Purification and characterization of a novel extracellular protease from Beauveria bassiana

180

Mycol. Res. 104 (2) : 180–186 (February 2000). Printed in the United Kingdom.

Purification and characterization of a novel extracellular protease from Beauveria bassiana

B. E. URTZ† and W. C. RICE* USDA Agricultural Research Service, Louisiana State University Rice Research Station, P.O. Box 1429, Crowley, LA 70527-1429, USA. Accepted 11 May 1999.

An extracellular protease designated BBP (Beauveria bassiana protease) was purified from a B. bassiana isolate and subsequently characterized. It was produced in the late stages of growth in a gelatin medium, and was purified using a combination of ionexchange chromatography and preparative gel electrophoresis. Inhibition by phenylmethylsulphonyl fluoride and chymostatin indicates that BBP is a serine type with chymotrypsin activity. Substrate specificity indicates that the protease has elastase activity as well. The protease was stable at 25 mC and had an alkaline pH optimum (7n5–9n5). Another protease was purified from the same isolate that produced BBP, and was identified as Pr1 based on its isoelectric point, amino terminal sequence, substrate specificity and cuticle degrading ability. A comparison between the two showed that BBP had a lower isoelectric point (pI 7n5) than Pr1 (pI  10) and was 0n5 kDa smaller. The proteases also exhibited differences in substrate specificity with Pr1 being most active against Suc-AlaAla-Pro-Phe-pNA, and BBP being most active against MeOSuc-Ala-Ala-Pro-Met-pNA. The two proteases had equal cuticledegrading activity and both were expressed in cuticle media, although the timing of their expression differed.

Fungal entomopathogens such as Beauveria bassiana and Metarhizium anisopliae are capable of infecting and killing a variety of insects. Consequently, these fungi can be used as biological control agents to protect plants against insect pests. In order for a fungal entomopathogen to infect an insect it must first penetrate the cuticular layers of the insect. Insect cuticle consists mostly of protein (Hepburn, 1985), and fungal proteases are believed to play an important role in cuticle penetration (St Leger, 1995). Metarhizium anisopliae has served as the model system for studying fungal entomopathogen proteases. A variety of endoproteases and exoproteases has been isolated from M. anisopliae (for a review see St Leger, 1995). Most of the endoproteases have been given Pr (protease) designations. Evidence suggests that Pr1 performs a major role in insect cuticle penetration and subsequent pathogenicity (St Leger, Charnley & Cooper, 1987 a ; St Leger et al., 1988 ; Goettel et al., 1989 ; St Leger et al., 1996). Pr1 is a serine protease that has chymoelastase activity, a basic pI, and is capable of degrading insoluble cuticle (St Leger, Charnley & Cooper, 1987 b). Two Pr1 genes have been cloned from M. anisopliae and sequenced, Pr1a and Pr1b (St Leger et al., 1992 ; Joshi, St Leger & Roberts, 1997). Since the deduced amino terminal sequence of the recently isolated Pr1b gene (Joshi et al., 1997) lacked homology with the previously described Pr1b protease (St Leger, Bidochka & Roberts, 1994 ; St Leger, 1995), a third Pr1

protease, Pr1c, may exist. Currently, only Pr1a and its isoforms have been purified and characterized (St Leger et al., 1987 b ; St Leger et al., 1994). Pr1 has also been purified from different B. bassiana isolates (Bidochka & Khachatourians, 1987 ; St Leger, Cooper & Charnley, 1987 c ; Shimizu, Tsuchitani & Matsumoto, 1992). Furthermore, a Pr1 gene from B. bassiana was cloned and sequenced (Joshi, St Leger & Bidochka, 1995). As with M. anisopliae, Pr1 appears to be a virulence factor given its ability to degrade cuticle and the observation that a protease deficient mutant of B. bassiana exhibited decreased virulence with grasshoppers (Melanoplus sanguinipes ; Bidochka & Khachatourians, 1990). We are evaluating B. bassiana as a biological control agent for the rice water weevil (Lissorhoptrus oryzophilus), a major insect pest of rice, and isolates were obtained from infected weevils. In an attempt to purify Pr1 and study its role in rice water weevil pathogenicity, we discovered another protease which to our knowledge has not been purified and characterized. In this paper we describe the expression, purification, and characterization of this novel protease which we have designated BBP (Beauveria bassiana protease). Furthermore, a comparison is made between BBP and a Pr1 protease purified from the same B. bassiana isolate. MATERIALS AND METHODS Fungal isolates

* Corresponding author. † Present address : Nalco Chemical Co., One Nalco Center, Naperville, IL 60563-1198.

