Agric. Biol. Chem., 49 (6), 1611-1619, 1985 1611

Substrate Specificity of the Milk-clotting 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 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 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. .2)

Calf chymosin is the ideal enzyme for cheese MATERIALS AND METHODS manufacture due to its high milk-clotting ac- tivity and its limited 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- 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. As shown in Fig. 5, a main peak, III, and a "1.0 minor one were obtained and the homogenei- ty of III was confirmed by PAGE and RP- HPLCwith TMS-250 column. Fraction D in Fig. 2 was chromatographed <.5 I -100 on RP-HPLC (Ultrosphere-octyl) and two main peaks, IV and V, were obtained as shown in Fig. 6. s' 50 % Similarly, fraction E in Fig. 2 was separated if 1 into many peaks by RP-HPLC(Ultrosphere- °0 10 20 30° ODS), giving two main peaks, VI and VII (Fig. Elution Volume (ml) 7). Each fraction indicated with a bracket was Fig. 4. Reverse Phase-High Performance Liquid collected, dried, and identified. Chromatography (RP-HPLC) of Fraction B Partially Purified by DEAE-cellulose after Gel Filtration. Identification of degradation products from asl - casein Partially purified fraction B was adsorbed on a column of TMS-250 (7.5 x75mm) and eluted at a flow rate of 1 ml/ As shown in Table I, the TV-terminal amino min by a linear gradient from 0.01 M trifluoroacetic acid to acids of I, II, and III were phenylalanine, 90% acetonitrile containing 0.01 m trifluoroacetic acid. and tyrosine, respectively, by dan- The column was operated at 28°C. sylation and their corresponding C-terminal amino acid sequences were -Leu-Trp, -Leu- Lys-Lys, and -Leu-Trp. The molecular weights

1.0 I 1

III ~ £ à"-å S

sCM % '+-§ ...5 å _ S

OJ C CD , -^ «

c 'å ^- _^ o s,l^^>A 1* o 50 10a Fraction Number Fig. 5. Ion Exchange Chromatography of Fraction C after Gel Filtration (Fig. 2) on DEAE-cellulose. Fraction C, indicated with a bracket in Fig. 2, was dialyzed against 0.005 mTris-HCl buffer containing 0.03 m NaCl and 6m urea, pH 8.2 and put on a DEAE-cellulose column (0.9 x 25 cm, Whatman DE-52) equilibrated with the same buffer. Degradation products were eluted by a linear NaCl gradient generated between 500 ml each of the initial buffer and of0.33 mNaCl in the buffer. The flow rate was 30ml/hr and 4.5 ml fractions were collected. #, absorbance at 280nm; -, NaCl concentration. Rennet from Irpex lacteus 1615 IV" were estimated to be 20,000, 12,000, and 1.5 ll,000 by SDS-PAGE for I, II, and III, re- spectively, and their amino acid compositions are shown in Table I, together with the com- positions of known asl-casein B segments.

1.0 1 1 VI

"no E c

|.5 V .100 *.5 . 100 | T ? = ?

J ^<

Table I. Identification of Peptides Formed from asl-Casein by Irpex lacteus Milk-clotting Enzyme

A m i n o ac i d p he(24 )-Trp ( 1 99) IT A rg ( l)-L y s(10 3 ) I ll T y r (10 4 )-T rp ( 19 9 )

A sp 1 4 1 3 7 8 7 7 T h r 5 5 1 1 4 4 S er 17 1 6 7 9 7 7 G lu 3 7 3 5 2 7 2 2 17 16 P ro 14 1 4 7 7 10 10 G ly 9 8 5 5 5 5 A la 9 9 3 3 6 6 C y s 0 0 0 0 0 0 V a l 10 6 7 5 4 M e t 4 5 2 2 3 3 Tie 10 5 6 4 5 L e u 14 13 9 1 0 8 7 T y r 10 2 2 8 P h e 6 7 4 4 4 4 H is 3 3 3 3 2 2 L y s 12 12 l l 1 0 4 4 A re 4 4 4 4 2 2 T rp 2 0 2 T o ta l resid u e s 17 4 17 6 10 3 1 0 3 9 4 9 6 M W 2 0 , 0 0 0 2 0 , 0 0 0 12, 00 0 1 2, 00 0 1 1,0 0 0 1 1,0 0 0 N -T erm in a l P h e P h e A r e A r e T y r T y r C -T e rm in a l -L e u -T rp -L e u -T rp -L y s-L y s -L y s-L y s -L eu -T rp -L eu -T rp

