1Department of Biology, Science Faculty, Dicle University, 21280, Diyarbakir, Turkey2Department of Primary Education, Ziya Gökalp Education Faculty, Dicle University, 21280, Diyarbakir, Turkey

*Corresponding author: fatmatpan@hotmail.com

Production, purification and characterisation ofthermostable metallo-protease from newly isolatedBacillus sp. KG5

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EurAsian Journal of BioSciencesEurasia J Biosci 9, 1-11 (2015)http://dx.doi.org/10.5053/ejobios.2015.9.0.1

AbstractBackground: Due to the importance of microbial proteases in biotechnological applications, anumber of microorganisms are being explored. The production, purification and characterisation ofextracellular metallo-proteases by producing Bacillus sp. KG5 was studied.Material and Methods: Bacterial strain KG5 was isolated from Kös (Bingöl) hot spring. The strainKG5 was identified by morphological, physiological, biochemical and 16S rRNA gene sequencing.The effects of various parameters on protease production, such as time, temperature, pH, carbonand nitrogen sources and CaCl2 were studied. The enzyme was purified by ammonium sulphateprecipitation and Sephadex G-75 gel permeation chromatography. Molecular weight was calculatedby sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymographicanalysis. The effects of some metal ions, chelators and inhibitors on enzyme activity weredetermined.Results: The optimum temperature, pH and incubation period for protease production were 40-45°C, 7.0 and 24 h, respectively. It was determined that the best nitrogen sources were yeast extractand urea, while the best carbon sources were lactose and galactose. However, glucose as a sourceof carbon was found to inhibit the production of the enzyme. The maximum enzyme productionwas increased in the presence of CaCl2. The molecular weight of purified enzyme was found to beapproximately 48 kDa. It was found that the enzyme was fully stable in the presence of 2 mM CaCl2

at 50°C after 120 min. Purified protease was significantly activated by Ca2+ and Mg2+, while it wasgreatly inhibited by Cu2+, Zn2+, Hg2+ and SDS as well as by the metal ion chelatorsethylenediaminetetraacetic (EDTA) and 1,10-phenanthroline. Phenylmethylsulfonyl fluoride (PMSF)had a little effect on the enzyme.Conclusions: Our findings suggest the potential of this isolate for protease production and that thisenzyme may be suitable for biotechnological applications.Keywords: Bacillus sp. KG5, biotechnology, protease production and characterisation.

Abbreviations: BM: Basal medium; EDTA: Ethylenediaminetetraacetic; NB: Nutrient broth; OD: Optical density;phen: 1,10-phenanthroline; PMSF: Phenylmethylsulfonyl fluoride; SDS-PAGE: sodium dodecyl sulphate-polyacrylamide gel electrophoresis; TCA: trichloroacetic acid.

Ahmetoglu N, Matpan Bekler F, Acer O, Guven RG, Guven K (2015) Production, purification andcharacterisation of thermostable metallo-protease from newly isolated Bacillus sp. KG5. Eurasia JBiosci 9: 1-11.

http://dx.doi.org/10.5053/ejobios.2015.9.0.1

Nazenin Ahmetoglu1, Fatma Matpan Bekler1*, Omer Acer1, Reyhan Gul Guven2,Kemal Guven1

©EurAsian Journal of BioSciences

Proteases are by far the most important groups of commercially and biotechnological enzymes, produced by various organisms such as bacteria, yeasts, moulds, plants and animal tissues, accounting for nearly 65% of the global industrial enzyme market (Anwar and Saleemuddin 1997, Banik and Prakash 2004, Annamalai et al. 2014). Microbial proteases, especially from Bacillus species, are the major industrial workhorses, and the use of

proteases in several applications has increased in the last decade (Joo and Chang 2006). Proteases are used in a number of applications such as bioreme-

diation, biosynthesis and biotransformation, brewing, dairy industries, detergent, diagnostics, food, meat, leather, photographic, and

INTRODUCTION

Received: January 2015Received in revised form: February 2015

Accepted: February 2015Printed: March 2015

pharmaceutical industries, in the production of protein hydrolysates, silk, and silver recovery, and in X-ray films (Joo and Chang 2006, Hmidet et al. 2009, Jellouli et al. 2011).

