Production of an extracellular protease by an Antarctic bacterial isolate (Bacillus sp. JSP1) as a potential feed additive¤

Proteasa extracelular de un aislado bacteriano Antartico (Bacillus sp. JSP1) con uso potencial como aditivo alimenticio para animales

Produção de proteasa extracelular por bactérias antárcticas isoladas, Bacillus sp. JSP1 como um aditivo potencial em concentrado


Inkyung Park1, Animal Scientist, PhD ; Jaiesoon Cho2*, Feed Biotechnologist, PhD.

1Animal Resources Research Center, College of Animal Bioscience and Technology, Konkuk University, 1 Hwayang-dong,  Gwangjin-gu, Seoul 143-701, Korea

2Department of Animal Sciences and Environment, College of Animal Bioscience and Technology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Korea.

(Recibido: 29 julio, 2010; aceptado: 18 enero, 2011)



Extracellular proteolytic activity was found in JSP1, an Antarctic bacterial isolate. The strain was related to Bacillus sp, based on 16S rRNA gene sequence analysis. The JSP1 protease was partially purified by ammonium sulfate precipitation. Optimal enzyme activity occurred at 40 oC and pH 7.4. Enzyme activity was significantly enhanced in the presence of Mg2+ and Ca2+ and was completely inactivated in presence of Cu2+, Zn2+, Hg2+, EDTA and SDS. The enzyme hydrolyzed casein the most effectively among the protein substrates tested. The enzyme also exhibited relatively high activity on keratin and gluten, and was active against peptidyl conjugates such as  L-Leu-p-Nitroanilide and N-Succinyl-L-Phe-p-Nitroanilide. This study suggests that JSP1 protease could be utilized as a potential environmentally-friendly feed additive in animal production.

Key words: antarctic, Bacillus sp., feed additive, keratin, livestock production, protease.



Se reporta haber encontrado actividad proteolítica extracelular en una bacteria antártica denominada JSP1. Con base en el análisis de secuencia genética 16S ARNr, la cepa fué relacionada con Bacillus sp. La proteasa JSP1 fué parcialmente purificada por precipitación con sulfato de amonio. La actividad óptima de la enzima se produjo a 40 ºC y pH 7,4. La actividad enzimática fue significativamente mayor en presencia de Mg2+ y Ca2+ y se inactivó completamente en presencia de Cu2+, Zn2+, Hg2+, EDTA y SDS. Entre todos los sustratos ensayados, el más eficientemente hidrolizado por la enzima fue la caseína. La enzima tuvo actividad relativamente alta sobre la queratina y el gluten, y participó activamente contra conjugados peptídicos como la L-Leu-p-nitroanilida y la N-succinil-L-fenilalanina-p-nitroanilida. La enzima podría ofrecer potencial para su uso como aditivo alimenticio ecológico en producción animal.

Palabras clave: antártida, aditivo alimenticio, Bacillus sp., producción animal, proteasa queratina.



A actividade proteolítica extracelular foi encontrada a partir de uma bactéria antárctica denominada JSP1, mediante uma análises de sequencia do gene 16S rRNA, a cepa foi relacionada para Bacillus sp. A proteasa JSP1 foi parcialmente purificada por precipitação com sulfato de amônia. Uma óptima actividade enzimática ocorreu em 40 oC e pH 7,4. A actividade da enzima foi significativamente maior na presença de Mg2 + e Ca2 +, e foi completamente  inactivada em presença de Cu2 + Zn2 +, Hg2 +, EDTA e SDS. A enzima hidrolisada de caseína foi mais eficaz entre os substratos de proteína testados, apresentando maior actividade na queratina e no glúten e foi activo contra os peptidil conjugados como L-Leu-p Nitroanilida e N-succinil-L-Phe-p-Nitroanilida. A enzima pode ser utilizado como aditivo ambiental na alimentação animal.

Palavras chave: antarctic, Bacillus sp., feed additive, keratin, livestock production, protease.


