Papain, chymotrypsin and related

proteins- a comparative study of their

beer chill-proofing abilities
and characteristics
John F. Kennedy and Victor W. Pike

Research Laboratory f o r Bioactive Carbohydrates and Proteins, Department o f Chemistry,

University o f Birmingham, Birmingham B15 2TT, UK

(Received 2 June 1980)

The role o f pro teolysis in the beer chill-proofing action o f the proteolytic e n z y m e papain {EC 3.4.22.2)
has been investigated by comparing the chill-proofing ability o f papain with that o f a proteolytieally
inactive derivative, S-earboxymethylpapain. The latter was prepared by treating papain with bromo-
acetic acid to carboxymethylate selectively the single essential sulphydryl group o f the enzyme, that o f
L-eysteine-25. Both papain and S-carboxymethylpapain were found to exhibit increasing chill-proofing
ability with increasing concentration in beer; at a protein concentration in beer o f 30 I.tg/ml the chill-
proofing effect o f each protein proved to be substantial. Papain, either in the presence or absence o f
sodium bisulphite, was, however, f o u n d to be more effective than S-earboxymethylpapain at all
protein concentrations. It is concluded that the chill-proofing action o f papain originates largely, but
not wholly, from its proteolytie action. Similarly, the chill-proofing ability o f the proteolytie enzyme
ehymotrypsin (EC 3.4.21.1) has been compared with that o f i t s proteolytieally inactive zymogen,
chymotrypsinogen A. Both proteins were f o u n d to exhibit increasing chili-proofing ability with increas-
ing concentration in beer. The chill-proofing effect o f ehymotrypsin was, however, f o u n d to be greater
than that o f ehymotrypsinogen A at all protein concentrations in beer. On the basis o f these results and
the close similarities in the molecular strueUgres o f chymotrypsin and chymotrypsinogen A, it is con-
cluded that the chill-proofing action o f chymotrypsin also originates largely, but not wholly, in its
proteolytic action. The results from this study collectively demonstrate that no straightforward corre-
lation exists between the proteolytic activity added to beer and its resistance to chill-haze formation.

Introduction would have the serious consequence of negating all attempts

to use water-insoluble papains as catalytic chill-proofing
The use of native papain (EC 3.4.22.2) for the prevention of agents. The results of this investigation are reported here.
chill-haze formation in beer, the process of beer chill-
proofing, is well established; Wallerstein 1 first described
such an application of papain in 1912. The ability of papain
to chill-proof beer has since been assumed by most to be Experimental and results
associated with the proteolytic action of the enzyme, and
to no other factor. Unequivocal evidence supporting this Materials
assumption has not, however, been reported hitherto,
probably because brewers have been satisfied with the Beer was supplied for this study by the Sub-department
efficacy of papain as a chill-proofing agent and have con- of Malting and Brewing at the University of Birmingham,
sidered the exact elucidation of its chill-proofing mechanism UK. The beer (specific gravity, 1.080; pH 4.36) had been
unnecessary. produced by two yeasts [Saccharomyces bayanus (NCYC
Our investigations 2-4 of several proteolytically-active 374) and Saccharomyces cerevisiae (NCYC 738)] acting
water-insoluble papains s- 7 as beer chill-proofing agents on wort containing a mixture of o-glucose syrup and malt
have revealed that they do not chill-proof enzymically but (3:7) in continuous fermentation at 20C with a residence
rather that both the water-insoluble enzymes and their time of 48 h. On collection, each beer batch was centrifuged
parent supports chill-proof efficiently by adsorbing beer for 30 rain at 6000 rev/min and 4C, decanted off and then
constituents. In a search for possible explanations of these stored at 5-10C. Chill-proofing experiments were per-
observations, we were prompted to examine more closely formed within three days of the onset of storage.
the chill-proofing mechanism of native papain. Our aim Papain (EC 3.4.22.2; batch 56225) and N-a-benzoyl-L-
was to assess the role of proteolysis, since an absence of arginine ethyl ester hydrochloride (BAEE) were purchased
proteolysis in the chill-proofing action of native papain from Koch-Light Laboratories Ltd.

