purification and characterization of novel organic-solvent-tolerant β-amylase and serine protease...

R E S EA RCH L E T T E R

Purification and characterization of novel organic-solvent-tolerant b-amylase and serine protease from a newly isolated

Salimicrobium halophilum strain LY20

Xin Li & Hui-Ying Yu

Life Science College, Yuncheng University, Yuncheng, China

Correspondence: Xin Li, Life Science

College, Yuncheng University, Yuncheng

044000, China. Tel./fax: +86 (0)

359 2090324; e-mail: [email protected]

Received 15 January 2012; revised 5 February

2012; accepted 6 February 2012.

DOI: 10.1111/j.1574-6968.2012.02522.x

Editor: Aharon Oren

Keywords

b-amylase; organic solvent tolerance;

purification; Salimicrobium; serine protease.

Abstract

A halophilic isolate Salimicrobium halophilum strain LY20 producing extracellu-

lar amylase and protease was isolated from Yuncheng, China. Production of

both enzymes was synchronized with bacterial growth and reached a maximum

level during the early-stationary phase. The amylase and protease were purified

to homogeneity with molecular weights of 81 and 30 kDa, respectively. Opti-

mal amylase activity was observed at 70 °C, pH 10.0% and 10% NaCl. Com-

plete inhibition by EDTA, diethyl pyrocarbonate (DEPC), and phenylarsine

oxide (PAO) indicated that the amylase was a metalloenzyme with histidine

and cysteine residues essential for its catalysis. Maltose was the main product

of starch hydrolysis, indicating an b-amylase activity. The purified protease

from LY20 showed highest activity at 80 °C, pH 10.0% and 12.5% NaCl. Com-

plete inhibition was shown by phenylmethylsulfonyl fluoride, DEPC, and PAO,

indicating that the enzyme probably belonged to the subclass of the serine pro-

teases with histidine and cysteine residues essential for catalysis. Furthermore,

both enzymes were highly stable over broad temperature (30–80 °C), pH (6.0–12.0) and NaCl concentration (2.5–20%) ranges, showing excellent thermosta-

ble, alkalistable, and halotolerant nature. The surfactants (SDS, Tween 80, and

Triton X-100) did not affect their activities. In addition, both enzymes from

LY20 displayed remarkable stability in the presence of water-soluble organic

solvents with log Pow � �0.24.

Introduction

As important hydrolytic enzymes, amylase and protease

represent the two largest groups of industrial enzymes and

account for approximately 85% of total enzyme sales all

over the world (Rao et al., 1998). At present, more than

3000 different enzymes have been characterized and many

of them found their way into biotechnological and indus-

trial applications (van den Burg, 2003). However, owing to

the harsh conditions during the industrial processes, many

of the commercially available enzymes do not withstand

industrial reaction conditions; therefore, isolation and

characterization of novel enzymes with desirable properties

such as thermostability, alkaline stability, and halophilicity

are important to meet the industrial demands. Recently,

considerable interest has been drawn on extremophiles,

which are the valuable source of novel enzymes (Antrani-

kian et al., 2005). Among the extremophiles, halophiles

are microorganisms that live, grow, and multiply in highly

saline environments. Extracellular enzymes from these

organisms with polymer-degrading ability at low water

activity are of interest in many harsh industrial processes

where concentrated salt solutions would inhibit enzymatic

conversions (Mellado et al., 2004).

The ability of enzymes to remain active in the presence

of organic solvents has received a great deal of attention

over the past two decades. In contrast to in water,

numerous advantages of using enzymes in organic sol-

vents or aqueous solutions containing organic solvents

have been observed, such as increased solubility of

FEMS Microbiol Lett && (2012) 1–8 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

MIC

ROBI

OLO

GY

LET

TER

S

nonpolar substrates and elimination of microbial contam-

ination in the reaction mixture (Ogino & Ishikawa,

2001). Generally, enzymes are easily denatured and their

activities disappear in the presence of organic solvents.