Many bacteria in the genus Bacillus, produce large amounts of protease extracellularly during the post-exponential and stationary growth phases into the culture medium (Lin et al. 2015). Several Bacillus species are involved in protease production i.e., Bacillus clausii (Christiansen and Nielsen 2002, Joo and Chang 2006, Oskouie et al. 2008), Bacillus cereus (Banik and Prakash 2004, Uyar et al. 2011), Bacillus circulans (Subba Rao et al. 2008, Benkiar et al. 2013), Bacillus lehensis (Joshi and Satyanarayana 2013), Bacillus licheniformis (Haki and Rakshit 2003, Sellami-Kamoun et al. 2008, Jellouli et al. 2011, Lin et al. 2015), Bacillus megaterium (Asker et al. 2013), Bacillus mojavensis (Beg and Gupta 2003, Haddar et al. 2009), Bacillus pumilus (Jaouadi et al. 2008), Bacillus sterothermophilus (Sookkheo et al. 2000), and Bacillus subtilis (Akcan and Uyar 2011, Maruthiah et al. 2013).

Extracellular microbial protease production is

known to be highly affected by media components

and conditions. In the present study, we describe the

optimal production, purification and characte-

risation of a new extracellular protease produced by

newly isolated Bacillus sp. KG5 from hot water spring

in Kös (Bingöl ) Turkey, which has not been previously

reported.

Bacterial strain and cell cultivation

The samples were collected from the mud of Kös

hot spring in Bingöl which is a city in the southeast of

Turkey and identified by Ahmetoglu et al. (2011).

Cultures were purifed from samples in the

enrichment media at 80°C, using the serial dilution

technique. In order to screen a potent proteolytic

bacteria, the selected colonies, which appeared on

the plates after incubation, were patched on skim

milk agar plate consisting of 0.1% peptone (w/v),

0.5% NaCl, 10% skim milk and 2% agar (pH 7.0) and

incubated at 60°C. Colonies with a surrounding clear

zone in skim milk agar were selected as protease

positive and streaked in a new skim milk agar plate

for further study. The isolate KG5 was characterised

morphologically, physiologically and biochemically

and using 16S rRNA gene sequence analysis. To

determine the temperature and pH range for

growth, the isolate was incubated at 15 to 75°C and

4.0-11.0 respectively. Cellular morphology was

determined by phase-contrast microscopy (Zeiss),

and colony morphology was determined by a Leica

M8 stereomicroscope using cultures grown on

Nutrient Broth (NB) agar (2%) plates for 24 h at 50°C.

Gram staining was carried out according to Dussault

(1955). Unless otherwise stated, the strain was

characterised using the modified methods of Gordon

and Pang (1973).

Genomic DNA extraction, PCR-mediated amplification of the 16S rDNA and purification of the PCR products were carried out as described by Rainey et al. (1994).

Cultures were grown in 100 mL Erlenmeyer flasks

containing 25 mL of basal medium (BM) containing

2% soluble starch, 0.2% yeast extract, 1% beef

extract, 0.02% CaCl2, and 0.01% MgSO4.7H2O. The

flasks were inoculated with 250 μL of a cell

suspension (1.1x108 cells/mL) and stirred in a water

bath at 120 rpm at 40°C for 24 h. After the desired

growth time, the cells were harvested by

centrifugation at 8,200×g for 10 min at 4°C. The

supernatant served as the enzyme source.

Enzyme assay

Proteolytic activity with azocasein (Sigma-

Aldrich) was determined according to the method of

Leighton et al. (1973). Here, 150 μL enzyme was

mixed with 250 μL 0.5% azocasein solution prepared

in 0.1 M Tris-HCl buffer pH 7.0. Then reaction mixture

was incubated at 45°C for 30 min. After the end of

incubation period, the reaction was stopped by

adding 1 mL of 10% trichloroacetic acid (TCA). After

the mixture was incubated for 15 min at 4°C, the

samples were centrifuged at 8,200×g for 10 min at

4°C. One milliliter supernatant was added in 500 μL

of 1.8 N NaOH solution and measured at 420 nm.

