Agric. Biol. Chem., 49 (6), 1611-1619, 1985 1611 Substrate Specificity of the Milk-clotting Enzyme from Irpex lactens on asl-Casein Hideyuki Kobayashi, Isao Kusakabe and Kazuo Murakami Institute of Applied Biochemistry, The University of Tsukuba, Ibaraki 305, Japan Received November 5, 1984 Milk-clotting enzymes may be classified into two groups according to their degradation pattern of asl-casein in solution at pH 6.0. On the one hand, calf chymosin and Mucor miehei enzyme produced only one degradation product corresponding to asl-I under the conditions we employed. Onthe other hand, Irpex lacteus and Endothia parasitica enzymes produced several degradation products accompanied by a product corresponding to asl-I. The Irpex milk-clotting enzyme hydrolyzed asl-casein at the positions of His(8)-Gln(9), Phe(23)-Phe(24), Lys(103)-Tyr(104), and Phe(153)-Tyr(154). Irpex enzyme has only one commoncleaving site with calfchymosin, that is, the Phe(23)-Phe(24) bond of asl-casein. Two milk-clotting enzymes excreted by a the proteolytic specificity of the two coagu- basidiomycete, Irpex lacteus, have been pu- lants on caseins and their degradation prod- rified and characterized.1* The major enzyme ucts. It has been shown that there are six has a maximumproteolytic activity on hemo- chymosin-susceptible bonds in asl-casein and globin at pH3.0 and exhibits almost the same it is hydrolyzed at pH>5.8 by chymosin to ratio of milk-clotting activity to proteolytic form, in this order, asl-I, asl-II, and asl-III.5) activity as the commercial rennet substitutes The proteolytic specificity of microbial milk- from Mucor pusillus and Mucor miehei. The clotting enzymes on asl-casein has not yet been specificity of Irpex enzyme on the oxidized established. insulin B-chain is distinct from other com- This paper deals with the proteolytic speci- mercial microbial milk-clotting enzymes and is ficity of the milk-clotting enzyme from Irpex more restricted than chymosin and porcine lacteus on asl-casein in solution at pH 6.0. pepsin.2) Calf chymosin is the ideal enzyme for cheese MATERIALS AND METHODS manufacture due to its high milk-clotting ac- tivity and its limited proteolysis of caseins. asl- Dansyl chloride was purchased from Pierce, and car- Casein is reported to be extensively hydrolyzed boxypeptidase A-DFP and carboxypeptidase B-DFP by calf chymosin during cheese ripening, while from Sigma. Molecular weight markers (ranging from /?-casein remains almost unchanged.3* 2,500 - 17,000) for SDS-PAGE were obtained from BDH Biochemicals. Pepstatin was secured from the Protein A cheese-making trial was carried out using Research Foundation of Japan. Reagent grade chemicals Irpex milk-clotting enzyme fraction obtained were used. by affinity chromatography.4) Altough there was no significant difference in electrophoretic asl-casein. Crude asl-casein was prepared from acid patterns between the cheese made with the precipitated whole casein by the method of Zittle and Irpex enzymefraction and calf rennet, there Custer.6) It was purified by ion-exchange chromatography on DEAE-cellulose column according to the method of was a significant difference in the composition Davies and Law.7) of free amino acids produced during ripening. These facts are attributed to the differences in Enzymes. Rennets from calf, Mucor miehei, and 1612 H. Kobayashi, I. Kusakabe and K. Murakami Endothia parasitica were purchased from Chr. Hansen gradient from 0.06 m to 0.3 m. Furthermore, all fragments Lab., Miles Lab., and the Pfizer Co., respectively. These from the fractions were purified with RP-HPLC[Altex, enzymes were purified by affinity chromatography.8'9) The Ultrosphere-ODS, Ultrosphere-octyl (4.6 x 250mm); Irpex lacteus milk-clotting enzyme B was also purified Toyo Soda, TMS-250 (7.5 x 75mm); Beckman model 340] as described previously.1} and identified. Milk-clotting activity. Milk-clotting activity was de- Amino acid analysis. Each of the asl-casein fragments termined according to the method described previously8) was hydrolyzed in vacuo in 6n HC1 at 110°C for 24hr. The and expressed as Soxhlet units (s.u.) per ml of enzyme amino acid compositions of the hydrolyzates were de- solution. termined on a Durrum amino acid analyzer, model D-5. Action of milk-clotting enzymes on asl-casein. A solution C-terminal amino acid analysis. asl-Casein fragments of asl-casein (1.5ml, 0.1%, w/v) in 0.02m phosphate dissolved in 0.2 mN-ethylmorpholine acetate buffer, pH buffer, pH 6.0 was mixed with 0.03ml ofenzyme solution 8.5, were incubated with