Therefore, enzymes that remain stable in the presence of

organic solvents might be useful for biotechnological

applications in which such solvents are used (Shafiei

et al., 2011). Because salt reduces water activity, a feature

in common with organic solvent systems, halophilic

enzymes are thought to be valuable tools as biocatalysts

in other low-water-activity environments, such as in

aqueous/organic and nonaqueous media (Marhuenda-

Egea & Bonete, 2002). Recently, halophilic proteases with

organic-solvent-tolerant properties have been obtained

from Salinivibrio sp. strain AF-2004 (Karbalaei-Heidari

et al., 2007a, b) and Natrialba magadii (Ruiz & De Cas-

tro, 2007). Also, Fukushima et al. (2005) and Shafiei

et al. (2011) reported organic-solvent-tolerant halophilic

a-amylase from Haloarcula sp. strain S-1 and Nesterenko-

nia sp. strain F. However, to the best of our knowledge,

there are no reports on organic-solvent-tolerant b-amy-

lases from halophiles.

The halophile Salimicrobium sp. has been studied with

regard to its ecology, physiology, biochemistry, and more

recently, its genetics (Yoon et al., 2007, 2009). However,

the microorganism’s biotechnological possibilities have

not been extensively exploited, and no reports about the

enzyme production from Salimicrobium sp have been

published. In this study, we report the purification and

characterization of b-amylase and protease from a newly

halophilic strain LY20, including organic solvent tolerance

of the enzymes.

Materials and methods

Bacterial isolation, identification, and enzyme

production

The strain LY20 was isolated from the saline soil of

Yuncheng, China, and cultivated aerobically at 37 °C in

the complex medium (CM) with the following composi-

tion (g L�1): casein peptone 7.5; yeast extract 10.0; solu-

ble starch 10.0; sodium citrate 3.0; MgSO4·7H2O 20.0;

KCl 2.0; FeSO4·7H2O 0.01; NaCl 120.0 and pH 7.5.

The strain was identified based on typical cultural,

morphological, and biochemical characteristics and 16S

rRNA gene sequencing. The organism was deposited at

China Center of Industrial Culture Collection with the

accession number CICC 10482. The 16S rRNA gene

sequence was submitted to GenBank with the accession

number HQ683738.

The kinetics of bacterial growth and extracellular

enzymes production were determined at different time

intervals. Bacterial growth, along with enzyme activity,

was measured by spectrophotometric method (Shimadzu

model UV-160A).

Extracellular enzymes purification

After cultivation of the strain LY20 in CM broth for 60 h,

cell-free supernatant was harvested by centrifugation at

12 000 g for 15 min at 4 °C and used for enzyme purifica-

tion. Ammonium sulfate was added to the supernatant up

to 75% concentration with continuous overnight stirring.

The precipitate collected by centrifugation (12 000 g for

25 min) was dissolved in a minimum volume of buffer A

(20 mM Tris–HCl containing 10% NaCl, pH 10.0) and

dialyzed against the same buffer overnight. The concen-

trated sample was loaded on a Q-Sepharose HP column

(1.6 9 14 cm) pre-equilibrated with buffer A. Bound pro-

teins were eluted by applying a linear gradient of 0.1–0.8 M

NaCl. Fractions containing amylase and protease activity

were pooled and concentrated by freeze-drying, respec-

tively. Each resulting concentrate was dissolved in a mini-

mal volume of buffer B (50 mM glycine–NaOH containing

10% NaCl, pH 10.0) and then loaded on a Sephacryl S-200

column (1.6 9 60 cm). The samples were eluted with buf-

fer B at a flow rate of 0.5 mL min�1 (2 mL per fraction).

Active fractions containing the extracellular amylase and

protease were pooled and used for further analysis.

Gel electrophoresis and zymography

Molecular weights of the amylase and protease were ana-

lyzed by SDS-PAGE using 12% acrylamide, according to

the method of Laemmli (1970). After electrophoresis, gel

was stained with Coomassie Brilliant Blue R-250. For activ-

ity staining, zymographic analysis of the protease was per-

formed using gelatin (0.1%) as the substrate as described

by Karbalaei-Heidari et al. (2009). Zymographic analysis

for the amylase was performed on nondenaturing electro-

phoresis slab gels (10% polyacrylamide) prepared with

10% of sucrose, as described by Cadenas & Engel (1994).

