ∙ min(-1), respectively.
Insights
A novel serine protease was isolated from the wild mushroom Termitomyces albuminosus. This enzyme exhibits optimal activity at pH 10.6 and 60°C, showing stability under various conditions.
Area of Science:
- Biochemistry
- Enzymology
- Mycology
Background:
- Wild edible mushrooms are a source of bioactive compounds.
- Proteases play crucial roles in various biological processes.
- Termitomyces albuminosus is a mushroom species with potential biotechnological applications.
Purpose of the Study:
- To isolate and characterize a novel protease from Termitomyces albuminosus.
- To determine the biochemical properties and potential applications of the purified protease.
Main Methods:
- Protease isolation using ion exchange chromatography (DEAE-cellulose, Q-Sepharose, SP-Sepharose) and FPLC-gel filtration (Superdex 75).
- Determination of optimal pH, temperature, and stability under various chemical conditions.
- Enzyme inhibition studies using PMSF, DTT, pepstatin, and lima bean trypsin inhibitor.
- Kinetic analysis (K(m) and V(max)) using casein as a substrate.
Main Results:
- A 30 kDa protease was purified with the N-terminal sequence GLQTNAPWGLARSS.
- Optimal activity was observed at pH 10.6 and 60 °C.
- The enzyme demonstrated stability in Tween 80 and urea, and retained significant activity in Triton X 100, EDTA, and SDS.
- Phenylmethylsulfonyl fluoride (PMSF) strongly inhibited the enzyme, indicating it is a serine protease, but it was not inhibited by DTT, pepstatin, or lima bean trypsin inhibitor.
- The protease was inhibited by heavy metal ions (Hg(2+), Cu(2+), Fe(3+)).
- Kinetic parameters for casein hydrolysis were determined (K(m) = 8.26 mg/ml, V(max) = 0.668 mg/ml/min).
Conclusions:
- A novel alkaline serine protease was successfully isolated and characterized from Termitomyces albuminosus.
- The enzyme's stability and kinetic properties suggest potential applications in industrial processes requiring high pH and temperature conditions.
- Further research could explore its specific substrate range and potential uses in biotechnology.