R ec o v er y (% ) 2 6 3 1 7

The recovery of fragments was determined by amino acid analysis and expressed as the percentage of total amounts of asl-casein used as the substrate. 1616 H. Kobayashi, I. Kusakabe and K. Murakami

Table II. Identification of Peptides Formed from asl-Casein by Irpex lacteus Milk-clotting Enzyme A rg (l)- G ln (9 )- Tyr(1 54)-Trp(199) Tyr(104)-phe(1 53) V I V II A m i n o a ci d H is(8 ) P h e(2 3 )

A sp 5 5 3 2 0 0 1 1 T h r 4 4 0 0 0 0 0 0 S e r 5 5 2 2 0 0 0 0 G lu 4 4 10 12 0 0 4 4 P ro 6 6 4 4 2 2 1 1 G ly 3 3 3 2 0 0 2 2 A la 3 3 3 3 0 0 0 0 C y s 0 0 0 0 0 0 0 0 V a l 1 1 3 3 0 0 1 1 M et 1 1 2 2 0 0 0 0 Tie 2 2 3 3 1 1 0 0 L eu 3 3 4 4 0 0 4 4 T y r 4 4 3 3 0 0 0 0 P h e 1 1 3 3 0 0 1 1 H is 0 0 2 2 2 2 0 0 L y s 1 1 3 3 2 2 0 0 A rg 0 0 2 2 1 1 1 1 T rp 2 0 0 0 T o tal re sid u es 4 3 4 5 5 0 5 0 8 8 15 15 N -T erm in a l T y r T y r T y r T y r A rg A rg G lu G lu C -T erm in al -L eu -T rp -L eu -T rp -G ln -P h e -G ln -P h e -L y s-H is -L y s-H is -A rg -P h e -A rg -P h e R e co v ery (% ) 6 4 10 8

The recovery of fragments was determined by amino acid analysis and expressed as the percentage of total amounts of asl-casein used as the substrate. Therefore, these fragments, I, II, and III, were degradation product corresponding to asl-I identified as Phe(24)-Trp(199), Arg(l)-Lys from asl-casein. The second group consists of (103), and Tyr(104)-Trp(199) of asl-casein, re- Endothia and Irpex enzymes, which produce spectively. several degradation products accompanied by As shown in Table II, the TV-terminal a product corresponding to asl-I. Therefore, amino acid of IV was tyrosine by dansyla- the first group is less proteolytic and shows tion, and the C-terminal sequence to be more restricted specificity on asl-casein than -Leu-Trp. Taking the amino acid composition the second under the conditions employed. into consideration, fragment IV was identi- Although the similarities of their specificity on fied as Tyr(154)-Trp(199) of asl-casein. In asl-casein by both enzymes in either group had the same manner, fragments V, VI, and VII been pointed out, the four enzymes differed were identified as Tyr(104)-Phe(153), Arg(l)- significantly from each other in their specificity His(8), and Gln(9)-Phe(23) of asl-casein, on oxidized insulin B-chain. This indicates that respectively. the specificity on protein substrates is affected not only by the amino acids adjacent to the DISCUSSION cleavage point but also by the conformation of the substrate surrounding the cleavage point. Milk-clotting enzymes may be classified into The specificity of Irpex enzymeon asl-casein two groups according to their degradation is summarized in Fig. 8 and is compared to pattern of asl-casein as shown in Fig. 1. Calf chymosin. Irpex enzyme and calf chymosin chymosin and Mucor enzyme belong to the have only one commoncleaving point, that is, first group, which produces almost entirely one at Phe(23)-Phe(24), and differ on subsequent Rennet from Irpex lacteus 1617

1 8 9 23 24 103104 H-Arg-Pro-Lys--Ile-Lys-His-Gl n-Gly-Leu--Leu-Arg-Phe-Phe-Val -Ala-Leu-Lys-Lys-Tyr-Lys-Val - _L lacteus ^ ^ A calf chymosin ^

149 150 153154 169 170 199 -Pro-Glu-Leu-Phe-Arg-Gln--Arg-Gl n-PheTyr-Gl n-Leu--Val -Pro-Leu-Gly-Thr-Gl n--Pro-Leu-Trp-OH _Llacteus ^ calf chymosin A A

Fig. 8. Comparison of Cleavage Sites of asl-Casein by Milk-clotting Enzymefrom Irpex lacteus and Calf Chymosin.