Amylase and protease activity assay

The amylase activity, with soluble starch as the substrate,

was determined using DNS (3,5-dinitrosalicylic acid)

method (Miller, 1959). One unit (U) of amylase activity

was defined as the amount of enzyme necessary to pro-

duce 1 lmol of reducing sugar per minute under the

assay conditions. Protease activity was measured as

described previously (Karbalaei-Heidari et al., 2009). One

unit (U) of protease activity was defined as the amount

of enzyme yielding 1 lmol of tyrosine per minute under

the assay conditions.

ª 2012 Federation of European Microbiological Societies FEMS Microbiol Lett && (2012) 1–8Published by Blackwell Publishing Ltd. All rights reserved

2 X. Li & H.-Y. Yu

Effects of temperature, pH, and NaCl on

enzyme activity and stability

The effect of pH on enzyme activity was studied over a pH

range of 4.0–12.0. The pH stability of the enzymes was

determined by incubation with different buffer systems at

30 °C for 24 h. The following buffer systems (100 mM)

were used: glycine-HCl buffer, pH 4.0; sodium acetate buf-

fer, pH 5.0–6.0; potassium phosphate buffer, pH 7.0; Tris–HCl buffer, pH 8.0–8.5; glycine–NaOH buffer, pH 9.0–12.0.

To investigate the effect of temperature, the assay was

conducted under different temperatures from 30 to 90 °C.The thermostability of the enzyme was determined by

pre-incubating the enzyme sample at various tempera-

tures for 24 h, and residual activity was measured using

the standard assay.

The activity of the purified enzyme was measured in

enzyme reaction mixture containing 0–20% NaCl. Salt

stability of the enzyme was determined by incubating the

enzyme with different concentrations of NaCl for 24 h,

and the remaining activity was determined under stan-

dard assay conditions.

Effects of organic solvents on enzyme activity

and stability

The effect of organic solvents with different log Pow val-

ues at 50% (v/v) concentration on the purified enzyme

was determined by incubating the enzyme solution in dif-

ferent organic solvents at 30 °C. Residual activity was

measured under the standard conditions. If residual activ-

ity was more than 50% after 10 days, half-life was taken

as ‘> 10 days’. While activity was < 50% after 1 h, half-

life was taken as ‘< 1 h’.

Effects of metal ions and chemical reagents

Effects of different metal ions and chemical reagents

[ethylenediaminetetraacetic acid (EDTA), phenylmethyl-

sulfonyl fluoride (PMSF), phenylarsine oxide (PAO),

diethyl pyrocarbonate (DEPC), b-mercaptoethanol, SDS,

Triton X-100, and Tween-80] on the activity of purified

enzymes were examined after they had been pre-incu-

bated with them at 30 °C for 12 h, respectively, and the

residual activity was determined under optimal assay con-

ditions. Activity of the enzyme assayed in the absence of

any additives was taken as 100%.

Analysis of hydrolysis products by the purified

amylase

The hydrolysis products of soluble starch were analyzed

by high-performance liquid chromatography (HPLC).

Amylase solution (1 mL) was incubated at 70 °C with

0.5% soluble starch in Tris–HCl buffer (pH 10.0) con-

taining 10% NaCl. Aliquots were drawn at different time

intervals, and hydrolysis was stopped by boiling at 100 °C. After centrifugation at 12 000 g for 15 min, each sam-

ple was analyzed by HPLC analysis on a micro Bond pack

Amino Carbohydrate column (4.1 9 300 mm). Samples

(15 lL) were injected and eluted with acetonitrile/water

(70 : 30 ratio) at a flow rate of 1 mL min�1. The hydro-

lyzed products were detected using a refractive index

detector. Glucose, maltose, maltotriose, and maltopenta-

ose (Sigma) were used as standards.

