J Sci Food Agric 1995,69,423-428

Partial Inactivation of Microbial Proteinases with Soybean Kunitz and Bowman-Birk I n h bi t or s Stefan0 Marchetti,a* Anna Pitotti,* Annalisa Giordano,O Cristina Chiaba,ll and Corrado Fogherc a Dipartimento di Produzione Vegetale e Tecnologie Agrarie, Universita di Udine, Udine, Italy

Dipartimento di Scienze degli Alimenti, Universita di Udine, Udine, Italy Istituto di Botanica e Genetica Vegetale, Universita Cattolica, Piacenza, Italy

(Received 26 August 1994; revised version received 18 April 1995; accepted 23 June 1995)

Abstract: It was previously demonstrated that some plant inhibitors are able to inactivate the bacterial proteinase subtilisin; since information concerning the effect of plant inhibitors on other microbial proteinases remains limited, we decided to determine the activity of the soybean Kunitz and Bowman-Birk inhibitors (KI and BBI, respectively) on 14 proteinases of fungal and bacterial origin. The results show that microbial proteinases are frequently inhibited by KI and BBI and that proteinases with the same EC number (eg subtilisin Carlsberg and subtilisin BPN’) may equally give different responses to the inhibitors. In particular, all serine proteinases examined were affected by both KI and BBI while metalloproteinases were not. Inhibition was also achieved on a range of microbial proteinases for which the mechanistic class is yet to be established; the data suggest that they belong to the serine type. In one instance, activation instead of inhibition was noted.

Key words: microbial proteinases, subtilisin, Kunitz inhibitor, Bowman-Birk inhibitor

INTRODUCTION (1993) for a review); results were confirmed by in uitro tests where both KI and BBI inactivated the main

Proteinase inhibitors (PI) are antinutritional factors digestive enzyme of these insects (Christeller et a1 1992). which occur in many different plants (Richardson 1980). When genes encoding PI similar to the soybean BBI In soybean (Glycine max Merr), there are two major PI were engineered into tobacco plants, resistance to classes headed by the Kunitz inhibitor (KI) and the tobacco hornworm was clearly achieved (Hilder er a1 Bowman-Birk inhibitor (BBI), respectively; members of 1987). Successful plant protection against this pest was each class are accumulated in the seed and for this also observed when genes encoding the proteinase reason they are regarded as typical storage proteins inhibitor I1 of tomato and potato were inserted (Perez-Grau and Goldberg 1989). The presence of both (Johnson et al 1990). inhibitors in the water surrounding young soybean With regard to the relation between pathogens and seedlings suggested that these substances may confer PI synthesis, tomato plants infected by Phytophthora protection not only from small mammals but also from infestans showed a remarkable increase in inhibitor insects or pathogenic microorganisms (Hwang et al content (Peng and Black 1976). Rickauer et al (1989) 1978) found that elicitors of Phytophthora parasitica var nico-

Actually, several lepidopteran insects showed poor tianae induce the accumulation of a trypsin inhibitor in growth and an increased rate of larval mortality when tobacco cells. A mechanism of protection induced by fed on a KI- or BBI-supplemented diet (see Boulter some pathogens and involving the inhibition of their

extracellular proteinases was proposed by Mosolov et a1 * To whom correspondence should be addressed. (1976). Indeed, inhibitors of microbial enzymes, and

J Sci Food Agric 0022-5142/95/%09.00 0 1995 SCI. Printed in Great Britain 423

424 S Marcherti et a1

notably the serine protease subtilisin, were identified in cereals (Richardson 1980) and grain legumes (Chavan and Hejgaard 1981). Bi-functional (chymotrypsin- or a- amylase-subtilisin) inhibitors were also found in plants.

I t is well recognised that inhibitors generally differ in their ability to inactivate standard proteinases of a certain mechanistic class (Gatehouse et al 1994). Likely enough, this conclusion can be extended also to systems in which the same enzyme inhibitor type (eg a Kunitz serine protease inhibitor from different plant species) is used (Christeller and Shaw 1989).

This report describes in oirro activity of KI and BBI against several fungal and bacterial proteinases, as a preliminary step before analysing inhibition directly on microorganisms.

EXPERIMENTAL

Proteinases, proteinase inhibitors (soybean Kunitz trypsin inhibitor type I-S and soybean Bowman-Birk trypsin-chymotrypsin inhibitor) and substrates (azocoll, haemoglobin) were purchased from Sigma Chemical Co, St Louis, USA; other reagents, all analytical grade, were from Farmitalia Carlo Erba (Milan, Italy). The list of tested proteinases and their microbial origin are reported in Table 1.