Beauveria bassiana (Bals.-Criv.) Vuill. 111892A was isolated from an infected rice water weevil (Rice et al., 1993), and

B. E. Urtz and W. C. Rice isolates ARSEF 149, ARSEF 336, and ARSEF 413 were acquired from the USDA-ARS entomopathogenic fungi (ARSEF) culture collection (Ithaca, NY). Isolates 74040 and 726 were obtained from J. E. Wright (Troy Biosciences, Phoenix, AZ) and Mycotech Inc. (Butte, MT), respectively.

Cuticle preparation Cotton boll weevils (Anthonomus grandis Boheman) were provided by the USDA-ARS Biological Control and Mass Rearing Research Unit (Mississippi State University, MS). Adult insects were frozen at k20 mC and ground with a mortar and pestle. The ground insects were washed several times with 1 % potassium tetraborate, dried, and the remaining cuticle ground to a fine powder ( 177 µm) (Andersen, 1980). House cricket (Acheta domesticus (L.) cuticle from Carolina Biological Supply) was prepared in a similar fashion after initial homogenization in a Waring blender.

Growth media and inocula Cultures were grown in 1 l flasks containing 250 ml of medium and incubated on a shaker (130 rpm) at 28 mC. All media contained basal salts plus trace elements as previously described (Paterson et al., 1994) except that trace elements were added at 1 ml l−". Gelatin and casein were added to a final concentration of 1 % (w\v) and autoclaved. Powdered cuticle was added to 1 % potassium tetraborate, heated for 5 min at 115 mC, washed several times with sterile water, and then added to a sterile medium (1 % w\v). Inocula (10( conidia) and dry weight measurements were made as previously described (Bidochka & Khachatourians, 1990).

Azocoll assay Proteolytic activity was measured using a modified azocoll assay (Bidochka & Khachatourians, 1990). Samples were added to 2n5 ml of 2n5 mg ml−" azocoll (
100 mesh ; Calbiochem) in 0n2  glycine (pH 8n5) and incubated on a shaker platform (450 rpm) at 37 mC for 30 min. The samples were then centrifuged and the A of the supernatant measured. &#! One proteolytic unit was defined as the amount of enzyme needed to produce an A of 1n0. &#! Protease purification Isolate 111892A was grown in gelatin medium for 5 d. The culture was centrifuged and the supernatant filtered through a 0n45 µm filter. The filtrate was concentrated with an Amicon ultrafiltration cell containing a YM-10 filter. The concentrate was diluted (1 : 10) twice with a low salt buffer (25 m MES, 10 m NaCl, pH 6) and concentrated to a final volume of 5–10 ml. The final concentrate was applied to a CM Sepharose CL-6B column (9i1n5 cm), washed with low salt buffer, and eluted in a NaCl gradient (10–200 m) in 25 m MES (pH 6). Column fractions were assayed for azocoll-degrading activity, and peak fractions were concentrated with a