Results

Strain identification and production of

extracellular enzymes

Based on morphological, physiological, and biochemical

characteristics, the isolate LY20 is a Gram-positive,

motile, rod-shaped and aerobic bacterium. Colonies are

light yellow, uniformly round, circular, and convex on

CM agar plate. It was able to grow in medium containing

0.5–25% (w/v) NaCl and grew optimally at 10% (w/v)

NaCl. No growth was observed in the absence of NaCl.

Thus, this bacterium can be considered as a moderately

halophilic microorganism (Ventosa et al., 1998). Optimal

temperature and pH for bacterial growth were 37 °C and

10.0. H2S production, methyl red, and Tween-80 hydroly-

sis were negative, while Voges–Proskauer test, nitrate

reduction, oxidase, catalase, and gelatin hydrolysis were

positive. Acid is produced from maltose, fructose,

sucrose, and glucose. Phylogenetic analysis based on 16S

rRNA gene sequence comparisons revealed that the isolate

LY20 belonged to Salimicrobium species and was most

closely related to Salimicrobium halophilum DSM 4771T

(98.9% 16S rRNA gene sequence similarity; Fig. 1).

As shown in Fig. 2, both enzymes started to produce

from the early-exponential phase of bacterial growth (4 h

for amylase and 10 h for protease) and reached a maxi-

mum level during the early-stationary phase (42 h).

Enzyme purification

Both enzymes were purified by ammonium sulfate precip-

itation, Q-Sepharose ion exchange, and Sephacryl S-200

gel filtration chromatography. The amylase was purified

21.5-fold with recovery of 31.9% and specific activity of

573.5 units mg�1 protein, while protease was purified

27.5-fold with recovery of 32.4% and specific activity of

832.7 units mg�1 protein. Molecular weights of the

b-amylase and protease were determined to be 81 and

30 kDa, respectively (Fig. 3, lanes 2 and 3), correspond-


Organic-solvent-tolerant b-amylase and protease from LY20 3

ing with those determined by gel filtration. These results

indicated that both enzymes from LY20 were monomeric

ones. Also, zymographic activity staining revealed the

activity bands for purified samples at corresponding posi-

tions on SDS-PAGE (Fig. 3, lanes 4 and 5).

Properties of the purified b-amylase

The amylase hydrolyzed soluble starch to form maltose as

the main product. This product was readily apparent dur-

ing the early stages of the reaction and increased in con-

centration along with the time course of the reaction.

Trace amounts of longer oligosaccharides (maltotriose

and maltopentaose) and glucose were also produced (data

not shown). These results provide an insight into the deg-

radation mode of the enzyme, which may preferentially

cleave at the a-1,4-linkage adjacent to nonreducing ends,

showing the b-amylase activity.

The b-amylase showed a activity over a wide tempera-

ture range (30–90 °C), pH range (4.0–12.0), and NaCl

concentrations (0–20%), with an optimum at 70 °C, pH10.0% and 10% NaCl (Fig. 4). The thermal stability pro-

file indicated that the enzyme was highly stable at tem-

peratures below 70 °C after 24-h incubation, but was

inactivated at 90 °C (Fig. 4a). Also, the b-amylase showed

good pH stability retaining more than 80% activity in the

pH range 6.0–11.0 (Fig. 4b). Furthermore, it was highly

stable at NaCl concentrations between 2.5% and 20%,

and more than 70% activity retained after dialysis in the

absence of NaCl (Fig. 4c).

As shown in Table 1, the metal ions tested did not

affect or slightly inhibit the amylase activity. The effect of

enzyme inhibitors indicated that DEPC, PAO and EDTA

completely inactivated the enzyme, but PMSF and b-mer-

captoethanol had no significant effect on its activity.

Moreover, more than 78% activity of the amylase

retained after incubation with surfactants, such as SDS,

Triton X-100, and Tween-80.

