Proteolytic Enzymes in Green Asparagus, Kiwi Fruit and Miut

Agric. Biol. Chem., 46 (8), 1983-1986, 1982 1983 Proteolytic Enzymes in Green Asparagus, Kiwi Fruit and Miut: Occurrence and Partial Characterization Tsutomu Yamaguchi, Yukiko Yamashita, Imao Takeda* and Hisashi Kiso* Department of Food and Nutrition, Yamaguchi Women's University, Yamaguchi, Yamaguchi 753, Japan *Research and Development Laboratory, Shimaya Co., Ltd., Shinnanyo, Yamaguchi 746, Japan Received July 22, 1981 For the screening of potent proteases of plant origin, gelatinolytic activities were measured for various vegetables and fruits. Green asparagus, kiwi fruit and miut were found to possess high proteolytic activities. Optimumtemperatures for the activities of green asparagus and miut were 40 to 45°C and that of kiwi fruit was 60°C. OptimumpHs for the three activities were in neutral or slightly alkaline regions. The proteolytic enzymesof kiwi fruit and miut were stimulated by cysteine and EDTAbut that of green asparagus was unaffected by them. Proteases of plant origin, such as papain,1} Each sample was homogenized with a Waring blender at chymopapain,2) ficin,3) bromelain,4) as- 10,000 rpm for 5 min in an ice bath, filtered with guaze and centrifuged at 10,000xg for 20 min. The supernatant clepain,5) mexicain,6) euphorbain,7) solanin,8) fraction (juice sample) was retained and stocked at -20°C flicin9) and white gourd protease,10) have after adjusting its pH to 5.6 with 0.1 n NaOHor 0.1 n HC1. been reported by many workers. Recently, it Assay ofgelatinolytic activity. Gelatin (9.0 g) suspended presenthas also beenin ginger11}reported thatandsomeprincemelon.12)proteases are in 100ml of distilled water was kept at roomtemperature Papain, bromelain or asclepain have already for 30 min and then at 40°C for 1 hr. The resultant gelatin solution (5.0ml) was added to a preincubated mixture of been used in the fields of food and medicine, as 4.0ml of juice sample and 1.0 ml ofm/15 phosphate buffer, meat tenderizers, digestive and anti- pH 5.6, containing 20mM EDTA. After incubation at inflammatory agents and preventers of tur- 40°C for 15min, the reaction mixture was cooled in ice- water (ca.l : 1) for 10 min, then the prevention of gelati- bidity in beer etc. nation was compared with the control for which distilled Weattempted, here, to find newproteases water was used instead of a juice sample. in plants applicable in many field. A broad screening test employing a gelatin digestion Assay of proteolytic activity. Proteolytic activity was method has been done on various vegetables assayed by the method of Kunitz13) using casein as a and fruits. It has been thus found that green substrate. An enzyme solution made up to 1.0ml with 0.05m Tris-HCl buffer, pH 8.0, was added to 1.0ml of a asparagus, kiwi fruit and miut have high pro- solution of 1.0% (w/w) casein in the same buffer at 40°C. teolytic activities. Some properties of these Incubation was continued for 20 min and then the reaction activities were also examined in the present was stopped by the addition of 3.0ml of 5.0% trichlo- work. roacetic acid (TCA). After standing for 30 min at room temperature, the precipitate was removedby filtration through Toyo filter paper No. 5B and the absorbance at MATERIALS AND METHODS 280nmof the TCA-soluble peptides released was determined with a Shimadzu UV-100-02 spectrophotometer. Preparation of vegetable and fruit samples. Vegetable One unit of activity was defined as the amount which and fruit samples were obtained in the harvesting season. yielded a 0.001 A2S0nm change per minute under the 1984 T. Yamaguchi et al. conditions mentioned above. RESULTS AND DISCUSSION Materials. Casein was a product of E. Merck, Darmstadt, West Germany. Cysteine and EDTAwere Gelatinolytic activities of various juice samples purchased from Nakarai Chemicals, Ltd., Kyoto. Gelatin Table I shows the results on the gelatinolytic was purchased from WakoPure Chemicals Industries, activities of various vegetable and fruit juices Ltd., Osaka. All other chemicals were of reagent grade. measured at pH 5.6. Weconfirmed that all of the plants which had been reported to have high protease ac- Table I. Gelatinolytic Activities tivities, such as ginger, princemelon, banana, of Juice Samples of Various fig, papaya and pineapple, also showed Vegetables and Fruits (A) Vegetables high activities with this method. Further, we newly found that green asparagus, kiwi Sample Activity5 Sample Activity* fruit and miut also showed the same degrees Azuki bean0 - Miut + + of activities as the plants mentioned above. Broccoli - Mungbeansprouts + Mungbean sprout and sweet potato also Cabbage - Onion - were newly found to show moderate activities Carrot - Parsley - Cauliflower - Pimiento - by this method. Celery - Potato - Someproperties of the proteolytic activities of Cucumber - Princemelon + + Eggplant - Pumpkin - green asparagus, kiwi fruit and miut Elizabeth melon - Radish - The proteolytic