181 Centricon-10 (Amicon) concentrator. The concentrate was washed with 0n2iHis-MES (pH 6n1) and subjected to preparative gel electrophoresis using a 1n5 cm polyacrylamide gel (6 %) in a Bio-Rad Model 491 Prep Cell. A His-MES (pH 6n1) continuous buffer system (McLellan, 1982) was used and the electrodes reversed. Prep cell fractions with peak proteolytic activity were concentrated with Centricon-10 concentrators, washed with 50 m K phosphate (pH 7), filter sterilized, and stored at 4 mC. Pr1 was purified using a combination of preparative isoelectric focusing (IEF) and gel filtration chromatography. Isolate 111892A was grown for 3 d in Sabouraud dextrose broth containing 0n2 % yeast extract. Cells were centrifuged, washed with a solution of basal salts plus trace elements, and added to boll weevil cuticle medium. After 4 d the medium was centrifuged and the supernatant removed. The pellet was washed with 0n2  K phosphate (pH 7) to remove bound proteases and centrifuged again. The two supernatants were combined, filtered through a 0n45 µm filter, and concentrated as described above. The concentrate was diluted (1 : 10) with 10 m K phosphate (pH 7) and concentrated again. The final concentrate was subjected to preparative IEF using a Bio-Rad Rotorfor and pH 9–11 ampholytes (Sigma). Fractions were assayed for azocoll-degrading activity, and peak fractions were concentrated with a Centriplus-10 (Amicon) concentrator. The concentrate was applied to a Sephadex G-75 superfine column (1n5i50 cm) and eluted with 50 m K phosphate (pH 7). Peak proteolytic fractions were combined, filter sterilized, and stored at 4 mC. Molecular weight and pI determinations Molecular weight determinations were made using a 12 % SDS–PAGE gel (Laemmli, 1970) in a Bio-Rad Mini-Protean II cell. Phenylmethylsulphonyl fluoride (PMSF) (final concn l 1 m) was added prior to denaturation to prevent selfdegradation. The mol. wt of BBP was also determined using a Sephadex G-75 superfine column (1n5i50 cm) calibrated with protein standards ranging from 6n5 to 66 kDa (Sigma). Isoelectric point (pI) determinations were made using a BioRad Model 111 Mini-IEF Cell with pH 3–10 ampholytes and Bio-Rad IEF standards (pI 4n5–9n6). Amino terminal sequencing N-terminal amino acid sequencing was performed by automated Edman degradation (Commonwealth Biotechnologies, Inc.). Samples were subjected to twenty sequence cycles. Alignment of amino acid sequences was done using a global alignment program from Align Plus 3 (Sci-ed software, Durham, NC). pH optimum and temperature stability The pH optimum was determined using azocoll and MeOSucAla-Ala-Pro-Met-pNA in Bis-Tris-Propane buffer (pH 6n5– 9n9). The protease (2 µg) was added to 2 ml of 5 mg ml−" azocoll in 0n1  Bis-Tris-Propane and incubated on a shaker

Beauveria bassiana protease

Substrate specificity and cuticle degradation Substrate specificity was determined using nitroanilide substrates (Sigma). BBP (0n1 µg) and Pr1 (0n02 µg) were diluted in 1n65 ml of 0n1  Tris (pH 8). After addition of 150 µl of substrate (2n4 m in DMSO), the A was monitored. An %"! extinction coefficient of 8800 was used for calculating reaction rates (Erlanger, Kokowsky & Cohen, 1961). Cuticle degradation was determined by measuring the release of soluble protein from insoluble cuticle. Ground boll weevil cuticle was prepared as described above. The cuticle powder was washed once with 1 % potassium tetraborate, twice with water, and suspended (20 mg ml−") in 50 m Tris (pH 8n5). One µg of protease was added to 2 ml of cuticle suspension and incubated on a shaker (400 rpm) for 1 h at 25 mC. The mixture was then centrifuged and the supernatant filtered through a 0n45 µm Acrodisc (Gelman) filter. The amount of protein in the supernatant was determined using the Bio-Rad protein assay (Bradford, 1976) and bovine gamma globulin standards. Control reactions contained cuticle without enzyme. Inhibitors Various protease inhibitors (Beynon & Salvesen, 1989 ; Barrett, 1994) were incubated with 50 ng of protease in 1n5 ml of 0n1  Tris (pH 8) for 15 min at 25 mC. After the addition of 40 µl of 10 m MeOSuc-Ala-Ala-Pro-Met-pNA (in DMSO), and a 15 min incubation at 25 mC, the A was measured. %"!

for the concentrates obtained from various B. bassiana isolates, and a pH 9–11 gradient was used for concentrates obtained from the various media. The gels were blotted with a membrane impregnated with MeOSuc-Ala-Ala-Pro-Met-7amino-4-trifluoro-methyl-coumarin (AFC) (Enzyme Systems Products, Dublin, CA).

Miscellaneous All data given are the means of at least two experiments. Unless otherwise stated, protein concentrations were determined using the Bio-Rad DC Protein Assay (Lowry et al., 1951) and bovine gamma globulin standards.