Table III. Comparison of Cleavage Sites of asl-Casein by Irpex lacteus Milk-clotting Enzymeand Calf Chymosin on the Basis of the Hydrophobicities of Side Chain of Amino Acid Residues Adjacent to the Bonds to be Split p * p 3 p 2 P i P i p 2 p 3. t

(A ) P ro Tie L y s H is G in G ly L e u *2.6 3.0 1. 5 0.5 - 0 .1 0 1. 8

L eu L eu A rg P h e P h e V a l A la * 1. 8 1.8 0.7 2.5 2. 5 1.5 0.5

A rg L eu L y s L y s T y r L y s V a l *0.7 1.8 1. 5 1. 5 I 2.3 1.5 1. 5 P h e A r g G in P h e T y r G in L e u 2.5 0.7 - 0. 1 2. 5 2.3 - 0. 1 1. 8

(B ) L eu L eu A rg P h e P h e V a l A la * 1.8 1. 8 0. 7 2.5 I 2.5 1. 5 0.5

T y r V a l P ro L e u G ly T h r G in * 2.3 1.5 2.6 1. 8 蝣 0 0.4 - 0.1

T y r P ro G lu L e u P h e A r g G in * 2.3 2.6 0.5 1. 8 2.5 0.7 - 0. 1

* Agt, hydrophobicities of side chain of amino acid residues (kcal/mol) cited from the refs. 13, 14. The arrow

