Influence of growth conditions on the production of extracellular ...

Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Influence of growth conditions on the production of extracellular proteolytic enzymes in Paenibacillus peoriae NRRL BD-62 and Paenibacillus polymyxa SCE2 V.M. Alvarez, I. von der Weid, L. Seldin and A.L.S. Santos Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Goés (IMPPG), Centro de Cieˆncias da Sau´de (CCS), Universidade Federal do Rio de Janeiro (UFRJ), Ilha do Fundaõ, Rio de Janeiro, Brazil

Keywords extracellular proteolytic enzymes, growth conditions, metalloproteases, Paenibacillus peoriae, Paenibacillus polymyxa. Correspondence A.L.S. Santos, Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Goés (IMPPG), Centro de Cieˆncias da Sau´de (CCS), Universidade Federal do Rio de Janeiro (UFRJ), Ilha do Fundaõ, Rio de Janeiro RJ 21941-590, Brazil. E-mail: andre@ micro.ufrj.br

2006/0099: received 24 January 2006, revised 28 June 2006 and accepted 3 July 2006 doi:10.1111/j.1472-765X.2006.02015.x

Abstract Aims: To analyse the extracellular protease profile of two Paenibacillus species, Paenibacillus peoriae and Paenibacillus polymyxa, as well as how different growth media influenced its expression. Methods and Results: Both bacteria were cultured in five media [Luria–Bertani broth, glucose broth, thiamine/biotin/nitrogen broth (TBN), trypticase soy broth and a defined medium] for 48 h at 32C. Our results showed a heterogeneous protease secretion pattern whose expression was dependent on medium composition. However, TBN induced the most quantitative and qualitative protease production on both Paenibacillus. The proteases were detected in neutral-alkaline pH range, being totally inhibited by 1,10-phenanthroline, a zinc-metalloprotease inhibitor. We also analysed the protease expression during the growth and, at least to P. peoriae, the most elevated protease activity was measured at 96 h, in which the highest number of spores and a low concentration of viable cells were observed. Conclusions: The results presented add P. peoriae and P. polymyxa to the list of neutral-alkaline extracellular protease producers. Significance and Impact of the Study: Paenibacillus species are ubiquitous in nature, are capable to form resistant spores and to produce several hydrolytic enzymes, including proteases. However, only few data concerning the production of these enzymes are available. Proteases produced by Paenibacillus strains may represent new sources for biotechnological use.

Introduction The soil-inhabiting bacteria belonging to the genus Paenibacillus can act as a plant growth-promoting rhizobacteria, affecting the plant development directly through the atmospheric nitrogen fixation, mineral solubilization, siderophores or phytohormone production (Glick et al. 1999; Timmusk 2003). Moreover, different Paenibacillus strains were reported to have inhibitory effect on bacteria and/or fungi (Kajimura and Kaneda 1997; Seldin et al. 1999; von der Weid et al. 2003), which was attributed to the biologically active antagonistic substances produced by these strains such as antimicrobial substances

(antibiotics, bacteriocins and/or small active peptides) and cell wall degrading enzymes (b-1,3-glucanases, cellulases, chitinases and proteases) (Dunn et al. 1997; Budi et al. 2000). Micro-organisms belonging to the genus Bacillus produce most commercial neutral and alkaline proteases. Neutral proteases from Bacillus are being used in food industry while alkaline proteases have properties, like high activity in alkaline pH, broad substrate specificity and optimal temperature around 60C that make them suitable for use in the detergent industry (Rao et al. 1998; Gupta et al. 2002). Other industrial uses of proteases are in food processing, pharmaceuticals, peptide synthesis,

ª 2006 The Authors Journal compilation ª 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 43 (2006) 625–630