Properties of the purified protease

Optimal activity of the protease was found to be at 80 °C,pH 10.0% and 12.5% NaCl (Fig. 4). It was highly stable

at temperatures below 70 °C after 24-h incubation, but

was inactivated at higher temperatures (Fig. 4a). Mean-

while, the protease showed good stability in a broad pH

range (6.0–11.0), which retained more than 70% activity

(Fig. 4b). As shown in Fig. 4c, about 82% activity lost in

the absence of NaCl, but 70% activity retained under

high salinity conditions (20%). Moreover, the protease

was highly stable at NaCl concentrations between 2.5%

and 20%.

None of the metal ions was found to enhance the pro-

tease activity, and about 80% activity lost in the presence

Fig. 1. Neighbor-joining phylogenetic tree showing the position of

isolate LY20 to other members of the genus Salimicrobium. Accession

numbers of the sequences used in this study are shown in

parentheses after the strain designation. Numbers at nodes are

percentage bootstrap values based on 1000 replications. Bar 0.005

substitutions per nucleotide position.

Fig. 2. Kinetics of bacterial growth and extracellular enzyme

production of strain LY20 in CM broth containing 12% (w/v) NaCl at

37 °C. Results represent the means of three separate experiments.

Fig. 3. SDS-PAGE and zymographic analysis of the purified enzymes.

Lane 1: molecular mass markers; lanes 2 and 4: protease; lanes 3 and

5: b-amylase.


4 X. Li & H.-Y. Yu

of Hg2+. EDTA and b-mercaptoethanol had no significant

effect on the enzyme activity. However, complete inhibi-

tion of the protease was shown by PMSF, DEPC, and

PAO. In addition, more than 85% activity retained after

incubation with surfactants tested (Table 1).

Effects of organic solvents on enzyme activity

and stability

As shown in Table 2, no complete inactivation of both

enzymes was observed in the presence of organic solvents

tested. More than 90% of the enzyme activity retained

after incubation with DMSO, acetonitrile, ethanol, and

acetone. Interestingly, ethanol and acetone even increased

the amylase activity to 117.4% and 118.9%, respectively,

and DMSO and ethanol also stimulated the protease

activity (110.8% and 110.2%). The half-lives of both

enzymes were drastically decreased in the presence of

organic solvents with log Pow � �0.24, but in the pres-

ence of organic solvents with lower log Pow, their half-

lives were longer than in the absence of the solvents.

Discussion

The ability of the moderately halophilic bacteria to grow

over a very wide range of salinities makes them very

attractive for screening of novel enzymes with unusual

properties. In this investigation, the isolate S. halophilum

strain LY20 was selected for further study because it

appeared to be the best producer of extracellular amylase

and protease. To date, there are no reports for amylase

Fig. 4. Effect of temperature (a), pH (b), and NaCl concentration (c)

on activity (solid lines) and stability (dotted lines) of the purified

b-amylase and protease. Relative activity was defined as the

percentage of activity detected with respect to the maximum enzyme

activity. For determining the stability, the activity of the enzyme

without any treatment was taken as 100%. Data are the average of

three independent experiments.

Table 1. Effects of metal ions and chemical reagents on the activity

of purified enzymes

Substances

Concentration

(mM)

Residual

activity of

b-amylase*

Residual

activity of

protease*

Control – 100 ± 1.0 100 ± 1.6

Ca2+ 5 98.9 ± 3.5 98.2 ± 2.0

Zn2+ 5 98.4 ± 1.7 97.0 ± 2.6

Fe2+ 5 99.1 ± 2.6 98.9 ± 1.7

Fe3+ 5 74.5 ± 1.4 97.6 ± 1.6

Cu2+ 5 100 ± 3.1 95.1 ± 2.1

Mn2+ 5 98.7 ± 2.3 90.7 ± 2.7

Hg2+ 5 73.8 ± 2.6 20.1 ± 1.0

Mg2+ 5 98.6 ± 3.3 99.4 ± 2.7

EDTA 10 0 96.5 ± 2.5

PMSF 10 90.1 ± 1.9 0

DEPC 10 0 0

PAO 10 0 0

b-Mercaptoethanol 10 87.1 ± 3.2 98.1 ± 3.8

SDS 20 78.3 ± 3.8 85.1 ± 1.1

Triton X-100 20 98.1 ± 3.4 97.2 ± 1.9

Tween-80 20 99.8 ± 2.7 98.9 ± 2.9

*Residual activity was determined as described in ‘Materials and

methods’ and expressed as the percentage of the control value (with-

out any additives). The average of relative values (N = 3) and standard

deviations (SD) are shown.