RESULTS Protease purification Beauveria bassiana isolate 111892A produced an extracellular protease with azocoll-degrading activity in the late stages of growth in a gelatin medium (Fig. 1). The protease was designated BBP and was purified using a combination of ionexchange chromatography and preparative gel electrophoresis (Table 1). Another protease was purified and identified as Pr1 based on its high pI, substrate specificity, cuticle degrading activity, and amino terminal sequence (see below). Both protease preparations were free of contaminations (Fig. 2).

Molecular weight and pI The estimated molecular weight of BBP was 31n5 kDa based on SDS–PAGE and 22 kDa based on gel filtration chromatography. It is unknown why the two procedures gave different results. It is possible that the structure of the protease is such that it elutes slower than expected in the gel filtration column. Based on SDS–PAGE, BBP was slightly smaller than Pr1 (32 kDa) (Fig. 2). The pI of BBP was 7n5 based on analytical IEF. In contrast, the pI of Pr1 was estimated to be
10 based on preparative IEF runs used to purify the enzyme.

Enzyme overlay membranes

16 Dry weight (mg ml–1)

Beauveria bassiana isolates were grown in gelatin or casein media until peak proteolytic activity was observed based on azocoll degradation. Isolate 111892A was also grown for 4 d in gelatin medium then transferred to a medium containing only basal salts plus trace elements and incubated for 1 d. All cultures were centrifuged and the supernatants filtered through a 0n45 µm filter. The culture filtrates were concentrated until the proteolytic activity was approximately 0n5 PU µl−". Boll weevil and cricket cuticle media were processed in the same manner except that after the first centrifugation the pellet was washed with 0n2  K phosphate (pH 7). The cuticle media were sampled at days 3 and 6. One µl samples were run on a polyacrylamide gel in a BioRad Model 111 Mini-IEF Cell. A pH 3–10 gradient was used

5 4 12 3 8

2

4

1 0

0

1

2

3

4

5

6

7

Proteolytic U ml–1

(400 rpm) for 30 min at 25 mC. After centrifugation, the A of &#! the supernatant was measured. A microplate assay (200 µl) was set up with 10 ng of protease and 0n25 m MeOSuc-AlaAla-Pro-Met-pNA in 75 m Bis-Tris-Propane buffer. The reaction was incubated for 15 min at 25 mC and the A %!& measured. Aliquots of protease were stored at 4 mC, 25 mC, 37 mC and 42 mC. At various times samples were taken and the amount of activity was measured using a microplate assay as described above with the exception that 0n1  Tris (pH 8) was used as a buffer.

182

0

Day

Fig. 1. Relationship between the growth of Beauveria bassiana 11892A, as measured by .. ($), and protease activity, as measured by azocoll degradation (#), in a gelatin medium over time.

B. E. Urtz and W. C. Rice

183

Table 1. Purification of B. bassiana protease BBP

Step Culture filtrate Ultrafiltration CM Sepharose Preparative electrophoresis " Proteolytic units (A

&#!

Volume (ml)

Total activity (PU)"

Total protein (mg)

Specific activity (PU mg−")

Yield (%)

Purification (fold)

217n0 7n4 12n0 1n1

3365 2773 2002 494

278n9 25n5 3n45 0n44

12 109 580 1123

100n0 82n4 59n5 14n7

1n0 9n0 48n0 92n8

of 1n0 with azocoll assay).

1

2

3

100

Relative activity (%)

97·4 66·2

45·0

25°

80 60

37°

40 20 45°

0 0

31·0

4 Time (h)

6

8

Fig. 4. Effect of temperature on Beauveria bassiana protease BBP activity using MeOSuc-Ala-Ala-Pro-Met-pNA as a substrate. Activities are relative to values obtained with enzyme held at 4 mC.

21·5

Table 2. Effect of various inhibitors on the activity of B. bassiana protease BBP

14·4

Fig. 2. SDS–PAGE of purified Beauveria bassiana proteases Pr1 and BBP stained with Coomassie blue. Lane 1, Bio-Rad low range standards whose sizes (kDa) are indicated on the left ; lane 2, BBP (10 µg) ; lane 3, Pr1 (10 µg).

100 80 Relative activity (%)

2

60

Inhibitor

Concentration (m)

Residual activity (%)

3,4-DCI PMSF E-64 Iodoacetamide Pepstatin 1,10-phenanthroline Phosphoramidon Chymostatin Elastatinal Leupeptin

0n1 1n0 0n1 1n0 0n01 1n0 0n02 0n1 0n1 0n1

96n1 3n8 100n1 100n9 101n8 99n0 99n2 1n5 102n2 68n9

Abbreviations : 3,4-DCI, 3,4-dichloroisocoumarin ; PMSF, phenylmethylsulphonyl fluoride ; E-64, -trans-epoxysuccinyl-leucylamide-(4-guanidino)butane.