625

Extracellular proteases in P. peoriae and P. polymyxa

meat tenderization, medical diagnosis, baking, brewing (Gennari et al. 1998), deproteinization of shrimp and crabshell waste (Yanga et al. 2000) and dehairing of hides and skins (Varela et al. 1997). Many companies worldwide have successfully launched several products based on proteases produced by Bacillus species such as Bacillus licheniformis and Bacillus lentus (detergent, food and silk degumming) and Bacillus subtilis (cosmetics and pharmaceuticals) (Gupta et al. 2002). The importance of proteases produced by Paenibacillus in industry was first described by Matta and Punj (1998), which established that the milk and milk products might be affected by these Gram-positive spore-formers because they secrete extracellular heat-stable proteases that have a deleterious effect on milk quality. However, few data are available concerning proteases produced by Paenibacillus species that are ubiquitous in nature. These proteases may represent new sources for biotechnological use. In this context, we determined the extracellular proteolytic profile of two genetically and phenotypically related Paenibacillus species (von der Weid et al. 2002), Paenibacillus polymyxa strain SCE2 (Ferreira and Seldin 1993) and Paenibacillus peoriae strain NRRL BD-62 (Montefusco et al. 1993), as well as the influence of the medium composition in their expression. Materials and methods Micro-organisms, growth conditions and cell-free culture supernatants Two species of the genus Paenibacillus were used: the strain NRRL BD-62 of P. peoriae and the strain SCE2 of P. polymyxa. Both Paenibacillus species were initially cultivated in 100-ml Erlenmeyer flasks containing 25 ml of the following culture media: glucose broth (GB; Seldin et al. 1983), thiamine/biotin/nitrogen broth (TBN; Seldin et al. 1983), Luria–Bertani broth (LB; Schleif and Wensink 1981), trypticase soy broth (TSB; Difco) and a chemically defined medium (DM; von der Weid et al. 2003) for 48 h at 32C without agitation. The cultures were centrifuged (15 000 g for 20 min at 4C) and the supernatants (crude preparations) were filtered in 0Æ22-lm pore filter (Millipore). Then, the cell-free culture supernatants were lyophilized, then resuspended (to a 20-fold concentration) in phosphate-buffered saline (PBS; 150 mmol l)1 NaCl, 20 mmol l)1 phosphate buffer, pH 7Æ0) and stored at )20C until use. Extracellular proteolytic activity and sporulation Extracellularly released proteolytic activity was monitored during the growth on both Paenibacillus species in TBN 626

V.M. Alvarez et al.

medium for 24–96 h at 32C. Viable counts and spores (heat-treated counts after 80C for 10 min) as well as the supernatant fluids were recorded every 24 h, to evaluate the growth kinetics and the extracellular protease production. The cell-free culture supernatants were lyophilized, concentrated and tested for proteolytic activity as described below. Quantitative proteolytic assay The extracellular proteolytic activity was measured spectrophotometrically using the substrate gelatin. Briefly, 10 ll of each concentrated supernatant, 270 ll of 10 mmol l)1 Tris–HCl buffer, pH 7Æ0, and 70 ll of 1% gelatin were added to a microcentrifuge tube and incubated for 1 h at 37C. Then, three aliquots (100 ll each) of the reaction mixture were transferred to wells on a microtitre plate containing 50 ll of water and 100 ll of a Coomassie solution (0Æ025% Coomassie brilliant blue G-250, 11Æ75% ethanol and 21Æ25% phosphoric acid). After 10 min to allow dye binding, the plate was read at 595 nm. One unit of enzyme activity was defined as the amount of enzyme that caused an increase of 0Æ001 in absorbance unit, under standard assay conditions (Buroker-Kilgore and Wang 1993). A control, where the substrate was added just after the reactions were stopped, was used as blank. Protein concentration was determined by the method described by Lowry et al. (1951), using bovine serum albumin as standard. To calculate the specific proteolytic activity, protein concentration of each sample was divided by its respective enzymatic activity. All experiments were performed in triplicate, with similar results obtained in three separate cell suspensions. Gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis The secretory proteolytic activity was also assayed and characterized by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 0Æ1% gelatin incorporated into the gel as substrate (Santos et al. 2005). The gels were loaded with 90 lg of protein of each concentrated supernatant per slot. Electrophoresis was performed at a constant current of 120 V at 4C and SDS was removed by incubation for 1 h with 1% Triton X-100. The gels were incubated in 10 mmol l)1 PBS, pH 7Æ0, for 24 h at 37C, to promote the proteolysis. The gels were stained for 2 h with 0Æ2% Coomassie brilliant blue R-250 in methanol–acetic acid–water (50 : 10 : 40) and destained in the same solvent. Molecular masses of the proteases were calculated by the comparison of the mobility of Gibco SDS-PAGE standards. The gels were dried, scanned and digitally processed.


V.M. Alvarez et al.

