Antifungal and Anticancer Activities of a Protein from the Mushroom Cordyceps militaris

Byung Tae Park; Kwang Heum Na; Eui Cha Jung; Jae Wan Park; Ha Hyung Kim

doi:10.4196/kjpp.2009.13.1.49

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Park, Na, Jung, Park, and Kim: Antifungal and Anticancer Activities of a Protein from the Mushroom Cordyceps militaris

Original Article

Korean J Physiol Pharmacol 2009;13(1):49-54.

Published online: 17 February 2009

DOI: https://doi.org/10.4196/kjpp.2009.13.1.49

Antifungal and Anticancer Activities of a Protein from the Mushroom Cordyceps militaris

Byung Tae Park, Kwang Heum Na, Eui Cha Jung, Jae Wan Park, Ha Hyung Kim

Physical Pharmacy Laboratory, College of Pharmacy, Chung-Ang University, Seoul 156-756, Korea

Corresponding to: Ha Hyung Kim, Physical Pharmacy Laboratory, College of Pharmacy, Chung-Ang University, 221, Huksuk-dong, Dongjak-gu, Seoul 156-756, Korea. (Tel) 82-2-820-5612, (Fax) 82-2-823-5612, (E-mail) hahyung@cau.ac.kr

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The mushroom Cordyceps militaris has been used for a long time in eastern Asia as a nutraceutical and in traditional Chinese medicine as a treatment for cancer patients. In the present study, a cytotoxic antifungal protease was purified from the dried fruiting bodies of C. militaris using anion-exchange chromatography on a DEAE-Sepharose column. Electrophoretic analyses indicated that this protein, designated C. militaris protein (CMP), has a molecular mass of 12 kDa and a pI of 5.1. The optimum conditions for protease activity were a temperature of 37°C and pH of 7.0~9.0. The enzyme activity was specifically inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride. Amino acid composition of intact CMP and amino acid sequences of three major peptides from a tryptic digest of CMP were determined. CMP exerted strong antifungal effect against the growth of the fungus Fusarium oxysporum, and exhibited cytotoxicity against human breast and bladder cancer cells. These results indicate that C. militaris represents a source of a novel protein that might be applied in diverse biological and medicinal applications.

Keywords: Mushroom, Cordyceps militaris, Protease, Antifungal activity, Cytotoxicity

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Fig. 1.

Elution profile of C. militaris protein extract applied to DEAE-Sepharose anion-exchange column chromatography, showing absorbance at 280 nm and NaCl concentration gradient.

Fig. 2.

(A) SDS-PAGE and (B) IEF gel of purified CMP. Lane 1, standard protein markers; lane 2, CMP without 2-mercaptoethanol; lane 3, CMP with 2-mercaptoethanol; lane 4, CMP stained with silver nitrate; lane 5, standard pI markers; lane 6, CMP.

Fig. 3.

(A) Comparison of the protease activities of CMP (•) and trypsin (▪) using casein as a substrate. Protease activity is expressed as the fluorescence change per minute. (B) Lineweaver-Burk plots of casein hydrolysis by CMP (•) and trypsin (▪).

Fig. 4.

Antifungal activity of CMP against the growth of (A) F. oxysporum and (B) B. cinerea. Discs contained either buffer alone (10 mM Tris-HCl with 0.1 M NaCl, pH 7.0) or following concentrations of CMP in this buffer: disc 1, 0 μM; disc 2, 200 μM; disc 3, 40 μM; disc 4, 8 μM; disc 5, 1.6 μM; and disc 6, 0.32 μM.

Table 1.

Summary of CMP purification from C. militaris

Fraction	Protein (mg/ml)	Volume (ml)	Protease activity^a	Specific activity^b	Relative purification	Yield (%)
Crude protein extract	12.6	7	471.1	37.4	1	100.0
CMP	0.2	15	130.4	652.0	17.4	3.4

^a Protease activity is expressed as the fluorescence change per minute.

^b Specific activity is expressed as the ratio of protease activity per protein (mg/ml).

Table 2.

Effects of pH, temperature, and inhibitors on the protease activity of CMP

	Condition	Residual protease activity (%)
pH	4.0	10.1
	5.0	35.5
	6.0	53.3
	7.0	100
	8.0	98.2
	9.0	98.7
	10.0	51.6
	11.0	41.7
Temperature (^oC)	4	37.8
	25	54.2
	37	100
	45	82.9
	55	37.5
	65	31.6
Inhibitor	None	100
	DMSO (1%)	87.0
	PMSF (1 mM)	33.6
	PMSF (0.125 mM)	46.1
	PMSF (0.001 mM)	83.1
	EDTA (10 mM)	101.5
	Pepstatin A (10 μg/ml)	104.3

Table 3.

Amino acid composition of CMP

Amino acid	Molar ratio (%)
Asp/Asn	9.63
Glu/Gln	11.01
Ser	6.22
Thr	9.43
Gly	10.74
Val	6.34
Leu	5.54
His	3.61
Arg	4.46
Met	1.97
Tyr	1.41
Trp	6.27
Phe	4.49
Cys	1.93
Total	100

Table 4.

Cancer cell viability after treatment with CMP

Dose (μM)	Cancer cell viability (%)
Dose (μM)	MCF-7	5637	A-549
15	33.41±3.81	39.06±15.60	64.68±13.23
5	57.16±16.19	48.64±17.71	77.77±10.47
1.7	107.94±19.00	67.27±20.49	114.14±22.1
0.6	98.8±14.53	74.73±22.24	109.56±8.4

Data are expressed as mean±SEM (n=3).

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