40 20 0 6·5

7·0

7·5

8·0

8·5

9·0

9·5

10·0

pH

Fig. 3. Effect of pH on Beauveria bassiana protease BBP activity using azocoll ($) and MeOSuc-Ala-Ala-Pro-Met-pNA (X) as substrates.

pH optimum and temperature stability The pH optimum of BBP was 9n5 with azocoll as the substrate and 7n5 with MeOSuc-Ala-Ala-Pro-Met-pNA as the substrate (Fig. 3). Over an eight hour period, the protease was fairly

stable at room temperature (25 mC) (Fig. 4). It gradually lost activity at 37 mC, however, and was completely inactivated after 4 h at 45 mC. Protease inhibition Even though 3,4-DCI had no effect, PMSF did inhibit BBP indicating that it is a serine type protease (Table 2). Cysteine (E-64 and iodoacetamide), aspartic acid (pepstatin), and metalloprotease (1,10-phenanthroline and phosphoramidon) inhibitors had no effect. The protease was also strongly

Beauveria bassiana protease

184

Table 3. Substrate specificities of B. bassiana proteases Pr1 and BBP. Relative activity (%)

MeOSuc-Ala-Ala-Pro-Met-pNA Suc-Ala-Ala-Pro-Leu-pNA MeOSuc-Ala-Ala-Pro-Val-pNA Suc-Ala-Ala-Val-Ala-pNA Suc-Ala-Ala-Ala-pNA Suc-Ala-Ala-Val-pNA Suc-Tyr-Leu-Val-pNA Suc-Ala-Ala-Pro-Phe-pNA Suc-Val-Pro-Phe-pNA Suc-Gly-Gly-Phe-pNA Boc-Leu-Ser-Thr-Arg-pNA Tos-Gly-Pro-Lys-pNA Bz-Pro-Phe-Arg-pNA Bz-Phe-Val-Arg-pNA Max. activity (µmol NA min−" ml−" mg−" protein

Pr1

BBP

26n4 9n8 0 12n4 0 0 1n7 100 0 0 25n8 0 0n7 3n0 18n86

100 8n1 0 21n0 0 0 0 46n7 6n1 0 0n7 5n1 0 5n6 4n09

1

2

3

4

5

6

7

8

Cathode

Anode

5 1

2

3

4

5

6

7

8

Cathode

inhibited by chymostatin, a chymotrypsin inhibitor, but elastatinal, an elastase inhibitor, had no effect. Substrate specificity and cuticle degradation BBP exhibited strong activity against chymotrypsin and elastase type substrates with its best activity against MeOSucAla-Ala-Pro-Met-pNA (Table 3). This differed with the substrate specificity of Pr1 which it exhibited its strongest activity against Suc-Ala-Ala-Pro-Phe-pNa. The max. activity of Pr1 was more than 4i greater than BBP with the nitroanilide substrates. The two proteases had identical rates of release of soluble protein, however, 0n48 mg ml−" h−", from insoluble boll weevil cuticle. This suggests that the two protease have comparable cuticle-degrading activity.

Anode

6

Figs 5, 6. Enzyme overlay membrane analysis of concentrates from Beauveria bassiana isolates and culture media. Samples were run on an analytical IEF gel with ampholytes and blotted with a membrane impregnated with MeOSuc-Ala-Ala-Pro-Met-AFC. Fig. 5. B. bassiana, pH 3–10 ampholytes. Lane 1, purified Pr1 ; lane 2, ARSEF 336 ; lane 3, 726 ; lane 4, ARSEF 413 ; lane 5, 74040 ; lane 6, ARSEF 149 ; lane 7, 111892A ; lane 8, purified BBP. Fig. 6. Culture media, pH 9–11 ampholytes. Lane 1, purified BBP ; lane 2, cotton boll weevil cuticle day 3 ; lane 3, cotton boll weevil cuticle day 6 ; lane 4, cricket cuticle day 3 ; lane 5, cricket cuticle day 6 ; lane 6, basal salts plus trace elements ; lane 7, casein ; lane 8, purified Pr1.

