Identification and Functional Characterization of Cysteine Protease from Nine Pear Cultivars (Pyrus pyrifolia)

Figures & data

FIGURE 1 Total protein contents in a: whole fruit; b: flesh; and c: peel of nine pear cultivars in mg/g dry weight. Value are means ± SD (n = 3). Different letters (a–h) of each indicate significant difference within column at p < 0.05 by DMR test. The cultivars used were Chuwhang (CW), Shinwha (SW), Whasan (WS), Gamcheon (GC), Manpung (MP), Mansu (MS), Wonwhang (WW), Nikita (NK), and Hanareum (HA).

FIGURE 2 SDS-PAGE; a: analysis; and b: native-PAGE zymography of nine pear cultivars. M: molecular weight marker. Arrow indicates the protease bands of pear cultivars with the expected molecular weights of 36 and 38 kDa. The cultivars used were Chuwhang (CW), Shinwha (SW), Whasan (WS), Gamcheon (GC), Manpung (MP), Mansu (MS), Wonwhang (WW), Nikita (NK), and Hanareum (HA).

TABLE 1 Esterase activity of nine pear cultivars

Pear cultivars	Whole fruit	Flesh	Peel
	Activity (U/mg protein)^1
Chuwhang	55.8 ± 2.7^c	58.3 ± 2.6^c	25.2 ± 1.2^d
Shinwha	58.8 ± 2.9^c	120.2 ± 20.9^b	40.2 ± 2.1^bc
Whasan	99.1 ± 4.1^b	150.5 ± 13.29^ab	43.6 ± 9.4^ab
Gamcheon	94.1 ± 17.5^b	125.8 ± 14.2^ab	50.6 ± 1.0^a
Manpung	58.0 ± 1.4^c	73.0 ± 3.0^c	39.1 ± 6.1^bc
Mansu	58.0 ± 1.8^c	64.9 ± 5.1^c	38.1 ± 1.6^bc
Wonwhang	60.5 ± 0.8^c	65.5 ± 3.6^c	33.4 ± 1.0^c
Nikita	126.6 ± 11.7^a	161.9 ± 24.9^ab	45.6 ± 1.9^ab
Hanarum	134.1 ± 3.5^a	172.4 ± 29.8^a	42.3 ± 1.1^b

¹Esterase activity was assayed using CBZ-Lys-p-nitrophenyl (CBZ Lys-ONp) ester as the substrate in 50 mM Na-phosphate buffer (pH 6.0). One unit of enzyme activity (U) was defined as the amount of enzyme causing an increase in the absorbance by one unit at 410 nm under the above conditions. Results are expressed as means ± SD (n = 3). Average values followed by different letter are significantly within column (p < 0.05) by DMR test.

Superscript letters (a–d) represent statistical significance among data.

TABLE 2 Caseinolytic activity of nine pear cultivars

Pear cultivars	Whole fruit	Flesh	Peel
	Activity (U/mg protein)^1
Chuwhang	12.1 ± 1.6^d	14.9 ± 5.5^f	10.8 ± 0.9^f
Shinwha	55.9 ± 3.0^b	70.4 ± 1.8^bcd	18.1± 4.7^bcd
Whasan	72.0 ± 2.7^a	80.0 ± 6.9^b	22.1 ± 2.8^b
Gamcheon	70.1 ± 5.6^a	75.5 ± 9.4^bc	27.3 ± 3.0^a
Manpung	41.3 ± 6.7^c	66.3 ± 5.1^cd	20.5 ± 0.3^ab
Mansu	36.0 ± 3.5^c	42.8 ± 5.8^e	14.1 ± 1.7^def
Wonwhang	61.2 ± 3.4^b	62.0 ± 7.1^d	13.1± 1.5^ef
Nikita	72.2 ± 5.6^a	80.9 ± 9.0^b	26.7 ± 1.7^a
Hanarum	74.8 ± 1.4^a	96.9 ± 5.5^a	16.3± 1.8^cde

¹Caseinolytic activity was assayed using succinlyated casein as substrate in 50 mM Na-borate buffer (pH 8.5). One unit of enzyme activity (U) was defined as the amount of enzyme causing an increase in the absorbance by one unit at 450 nm under the above conditions. Results are expressed as means ± SD (n = 3). Average values followed by different letter are significantly within column (p < 0.05) by DMR test.

