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Isolation of a psychrotrophic Azospirillum sp. and characterization of its extracellular protease

Kun-Hee Oh, Chang Soo Seong, Soo Woong Lee, O-Seob Kwon, Young Shik Park
DOI: http://dx.doi.org/10.1111/j.1574-6968.1999.tb13565.x 173-178 First published online: 1 May 1999


A novel psychrotrophic bacterium secreting a protease was isolated from a mountain soil in Korea. On the basis of a 16S rDNA sequence analysis and physiological properties, the isolate was identified as an Azospirillum sp. The protease purified from the culture supernatant was a monomer in its native form with an apparent molecular mass of 48.6 kDa on SDS-PAGE. The protease was active in a broad pH range around 8.5 and at temperatures up to 40°C and stable at temperatures below 30°C for 3 days. The proteolytic activity was inhibited by iodoacetamide and EDTA. The Mg2+ ion did not activate the enzyme much but reversed the inhibition by EDTA, suggesting that the protease belongs to a cysteine protease stabilized by the Mg2+ ion.

  • Psychrotroph
  • Azospirillum sp.
  • 16S rRNA sequence
  • Extracellular protease
  • Purification

1 Introduction

Microorganisms growing at low temperatures are important for their metabolic contribution in the ecosphere as well as for their enzymes with potential industrial applications [1, 2]. Considerable attention has been given to proteases and lipases because of their commercial value. Their relatively low optimal temperatures are potentially useful in some industrial applications for food processing, detergent additives and biotransformation of chemicals [2]. Extracellular proteases have been found in several kinds of psychrophilic or psychrotrophic organisms, which include Escherichia freundii [3], Xanthomonas maltophilia [4], Candida humicola [5] and Pseudomonas [68]. In the course of screening useful cold-adapted bacteria, we have isolated from alpine soil another psychrotrophic bacterium of Azospirillum sp. producing a protease. In this paper, we describe the characteristics of the isolate as well as the purification and properties of the protease. This is the first report on the psychrotrophic Azospirillum sp. producing protease.

2 Materials and methods

2.1 Isolation of a psychrotrophic bacterium producing extracellular protease

A soil sample was collected from the mountain valley called Ulumgol (meaning ice-valley in Korean) (Milyang, Korea), where the temperature is maintained below 4°C all the year along. The sample was diluted in ice-cold water, spread on LB medium plates and incubated in a cold room for 2 weeks. The colonies were examined for the secretion of protease by inoculating on a separate LB agar medium containing 10% skim milk. Bacterial colonies exhibiting the largest clear zones were isolated and further examined. All the manipulations were performed in a cold room. The isolate was maintained on a LB agar plate at 4°C.

2.2 Identification of the isolate

Taxonomic properties were examined by methods previously described [9]. N2-fixation was examined in semi-solid nitrogen-free malate medium supplemented with bromothymol blue. The molecular identification of the isolate was achieved by 16S rRNA sequencing. The 16S rRNA gene was amplified by PCR using degenerate primers designed by Moyer et al. [10]. The forward primer sequence was AGAATTCTNANACATGCAAGTCGAICG, the reverse primer was GTGGATCCGGYTACCTTGTTACGACTT. EcoR1 and BamH1 recognition sequences (underlined) are attached to the 5′-ends of both primers, respectively. Amplified rDNA was cloned in pBS SK+ (Stratagene), subcloned after restriction enzyme digestion and sequenced by the dideoxy method at both ends.

2.3 Purification of protease

For protease production, batch cultures in LB broth were grown at 20°C for 24 h with shaking at 200 rpm. The extracellular protease in the cell-free culture supernatant was obtained by ammonium sulfate precipitation to a final concentration of 80%. The protein precipitate was dissolved in a minimal volume of 50 mM Tris-HCl, pH 8.0 and applied to a column (2.5×41 cm) of Sephadex G-100 which was equilibrated with 20 mM Tris-HCl, pH 8.0. The column was developed with the same buffer at a flow rate of 15 ml h−1. Active fractions for the proteolytic activity were combined and applied to a DEAE-cellulose column (2.5×6 cm), pre-equilibrated with the same buffer. The column was washed with 50 mM NaCl in the buffer until the absorbency at 280 nm reached zero and eluted with 0.15 M NaCl at a flow rate of 15 ml h−1. Active fractions were analyzed for their purity by SDS-polyacrylamide gel electrophoresis (PAGE) and combined for concentration by ultrafiltration (Amicon, YM30 membrane). The concentrated protein (0.32 mg ml−1) was dialyzed twice against 2 l of 20 mM Tris-HCl, pH 8.0 and stored at −70°C. All the procedures were performed in a cold room. Protein was measured with the Bradford reagent [11] using bovine serum albumin as a standard.