Proteases have attracted a great deal of attention due  to  their  broad  range  of  applications  in  the detergent,  food,  pharmaceutical,  chemical,  leather, paper,  and  pulp,  and  silk  industries  (Rai  and Mukherjee,  2009).  Microbial  proteases  comprise  approximately  40%  of  the  worldwide  production of  enzymes  (Jaouadi  et al.,  2008).  In  the  field  of animal  nutrition,  exogenous  proteases  have  been used  as  feed  additives.  For  instance,  proteases help  degrade  soybean  proteins  such  as  glycinin and  "-conglycinin,  along  with  some  protein  anti-nutritional  factors  (lectins  and  trypsin-inhibitors) in  inadequately  processed  soybean  meal  (Thorpe and  Beal,  2001).  Moreover,  the  combined  use  of proteases  together  with  carbohydrate-degrading enzymes such as xylanase and amylase was found to enhance the nutritional value of a corn and soybean meal-based  diet  for  poultry  (Hong  et al.,  2002; Marsman et al., 1997).

In  particular,  Bacillus species,  a  type  of exogenous  spore-forming  bacteria,  are  proli! c producers  of  extracellular  proteases  (Rao  et al., 1998).  Currently,  Bacillus licheniformisBacillussubtilis,  and  Bacillus pumilus  are  well-known species  employed  industrially  for  alkaline  protease production  (Gupta  et al.,  2002). Although  they do not commensally  colonize  the  gastrointestinal  tract, Bacillus species have been  shown  to be  effective  in maintaining a  favorable balance of micro# ora  in  the gastrointestinal tract and in improving the production performance  of  farm  animals  (Alexopoulos  et al., 2004; Kritas and Morrison, 2005).

Some  useful  and  unusual  enzymes  have  been reported  from  so-called  extremophiles  inhabiting Antarctica  (Demirjian et al., 2001). Considering  that the number of microbes cultured to date remains only  a  tiny  fraction of  all microbial  species on  earth,  the  number  of  novel  enzymes  is  expected  to  increase continuously  (Park  et al.,  2007).  In  this  report,  data are  presented  concerning  general  properties  of  extracellular  proteolytic  activity  derived  from  an  Antarctic bacterial isolate, Bacillus sp. JSP1.


Materials and methods
Bacterial strain and culture conditions A bacterial isolate, JSP1, derived from Antarctic soil  samples  was  supplied  from  Korea  Polar  Research  Institute,  operating  the  King  Sejong  Station (South Korea) in Antarctica. Screening for protease activity was performed on selective agar plates [1.0% skim milk (Sigma), 0.45% (NH4)2SO4, 0.05% yeast extract (Difco), 0.07% KH2PO4, 0.01% MgSO47H2O, 0.01% NaCl, 0.01% CaCl22H2O, 0.001% MnSO44H2O, 0.001% FeSO47H2O, and 1.5% bacto agar (Difco), pH 7.4] at 28 oC by observing a clear zone of hydrolyzed casein around the colonies, as previously described (Hutadilok- Towatana et al., 1999).

Growth and protease production were investigated in 100 mL of protease production medium [0.5% skim milk, 0.5% yeast extract, 0.07% KH2PO4, 0.01% NaCl, 0.01% CaCl22H2O, 0.01% MgSO47H2O, 0.001% MnSO44H2O and 0.001% FeSO47H2O (pH 7.4)] in a 500 mL Erlenmeyer ! ask, aerobically incubated with vigorous shaking (220 rpm), by monitoring the absorbance (O.D.600nm) for the cell growth and protease activity of the culture supernatant at 28 oC at various time points.

Taxonomic identification of strain JSP1

Genomic DNA was extracted from strain JSP1 using a FastDNA kit (Qbiogene) according to the manufacturer.s protocol. The 16S rRNA gene was ampli" ed from genomic DNA by PCR using the universal primers 27F (5.-AGAGTTTGATCCTGGCTCAG-3.) and 1492R (5.-GGTTACCTTGTTACGACTT-3.) (William et  al., 1991). The ampli" ed 1,427 bp sequences were determined by an automated ABI PRISM 3730 XL DNA analyzer (Applied Biosystems). The resulting sequences were compared with the GenBank database (NCBI) using BLAST (Altschul et  al., 1990). Sequences showing a relevant degree of similarity were imported into the CLUSTAL W program (Thompson et  al., 1994) and aligned. The evolutionary distances to other Bacillus  strainswere computed using the Maximum Composite Likelihood method (Tamura et  al., 2004), and the phylogenetic relationships were determined using the software MEGA, version 4.0 (Tamura et al., 2007).