Effect of pH, temperature and proteolytic inhibitors on the extracellular proteolytic activity This set of experiments was evaluated using the standard SDS-PAGE assay conditions described above. The effect of pH was determined incubating the gel strips at 37C for 24 h in the following buffers: 50 mmol l)1 sodium phosphate buffer, pH 5Æ0, 10 mmol l)1 PBS, pH 7Æ0, and 20 mmol l)1 glycine-NaOH, pH 10Æ0. To determine the proteolytic class, the gel strips containing the released proteases were incubated in PBS, pH 7Æ0, for 24 h at 37C, in the absence and presence of the following proteolytic inhibitors (Sigma): 10 mmol l)1 phenylmethylsulfonyl fluoride (PMSF), 10 mmol l)1 1,10-phenanthroline, 10 mmol l)1 EDTA, 10 mmol l)1 EGTA and 10 lmol l)1 E-64. Extracellular proteolytic enzymes were also analysed on gelatin-SDS-PAGE for 24 h at different temperatures (4, 25, 37, 40, 50 and 65C) in PBS, pH 7Æ0. Alternatively, for measuring the thermal stability of the proteolytic enzymes, the concentrated supernatants were incubated in 10 mmol l)1 Tris–HCl buffer, pH 7Æ0, for 60 min, at 4, 25, 37, 40, 50 and 65C. Immediately after, the supernatants were applied on gelatin-SDS-PAGE (Santos et al. 2005). Results The secretion of proteolytic enzymes was investigated in the culture supernatants of two Paenibacillus species, P. polymyxa and P. peoriae, after their growth in five different media through gelatin-SDS-PAGE. Regarding each Paenibacillus species, the proteolytic profile was dissimilar in the five distinct media, and great quantitative and qualitative changes were observed (Fig. 1). For instance, P. peoriae produced two weak extracellular proteolytic enzymes when grown in GB in comparison with five proteases in TSB (Fig. 1). Paenibacillus polymyxa and P. peoriae presented similar proteolytic profiles when cultured in TBN. These profiles were composed of at least four intense proteolytic halos with molecular masses of 20, 35, 50 and 210 kDa (Fig. 1). When both strains were grown in LB no extracellular proteases were observed. Similarly, no proteolytic activity was detected in P. polymyxa grown in DM and GB (Fig. 1). All these proteolytic activities were detected in neutral pH value. Corroborating with these findings, the quantitative proteolytic measurement demonstrated that the two strains belonging to Paenibacillus species produced a great amount of extracellular protease activities in TBN when compared with the other four media (data not shown). Therefore, TBN was chosen to carry out the further tests. The growth kinetics of the two Paenibacillus species showed great differences in cell numbers (viable counts) and spores (Fig. 2). For the determination of the growth


(a) 209

47 34 29 18 (b)

GB

LB

TBN

TSB

DM

209

47 34 29 18 Figure 1 Gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis showing the extracellular proteolytic profiles of Paenibacillus peoriae (a) and Paenibacillus polymyxa (b) grown in five different culture media: glucose broth (GB), Luria–Bertani broth (LB), thiamine– biotin–nitrogen broth (TBN), trypticase soy broth (TSB) and a chemically defined medium (DM) for 48 h at 32C without agitation. The gel strips containing concentrated supernatant were incubated in 10 mmol l)1 phosphate-buffered saline, pH 7Æ0, for 24 h at 37C. The numbers on the left indicate the molecular masses.

stage in which P. peoriae and P. polymyxa produce high amount of secretory proteolytic enzymes, concentrated supernatant samples were obtained at various stages during their growth in TBN, and were tested for their hydrolytic activity using soluble gelatin as substrate. In the first 24 h, during which time a high number of viable counts and a low number of spores were observed in P. peoriae, the lowest proteolytic activity was also measured (Fig. 2a). At 48 h of growth, P. peoriae reached the late log phase and the greatest viable count (1Æ6 · 107 CFU ml)1). The spore numbers suffered a significant increase at 72 h, while the cell number decreased in a severe way (Fig. 2a). For this P. peoriae strain, the most elevated protease activity was detected at 96 h, when the highest number of spores (2Æ0 · 104 CFU ml)1) and a low concentration of viable cells (5Æ0 · 106 CFU ml)1) were observed (Fig. 2a). Paenibacillus polymyxa reached the end of the log phase at 48 h and recorded a number of viable cells equal to 4Æ4 · 107 CFU ml)1 (Fig. 2b), however, only 1Æ4 · 104 CFU ml)1 of spores were counted at the same time. At 96 h of cultivation, P. polymyxa reached the highest


627


15

20 15

10

10 5

5 0

0

800 600 400 200

Proteolytic activity

1000

25

Spores (CFU ml–1 × 103)

Viable counts (CFU ml–1 × 106)

(a)

V.M. Alvarez et al.