and protease production at the same time from one iso-

late, because the protease can hydrolyze other proteins

such as amylase. However, maximal production of both

enzymes was observed simultaneously during the station-

ary growth phase of LY20 (Fig. 2). This particular phe-

nomenon could be explained that the amylase was not

the substrate of the protease, which was confirmed by

SDS-PAGE after incubating the two enzyme solutions

(80 °C and pH 10.0) for 30 min (data not shown).

There are many reports on isolation of amylases from

halophiles (Mellado et al., 2004; Litchfield, 2011), but

pure preparation of halophilic b-amylase has not been

obtained. In this study, purification of an b-amylase from

LY20 was reported. Similar enzyme was previously

described from Halobacillus sp. LY9 (Li & Yu, 2011), but

its enzymatic properties were mostly obtained from crude

extracts. Molecular weight of the b-amylase was deter-

mined to be 81 kDa (Fig. 3, lane 2). The value was higher

than other b-amylases from nonhalophiles (Shen et al.,

1988; Young et al., 2001). The enzyme showed an optimal

activity at 70 °C and excellent thermostability under high

temperatures. These characteristics made it obviously dif-

ferent from other b-amylases, which were neither active

nor stable at temperatures above 65 °C (Shen et al., 1988;

Young et al., 2001). It is desirable that amylases should

be active at high temperature for gelanization (100–110 °C),liquefaction (80–90 °C), and saccharification (60–65 °C)for the application in the starch industry. Until today,

amylases from bacteria belonging to genus Bacillus are

heavily used in the starch-processing industry (Mamo &

Gessesse, 1999; Demirkan et al., 2005). As thermostability

is an important feature for amylolytic enzymes, the

b-amylase from LY20 might be industrially exploited for

starch liquefaction and saccharification.

Molecular weight of the purified protease was esti-

mated to be 30 kDa on SDS-PAGE. Similar values pre-

sented other halophilic proteases previously characterized

(Karbalaei-Heidari et al., 2007a, b; Xiong et al., 2007).

The enzyme showed the optimal activity at 80 °C. In con-

trast to other proteases from halophiles (Amoozegar

et al., 2007; Karbalaei-Heidari et al., 2009), it required

relatively higher temperature to maintain the maximum

activity. Moreover, high thermostability over a wide tem-

perature range (30–80 °C) was observed. These properties

made it potential use in industrial applications that

require high temperatures.

The amylase and protease from LY20 were found to be

highly active and stable in the presence of higher concen-

trations of NaCl. This type of extreme halotolerance has

been reported in other halophilic enzymes from Halo-

monas meridiana (Coronado et al., 2000) and Chromoha-

lobacter sp. TVSP 101 (Prakash et al., 2009). Optimal pH

for the activity and stability of both enzymes ranged from

7.0 to 10.0. These results clearly indicated their haloalka-

line nature. Several researchers all over the world are now

trying to exploit microorganisms for the isolation of alka-

line enzymes because of their tremendous potentiality in

detergent industry (Chakraborty et al., 2011). Therefore,

the enzymes from LY20 may have widespread applications

in detergent, food, and other industrial processes contain-

ing high salt concentration.

Organic-solvent-tolerant halophilic enzymes appear to

be quite attractive for industrial applications such as bio-

remediation of carbohydrate-polluted salt marshes and

industrial wastewaters contaminated with organic sol-

vents. However, reports for halophilic enzymes with

organic solvent tolerance were scarce. Thus, the behavior

of the b-amylase and protease in the presence of organic

solvents was determined. As shown in Table 2, both

enzymes showed high activity, and obvious stimulation

by some organic solvents was observed. These behaviors

might be due to the residues of carried-over nonpolar

hydrophobic solvent providing an interface, thereby keep-

ing the enzyme in an open conformation and thus result-

ing in the observed activation (Zaks & Klibanov, 1988).