Amino terminal sequencing The N-terminal amino acid sequence of Beauveria bassiana 111892A Pr1 is shown in Table 4. Alignment of conserved residues clearly supports the identification of the Pr1 protease. The sequence differs, however, from a previously described B. bassiana Pr1 sequence, suggesting isoform variation (Joshi, St Leger & Bidochka, 1995). Either due to acylation and\or degradation, insufficient amino acid residues were detected to allow determination of the amino terminal sequence of BBP. Enzyme overlay membrane analysis All the B. bassiana isolates produced a protease with azocolldegrading activity within 4–5 d when grown in gelatin media.

Three of the isolates (ARSEF 149, 74040, and ARSEF 413) produced a protease active against MeOSuc-Ala-Ala-ProMet-AFC with a pI identical to BBP (Fig. 5). The two remaining isolates, 726 and ARSEF 336, produced a protease with a higher pI. Concentrates from 726 and ARSEF 336 were subjected to preparative IEF, and fractions containing peak proteolytic activity, based on azocoll degradation, were assayed with MeOSuc-Ala-Ala-Pro-Met-pNA and Suc-AlaAla-Pro-Phe-pNA (data not shown). The protease fractions from 726 and ARSEF 336 exhibited 2–4ihigher activity with MeOSuc-Ala-Ala-Pro-Met-pNA suggesting that the fractions contained an isoform of BBP.

Table 4. N-terminal amino acid sequences of Pr1 proteases from B. bassiana and M. anisopliae strains

B. bassiana Pr1 B. bassiana Pr1 M. anisopliae Pr1a M. anisopliae Pr1b * Probable amino acids G, V, A.

Amino acid sequence

Accession no.\source

AVVRQAGAPWGLGRI *VLPXQAGAPXGLGRI SGITEQSGAPWGLGRI NGFVEQKNAPWNLGRI

U16305 111892A P29138 U59484

B. E. Urtz and W. C. Rice When 111892A was grown in gelatin medium for 4 d and then shifted to a medium containing only basal salts plus trace elements, only Pr1 was detected (Fig. 6). Pr1 was also found in the late stages of growth in casein medium. Proteolytic activity, as measured by azocoll degradation, peaked at day 5 in cricket cuticle medium and days 6–7 in boll weevil cuticle medium. Similar proteolytic patterns were observed with the two media (Fig. 6). At day 3, activity was high from BBP, but little or no Pr1 activity was found. By day 6, Pr1 activity had greatly increased, but BBP showed very little activity. DISCUSSION An extracellular protease designated BBP was purified from B. bassiana 111892A and subsequently characterized. 111892A represents a common genotype found infecting the rice water weevil (Urtz & Rice, 1997). BBP was produced in the late stages of growth in a gelatin medium as well as in two types of cuticle media. The protease had an alkaline pH optimum, a Mr of 31n5 kDa (based on SDS–PAGE) and a pI of 7n5. Inhibition by PMSF and chymostatin indicates that the protease is a serine type with chymotrypsin activity. Substrate specificity data suggests elastase activity as well. St Leger et al. (1987c) reported that B. bassiana produces a trypsin-like Pr2 protease with a pI of 7n4. Despite the similarities in pI, it is unlikely that this protease is the same as BBP. The Pr2 protease exhibited its best activity against the substrate Bz-Phe-Val-Arg-pNA, a substrate which BBP showed minor activity against. Furthermore, Pr2 had trace activity against the substrates Suc-Ala-Ala-Pro-Phe-pNa and Suc-AlaAla-Val-Ala-pNa, two substrates BBP had relatively strong activity against. Finally, the Pr2 protease had no cuticledegrading activity. In contrast, BBP had very good cuticledegrading activity. A metalloprotease was identified in M. anisopliae (St Leger et al., 1994) which also has a pI, 7n3, similar to BBP. Since BBP was not inhibited by 1,10-phenanthroline or phosphoramidon it would not appear to be a metalloprotease. It seems more likely that BBP is a Pr1 type protease. Like previously described Pr1 proteases, it has an alkaline pI, chymoelastase activity, and is capable of degrading insoluble cuticle (St Leger, 1995). St Leger et al. (1987 c) reported that B. bassiana produced two Pr1 type activities, pI
10 and pI 9, when grown in a locust (Schistocerca gregaria) cuticle medium. Based on its high pI and substrate specificity, the pI
10 protease seems to be analogous to the Pr1 protease we isolated and the protease described by Bidochka & Khachatourians (1987, 1988). The pI 9 activity was not further characterized. Two Pr1 genes, Pr1a and Pr1b, have been cloned from M. anisopliae and sequenced (St Leger et al., 1992 ; Joshi et al., 1997). Pr1a shows similarity to the Pr1 we purified based on its high pI, preference for Suc-Ala-Ala-Pro-Phe-pNa and amino acid sequence (St Leger et al., 1994). Based on the deduced amino acid sequence, Pr1b has a predicted pI, 8n05, lower than Pr1a. Pr1b also has changes in its active site which may effect its catalytic activity (Joshi et al., 1997). BBP has a lower pI than Pr1 and also exhibits differences in substrate