Proteinase activity determination

For each enzyme, a stock solution was prepared and frozen ( - 20°C) in 100 p1 aliquots until use; enzymes were dissolved at concentrations and in buffers indi- cated in Table 2. Different quantities of each enzyme

were tested on a constant amount of substrate to deter- mine the maximum concentration showing linear activ- ity during incubation time (30 min for azocoll and 10 min for haemoglobin). The values obtained in these preliminary tests and adopted in determining the inhibi- tory activity of soybean KI and BBI are reported in Table 3.

Acidic proteinases (types XI11 and XVIII) were tested on haemoglobin, according to Ryle (1984). A 25 mg ml- ' solution of haemoglobin in 60 mM HCI (pH 3, adjusted with NaOH) was centrifuged for 10 min at 3000 x g and the sediment discarded; the super- natant (1 ml) was used as substrate. After equilibration at 37"C, incubation was started by adding the enzyme solution (7.5 pl for type XI11 and 120 p1 for type XVIII). The final reaction volume was adjusted to 1.15 ml with the enzyme solution buffer. After 10 min at 37"C, 2 ml of a 50 g litre-' trichloroacetic acid solution were added to denature the enzyme and precipitate the unhydrolysed substrate; samples were then cooled on ice for 10 min. After centrifugation (20 min at 3000 x g), the supernatant was read at 276 nm; absorb- ance was estimated by subtracting the value of the blank (enzyme-free incubation medium).

For all the remaining proteases, the chromogenic substrate azocoll was used (Chavira et a1 (1984), with minor modifications). The incubation medium con- tained 15 mg azocoll in a final volume of 1.5 ml. Volumes were adjusted with 10 mM Tris-HCl, pH 8 (except for protease XXI, tested in 10 mM Na tetra- borate buffer, pH 11). After equilibration at 37"C, incu- bation (30 min at 37°C) was started by adding the enzyme solution (from 2 to 10 p1 of a 1- to 120-fold

TABLE 1 Name and source of the proteases tested

Protease" Source Ocher designation Mechanistic class

I1 1v VIII

IX X

XI11

XIV

xv XVI XVIII XIX XXI XXIII XXVII

Aspergillus oryzae Streptomyces caespitosus Bacillus lichenformis

Bacillus polymyxa Bacillus thermoproteolyticus

Aspergillus saitoi rokko

Strepromyces griseus

Bacillus polymyxa Bacillus subtilis Rhizopus spp Aspergillus sojae Streptomyces griseus Aspergillus oryzae Bacillus subtilis

Subtilisin Carlsberg subtilopeptidase A

Thermophilic-bacterial protease; thermolysin

Aspergillus acid proteinase; aspergillopeptidase Molsin

Pronase E

Newlase

Nagarse subtilisin BPN'

Unknown Unknown Serine EC 3.4.21.14

Metallo EC 3.4.24.4 Metallo EC 3.4.24.4

Aspartic EC 3.4.23.6

Serine + metallo

Metallo EC 3.4.24.4 Unknown Unknown Unknown Unknown Unknown Serine EC 3.4.21.14

EC 3.4.21.4 + EC 3.4.24.4

Sigma type.

Partial inactivation of microbial proteinases

TABLE 2 Composition of the enzyme stock solutions

425

Protease" Concentration in the stock solution (mg ml- of buffer)

Buffer

I 1 IV VIII IX X XI11 XIV xv XVP XVIII XIX XXI XXIlI XXVII

5 4 0 5.00 0.10 0.40 0.02

10.00 0.33 1.00 0.19 0.50

10.00 0.10 0.14 0.10

~ ~ ~~

Na acetate 10 mM/Ca acetate 5 mM, pH 7.5 HCI 1 mM Na acetate 10 mM/Ca acetate 5 mM, pH 7.5 Na acetate 10 mM/Ca acetate 5 mM, pH 7.5 Na acetate 10 mM/Ca acetate 5 mM, pH 7.5 HCI 1 mM Na acetate 10 mM/Ca acetate 5 mM, pH 7.5 Na acetate 10 mM/Ca acetate 5 mM, pH 7.5 Na acetate 10 mM/Ca acetate 5 mM, pH 7.5 HCI 1 mM HCI 1 mM Na tetraborate 10 mM, pH 11 Na acetate 10 mM/Ca acetate 5 mM, pH 7.5 Na acetate 10 mM/Ca acetate 5 mM, pH 7.5

" Sigma type. Aqueous solution.

diluted stock solution, depending on protease activity). To stop the reaction, samples were put on ice for 10 min. Samples were then centrifuged at 16 OOO x g for 7 min; the supernatant was immediately removed and read for absorbance at 520 nm. The blank consisted of the enzyme-free incubation medium.