0141 --0229/81/010059--05 $02.00

1981 IPC Business Press Enzyme Microb. Technol., 1981, Vol. 3, January 59
Papers
Chymotrypsin (EC 3.4.21.1,3 x crystallized) was automated titrimetric method 12 with BAEE as substrate as
obtained from the Sigma Chemical Co. Ltd. Chymotryp- reported in detail previously.6 One unit of esterolytic
sinogen A (6 x crystallized) and Hammarsten casein were activity (E) is defined as the activity that hydrolyses
obtained from BDH Ltd. Visking dialysis tubing (19 mm 1 gmol BAEE/min at 25C.
diameter, inflated) was obtained from the Scientific Instru-
ment Co. Ltd, London. Preparation o f protein solutions
Papain and S-earboxymethylpapain. Crude papain
Nephelometry for the measurement (130.9 rag, batch 56225) was suspended in activating
o f beer haze buffer (25 ml) [sodium phosphate buffer (0.1M ; pH 8.0)
Nephelometry was performed as described previously 2 containing L-cysteine hydrochloride (10 -3 M) and H4
using EEL equipment (Unigalvo Type 20 galvanometer, EDTA (4 x 10 -4 M)], gently stirred and then centrifuged.
nephelometer head and 'B' filter) that had been calibrated Two portions (10 ml each) were taken from the clear
in European Brewing Convention (EBC) formazin units, s supernatant solution. To one portion was added activating
All beer samples were well shaken immediately before buffer (1.0 ml) and to the other was added a solution of
measurement. bromoacetic acid in activating buffer (1 0 ml; 1.0 mg/ml).
The absorbance at 280 nm (pathlength, 1.0 cm) of each
Test o f the susceptibility o f beer to mixed solution was measured against a reference of activ-
chill-haze formation ating buffer (Table 1). Each solution was incubated at
25C for 24 h and then separately dialysed against activ-
The susceptibility of a beer sample to chill-haze formation ating buffer for 48 h at 4C. The absorbance at 280 nm of
was tested by incubating beer for 7 days at 60C and then each solution was then remeasured against a reference of
for 72 h at 4C, as described previously. 2 A beer that activating buffer as a check against loss of protein during
produces a haze increase of less than 4 EBC formazin units dialysis (Table 1). Quantitative amino acid analyses of
during such incubation is considered to be chill-proofed for similarly prepared solutions of papain revealed that the
the most demanding of storage conditions. 9 Values of beer absorbance at 280 nm of a papain solution is linearly
haze reported are the mean of five determinations. Error related to protein concentration by the equivalence 1.02
bars on Figures extend over the range of mean + standard absorbance unit ~ 1.0 mg protein/ml. This equivalence was
error of the mean. used to calculate the final protein concentrations of both
solutions prepared (Table 1). Samples from each protein
solution were assayed and their specific esterolytic and
Assay o f proteolytic activity proteolytic activities calculated (Table 1).
The proteolytic activities of papain and S-carboxymethyl-
papain were determined at 37C and pH 6.5 with Hammar- Chymotrypsin and chymotrypsinogen A Chymotrypsin
sten casein (1%, w/v) as substrate. 1 The detailed method 6 (25.3 rag) and chymotrypsinogen A (27.4 rag) were dissolved
has been reported previously and uses Folin and Ciocalteu's in separate portions (25 ml each) of distilled water (Table 1).
phenol reagent n to detect spectrophotometrically, at The absorbance at 280 nm of each solution was measured as
660 nm, the acid-soluble products released during the a qualitative check of protein concentration (Table 1). Each
enzyme-catalysed reaction. One unit of proteolytic solution was then assayed and its specific proteolytic activity
activity (F) is defined as the activity that gives an initial calculated (Table 1).
~ l c m _
increase m a 6o0nm 1 per rain under the conditions of
-

assay
The proteolytic activities of chymotrypsin and
Determination o f the effects o f added
chymotrypsinogen A were determined by the same pro-
proteins and their associated proteolytic
cedure, except that H 4 EDTA and L-cysteine hydrochloride
activities on the chill-haze development
were omitted from the assay medium.
o f beer
Portions from each prepared protein solution were diluted
Assay for esterolytic activity to give a series of protein solutions, each of known concen-
tration. Five separate portions (0.5 ml) were taken from each
The esterolytic activities of papain and S-carboxy- dilute protein solution prepared and beer (7.5 ml) was
methylpapain at 25C and pH 6,5 were determined by an added to each. The resultant solutions of protein in beer
automated titrimetric method 12 with BAEE as substrate as were separately sealed under nitrogen, kept at 4C for
Table 1 Absorbances, protein concentrations, specific esterolytic activities and specific proteolytic activities o f protein solutions studied as
chill-proofing agents

Prepared solution Absorbance at Protein Specific Specific

280 nm a concentration esterolytic proteolytic
(mg/ml) activity, E activity, F
(units/rag) (units/mg)

Papainb 1.52 (1.49) 0.75 1.96 4.60x 10- :