0 350

140 120

40

100 30

80 60

20

40

10

20 0

0 24

48 72 Time (h)

300 250 200 150 100

Proteolytic activity

50

Spores (CFU ml–1 × 103)

Viable counts (CFU ml–1 × 106)

(b)

50 0

96

Figure 2 Production of extracellular proteolytic activity during the cellular growth curve of Paenibacillus peoriae (a) and Paenibacillus polymyxa (b) in thiamine–biotin–nitrogen broth medium for 24–96 h at 32C. (d) Viable counts, ( ) spores and (h) proteolytic activity [arbitrary units (AU) · mg of protein].

spore numbers, but the best proteolytic activity peak was detected at 72 h (Fig. 2b). In contrast to quantitative changes along the 96 h of cultivation of both strains of the Paenibacillus species studied, no qualitative modifica-

tion was observed in the extracellular proteolytic profile when assessed by gelatin-SDS-PAGE, as the 20, 35, 50 and 210 kDa proteolytic bands were observed during the 96 h of growth (data not shown). Aiming to characterize these proteases more fully, we tested their activities in gelatin-containing gels in the presence of proteolytic inhibitors. The results showed that the pattern of extracellular proteolytic activities expressed in both Paenibacillus strains grown in TBN is composed exclusively of metalloproteases, displaying total sensibility to 1,10-phenanthroline (a powerful zinc-metalloprotease inhibitor) (Fig. 3a). Conversely, two other metalloprotease inhibitors (EDTA and EGTA) did not significantly interfere with the protease activities. Likewise, cysteine (E-64) and serine (PMSF) protease inhibitors were also tested but did not present any inhibitory activity (Fig. 3a). Additionally, all the proteases detected in the other four media were also restrained by 1,10-phenanthroline (data not shown). The two major extracellular proteolytic activities (20 and 35 kDa), evidenced during P. polymyxa and P. peoriae growth, were observed over a broad pH range (5Æ0– 10Æ0) (Fig. 3b). Furthermore, in the range of temperature between 25 and 50C, the proteolytic enzymes released by the strains studied here were observed without any detectable loss of activity. Conversely, when the gels were incubated at 4 or 60C, no proteolytic activity was visualized (data not shown). Alternatively, the proteolytic enzymes remained active after incubation of the concentrated supernatants for 60 min at temperatures varying from 4 to 50C. After incubation of the supernatants at 65C for 60 min, a residual activity (c. 25%) of the proteolytic enzymes of 20 and 35 kDa was detected (data not shown).

(a)

(b)

209

209

47

47

34 29

34 29

18

18 control PHEN EDTA EGTA

E-64 PMSF

5·0

7·0

10·0

Figure 3 The effect of different proteolytic inhibitors and pH on the extracellular proteases of Paenibacillus peoriae after growth in thiamine–biotin–nitrogen medium for 48 h. (a) The gel strips were incubated in the absence (control) and presence of 10 mmol l)1 1,10-phenanthroline (PHEN), 10 mmol l)1 EDTA, 10 mmol l)1 EGTA, 10 mmol l)1 PMSF and 10 lmol l)1 E-64. (b) The gel strips were incubated in the following buffers: 50 mmol l)1 sodium-phosphate buffer supplemented with 2 mmol l)1 dithiothreitol (DTT), pH 5Æ0, 10 mmol l)1 phosphate-buffered saline, pH 7Æ0, and 20 mmol l)1 glycine-NaOH, pH 10Æ0. Similar results were observed when Paenibacillus polymyxa derived-culture supernatant was tested (data not shown). The numbers on the left indicate the molecular masses.

628


V.M. Alvarez et al.