Inhibitory activity determination

For both KI and BBI, a stock solution was prepared by dissolving 50 mg ml- of each inhibitor in Tris-HC1 10 mM buffer, pH 8.0 and frozen (-20°C) in aliquots. The inhibitory effect was determined using the same

method described for testing proteinase activity; differ- ent amounts of the stock solution were added in order to obtain various concentrations of the inhibitors in the same reaction volume. The content of both KI and BBI was expressed in terms of arbitrary units, one unit being the quantity of inhibitor needed to halve the maximum proteolytic activity of bovine trypsin in an incubation medium having the following composition: 20 pl 1 mM HCI containing 2 pg trypsin (bovine pancreas 2 x crystallised), 160 pl 1 mM BAPNA (Na-benzoyl-DL- arginine-p-nitroanilide) (Erlanger et al 1961 ; Kakade ec a1 1969) in 50 mM Tris-HC1 containing 20 mM CaCI,

TABLE 3 Enzyme concentration and buffer in the incubation media

Protease Enzyme concentration Reaction buffer (PQ m1- 9

I1 18.33 Tris HCI 10 mM, pH 8 IV 6.67 Tris HCI 10 mM, pH 8 VIII 0.67 Tris HCI 10 mM, pH 8 IX 2.67 Tris HCl 10 mM, pH 8 X 0.11 Tris HCI 10 mM, pH 8 XI11 65.22 HCl60 mM, pH 3 XIV 1.76 Tris HCI 10 mM, pH 8 xv 6.67 Tris HC1 10 mM, pH 8 XVP 0.32 Tris HCI 10 mM, pH 8 XVIII 52.17 HCI 60 mM, pH 3 XIX 26.67 Tris HCI 10 mM, pH 8 XXI 0.17 Na tetraborate 10 mM, pH 11 XXIII 0.37 Tris HCI 10 mM, pH 8 XXVII 0.27 Tris HCl 10 mM, pH 8

" Sigma type; see Table 1 for sources of proteases.

reaction buffer. Aqueous solution; concentration expressed as pg of protein ml-' of

426

1ooa 1

S Marchetti et a1

140 - Typs XVlll _ _ E 120 //-=-=-= > 100 x .-

0- 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

inhibitor Concn. (Fg mf')

Fig 1. Trypsin activity at different KI and BBI concentra- tions.

pH 8.2, 20 p1 Tris-HC1 10 mM pH 8.0. To establish the unit value for KI and BBI, different amounts of inhibi- tors (dissolved in Tris-HC1 10 mM pH 8.0) were tested in wells of micro-ELISA plate (Dynatech Instruments Inc, Chantilly, USA) After 20 min incubation at 20°C reaction was stopped by adding 50 pl acetic acid (300 ml litre in water) to each well and the absorbance at 405 nm was read using a micro-ELISA auto-reader (Dynatech Instruments Inc). Data of absorbance were regressed over KI and BBI doses (Fig 1) and the value of each inhibitor unit (IU) was extrapolated directly from the regression line (1 IU = 2.60 pg and 1.45 pg for K1 and BBI, respectively). Deviations from linearity were almost non-existent as demonstrated by the figures obtained for the correlation coefficients ( I > 0.999 for both KI and BBI, P < 0.001).

For each proteinase, observations regarding the effect of inhibitor content and type were combined in a facto- rial experiment with three replicates. Replication was achieved in separate experiments. Data were submitted to the analysis of variance using a model fitted to com- plete randomisation. Means were compared with the new Duncan's multiple range test at a probability level of P < 0.05; for ease of representation, results concern- ing the KI and BBI effect on microbial proteinases were reported in graphs where the confidence limits of each mean (P < 0.05) are shown.