Furthermore, half-lives of both enzymes were drastically

decreased in the presence of organic solvents with log

Pow � �0.24, but in the presence of organic solvents

with log Pow � �0.24, their half-lives were similar to or

much longer than in the absence of the solvents. Together

these results indicated that, in contrast to the organic sol-

vent stability of other proteases (Karbalaei-Heidari et al.,

2007a, b; Ruiz & De Castro, 2007) and amylases (Fuku-

shima et al., 2005; Shafiei et al., 2011), stability of the

Table 2. Activity and stability of purified enzymes in different organic

solvents

Organic solvents Log Pow

Residual activity (%)

b-Amylase Protease

Control – 100 (4 days) 100 (3 days)

DMSO �1.35 99.5 (> 10 days) 110.8 (> 10 days)

DMF �1.0 78.4 (5 days) 79.0 (> 10 days)

Methanol �0.76 79.1 (> 10 days) 68.3 (8 days)

Acetonitrile �0.34 94.7 (4 days) 93.8 (7 days)

Ethanol �0.3 117.4 (> 10 days) 110.2 (> 10 days)

Acetone �0.24 118.9 (> 10 days) 97.8 (> 10 days)

Benzene 2.13 47.9 (1 day) 55.4 (2 days)

n-Hexane 3.5 53.8 (1 day) 50.1 (1 day)

Isooctane 4.7 59.1 (1 day) 59.8 (1 day)

Dodecane 6.6 58.2 (< 1 h) 57.9 (< 1 h)

The log Po/w is the logarithm of the partition coefficient, P, of the sol-

vent between n-octanol and water and is used as a quantitative mea-

sure of the solvent polarity. The activity of the purified enzyme in the

absence of organic solvents was taken as controls. The numbers in

brackets are the half-lives of the enzymes in different organic sol-

vents.


6 X. Li & H.-Y. Yu

enzymes from LY20 was dependent on the polarity of the

solvents and was higher in the presence of water-soluble

solvents with lower log Pow values.

Enzyme inhibition studies showed that the b-amylase

was completely inhibited by DEPC (a histidine modifier)

and PAO (a cysteine modifier), indicating that the

histidine and cysteine residues were essential for enzyme

catalysis. Significant inhibition by EDTA suggested that

the b-amylase was a metalloenzyme. Similar finding has

not been observed in other halophilic amylases. However

for the purified protease, complete inhibition of proteo-

lytic activity was shown by PMSF, DEPC, and PAO,

indicating that the enzyme was a serine protease with

histidine and cysteine residues in its active site. Moreover,

high activity in the presence of EDTA suggested that the

protease might be very useful for application as detergent

additive because chelating agents are components of most

detergents (Haddar et al., 2009). In addition, both

enzymes from LY20 showed high activity in the presence

of surfactants at higher concentrations than those

reported for other halophilic enzymes (Dodia et al., 2008;

Chakraborty et al., 2009; Shafiei et al., 2011).

In the present study, the b-amylase and serine protease

from S. halophilum strain LY20 showed excellent thermo-

stable, alkalitolerant, halotolerant, and surfactant-stable

properties. Also, considering their high activity and stabil-

ity in the presence of organic solvents, they could be

potentially useful for practical applications in biotechno-

logical processes with nonconventional media.

Acknowledgements

This work was financially supported by Shanxi Provincial

Science and Technology Foundation (grants no.

20110021) and Natural Science Fund of Shanxi Province

(grants no. 2011021031-4).

References

Amoozegar MA, Fatemi AZ, Karbalaei-Heidari HR & Reza

Razavi M (2007) Production of an extracellular alkaline

metalloprotease from a newly isolated moderate halophile,

Salinivibrio sp. strain AF-2004. Microbiol Res 162:

369–377.Antranikian G, Vorgias CE & Bertoldo C (2005) Extreme

environments as a resource for microorganisms and novel

biocatalysts. Adv Biochem Eng Biotechnol 96: 219–262.Cadenas Q & Engel PC (1994) Activity staining of halophilic

enzymes: substitution of salt with zwitterions in non-

denaturing electrophoresis. Biochem Mol Biol Int 33: 785–792.