indicatesBerger.15) the splitting point. The positions (P4-P3 ) on the substrate are designated according to Schechter and (A), Irpex milk-clotting enzyme; (B), calf chymosin. cleavage sites. It is well knownthat chymosin in the same manner as chymosin to form asl-I, produces asl-I, asl-II, and asl-III sequentially at however, the next cleavage point is the pH 6.0. At the beginning, chymosin cleaves the Lys(103)-Tyr(104) bond of asl-I to form III, Phe(23)-Phe(24) bond of asl-casein to form Tyr(104)-Trp(199). Ill is hydrolyzed at asl-I[Phe(24)-Trp(199)], and then it acts on the Phe(153)-Tyr(154) forming IV, Tyr(104)- Leu(169)-Gly(170) bond of asl-I to form asl- Phe(153), and V, Tyr(154)-Trp(199). Yields of II[Phe(24)-Leu(169)], and finally it hydrolyzes fragments I, II that is Arg(l)-Lys(103), and III the Leu(149)-Phe(150) bond of asl-II to pro- are estimated to be 26%, 3%, and 17%, re- duce asl-III[Phe(24)-Leu(149)]. Irpex enzyme spectively. This may indicate that a certain cleaves the Phe(23)-Phe(24) bond of asl-casein fragment [Phe(24)-Lys(103)], which is not 1618 H. Kobayashi, I. Kusakabe and K. Murakami identified, is formed during proteolysis or is with pepsin.18) This indicates that the P3 site of hydrolyzed further. The two fragments VI, the substrate is important to the secondary Arg(l)-His(8), and VII, Gln(9)-Phe(23) were interaction between the enzyme and the sub- produced from the hydrolysis of Arg(l)- strate. As shown in Table III, hydrophobic Phe(23) by Irpex enzyme. amino acids located in the P4 and/or P3 site(s) As shown in Table III, chymosin always might be important in the secondary interac- requires a hydrophobic amino acid such as tion between the enzymeand the substrate in phenylalanine or leucine in the P1 site as both cases of chymosin and Irpex enzyme. mentioned by Schechter and Berger.15) Irpex A previous report5) stated that Cheddar enzyme is specific for the peptide bonds cheese madewith Irpex enzymeand calf rennet formed by two amino acids with large hydro- did not show any difference in the degradation phobic side chains such as Phe(23)-Phe(24) pattern of asl-casein. This indicates that the and Phe(1 53)-Tyr(1 54). A noticeable exception specificity of Irpex enzyme on asl-casein in is the hydrolysis by Irpex enzyme of the His(8)- cheese is identical to that of chymosin. The Gln(9) bond. different specificity of Irpex enzyme exhibited It is established that microbial enzymes that on asl-casein in solution and in cheese depend activate trypsinogen, such as the acid pro- on the amount of enzyme used in solution, teinases of Aspergillus niger, A. saitoi, and which was 100 times higher than that in cheese Rhizopus chinensis hydrolyze Lys-X bonds manufacture, and on the high level of NaCl in preferentially.16* Although the Irpex enzyme cheese. The effect of NaCl on the specificity (data not shown), as well as the Mucor mie- against asl-casein of Irpex enzyme will be hei enzyme, M. pusillus enzyme, and pepsin, published in another paper. did not activate trypsinogen, the Irpex en- zyme hydrolyzed the Lys(103)-Tyr(104) Acknowledgment. The authors thank KyowaHakko bond to form III, Tyr(104)-Trp(199), with Co., Ltd. for the generous supply of Irpex lacteus rennet powder. high yield. This might be attributed to the large hydrophobic region formed by the REFERENCES neighboring six amino acids located from 1) H. Hobayashi, I. Kusakabe and K. Murakami, the P3 to P3> sites. In addition, Tang report- Agric. Biol. Chem., 47, 551 (1983). ed that even low frequencies of hydrolysis at 2) H. Kobayashi, I. Kusakabe and K. Murakami, the Lys-X bond by pepsin were observed in Agric. Biol. Chem., 47, 1921 (1983). protein substrates.17) Furthermore, amino 3) P. H. D. Foster and M. L. Green, J. Dairy Res., 41, acids at the Px site were classified into three 259 (1974). groups in the order of susceptibility* as fol- 4) H. Kobayashi, I. Kusakabe and K. Murakami, lows: Phe, Leu and Tyr belonged to the first Agric. Biol. Chem., 49, 1605 (1985). 5) O. M. Mulvihill andP. F. Fox, /. Dairy Res., 46, 641 group of highly susceptible ones; Glu, Asp, (1979). His and Lys, to the second group of suscep- 6) C. A. Zittle and J. H. Custer, J. Dairy $ci., 46, 1183 tible ones; Gly, He, Pro and Arg, to the third (1963). group of non-susceptible ones. Therefore, the 7) D. T. DaiviesandA. J. R. Law,/. DairyRes.,44, 213 susceptibility of Lys(103)-Tyr(104) and (1977). His(8)-Gln(9) bonds, and the non-suscepti- 8) H. Kobayashi and K. Murakami, Agric. Biol. Chem., 42, 2227 (1978). bility of Ile(6)-Lys(7), Pro(5)-Ile(6) and 9) H. Kobayashi, I. Kusakabe and K. Murakami, Anal. Pro(12)-Gln(13) bonds by Irpex enzyme Biochem., 122, 308 (1982). coincided with the specificity of pepsin. 10) R. T. Swank and K. D. Munkres, Anal. Biochem.^ 39, 462 (1971). Morihara et al. reported that the elongation ll) B. J. Davis, Ann. New York Acad. Set, 111, 404 of the peptide chain length from Px to P3 (1964). resulted in a markedincrease of hydrolysis 12) B. S. Hartley, Biochem. J., 119, 805 (1970). with acid proteinases of microbial origin and 13) C. Tan ford, J. Amer. Chem. Soc, 84, 4240 (1962). Rennet from Irpex lacteus 1619

Y. Nozaki and C. Tan ford, /. Biol. Chem., 246, 221 1 157, 561 (1973). (1971). 17) J. Tang, Nature, 199, 1094 (1963). I. Schechter and A. Berger, Biochem. Biophys. Res. 18) T. Oka and K. Morihara, Arch. Biochem. Biophys., Commun., 27, 157 (1967). 156, 552 (1973). K. Morihara and T. Oka, Arch. Biochem. Biophys.,