One Unit (U) is defined as the amount of enzyme

Ahmetoglu et al.

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EurAsian Journal of BioSciences 9: 1-11 (2015)

MATERIALS AND METHODS

that breaksdown the amino acid of 1 micromole of

azocasein per minute. All protein content was

assayed according to the Lowry method (Lowry et al.

1951).

Effect of different incubation periods on

enzyme production

In order to determine the effect of different

incubation periods on enzyme production, the

cultures were grown in BM for 3-108 h. After

incubation, growth was measured at OD470 then the

samples were centrifuged at 8,200×g; the

supernatant was used for the protease activity.

Effect of various carbon and nitrogen sources

on enzyme production

In order to determine the effect of various carbon

and nitrogen sources on enzyme production, 250 μL

fresh medium with the isolate was inoculated into

soluble starch-free BM containing 2% concentration

of various carbon sources (glucose, sucrose, maltose,

lactose, galactose, fructose and glycerol ) and 1.2%

nitrogen sources (yeast extract, beef extract,

peptone, gelatine, urea, ammonium sulphate,

ammonium chloride and tryptone) in 100 mL

Erlenmeyer.

Effect of some metal ions on the enzyme

production

For the effects of some metal ions on the enzyme

production, the isolate was inoculated into BM

medium containing 0.5% CaCl2, NaCl, MgCl2, and

MnCl2 and incubated at 40°C in a shaker for 24 h.

Growth was followed by optical density

measurements at 470 nm. The cultures were

centrifuged at 8,200×g for 10 min at 4°C and the cell-

free supernatants were used for the proteolytic

enzyme activities.

Purification procedure of protease from

Bacillus sp. KG5

The crude extract with protease activity was

precipitated by ammonium sulphate added slowly

over period of time on ice with a constant stirring up

to a final concentration of 70% (w/v). The

centrifuged precipitate (8,200×g 20 min, 4°C) was re-

dissolved in a small volume of 0.05 M pH 7.0 Tris-HCl

buffer, dialysed twice at 4°C against 1 L volume of

the same buffer overnight. The dialysed enzyme

samples were loaded on a Sephadex G-75 (1.5X30

cm) column equilibrated with 0.1 M Tris-HCl, pH 7.0,

and eluted with the same buffer at a flow rate of 3

mL/min. The peaks exhibiting protease activity were

pooled together and the purified enzyme was used

for further characterisation. All purification

procedures were carried out at 4°C.

Properties of the purified protease

Sodium dodecyl sulphate-polyacrylamide gel

(SDS-PAGE) electrophoresis and zymography

SDS-PAGE was carried out for the determination

of purity and molecular weight of the protease as described by Laemmli (1970). SDS-denatured β-

galactosidase (116 kDa), fructose phosphate (80.5 kDa), and α-amylase (58 kDa) were used as standard protein molecular weight markers. For the zymogram of protease activity, the sample was not heated before electrophoresis. After electrophoresis, the gel containing 0.1% gelatine was submerged in 0.1 M Tris-HCl (pH 7.0) containing 2.5% Triton X-100 and 5mM CaCl2 for 45 min to

remove the SDS. Then, Triton X-100 was removed by

washing the gel with 0.1 M Tris-HCl buffer (pH 7.0).

Finally, the gel was stained with Coomassie Brilliant

Blue R-250. The development of clear zones on the

blue background of the gel indicated the presence of

protease activity.

Effect of pH and temperature on the activity of

the enzyme

The effect of pH on the purified protease activity

was performed at 45°C in 0.1 M Na-phosphate buffer

(6.0-6.5), 0.1 M Tris-HCl buffer (7.0-9.0) and 0.1 M

NaOH-Glycine (9.0-11.0) for 30 min. The effect of

temperature on activity was determined by assaying

enzyme activity between 20 and 70°C for 30 min.

Thermal stability of purified enzyme

The thermostability of the purified enzyme was

tested by pre-incubating the enzyme preparation in

the presence of 2 mM CaCl2 at 50°C for 120 min and

then determining the residual activity at regular

intervals of 30 min.