Discussion Proteases are a well-known group of hydrolytic enzymes that catalyse the hydrolysis of proteins and/or peptides. Besides their physiological importance, they constitute one of the important groups of industrial enzymes, as they account for at least one-quarter of the global enzyme production (Rao et al. 1998). Additionally, several Paenibacillus strains are being tested as potential biocontrol agents because they are soil-inhabiting bacteria capable to form resistant spores, fix molecular nitrogen and produce several hydrolytic enzymes (Dunn et al. 1997; Budi et al. 2000; von der Weid et al. 2003). Generally, proteases produced from micro-organisms are constitutive or partially inducible in nature and strongly influenced by medium components, such as variation in carbon/nitrogen ratio, presence of some easily metabolizable sugars, such as glucose (Beg et al. 2002) and metal ions (Varela et al. 1997). Protease synthesis is also affected by rapidly metabolizable nitrogen sources, such as amino acids in the medium. Besides these, several other physical factors, such as aeration, inoculum’s density, pH, temperature and incubation, also affect the amount of protease produced (Puri et al. 2002). Here, we showed that P. peoriae and P. polymyxa released distinct proteases that were directly dependent on the growth medium used. Proteases are largely produced during postexponential and stationary phases and thus are generally regulated by carbon and nitrogen stress. Besides, proteases are known to be associated with the onset of the stationary phase, which is marked by the transition from the vegetative growth to the sporulation stage in spore-formers. Therefore, protease production is often related to the sporulation stage in many Bacillus species, such as B. subtilis (O’Hara and Hageman 1990) and B. licheniformis (Hanlon and Hodges 1981). Our results also suggested the association between sporulation and extracellular metalloprotease expression in P. peoriae. Metalloproteases are the most diverse of the catalytic types of proteolytic enzymes. They are characterized by requiring a divalent metal ion for their activity. They include enzymes from a variety of origins such as collagenases from higher organisms and thermolysin from bacteria. Most metalloproteases are zinc-containing proteins. Zinc is an integral component of many proteins that are involved in virtually all aspects of metabolism of the different species of all phyla (Rao et al. 1998; Miyoshi and Shinoda 2000). The importance of thermostable proteolytic enzymes in a number of industrial processes is widely acknowledged (Rao et al. 1998). The metalloproteases produced by P. peoriae and P. polymyxa showed preferential neutral pH and were active at temperatures up to 50C. Similarly, strain P. polymyxa B-17 produced a thermostable extracel-


lular metalloprotease of 30 kDa, inhibited by EDTA, which presented an optimum temperature of 50C and shared significant activity at 70C (Matta and Punj 1998). Neutral-alkaline proteases constitute a very large and complex group of enzymes, with both nutritional and regulatory roles in nature. The major applications of these enzymes are in detergent formulation, food industry, leather processing, chemical synthesis and waste management. Looking into the depth of microbial diversity, there is always a chance of finding micro-organisms producing novel enzymes that have better properties and are suitable for commercial exploitation. The multitude of physicochemically diverse habitats has challenged nature to develop equally numerous molecular adaptations in the microbial world. Microbial diversity is a major resource for biotechnological products and processes. Bacteria are the most dominant group of neutral-alkaline protease producers, which has Bacillus as its most prominent source (Rao et al. 1998; Gupta et al. 2002). Hence, although microbial neutral-alkaline proteases already play an important role in industry, their potential is much greater and their application in future processes is likely to increase. In this context, the results described herein add P. peoriae and P. polymyxa to the list of neutral-alkaline protease producers. The purification and fine biochemical characterization of these enzymes are in progress in our laboratory. Acknowledgements This work was supported by grants from Fundaçaõ Universita´ria Jose´ Bonifaćio (FUJB), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and Fundaçaõ de Amparo a` Pesquisa do Estado do Rio de Janeiro (FAPERJ). References Beg, Q.K., Saxena, R.K. and Gupta, R. (2002) De-repression and subsequent induction of protease synthesis by Bacillus mojavensis under fed-batch operations. Process Biochem 37, 1103–1109. Budi, S.W., van Tuinen, D., Arnould, C., Dumas-Gaudot, E., Gianinazzi-Pearson, V. and Gianinazzi, S. (2000) Hydrolytic enzyme activity of Paenibacillus sp. strain B2 and effects of the antagonistic bacterium on cell integrity of two soil-borne pathogenic fungi. Appl Soil Ecol 15, 191–199. Buroker-Kilgore, M. and Wang, K.K.W. (1993) A Coomassie brilliant blue G-250-based colorimetric assay for measuring activity of calpain and other proteases. Anal Biochem 208, 387–392.