2.4 Assay of the proteolytic activity

The protease activity was determined with casein as a substrate. The reaction mixture contained 50 mM Tris-HCl, pH 8.5, 2 mg ml−1 of casein and an enzyme solution. The mixture was incubated at 30°C for 25 min and stopped by the addition of an equal volume of 10% trichloroacetic acid solution. After 5 min in ice-cold water, the acid-soluble fragments in the supernatant were measured for absorbency at 278 nm. One unit of enzyme activity was defined as the increase of 0.1 in absorbency min−1 under the standard assay conditions.

2.5 PAGE

SDS-PAGE was carried out by the method of Laemmli [12]. Isoelectric focusing was performed in 5% acrylamide containing 2% pharmalyte 2D (pH 3–10) and 10% glycerol in glass tubes. Purified protein was applied on the gel and run for 6 h at a constant voltage of 500. The focused gel was stained for protein or sliced in 1-cm lengths for measurement of the pH after 2 h in water. For visualization of the protease activity in the gel, casein-impregnated SDS-PAGE was done in 12.5% acrylamide gel co-polymerized with 0.5% casein [13]. After electrophoresis, the gel was immersed in 2.5% Triton X-100 for 1 h to remove SDS. The enzymatic reaction was allowed to occur by incubating the gel in 50 mM Tris-HCl, pH 8.5 for 2 h at room temperature. The gel was stained in 0.18% amido black and 0.2% Coomassie brilliant blue in 50% methanol, 12.5% acetic acid.

3 Results and discussion

3.1 Isolation and characterization of a psychrotrophic Azospirillum sp.

Among several colonies, one generating the largest clear zone of hydrolysis on the skim milk-supplemented LB medium at 4°C was further purified and characterized. The growth of the organism to the stationary phase was observed after 5 days at 4°C and after 15 h at 30°C. However, growth was not observed at all at 37°C. Therefore, the isolate belongs to the psychrotrophs according to the classification of microorganisms living at low temperatures [14].

By determining the 16S rRNA sequence of the isolate (GenBank accession number AF112477), a high homology with Azospirillum sp3 (GenBank number Z29622) (98.8% similarity within the determined sequence of 1457 nucleotides) was found as well as a significant level of homology with Pseudomonas 16S rDNA. The sequence of the isolate was then aligned and compared with previously published sequences of the genera Azospirillum and Pseudomonas and a neighbor-joining phylogenetic tree was constructed on the basis of the sequence similarity matrix (Fig. 1). Distinct from the other species of Azospirillum, the isolate constitutes a separate cluster together with Azospirillum sp3 within the group of Pseudomonas sp. The isolate was a motile Gram-negative vibroid form, fermentative for glucose but not lactose or sucrose, positive in catalase and oxidase tests and able to fix N2 in nitrogen-free malate medium. According to Bergy's manual [15], the genus Azospirillum has a weak fermentative ability, whereas strictly respiratory types of metabolism belong to the genus Pseudomonas. Therefore, it was concluded that the isolate is a novel species of Azospirillum. So far as we know, the isolate is the first psychrotroph identified in the genus Azospirillum.


Phylogenetic tree for the psychrotrophic Azospirillum sp. and its relatives constructed by using 16S rRNA gene sequences. The genus names Azospirillum and Pseudomonas are abbreviated as A. and P., respectively. GenBank accession numbers are described in the parentheses. The isolate (A. sp., AF112477) is indicated by an arrow. Numbers at the nodes are the bootstrap values obtained after 100 replicates. The scale bar indicates a distance corresponding to 0.1% difference between sequences.

3.2 Purification and characterization of the extracellular protease

The isolate constitutively expressed an extracellular protease independently upon the presence of skim milk in the medium and growth temperatures. Purification of the protease was achieved by two consecutive fractionations on Sephadex G-100 and DEAE-cellulose columns. The purification steps were analyzed by SDS-PAGE (Fig. 2A). From a culture of 1 l, approximately 1 mg of purified protein was routinely obtained in the final yields of 3–5%. The purified enzyme was extensively dialyzed against buffer to remove salts and used for the characterization performed in this study.