185 specificity. It is possible, therefore, that BBP is analogous to M. anisopliae Pr1b. Unfortunately, we were unable obtain an amino terminal sequence from BBP which would have provided more information with regard to its relationship to the Pr1 proteases. Current plans to clone and sequence the BBP gene should facilitate our understanding of how this protease relates to other entomopathogenic proteases. BBP does not appear to be unique to B. bassiana 111892A. Three additional B. bassiana isolates produced a protease with the same pI when grown in gelatin media. Two others produced a protease with a higher pI, but substrate specificity suggests that this may be an isoform of BBP. Since BBP was not detected in gelatin medium until the late stages of growth, the protease was probably not responsible for the initial breakdown and subsequent utilization of the gelatin. It was initially thought that the protease was produced in response to carbon and\or nitrogen deprivation. When cells at late stages of growth in gelatin medium were shifted to a medium containing only basal salts plus trace elements, however, Pr1 was generated. Pr1 was also produced in the late stages of growth in casein medium. The factor(s) responsible for the expression of BBP are, therefore, unknown. BBP had cuticle degrading activity that was comparable to Pr1. Furthermore, like Pr1, BBP was expressed in both cuticle media. The two enzymes appeared, however, to be differentially expressed with BBP in abundance early and Pr1 in abundance later. The presence of BBP in cuticle media combined with its cuticle degrading activity suggests that it may play a role in insect cuticle penetration. Now that BBP has been purified we can begin to develop tools (e.g. specific antibodies) that will help us study its role versus that of the previously described Pr1. This should enhance our knowledge of B. bassiana pathogenicity and facilitate its use as a biological control agent. REFERENCES Andersen, S. O. (1980). Cuticular sclerotization. In Cuticle Techniques in Arthropods (ed. T. A. Miller), pp. 185–215. Springer-Verlag, New York. Barrett, A. J. (1994). Classification of peptidases. In Methods in Enzymology (ed. A. J. Barrett) 244, pp. 1–15. Academic Press, San Diego. Beynon, R. J. & Salvesen, G. (1989). Commercially available protease inhibitors. In Proteolytic Enzymes : A Practical Approach (ed. R. J. Beyon & J. S. Bond), pp. 241–249. Oxford University Press : Oxford. Bidochka, M. J. & Khachatourians, G. G. (1987). Purification and properties of an extracellular protease produced by Beauveria bassiana. Applied and Environmental Microbiology 53, 1679–1684. Bidochka, M. J. & Khachatourians, G. G. (1988). N-acetyl--glucosaminemediated regulation of extracellular protease in the entomopathogenic fungus Beauveria bassiana. Applied and Environmental Microbiology 54, 2699–2704. Bidochka, M. J. & Khachatourians, G. G. (1990). Identification of Beauveria bassiana extracellular protease as a virulence factor in pathogenicity toward the migratory grasshopper, Melanoplus sanguinipes. Journal of Invertebrate Pathology 56, 362–370. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254. Erlanger, B. F., Kokowsky, N. & Cohen, W. (1961). The preparation and properties of two new chromogenic substrates of trypsin. Archives of Biochemistry and Biophysics 95, 271–278. Goettel, M. S., St Leger, R. J., Rizzo, N. W., Staples, R. C. & Roberts, D. W. (1989). Ultrastructural localization of a cuticle-degrading protease produced

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