Nucleotide sequence accession numbers

The nucleotide sequence of the 16S rRNA gene has been deposited in the GenBank database under Accession No. GU014529.

Partial purification of the enzyme

One liter of protease production medium [0.5% skim milk, 0.5% yeast extract, 0.07%
KH2PO4, 0.01% NaCl, 0.01% CaCl22H2O, 0.01% MgSO47H2O, 0.001% MnSO44H2O and 0.001% FeSO47H2O (pH 7.4)] in two 2 L Erlenmeyer ! asks was aseptically inoculated with a single colony of strain JSP1 and aerobically cultivated with vigorous shaking (220 rpm) for 96 h at 28 oC. The culture medium containing secreted protease was centrifuged at 9000 g for 30 min at 4 oC to remove the cells, and proteins in the supernatant were then precipitated with ammonium sulfate (75% saturation). The pellet was dissolved in 50 mM Tris- HCl (pH 8.0) and dialyzed overnight against 50 mM Tris-HCl (pH 7.4) at 4 oC. The dialyzed solution was used as the protease source throughout this work to examine its catalytic properties.


Native Polyacrylamide Gel Electrophoresis (PAGE) was carried out with a Modular Mini-Protein II Electrophoresis System (Bio- Rad, Hercules, CA, USA) according to the  manufacturer.s instructions. Zymograms (0.1% casein in 10% polyacrylamide) were run for 6 h at 4 oC and 60 V in buffer containing 25 mM Tris-HCl (pH 8.0) and 125 mM glycine. After electrophoresis, the gel was incubated overnight at room temperature in calcium proteolysis buffer (20 mM Tris-HCl, 20 mM CaCl2 ; pH 7.4) under gentle shaking. The gel was stained with SimplyBlueSafeStain (Invitrogen) for 30 min and destained overnight. The bands of caseinolytic activity appear white on a blue-stained background.

Preparation of keratin substrate

Keratin substrate was prepared from chicken feathers by the modi" cation method of Nam et  al. (2002). Brie! y, ground chicken feathers (1 g) in 50 mL of dimethyl sulfoxide, were solubilized by heat treatment on a hot plate at 90 oC for 2 h. Soluble keratin was then precipitated by addition of cold acetone (200 mL) at -70 oC for 2 h followed by centrifugation at 10000 g for 20 min. The precipitate was washed twice with distilled water and then dissolved in 10 mM Tris-HCl buffer (pH 9.0).

Enzyme assay and general catalytic properties

Unless otherwise stated, the assay was performed at 40 oC for 1 h in a reaction mixture containing 460 µL of 50 mM Tris-HCl (pH 7.4), 140 µL of 3% azocasein as a nonspecific substrate, and 100 µL of enzyme. The reaction was terminated by addition of 700 µL of 10% trichloroacetic acid. One unit of the azocaseinolytic activity was de ned as the amount of enzyme required to produce an increase in absorbance at 366 nm of 0.01 per minute under the given assay conditions. Protease substrate speci city was examined with case in (Sigma, St. Louis, MO, USA), gelatin (Sigma, St. Louis, MO, USA), collagen (Sigma, St. Louis, MO, USA), bovine serum albumin (Sigma, St. Louis, MO, USA), gluten (Sigma, St. Louis, MO, USA), and chicken feather keratin by a modi ed method of Wang et  al. (2005). Brie! y, 200 µL of enzyme was added to a reaction mixture containing 3% of each substrate in 360 µL of 50 mM Tris-HCl (pH 7.4) and incubated at 40 oC. The reaction was stopped by adding 700 µL of 10% trichloroacetic acid and centrifuged at 10000 g for 10 min.

The protein remaining in the supernatant was determined by Folin-phenol reagent (Folin and Ciocalteau, 1927). One unit of protease activity was de ned as the amount of nzyme that liberated 1µg of tyrosine per minute under the de ned assay conditions. Peptidolytic activity was also assayed at 40 oC using 2 mM each of L-Leu-p-Nitroanilide, N-Succinyl-L-Ala-Ala-Ala-p-Nitroanilide, and N-Succinyl-L-Phe-p-Nitroanilide as substrates in 50 mM Tris-HCl (pH 7.4). The amount of p-Nitroaniline liberated was determined from the  samples. absorbance at 405 nm.