SDS-PAGE and activity staining of the purified protease. (A) proteins obtained during purification procedures were electrophoresed on a 12.5% SDS-polyacrylamide gel: lane 1, ammonium sulfate fraction; lane 2, Sephadex G-100 gel chromatography fraction; lane 3, DEAE-cellulose ion exchange chromatography fraction; M, pre-stained marker proteins (Bio-Rad). (B) the purified protease was electrophoresed on a separate SDS-polyacrylamide gel containing casein, renatured and stained for activity.

The molecular mass of the purified protease on SDS-PAGE was estimated as 48.6 kDa. An in situ protease assay performed in the casein-impregnated SDS-PAGE showed a distinct single activity band (Fig. 2B), corresponding to the main band observed in the final purified fraction (Fig. 2A). In the gel permeation chromatography on Sephadex G-100, the protease was eluted in the middle of the fractionation range (Fig. 3). These results indicate that the native protease is composed of a single polypeptide chain of 48.6 kDa. The isoelectric point of the native protease was estimated as 4.5 by isoelectric focusing on the non-denaturing gel (data not shown).


The elution profile of Azospirillum protease on a column of Sephadex G-100. Ammonium sulfate precipitate of extracellular protease in the culture supernatant was applied on the column (2.5×41 cm) equilibrated with 20 mM Tris-HCl, pH 8.0 and then eluted with the same buffer at a flow rate of 15 ml h−1. Fractions of 2 ml were collected and assayed for the protein (open circle) and protease activity (closed circle).

The protease was most active at pH 8.0-8.5 towards casein with the half activity observed at pH 8.0±2.0. The effect of the temperature on the activity of the protease is shown in Fig. 4. The protease was active at temperatures up to 40°C. At 10, 20 and 30°C, the enzyme maintained 33, 55 and 75% of the activity shown at 40°C, respectively. However, the activity decreased after 5 min of incubation at 50°C. Thermal stability studies showed that the protease retained 100% of its activity when exposed for 3 days to 4, 10 and 20°C. After 24 h at 30°C, only 40% of the original activity was maintained. Thus, the protease is highly stable at temperatures below 30°C. Finally, the activity did not decrease at all after five cycles of freeze-thaw, supporting the robust nature of the protein at a low temperature.


The effect of temperature on the protease activity. The purified protease activity was measured at various temperatures (10°C, •; 20°C, ▿; 30°C, ■; 40°C, ◇; 50°C, ▲) for the indicated times.

The protease was incubated in 50 mM Tris-HCl (pH 8.0) with various chemicals at 4°C for 1 h and the remaining activity was measured with casein as the substrate at 30°C. The enzyme activity was not altered at all by 10 mM of serine protease inhibitor PMSF but was strongly inhibited by 10 mM EDTA, 0.1% SDS, 10 mM DTT and 10 mM iodoacetamide (Table 1). The protease was activated a little by Mg2+ and Ca2+ but was inhibited by Mn2+, Cu2+ and Zn2+ (Table 2). The protease was incubated with 1 mM EDTA as described above to remove endogenous metal ions in the protein and then assayed for activity with 10 mM of each metal ions (Table 2). The inhibitory effect of EDTA was reversed near completely by the addition of the Mg2+ ion. Although Ca2+ also reversed the inhibition, it was less effective than Mg2+ and furthermore, the inhibitory effect of the Ca2+ ion-specific chelator EGTA was not significant (Table 1). Therefore, it was suggested that the enzyme is a cysteine protease, stabilized by the Mg2+ ion.

View this table:

Effects of various compounds on the activity of the protease

View this table:

Effect of metal ions on the activity of protease

This is the first report on the isolation of a psychrotrophic Azospirillum sp. and its extracellular protease. The protease was active as well as stable at low temperatures below 30°C, which could be useful properties for a possible application in the industry. More investigation should be followed to better characterize the protein, such as substrate specificity, amino acid analysis, gene cloning, etc.


  • 1 Department of Genetic Engineering, Kyung Hee University, Yongin 449-701, Korea.


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