Superscript letters (a–f) represent statistical significance among data.

TABLE 3 Collagenase activity of nine pear cultivars

Pear cultivars	Whole fruit	Flesh	Peel
	Activity (U/mg protein)^1
Chuwhang	1.67 ± 0.29^d	2.80 ± 0.36^d	0.87 ± 0.15^d
Shinwha	4.16 ± 0.76^bc	3.86 ± 0.40^abcd	1.16 ± 0.23^bcd
Whasan	4.92 ± 0.24^ab	4.63 ± 0.51^abc	1.48 ± 0.34^ab
Gamcheon	4.42 ± 0.33^bc	4.15 ± 0.39^abcd	1.75 ± 0.16^a
Manpung	3.81 ± 0.47^bc	3.08 ± 0.32^bcd	1.39 ± 0.19^abc
Mansu	3.16 ± 0.31^c	2.95 ± 0.35^cd	0.99 ± 0.17^cd
Wonwhang	4.19 ± 0.51^bc	3.02 ± 0.43^cd	0.99 ± 0.41^cd
Nikita	5.21 ± 0.57^ab	4.73 ± 0.48^bc	1.72 ± 0.21^a
Hanarum	6.06 ± 0.22^a	4.88 ± 0.35^a	1.05± 0.19^bcd

¹Collagenase activity was assayed using azo-collagen as substrate in 50 mM Tris-Cl buffer (pH 7.4). One unit of enzyme activity (U) was defined as the amount of enzyme causing an increase in the absorbance by one unit at 520 nm under the above conditions. Results are expressed as means ± SD (n = 3). Average values followed by different letter are significantly within column (p < 0.05) by DMR test.

Superscript letters (a–d) represent statistical significance among data.

FIGURE 3 Identification of pear protease by in-gel trypsinization and MALDI-TOF analysis. a: Coomassie-stained gel of Nikita pear. Arrow indicates the bands excised for in gel digestion. b: Peptide mapping spectrum. Numbered peaks are matched mass peaks with expected sequence of cysteine protease. c: Peptide mass peak list. d: Sequence coverage through the peptide finger printing analysis by MALDI-TOF.

TABLE 4 Cysteine protease contents of nine pear cultivars

Pear cultivars	Whole fruit	Flesh	Peel
	(mg/g DW)^1
Chuwhang	0.08 ± 0.01^e	0.68 ± 0.03^h	0.07 ± 0.01^e
Shinwha	0.21 ± 0.04^d	2.00 ± 0.11^e	0.24 ± 0.04^d
Whasan	1.65 ± 0.16^a	3.67 ± 0.14^c	0.51 ± 0.06^b
Gamcheon	0.88 ± 0.08^b	2.57 ± 0.03^d	0.83 ± 0.09^a
Manpung	0.50 ± 0.02^c	1.69 ± 0.06^f	0.40 ± 0.08^c
Mansu	0.12 ± 0.01^de	1.27 ± 0.10^g	0.14 ± 0.03^e
Wonwhang	0.42 ± 0.07^c	0.89 ± 0.08^h	0.12 ± 0.01^e
Nikita	1.70 ± 0.11^a	4.03 ± 0.22^b	0.58 ± 0.03^b
Hanarum	2.36 ± 0.14^a	4.76 ± 0.16^a	0.35 ± 0.03^c

¹Cysteine protease contents in total protein extracts were quantified by SDS-PAGE analysis and densitometry scanning of protein bands with molecular weights of 36 kDa and 38 kDa using the NIH program. Protease content in Whasan, Nikita, Hanareum, Shinwha, and Gamcheon cultivars was the sum of two bands, while the protease content of Manpung, Mansu, Wonwhang, and Chuwhang cultivars was from one band. Value are means ± SD (n = 3). Different letters (a–h) of each indicate significant difference within column at p < 0.05 by DMR test.

Superscript letters (a–f) represent statistical significance among data.