activities of the juices from Garlic - Shiitake0 - green asparagus, kiwi fruit and miut were Ginger + + + Spinach - Green asparagus +++ Sweetpotato + Table II. Proteolytic Activities of Honewort - Taro - Juice Samples of Green Asparagus, Indian lotus - Tomato - Kiwi Fruit and Miut Lettuce - Water melon - Proteolytic (B) Fruits S ample activity0 (units/ml) Sample Activity^ Sample Activity1 Upper half 208 Apple Dark green Avocado Lower half 120 Banana0 Upper half 87 Fig Green Light green Grape, Delaware asparagus Lower half 55 Grapefruit Upper half 33 Japanese Light yellow persimmon Lower half 15 +++ LoquatMandarinMangoPapayaorange PeachPineapplePlum +++ +++ Kiwi fruit + Kiwi fruit Sarcocarp 1 50 Prepared by homogenization with an equal weight Inflorescence 75 of distilled water. Miut The degree of activity, measured as described in the Young rootstalk 50 text, was expressed as follows: + + +, gelatin was The sample juice (0.1ml) was added to 1.0ml of liquefied completely; + + , liquefied considerably; + , liquefied slightly; - , not liquefied. 0.05m Tris-HCl buffer, pH 8.0, containing 4.0mM The following vegetables and fruits were also studied cysteine and 4.0mMEDTA.The control was pre- but no activity was detected: Chinese cabbage, pared by incubating the sample juice in the buffer Chinese leek, corn, cow pea, edible burdock, green solution containing cysteine and EDTAwithout pea with pod, Japanese butterbur, kidney bean with casein. All the samples were incubated at 40°C for pod, okra, perilla leaf, red pepper, small turnip, 20 min. Other conditions were the same as described soybean, Welsh onion, yam, Japanese plum, lemon in the text. Activity was obtained by subtracting the and sour orange. value of each control. Proteolytic Enzymes in Asparagus, Kiwi Fruit and Miut 1985 green asparagus, kiwi fruit or miut was noted when the solutions were stored at - 15°C for 1 ingmonth.theseThermalenzymesstabilityin 0.05mwasTris-HCltested bybufferkeep- II).determined using casein as a substrate (Table (pH 7.5) at 20~90°C for 10 min. The activity III).of kiwiAlmostfruit nowaslossextremelyof activityheat-stableof these (Tablethree wereenzymeskeptwasat observed25°C for 20hr.at pHAs6~8,shownwhenin theyFig. All of them showed noticeable caseinolytic miut1, thewereproteolyticmarkedlyactivitiesstimulated ofbykiwicysteinefruit andat activities. Especially green asparagus has a high activity. The activity of green asparagus was affected by the growth-conditions. The dark green asparagus grown in the light showed the highest activity. The light-yellow asparagus grown in the absence of light showed the lowest activity. In all cases, the upper halves of asparagus showed approximately twice the activities of the lower halves. The proteolytic activity of kiwi fruit wasin the sarcocarp. The activity of miut was found in both the inflorescence and young rootstalk. The inflorescence and young rootstalk are used as flavor- ing vegetables in Japan. It is interesting that, although both ginger and miut belong to the Zingiber family, the proteolytic activity is present in the tuber of ginger and in the inflorescence or young rootstalk of miut. Weexamined some properties of these proteolytic activities to get information useful for the purification of the responsible enzymes. As 2 5 10 20 shownin Table III, the activities of green Cysteine Concn.(mM) asparagus, kiwi fruit and miut were highest Fig. 1. Effect of Cysteine on Proteolytic Enzymes of between pH 6.5 and 8.5. The optimal tempera- Green Asparagus, Kiwi Fruit and Miut. tures of green asparagus, kiwi fruit and miut The reaction mixture consisted of 0.1 ml of enzyme so- for the maximumrate of hydrolysis toward lution (juice sample dialysed against distilled water), 1.0 ml casein were in the ranges of 41°C, 60°C and of 1.0% casein in 0.05m Tris-HCl buffer, pH 8.0, and 1.0ml of the same buffer containing various concen- 43°C, respectively. It is a distinctive character- trations of cysteine. Other conditions were the sameas istic of kiwi fruit that its optimal temperature described in the text and Table II. O, green asparagus; A, is very high. No loss of proteolytic activity of kiwi fruit; #, miut. Table III. Properties of Proteolytic Enzymes of Green Asparagus, Kiwi Fruit and Miut The reaction mixture consisted of 0. 1 ml of enzyme solution (juice sample dialysed against distilled water), 1.0ml of buffer solution containing 4.0him cysteine and 4.0mM EDTAand 1.0ml of 1.0% casein. Other conditions were the same as described in the text and Table II. The buffer solutions used were: 0.2m acetate buffer, pH 2.5~4.5; 0.1 m phosphate buffer, pH 5.0~7.0; 0.05m Tris-HCl buffer, pH 7.5~ 10.0. Green asparagus Kiwi fruit Miut Optimum pH 7.8-8.5 7.3-7.6 6.5-7.3 Optimum temperature 38 -43°C 58 - 62°C 40 -45°C Thermal stability* 1 7% 98% 66% pH stabilityb 6-8 7- 10 5-8 Expressed as the remaining activity after 60°C-treatment for 10 min.

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