S-Carboxymethylpapain b 1.51 (1.49) 0.75 <0.02 6.15x 10 -4
Chymotrypsin c 1.70 1.01 n.d. 1.38x 10 J
Chymotrypsinogen A c 1.80 1.10 n.d. 8.89x 10 ~

a Values in parentheses are values before dialysis n .d., not determined

bprepared in activating b u f f e r
c Prepared in distilled water

60 EnzymeMicrob. Technol., 1981,Vol. 3, January

 Chill-proofing abilities of papain and chymotrypsin: John F. Kennedy and Victor W. Pike

seven days, and then tested for susceptibility to chill- 15

haze formation. The average increase in haze during the
test was then calculated for each set of five beer samples. g
Relationships between protein concentration and chill- c

haze formation are shown for papain and S-carboxy-

methylpapain in Figure I and for chymotrypsin and 9
chymotrypsinogen A in Figure 2. The relationship between rn

added proteolytic activity and chill-haze formation is shown

for each protein in Figure 3. =o

O
Determination o f the effects o f sodium 5

bisulphite on the chill-proofing abilities

o f papain and S-carboxymethylpapain 5 I I I J~
0 10 30 50 70
The above experiments with papain and S-carboxy- Concentretion of 0dded protein (/~g/ml)
methylpapain were repeated with the modification that
Figure 2 Effects of chymotrypsin (o) and chymotrypsinogen A (o)
sodium bisulphite was predissolved in the beer to a concentration on the haze developed by beer
concentration of 50 mg/1. The control experiment, using
beer containing no sodium bisulphite, was performed
simultaneously. The results are shown for papain and treatment of papain with bromoacetic acid irreversibly
S-carboxymethylpapain in Figures 4 and 5, respectively. inactivates the enzyme by selective carboxymethylation
of the L-cysteine-25 sulphydryl group is known.l 3,16
Discussion Furthermore, the enzyme, on exposure to bromoacetic
acid, is reported 16 to incur little alteration to its overall
In order to assess the role of proteolysis in the chill-proofing secondary and tertiary structures. Bromoacetic acid was
mechanism of native papain (EC 3.4.22.2) we set out to therefore the reagent selected to prepare catalytically
compare the chill-proofing ability of proteolytically active inactive S-carboxymethylpapain from native papain. The
papain with that of a structurally similar irreversibly- procedure devised for this preparation essentially involved
inactivated derivative of papain. For the comparison to be treating a portion of a stock solution of papain with a
meaningful it was essential that the method to be selected solution of bromoacetic acid in pH 8.0 buffer at 4C and
for the irreversible inactivation of papain caused as little as subsequently dialysing the protein solution to remove
possible alteration to other properties of the enzyme, such unreacted bromoacetic acid. Another equal portion of the
as, for example, its secondary and tertiary structures and stock papain solution was treated in a parallel procedure
overall charge. Many studies ~3-1s have shown that the that omitted bromoacetic acid. Thus two solutions, one of
single sulphydryl group of papain, that of L-cysteine-25, is papain and one of S-carboxymethylpapain, were obtained
essential to the catalytic activity of the enzyme. That the and these were virtually identical in all respects except
catalytic activity (Table 1).
Portions from each prepared solution were diluted to
12
give a series of protein solutions, each of known protein
concentration. The effect of each solution on the chill-haze
development of beer was then assessed identically using the
afore-mentioned standardized procedures. The results reveal
that the formation of chill-haze in beer decreases with
concentration of added protein for both papain and S-
~o carboxymethylpapain (Figure 1).
"~ 10 It has been suggested that added sulphur dioxide is able
.E
to maintain the proteolytic activity of papain in beerJ 7

i
E Other work indicates that beer alone protects papain from
(.9
loss of catalytic activityJ 8 The dissolution of sodium bisul-
ri
phite in beer (50 mg/1) before treatment of the beer with
Ld
v papain produced no significant difference in chill-proofing
az:
effect (Figure 4), indicating that inactivation of papain by
g oxidation of the essential thiol group had been insignificant
in the absence of bisulphite. S-Carboxymethylpapain was
similarly unaffected by bisulphite (Figure 5).
At all concentrations of added protein in beer, papain
was found to be significantly more effective as a chill-
proofing agent than was S-carboxymethylpapain (Figure 1).
From this result it is concluded that the proteolytic action
of papain plays a substantial role in the beer chill-proofing
I_ I action of the enzyme. This conclusion bolsters the view,
10 50 50 which is generally held, that the achievement of proteolysis
Concentrotion of odded prolein in beer (/~g/ml) in beer promotes chill-proofing by reducing the eventual
Figure 1 Effects of papain (o) and S-carboxyrnethylpapain concen- formation of insoluble protein-polyphenol complexes, the
tration (o) on the haze developed by beer major components of chill-haze.~9

