Papain, Chymotrypsin and Related Proteins-A Comparative Study of Their Beer Chill-Proofing Abilities and Characteristics
Papain, Chymotrypsin and Related Proteins-A Comparative Study of Their Beer Chill-Proofing Abilities and Characteristics
Papain, Chymotrypsin and Related Proteins-A Comparative Study of Their Beer Chill-Proofing Abilities and Characteristics
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.
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
O
Determination o f the effects o f sodium 5
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
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
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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
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17 Findlay'W'P'K'MdernBrewingTechnlgy Macmillan
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20 Fersht, A. R. J. Mot. Biol. 1972, 64,497
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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,
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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