Determination of pH and temperature optima on  protease activity

To study the temperature optimum and enzyme activity, the enzyme reaction mixture was  incubated at different temperatures from 0 to 80 oC in 50 mM Tris-HCl (pH 7.4) buffer  sing azocasein as a substrate. The pH optimum for protease activity with azocasein substrate was determined at 40 oC in 50 mM glycine-HCl (pH 3), 50 mM sodium acetate (pH 4-5), 50 mM Bis-Tris-HCl (pH 6-7), and 50 mM Tris-HCl (pH 7.4-9.0) buffers.

Effect of reagents on enzyme activity

The effects of metal ions and inhibitors on protease activity were examined with azocasein as a substrate. Each additive (5 mM) was pre-incubated with the enzyme for 30 min at 40 oC before the standard assay was performed, and the residual activity was measured.



Identification of isolated strain JSP1, protease production and partial purification of the enzyme

To identify the isolated strain JSP1 that shows protease activity (Figure 1), we cloned its 16S rRNA gene and compared the sequence with those available in the  database. A phylogenic tree based on the 16S rRNA gene sequences from 10 bacterial Bacillus  strains show ed that the JSP1 strain shared 99.7% sequence identity with the type strain, Bacillus megaterium IAM 13418 (Figure 2).

Therefore, it was named Bacillus sp. JSP1.

Time courses of cell growth and extracellular protease activity were shown in figure 3. Protease activity was nearly proportional to cell growth during cultivation. The enzyme was steeply produced after 16 h of incubation, showing a maximum activity (21.5 ± 0.1 U/mL) at 60 h of incubation.
The partial purification pro le of the extracellular protease produced by Bacillus sp. JSP1 was summarized in table 1.

Effect of pH and temperature on enzyme activity

Optimal protease activity occurred at pH 7.4, while over 60% of the peak activity was achieved between pH 6.0 and 8.0. However, activity was nearly completely inactivated at acidic pH (pH 3.0-5.0) and above pH 8.5 (Figure 4A). As shown in figure 4B, JSP1 protease showed optimal activity at 20 . 40°C and retained more than 35% of the activity at 5 . 50°C.


Substrate specificity

JSP1  protease  hydrolyzed  casein  the  most  effectively  among  the  protein  substrates  tested (Table 2). The  enzyme  also  showed  relatively high  activity  on  keratin,  which  is  the  most  abundant  structural  protein  in  skin,  hair,  wool,  and  feathers  (Tatineni et al., 2008) and gluten, which  is a useful  protein  source  in  cattle  feed  (Firkins  et  al.,  1985; Ohajuruka  and  Palmquist,  1989).  Low  levels  of hydrolysis  were  observed  with  gelatin,  collagen,  and  bovine  serum  albumin  (Table  2).  Among  the  peptidyl-p-Nitroanilide  substrates  tested,  the  enzyme  was  active  against  L-Leu-p-Nitroanilide and  N-Succinyl-L-Phe-p-Nitroanilide  which  is  cleaved  by  subtilisin-like  and  chymotrypsin-like  enzymes  (Hutadilok-Towatana  et  al.,  1999),  and  exhibited no activity on N-Succinyl-L-Ala-Ala-Ala- p-Nitroanilide.


Effect of various reagents on enzyme activity

The  protease  activity  in  the  presence  of  various metal  ions  or  chemicals  was  shown  in  figure  5. Amongst the metal ions, Mg2+ and Ca2+ were highly effective  at  stimulating  JSP1  protease,  increasing activity by 40% and 55%, respectively. In contrast, enzyme activity  was significantly  reduced  (50-78% of the control activity) in the presence of Fe2+, Co2+, and  Ni2+.  Although  Cu2+,  Zn2+,  and  Hg2+  almost completely  inactivated  the  enzyme,  no  important effect  on  the  activity was  observed with Mn2+  and Ba2+. The  enzyme was  also  completely  inactivated by EDTA  and  Sodium Dodecyl  Sulfate (SDS),  but moderately  inhibited  by  Phenyl  Methyl  Sulfonyl Fluoride (PMSF).



In  the  present  study,  the  extracellular  protease secreted  by  an  Antarctic  strain,  Bacillus  sp. JSP1,  was  partially  characterized,  and  most  of  its properties  were  found  to  be  distinct  from  those  of other proteases from Bacillus strains.