E n z y m e M i c r o b . T e c h n o l . , 1 9 8 1 , V o l . 3, J a n u a r y 61
Papers

T
50

g
25
E

J~
l
g

I I
o

-25 I [ 1 [_
i x 10-5 i x 10-4 i 10-3 Lxl0-2

Concentration of added proteolytic activity in beer ( Funits/mL )

Figure 3 Relationships between the concentration of added proteolytic activity in beer and percentage reduction in chill-haze formation.
Proteolytic activity f r o m : a, papain; o, S-carboxvmethylpapain; =, chvmotrypsin; e, chymotrypsinogen A

16 16

G
g
m
_c
g \\x x
E
E 5
O
5 [13
~O W.~
123 \Xx
t.tl

o 12
r~ ~ ~

L
0 20 40
I0 I I S - Corboxymethylpapain concentration ( / x g / m l )
20 40
Figure 5 Effect of S-carboxymethylpapain concentration on the
Papain concentration (/~g/ml)
haze developed by beer in the presence (~) and absence of (o) sodium
Figure 4 Effect of papain concentration on the haze developed by bisulphite
beer in the presence (e) and absence of (o) of sodium bisulphite

That the prepared S-carboxymethylpapain was found to ible for the chill-proofing ability of the enzyme. A non-
exhibit appreciable chill-proofing ability, despite having proteolytic contribution to the overall chill-proofing ability
negligible specific pmteolytic activity in comparison to the of papain seems to exist also.
specific proteolytic activity of papain (Table 1), is evidence In the light of the results obtained with papain and
that the proteolytic action of papain is not wholly respons- S-carboxymethylpapain, we were further interested to

62 E n z y m e M i c r o b . T e c h n o l . , 1 9 8 1 , V o l . 3, J a n u a r y
 Chill-proofing abilities of papain and chymotrypsin: John F. Kennedy and Victor W. Pike