Effect of metal ions, inhibitors and detergents

on protease activity

To investigate of some metal ions (Cu2+, Hg2+,

Mn2+, Zn2+, Ca2+, and Mg2+), various chelating agents

and inhibitors such as ethylenediaminetetraacetic

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Ahmetoglu et al.EurAsian Journal of BioSciences 9: 1-11 (2015)

acid (EDTA) and 1,10-phenanthroline (phen), phenyl-

methanesulfonylfluoride (PMSF) and some different

concentrations (0.1-1%) of detergents (SDS, Triton

X-100, Tween-80 and commercially detergent Alo)

on the purified protease activity, the enzyme was

pre-incubated with all agents for 15 min and then

the remaining activities were measured using the

enzyme assay under standard assay conditions.

Activity in the absence of the agents was taken as

the control (100%). Divalent metals, chelating

agents, chemicals, and detergents were dissolved in

0.1 M Tris-HCl buffer (pH 7.0). Only PMSF was

dissolved in ethanol and phen was dissolved in

methanol.

Isolation and characterisation of strain KG5

The strain KG5 was rod-shaped, Gram-positive, facultative anaerobic, motile and possessing oval and central endospores (Fig. 1). KG5 grows at a temperature of 15-45°C (optimum 40°C), meaning that it is considered a mesophilic strain. Due to the negative catalase and glucose utilisation, KG5 was found to be different from other Bacillus cereus strains (Table 1). The GenBank/EMBL/DDBJ accession number for the 16S rRNA sequence of Bacillus sp. KG5 is KP318029. The 16S rRNA gene sequence analysis of Bacillus sp. KG5 showed high pairwise sequence similarity to B. cereus ATCC 14579T (100%), Bacillus anthracis ATCC 14578T (99.9%) Bacillus thrungiensis ATCC 10792T (99.7%) and Bacillus toyonensis BCT-7112T.

Since no studies have been performed on the Kös

hot water spring, our study is significant for the

isolation of new microorganisms which are of

biotechnological importance. In addition, this study

presents novelty due to the isolation, purification

and characterisation of metallo-protease obtained

from Bacillus species.

Effects of different incubation periods on the

protease production

As can be seen from Fig. 2, the time course

experiments of protease synthesis in the cells show

that there is a sharp increase up to 24 h (7235.4

U/mL) at 40°C. Since it is important to produce

enzyme within a short time in the field of biotechnology, a 24 h incubation time is very important in terms of biotechnology. Shafee et al.

(2005), Sousa et al. (2007), Haddar et al. (2009) and Shivanand and Jarayaman (2009) reported the optimum protease production at 24 h for Bacillus cereus, Bacillus mojavensis A21 and Bacillus aquimaris VITP4, respectively.

Effects of various carbon and nitrogen sources

on the protease production

As shown in Fig. 3, lactose (8430.2 U/mL) as a carbon source increased the enzyme production to a great extent, while others except galactose inhibited enzyme production. It is already known that enzyme production is regulated by physiological mechanisms and the catabolites of glucose (catabolite repression) in liquid culture often repress the production of hydrolytic enzyme (Zambare et al. 2011). Similar results were reported by Johnvesly and Naik (2001) and VijayAnand et al.

(2009). Mabrouk et al. (1999), Oh et al. (2000) and Ibrahim and Al-Salamah (2009) all stated that lactose increases protease production. As shown in Fig. 4, yeast extract (9318 U/mL) increased the enzyme production to a great extent, while peptone (4729 U/mL) and tryptone (900 U/mL) inhibited enzyme production compared with the control (6420 U/mL). Among nitrogen sources, urea (7584 U/mL) caused a slight increase in enzyme activity. Johnvesly and Naik (2001), Frika et al. (2005) and Abidi et al. (2008) also reported that the maximum protease production was obtained in the presence of yeast extract.

The neutral protease produced from Bacillus sp.

KG5 can be conceived as a possible candidate for the

cost-effective enzyme due to the use of cheap

substrates such as lactose and yeast extract. In

addition, ammonium salts have no repressive effect

on protease production; this feature is important for

enzyme purification steps.

Effects of some metal ions on the protease

production

It has been found that protease production was

higly stimulated by CaCl2 (15400 U/mL), whereas it

was inhibited by NaCl (208 U/mL), MgCl2 (1742 U/mL)

Ahmetoglu et al.