The  JSP1  protease  is  nearly  a  neutral  protease, with an optimal pH of 7.4, while most known Bacillus species produce commercial proteases that are highly active at pH 7.0 and 11.0, with an optimum around pH 8.0-10.0  (Davail  et al., 1994; Hutadilok-Towatana  et al., 1999; Jaouadi et al., 2008).

The  optimum  temperature  of  40  oC  for  the protease  is  similar  to  the  temperature  (35  oC)  for the  serine  protease  of  the  psychrophilic  bacterium, Colwellia sp. NJ341 (Wang et al., 2005). Moreover, the  JSP1  protease  maintains  30%  of  its  highest activity  at  0  oC,  which  is  one  of  the  typical characteristics found in cold-active enzymes (Wang et al., 2005; Zhang and Zeng, 2008).

The  JSP1  protease  showed  the  highest  activity on  casein,  much  like  the  alkaline  serine  proteases from  Bacillus  pumilus CBS  (Jaouadi  et  al.,  2008), Bacillus  stearothermophilus  F1  (Rahman  et  al., 1994) and Bacillus sp. KSM-K16 (Kobayashi et al., 1995).  Unexpectedly,  the  enzyme  could  hydrolyze keratin  which,  like  other  insoluble  proteins,  is  an unacceptable  substrate  for  common  proteases  such as  trypsin  and  pepsin  (Letourneau  et  al.,  1998; Papadopoulos  et  al.,  1986). In  the  animal  feed industry, feather waste can be a potential alternative to  more  costly  dietary  ingredients  for  animal feedstuffs  (Shih,  1993).  Worldwide,  commercial poultry  processing  produces  millions  of  tons  of feathers  per  year, which  are  currently  converted  to feather  meal  through  steam  pressure  and  chemical treatment  (Shih,  1993).  Although  chemical treatment  renders  keratin  waste  more  digestible, it  is  high-priced  and  destroys  certain  amino  acids (Papadopoulos et al., 1986).

The nutritional enhancement of feather meal by the enzymatic treatment might signi! cantly improve amino acid availability of feather keratin (Odetallah et  al.,  2003).  Until  now,  known  keratinolytic enzymes have been mainly produced by mesophilic fungi  (Santos  et  al.,  1996),  actinomycetes  (B"ckle et  al.,  1995),  some  thermophilic  Bacillus  sp.  (Kim et  al.,  2001),  and  some  thermophilic  anaerobes (Nam  et  al.,  2002).  To our knowledge, little has been known about keratinolytic  activity  detected  in Antarctic Bacillus strains.

Calcium  ions  are generally known  to be  involved in  maintaining  the  activity  of  Bacillus  serine proteases  (Hutadilok-Towatana  et  al.,  1999;  Jaouadi et  al.,  2008).  However,  the  JSP1  protease  seems to  belong  to  the  metalloprotease  rather  than  to  the serine  protease  family,  because  the  activity  of  this enzyme was  strongly  inhibited  by  a  chelating  agent, EDTA, but only partially  inhibited by PMSF,  a well-known  inhibitor  of  serine  proteases  (Hutadilok-Towatana  et  al.,  1999).  This  reasoning may  be  also supported  by  the  observed Zn2+-dependent  inhibition, because  excess  zinc  inhibits  some  metalloproteases  (Auld,  1995).  It  is  interesting  that  the  enzyme  exhibited  keratinolytic  activity,  despite  the  fact  that  metalloproteases  are  not  frequently  associated with  keratinolytic  activity  (Tatineni  et  al.,  2008). The  enzyme  was  sensitive  to  anionic  SDS  addition, indicating that hydrogen bonds ay play a pivotal role in maintaining enzyme activity (Wang et al., 2005).

The  JSP1 protease may offer potential  for use as an environmentally-friendly feed dditive to improve the production performance of  farm  animals, due  to its broad substrate specificity and relatively desirable activity  levels  at  physiologically  relevant  pH  and temperature.  Additionally,  the  keratinolytic  activity of  the enzyme will help  to conduct biotechnological processes  of  the  keratinous  biomaterials  from poultry  and  leather  industries.  A  more  detailed characterization of the enzyme such as gene cloning, protein  engineering,  and  fermentation  technology  is warranted  to  maximize  the  catalytic  efficiency and productive yield of the enzyme.



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