examine the chill-proofing ability of another pair of proteins, An alternative, but highly improbable, interpretation of
chymotrypsin, which is a proteolytic enzyme (EC 3.4.21.1 ), the results presented throughout this study is that the catalytic
and its zymogen, chymotrypsinogen A, which is proteolytic- actions of the preparations of S-carboxymethylpapain and
ally inactive, s ,21 [The commercial supply of the zymogen chymotrypsinogen A differ substantially from those of
used in this study had, however, a small residual specific papain and chymotrypsin, respectively, and are especially
proteolytic activity (Table 1)]. Trypsin (EC 3.4.21.4) is efficient at effecting beer chill-proofing. It is assumed that
required to initiate the conversion of chymotrypsinogen A any differences in the substrate specificities of the various
into chymotrypsin. 22 The conversion has the result of proteins do not critically alter the conclusions of this
cleaving two dipeptides from the chymotrypsinogen A study.
molecule. Thus there is a close similarity in the primary The results of this study imply that proteolysis plays a
structures of these two proteins. X-ray crystallography 23-2s substantial role in the chill-proofing actions of both papain
has also revealed a considerable similarity in their secondary and chymotrypsin. Attempts to use water-insoluble
and tertiary structures. For example, both proteins possess derivatives of papain or of other proteolytic enzymes for
the charge-relay system 26,27 responsible for catalytic activity the chill-proofing of beer therefore remain feasible in
in chymotrypsin. principle.
A stock solution of each protein was prepared (Table 1)
and tested for chill-proofing ability by the procedure
previously described for stock solutions of papain and
S-carboxymethylpapain. The results show that the formation Acknowledgements
of chill-haze in beer decreases with increasing concentration The authors thank the Science Research Council and
of added protein for both chymotrypsin and chymotryp- Koch-Light Laboratories Ltd for a CAPS research student-
sinogen A. Also, it was found that at any particular con- ship to VWP. The authors also thank Professor J. S. Hough
centration of added protein the proteolytic enzyme chymo- of the Sub-department of Malting and Brewing at The
trypsin acts as a more effective chill-proofing agent than University of Birmingham for generously providing the beer
does the zymogen, chymotrypsinogen A (Figure 2). These
used in this study.
results are analogous to those obtained with papain and
S-carboxymethylpapain. If it may be assumed that small
differences in the molecular structures o f chymotrypsin
and chymotrypsinogen A are negligible with respect to References
their non-proteolytic influences on beer, it may be con-
1 Wallerstein, L. J. Inst. Brew. 1912, 18, 730
cluded that the chill-proofing ability of chymotrypsin, like 2 Kennedy, J. F., Pike, V. W. and Barker, S. A. Enzyme Microb.
that of papain, is derivable largely, but not wholly, from Techol. 1980, 2, 126
its proteolytic action. 3 Kennedy, J. F. and Pike, V. W. Enzyme Microb. TechoL 1980,
This study has demonstrated that two proteins, 2, 288
chymotrypsinogen A and S-carboxymethylpapain, which 4 Pike, V. W. PhD Thesis University of Birmingham (1976)
5 Kennedy, J. F., Barker, S. A. and Pike, V. W.Biochim. Biophys.
each have negligible specific proteolytic activity, are capable Acta 1977,484, 115
of appreciable beer chill-proofing action. These results were 6 Kennedy, J. F. and Pike, V. W.J. Chem. Soc. (Perkin Trans. 1)
unpredicted; since chill-haze is composed mainly of p r o t e i n - 1978, 1058
polyphenol complexes, formed between proteins and 7 Kennedy, J. F. and Pike, V. W. Enzyme Microb. Technol.
1979, 1, 31
polyphenols during the cold-storage of beer, it would have 8 Bishop, L. R. J. Inst. Brew. 1966, 66, 388
been reasonable to have predicted that tile addition of 9 Analytica EBC 2nd ed., Elsevier, Amsterdam, 1963, p. 66
proteolytically inactive protein to beer would promote 10 Kunitz, M.J. Gen. Physiol. 1947, 30, 291
rather than reduce chill-haze formation. 11 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall,
Comparisons of the effects of the proteins studied and R. J. J. Biol. Chem. 1951,193, 265
12 Sluyterman, L. A. AE Biochim. Biophys. Acta 1964, 85,305
their associated proteolytic activities on beer provide no 13 Light, A. Biochem. Biophys. Res. Commun. 1964, 17,781
evidence for a general correlation between the concentration 14 Whitaker, J. R. and Perez-Villasenfr, J. Arch. Biochem,
of added proteolytic activity in beer and its resistance to Biophys. 1968, 70, 124
chill-haze formation (Figure 3). It should, when considering 15 Banks, T. E. and Schafer, J. A. Biochemistry 1972, 11,110
the results in Figure 3, be noted that the concentrations o f 16 Yu-Kum, S. and Chem-Lu, T. Sci. Siniea 1963, 12, 1845
17 Findlay'W'P'K'MdernBrewingTechnlgy Macmillan
added protein and added proteolytic activity in beer are Press, London and Basingstoke, 1971, p. 280
linearly related for each protein and so the recorded 18 Gray, P. P.. Saletan, L. T. and Gantz, C. S. Prec. Eur. Brew.
reductions in chill-haze are probably not the results of Cony. (Brussels) 1963, 288
proteolysis alone. Indeed, the chill-proofing effects associated 19 Bengough, W. !. and Harris, G. J. Inst. Brew. 1955,61,134
20 Fersht, A. R. J. Mot. Biol. 1972, 64,497
with S-carboxymethylpapain and chymotrypsinogen A are 21 Gertler, A., Walsh, J. A. and Neurath, H. Biochemistry 1974,
probably not associated with proteolysis at all. Those for 13, 1302
papain and chymotrypsin, on the basis of previous discussion, 22 Miller, D. D., Herbert, T. A. and Teller, D. C. Biochemistry
are probably attributable to a combination of catalytic and 1971, 10,4641
non-catalytic effects. 23 Freer, S. T., Kraut, J., Robertus, J. D., Wright, H. T. and
Xuong, Ng. H. Biochemistry 1970, 9, 1997
Other workers ~8'2 8,29 have noted poor correlations 24 Wright, H.T.J. Mol.Biol. 1973,79,1,13
between the degree of proteolysis apparent in beer treated 25 Birktoft, J. J., Kraut, J. and Freer, S. T. Biochemistry 1976,
with papain and the resistance of the beer to chill-haze 15,4481
formation. The existence of a non-proteolytic contribution 26 Fersht, A. R. and Sperling, J. J. Mol. Biol. 1973, 74, 137
27 Robillard,G. andSchulman, R. (;.J. Mol. Biol. 1974,86,519
to the chill-proofing ability of papain may underly such 28 DcClerck, J.Brass. Malt. Fr.-belge. 1965, May, 128
observations. The precise nature of the non-proteolytic 29 Jones, M., Woof, J. B. and Pierce, J. S. Prec. Am. Soc. Brew.
chill-proofing factor must await further elucidation. Chem. 1967, 14

Enzyme Microb. Technol., 1981, Vol. 3, January 63