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EurAsian Journal of BioSciences 9: 1-11 (2015)

RESULTS AND DISCUSSION

and MnCl2 (150 U/mL) compared with the control

(6420 U/mL) (Fig. 5). It is most likely that the strain

uses Ca2+ for enzyme secretion. The previous studies

also showed that the protease production was

enhanced in the presence of Ca2+ (Mabrouk et al.

1999, Secades et al. 2001, Rahman et al. 2003, Frikha

et al. 2005, Shafee et al. 2005). These results

indicated that KG5 protease is Ca2+- dependent.

Purification of protease from Bacillus sp. KG5

The steps that used for the protease enzyme

extraction and purification from Bacillus sp. KG5 are

shown in Table 2. It can be clearly seen that protease

was purified up to 13-fold with a yield of 23% of the

original enzyme (Table 2).

Both SDS-PAGE and non-denaturing PAGE

analysis showed that the molecular weight of the

protease from Bacillus sp. KG5 was found to be

around 48 kDa determined by Commassie staining

and zymography (Fig. 6). Towatana-Hutadilok et al.

(1999), Nilegonkar et al. (2007), Sousa et al. (2007)

and Wang et al. (2009) reported that the molecular

weights of proteases from Bacillus sp. PS719, B.

cereus MCM B-326, B. cereus and B. cereus TKU006

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Ahmetoglu et al.EurAsian Journal of BioSciences 9: 1-11 (2015)

Fig. 1. Microscopic view of KG5.

Fig. 2. Effect of time course on the protease production andbacterial growth (■ OD, ♦ Protease production).

Fig. 3. Effects of carbon sources on the proteaseproduction. Activity in the absence of the carbon source was taken as control (7235

U/mL).

Fig. 4. Effects of nitrogen sources on the proteaseproduction.Activity in the absence of the nitrogen source was taken as control (7235

U/mL).

Fig. 5. Effects of some metals on the protease production. Activity in the absence of the metal was taken as control (7235 U/mL).

were 42, 45.6, 45 and 39 kDa, respectively.

Effect of pH on the purified enzyme

In Fig. 7, the optimum pH was 7.0-7.5. Jellouli et al. (2011) and Asker et al. (2013) similarly reported the optimum pH to be 7.0 for protease activity. The importance of neutral proteases are their use in the food industry, because they possess specific function reducing the bitterness of food protein hydrolysates by hydrolysing hydrophobic amino acid bonds at neutral pH (Sandhya et al. 2005).

Effect of temperature and thermal stability on

the purified enzyme

In our study, it was found that the protease was

very active at 40-45°C (Fig. 8). Temperature is one of

the most important factors affecting the enzyme production (Uyar et al. 2011). Similar results were reported by Sousa et al. (2007) and VijayAnand et al.

(2009). Fig. 9 shows the effect of temperature on the purified protease stability. It was found that the enzyme was fully stable in the presence of 2 mM CaCl2 at 50ºC even after 120 min. Thermostability is

a critical feature required of proteases for industrial

applications such as detergent and leather

processing. Due to an increase in the enzyme

stability in the presence of CaCl2, the enzyme can be

used in biotechnological applications. Adinarayana et al. (2003), determined that protease from B. subtilis PE-11 was highly stable in the presence of

Ahmetoglu et al.

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EurAsian Journal of BioSciences 9: 1-11 (2015)

Table 1. Some characters that distinguish Bacillus sp. KG5.

(+): positive, (-): negative, (w): weak, ND: Not determined

Table 2. Purification steps of protease.

CaCl2 up to 60ºC. For this reason, the stability of

Bacillus sp. KG5 protease may have an advantage in

its use in laundry detergent formulations.