629


Dunn, C., Delany, I., Fenton, A. and O’Gara, F. (1997) Mechanisms involved in biocontrol by microbial inoculants. Agronomie 16, 721–729. Ferreira, E.C.N. and Seldin, L. (1993) Characterization of Bacillus polymyxa isolates from different Brazilian soils. Rev Microbiol 24, 151–155. Gennari, F., Miertus, S., Stredansky, M. and Pizzio, F. (1998) Use of biocatalysts for industrial applications. Genet Eng Biotechnol 3–4, 14–23. Glick, B.R., Patten, C.L., Holguin, G. and Penrose, D.M. (1999) Biochemical and Genetic Mechanisms Used by Plant Growthpromoting Bacteria. London: Imperial College Press. Gupta, R., Beg, K.Q. and Lorenz, P. (2002) Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnol 59, 15–32. Hanlon, G.W. and Hodges, N.A. (1981) Bacitracin and protease production in relation to sporulation during exponential growth of Bacillus licheniformis on poorly utilized carbon and nitrogen sources. J Bacteriol 147, 427–431. Kajimura, Y. and Kaneda, M. (1997) Fusaricidins B, C, and D, new depsipeptide antibiotics produced by Bacillus polymyxa KT-8. Isolation, structure elucidation and biological activity. J Antibiot 50, 220–228. Lowry, O.H., Rozebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193, 265–275. Matta, H. and Punj, V. (1998) Isolation and partial characterization of a thermostable extracellular protease from Bacillus polymyxa B-17. Int J Food Microbiol 42, 139–145. Miyoshi, S. and Shinoda, S. (2000) Microbial metalloproteases and pathogenesis. Microb Infect 2, 91–98. Montefusco, A., Nakamura, L.K. and Labeda, P.D. (1993) Bacillus peoriae sp. nov. Int J Syst Bacteriol 43, 388–390. O’Hara, M.B. and Hageman, J.H. (1990) Energy and calcium ion dependence of proteolysis during sporulation of Bacillus subtilis cells. J Bacteriol 172, 4161–4170. Puri, S., Beg, Q.K. and Gupta, R. (2002) Optimization of alkaline protease production from Bacillus sp. by response surface methodology. Curr Microbiol 44, 286–290.

630

V.M. Alvarez et al.

Rao, M.B., Tanksale, A.M., Ghatge, M.S. and Deshpande, V.V. (1998) Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev 62, 597–635. Santos, A.L.S., Abreu, C.M., Alviano, C.S. and Soares, R.M.A. (2005) Use of proteolytic enzymes as an additional tool for trypanosomatid identification. Parasitology 130, 79–88. Schleif, R.F. and Wensink, P.C. (1981) Practical Methods in Molecular Biology. New York: Springer-Verlag. Seldin, L., van Elsas, J.D. and Penido, E.G.C. (1983) Bacillus nitrogen fixers from Brazilian soils. Plant Soil 70, 243–255. Seldin, L., Azevedo, F.S., Alviano, D.S., Alviano, C.S. and Bastos, M.C.F. (1999) Inhibitory activity of Paenibacillus polymyxa SCE2 against human pathogenic micro-organisms. Lett Appl Microbiol 28, 423–427. Timmusk, S. (2003) Mechanism of Action of the Plant Growth Promoting Bacterium Paenibacillus polymyxa. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology. Uppsala: Acta Universitatis Upsaliensiensis, p. 908. Varela, H., Ferrari, M.D., Belobradjic, L., Vasquez, A. and Loperena, M.L. (1997) Skin unhairing proteases of Bacillus subtilis: production and partial characterization. Biotechnol Lett 19, 755–758. von der Weid, I., Duarte, G.F., van Elsas, J.D. and Seldin, L. (2002) Paenibacillus brasilensis sp. nov., a novel nitrogenfixing species isolated from the maize rhizosphere in Brazil. Int J Syst Evol Microbiol 52, 2147–2153. von der Weid, I., Alviano, D.S., Santos, A.L.S., Soares, R.M.A., Alviano, C.S. and Seldin, L. (2003) Antimicrobial activity of Paenibacillus peoriae strain NRRL BD-62 against a broad spectrum of phytopathogenic bacteria and fungi. J Appl Microbiol 95, 1143–1151. Yanga, J.K., Shihb, I.L., Tzengc, Y.M. and Wanga, S.L. (2000) Production and purification of protease from Bacillus subtilis that can deproteinize crustacean wastes. Enzyme Microb Technol 26, 406–413.