RESULTS AND DISCUSSION

Eight proteases out of 14 were affected by both inhibi- tors, while five of them (types IV, IX, X, XIII, XV) showed maximum proteolytic activity even at KI or BBI concentrations as high as 230 IU. Unexpectedly, one proteinase (type XVIII) was activated. Through repeated trials, we confirmed the occurrence of this phe- nomenon and demonstrated that activation is dose dependent (Fig 2); no difference was noted between KI and BBI at any dose. The possible interactions between components of the incubation medium were studied in order to check for the occurrence of a real activation. In particular, a 10 min preincubation of the substrate with the inhibitors did not alter the results, showing that there is no direct interaction between inhibitors and

3 80

c 0 BBI 2 40 A KI n

2 0 1 . ' , ' . ' . ' . ' . ' ' 0 100 200 300 400 500 800 700

Inhibitor Units

Fig 2. Enhanced proteolytic activity at different concentra- tions of BBI and KI.

substrate. When the enzyme was preincubated for 10 min with Tris-HC1 10 mM pH 8.0 or alternatively with the inhibitors dissolved in the same quantity of Tris-HC1 buffer (in this case, the reaction was started with substrate addition), different absorbances were read. More precisely, in presence of inhibitors, the level of activation was the same as that recorded without preincubation; contrariwise, in absence of inhibitors, proteolytic activity was reduced to 91.9%.

This evidence would suggest that inhibitors may prevent enzyme autodigestion or denaturation and this protective role would result in activation; however, more detailed studies are needed to clarify the mecha- nisms involved.

Among inhibited enzymes, there were typical serine proteinases such as subtilisin Carlsberg (type VIII) and subtilisin BPN' (type XXVII). Previous work showed a slight inhibitory activity of soybean KI or related sub- stances (potato and barley Kunitz-type inhibitors) on subtilisin Carlsberg (Tai et al 1991; Walsh and Twitchell 1991). According to our results, both KI and BBI are strong inhibitors of subtilisin Carlsberg and mild inactivating agents of subtilisin BPN' (Fig 3a and

With regard to pronase E (type XIV), which is not a single proteinase but a mixture of a serine plus a metal- loproteinase, it should be noted that its proteolytic activity was reduced about 50% even at the lowest KI and BBI concentration (Fig 3c). Likely enough, the serine proteinase was the only inactivated component; according to this hypothesis, the serine enzyme of pronase E should be the most KI- and BBI-sensitive proteinase among those on trial. Actually, the metallop- roteinase of pronase E shares the same EC number of other three metalloproteinases (type IX, type XV and type X or thermolysin) that were completely unaffected by both KI and BBI.

Inhibition was also noted for proteinases of unknown classification, like type XXIII and type I1 from Asper- gillus oryzae (Figs 3d and 3e, respectively), type XVI from Bacillus subtilis (Fig 3f), type XIX from Aspergillus sojae (Fig 3g), and type XXI from Streptomyces griseus (Fig 3h). Inhibition was particularly strong for type XVI and type XXI; in the latter case, a significant difference between KI and BBI was observed at low IU values

b).

Partial inactivation of microbial proteinases

120 120 - Type Vlll (bl -

5 1001 s .e

9 .- 0 8 0 . - 2 4 0 - 0)

20 - A KI 5 20 a a

0 ' . " ' " " ' 0

427

Type XXVll

80:7-=+ ' 0 BBI - A KI

' ' ' ~ " " - ' ~ ~ ' -

120 Type X N 1

80

40

0 ' . ' . I 0 100 200 3M) 400 500

120 Tvpe XXlll 1

40 1 0 BBI 20 A KI

0 0 100 200 300 400 500 800 700

Inhibitor Units Inhibitor Units

o 100 200 300 400 500

Inhibitor Units

120 Type XIX I

0 0 100 200 300 400 500 (Loo 700

Inhibitor Units

120 Type XVI

0 50 100 150 200 250

Inhibitor Units

"i 20

0 1 ' I . ' . ' 0 100 200 300 400 500

Inhibitor Units

Fig 3. Effect of BBI and KI on the proteolytic activity of eight microbial proteinases.

(Fig 3h), BBI being more effective. In all other instances, the analysis of variance did not reveal statistical differ- ences between inhibitors or significant inhibitor x dose interactions.

CONCLUSIONS

Soybean KI and BBI are well-known inhibitors of bovine trypsin, chymotrypsin and elastase as well as of lepidopteran trypsin-like digestive enzymes. Our experi- ments showed that these substances are also able to

inactivate a fairly large number of microbial protein- ases. Furthermore, we demonstrated that proteinases sharing the same EC number (eg subtilisin Carlsberg and subtilisin BPN') may well give different responses to the inhibitors and that sometimes KI and BBI are characterised by different activities against the same enzyme. However, all serine proteinases examined were affected by both KI and BBI while metalloproteinases were not. Inhibition was also achieved on a range of microbial proteinases for which the mechanistic class is yet to be established; our data suggest that they belong

428 S Marchetti et a1

to the serine type. One acidic proteinase (type XVIII or newlase) was unexpectedly activated.

ACKNOWLEDGEMENTS

This work was financially supported by the Italian National Council of Research (CNR).

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