Effects of metal ions, inhibitors and

detergents on the purified protease activity

As shown in Table 3, protease was significantly activated by Ca2+ (242% at 2 mM ), Mg2+ (189% at 5 mM) and Mn2+ (129% at 2 mM), while strongly inhibited by Cu2+, Hg2+ and Zn2+ (at 10 mM). Adinarayana et al. (2003), Hmidet et al. (2009) and Annamalai et al. (2014) stated that Ca2+ acts as an activator for proteases. An increase in the activity in the presence of Ca2+ may be due to stabilisation of enzymes in its active conformation rather than it being involved in the catalytic reaction. It probably acts as a salt or an ion bridge via a cluster of carboxylic groups (Divakar et al. 2010). EDTA (96% at 10 mM) and 1,10 phenantroline (95% at 10 mM), which are metal chelating agents, heavily inhibited the protease activity. These data indicate that divalent metal ion cofactors are necessary for the

activity of protease; in other words, the enzyme is belonging to the metallo-protease family. In previous studies, Matta and Punj (1998), Kim et al.

(2001), Secades et al. (2001), Doddapaneni et al.

Ahmetoglu et al.EurAsian Journal of BioSciences 9: 1-11 (2015)

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Table 3. Effects of metal ions, inhibitors and detergents onthe purified protease activity.

*Relative activity was determined as percentage of control with noadditions.ND: Not defined, -: Not tested

Fig. 6. a): SDS-PAGE(Lane 1: protein molecular weight of the standarts [β-galactosidase (116 kDa), fructose phosphate (80.5 kDa), α-amylase

(58 kDa)], Lane 2: Sephadex G-75 gel permeation chromatography.

b): Zymogram with the nondenatured polyacrylamide gelelectrophoresis containing 0.1% gelatine(Sample: Sephadex G-75 gel permeation chromatography).

Ahmetoglu et al.EurAsian Journal of BioSciences 9: 1-11 (2015)

(2009), Wang et al. (2009) and Wu et al. (2011) also found that Ca2+ activated and EDTA inhibited the protease activity. They stated the enzyme was a member of the metallo-proteases family. PMSF was also slightly inhibited the protease activity. Furthermore, it was described that Hg2+ (Jaouadi et al. 2008, Jellouli et al. 2011, Benkiar et al. 2013), Cu2+

and Zn2+ (Nilegonkar et al. 2007, Hmidet et al. 2009) had inhibitory effects on protease activity. Our results, related to the inhibition of protease production by Hg2+, Cu2+ and Zn2+, were consistent with previously published studies. The enzyme activity was strongly inhibited by 1% SDS and Alo, while Triton X-100 and Tween-80 had a little effect on the protease activity. Wang et al. (2009) stated that the enzyme was stable in the presence of Triton

X-100. Many of the available proteases exhibited low activity and stability towards anionic surfactants like SDS (Uyar et al. 2011). Sellami-Kamoun et al. (2008) determined the inhibitory effect of SDS on the protease activity.

Commercial and biotechnological enzymes such

as neutral proteases produced by Bacillus species

are of importance in the food industry, because they

possess specific functions reducing the bitterness of

food protein hydrolysates. In the present study, the

optimal production, purification and

characterisation of a new extracellular protease

produced by newly isolated and identified Bacillus

sp. KG5 from a hot water spring in Kös (Bingöl),

Turkey were studied. The increased production of

metalloprotease by the bacteria in yeast extract,

urea, lactose and galactose, was seen alongside high

activation in the presence of CaCl2 and temperature

stability. The production process can therefore be

commercialised. Future applications of Bacillus sp.

KG5 neutral protease shows the potential use of this

enzyme in the food and detergent industry.

8

Fig. 7. Effect of pH on the protease activity.

Fig. 8. Effect of temperature on the protease activity.

Fig. 9. Thermal stability of the purified protease.

CONCLUSION

Abidi F, Limam F, Nejib MM (2008) Production of alkaline proteases by Botrytis cinerea using economic raw materials:assay as biodetergent. Process Biochemistry 43: 1202-1208. http://dx.doi.org/10.1016/j.procbio.2008.06.018

Adinarayana K, Ellaiah P, Prasas DS (2003) Purification and partial characterization of thermostable serine alkalineprotease from a newly isolated Bacillus subtilis PE-11. American Association of Pharmaceutical Scientists 4: 440- 448. http://dx.doi.org/10.1208/pt040456

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Ahmetoglu et al.EurAsian Journal of BioSciences 9: 1-11 (2015)