DFP-treated carboxypeptidases (500 s.u./ml) and incubated at 35°C. Samples (0.2ml) A and B for 0, 0.5, 1, and 2hr. The released amino acids were taken at various times, and the reaction was stop- were determined by the amino acid analyzer. ped by mixing the samples with 0.2ml of 9m urea con- taining 5x 10~5m pepstatin. Then the samples were N-terminal amino acid analysis. The N-terminal amino analyzed by PAGE. acid of each fragment was isolated by the method of Hartley.12* Gel electrophoresis. SDS-PAGE was performed with RESULTS 12.5% acrylamide in the presence of 8m urea and 0.1% SDS by the method of Swank and Munkres.10) PAGEwas Degradation of asl-casein by some microbial carried out with 7.5% gel at pH 8.9 in the presence of4.5 m milk-clotting enzymes and calf chymosin urea by the method of Davis.11] The protein was stained As shown in Fig. 1, although the Mucor with 0.05% Coomassie brilliant blue R-250 in acetic acid- methanol-water (1 : 1 : 5) mixture. enzyme had slightly higher proteolytic activity than calf chymosin, their proteolytic patterns Isolation of ot,sl-casein degradation products. One hun- on asl-casein showed no significant difference dred milliliters of a 0.1% asl-casein solution in 0.02m between the two. The proteolytic pattern of phosphate buffer, pH 6.0, was incubated with the Irpex Irpex enzyme resembled that of Endothia en- enzyme at 35°C (enzyme-substrate= 1 : 300mol/mol). zyme but both enzymes' patterns were dif- After 30 min of incubation, the reaction was terminated by heating in a boiling water bath for 5min. The sample was ferent from those of calf chymosin and Mucor dried in a rotary evaporator, dissolved in 10ml of 0.01 m enzyme. Tris-HCl buffer containing 0.06m NaCl and 6m urea, pH 8.6, and put on a Sephadex G-100 column equilibrated with the same buffer. Each of the separated fractions was Isolation of degradation products by Irpex milk- chromatographed on a DEAE-cellulose column equili- clotting enzymefrom ocsl -casein brated with the same buffer, then eluted by a linear NaCl As shown in Fig. 2, five fractions designated (A) (B) (C) (D) iBiii*ia* ( I*l**~ 'I*l I**l f. ' [ j ! ifsfib å ;*å å å ddl^5**à"«å *n . t f 1T 4*--*99: ~ . ~ ^)%4*I*|m, isiIiU^I*) L^?A~t~l* -" ~l5l?fe\ .5 1 2 4 6 .5 1 2 4 6 .5 1 2 4 6 .5 1 2 4 6 Time (hr) Fig. 1. Disc Gel Electrophoretic pattern of asl-Casein Incubated with Calf Chymosin (A), Irpex lacteus Milk-clotting Enzyme (B), Mucor miehei Milk-clotting Enzyme (C) and Endothia parasitica Milk-clotting Enzyme (D). Rennet from Irpex lacteus 1613 II A B C D E 1 I rl-lr '.I IIAa"1 0 50 100 150 Fraction Number Fig. 2. Gel Filtration of the Reaction Mixture on a Sephadex G-100 Column (4x 100cm) in 0.01 m Tris- HC1 Buffer Containing 0.06m NaCl and 6m Urea, pH 8.6. The flow rate was 40ml/hr and ll ml fractions were collected. #, absorbance at 280nm. 1.0| i "-n-----_______5:s- S f ! *u * \ å A wI,,i-'^n, ^ \m~~S >^> 0 50 100 150 Fraction Number Fig. 3. Ion Exchange Chromatography of Fraction A after Gel Filtration (Fig. 2) on DEAE-cellulose. Fraction A from the gel filtration on Sephadex G-100, indicated with a bracket in Fig. 2, was put on a DEAE- cellulose column (0.9 x 20cm, Whatman DE-52) equilibrated with 0.01 MTris-HCl buffer containing 0.06 m NaCl, 6m urea, pH 8.6. Degradation products were eluted by a pH gradient decreased from pH 8.6 to 4 generated between 400ml each of the initial buffer and of 0.3 m acetic acid containing 6 m urea. The flow rate was 30ml/hr and 4.5ml fractions were collected. #, absorbance at 280nm; -, pH. as A, B, C, D, and E in the order ofelution judged homogeneous by PAGE(Fig. 3) and by were obtained by Sephadex G-100 column reverse phase high performance liquid chro- chromatography. Judging from the PAGE matography (RP-HPLC) with a TMS-250 (Fig. 2), fraction A had two components which column. were eluted from a DEAE-cellulose column Fraction B in Fig. 2 was separated into two with a pH gradient from 8.6 to 4, as shown in fractions using ion-exchange chromatography Fig. 3. The first fraction was found to be on a DEAE-cellulose column with a linear unchanged asl-casein itself by PAGE and NaCl gradient. A main peak appeared homo- amino acid analysis. The second peak, I, was geneous in PAGEbut wasseparated into two 1614 H. Kobayashi, I. Kusakabe and K. Murakami peaks by RP-HPLC (TMS-250) as shown in Fig. 4. The main peak, II, indicated with a 1.5 bracket, was dried and identified. Fraction C in Fig 2 was also separated into two fractions by DEAE-cellulose column E chromatography with NaCl gradient elution.