Chakraborty S, Khopade A, Kokare C, Mahadik K & Chopade

B (2009) Isolation and characterization of novel a-amylase

from marine Streptomyces sp. D1. J Mol Catal B Enzym 58:

17–23.Chakraborty S, Khopade A, Biao R, Jian W, Liu XY, Mahadik

K, Chopade B, Zhang LX & Kokare C (2011)

Characterization and stability studies on surfactant,

detergent and oxidant stable a-amylase from marine

haloalkaliphilic Saccharopolyspora sp. A9. J Mol Catal B

Enzym 68: 52–58.Coronado MJ, Vargas C, Hofemeister J, Ventosa A & Nieto JJ

(2000) Production and biochemical characterization of an

a-amylase from the moderate halophile Halomonas

meridiana. FEMS Microbiol Lett 183: 67–71.Demirkan ES, Mikami B, Adachi M, Higas T & Utsumi S

(2005) a-Amylase from B. amyloliquefaciens: purification,

characterization, raw starch degradation and expression in

E. coli. Process Biochem 40: 2629–2636.Dodia MS, Rawal CM, Bhimani HG, Joshi RH, Khare SK &

Singh SP (2008) Purification and stability characteristics of

an alkaline serine protease from a newly isolated

Haloalkaliphilic bacterium sp. AH-6. J Ind Microbiol

Biotechnol 35: 121–131.Fukushima T, Mizuki T, Echigo A, Inoue A & Usami R (2005)

Organic solvent tolerance of halophilic a-amylase from a

Haloarchaeon, Haloarcula sp. strain S-1. Extremophiles 9:

85–89.Haddar A, Bougatef A, Agrebi R, Sellami-Kamoun A & Nasri

M (2009) A novel surfactant-stable alkaline serine-protease

from a newly isolated Bacillus mojavensis A21. Purification

and characterization. Process Biochem 44: 29–35.Karbalaei-Heidari HR, Ziaee AA & Amoozegar MA (2007a)

Purification and biochemical characterization of a protease

secreted by the Salinivibrio sp. strain AF-2004 and its

behavior in organic solvents. Extremophiles 11: 237–243.Karbalaei-Heidari HR, Ziaee AA, Schaller J & Amoozegar MA

(2007b) Purification and characterization of an extracellular

haloalkaline proteinase produced by the moderately

halophilic bacterium, Salinivibrio sp. strain AF-2004. Enzyme

Microb Technol 40: 266–272.Karbalaei-Heidari HR, Amoozegar MA, Hajighasemi M, Ziaee

AA & Ventosa A (2009) Production, optimization and

purification of a novel extracellular protease from the

moderately halophilic bacterium Halobacillus karajensis.

J Ind Microbiol Biotechnol 36: 21–27.Laemmli UK (1970) Cleavage of structural proteins during the

assembly of the head of bacteriophage T4. Nature 227: 680–685.

Li X & Yu HY (2011) Extracellular production of beta-amylase

by a halophilic isolate, Halobacillus sp. LY9. J Ind Microbiol

Biotechnol 38: 1837–1843.Litchfield CD (2011) Potential for industrial products from the

halophilic Archaea. J Ind Microbiol Biotechnol 38: 1635–1647.

Mamo G & Gessesse A (1999) Effect of cultivation conditions

on growth and a-amylase production by a thermophilic

Bacillus sp. Lett Appl Microbiol 29: 61–65.



Marhuenda-Egea FC & Bonete MJ (2002) Extreme halophilic

enzymes in organic solvents. Curr Opin Biotechnol 13: 385–389.

Mellado E, Sanchez-Porro C, Martın S & Ventosa A (2004)

Extracellular hydrolytic enzymes produced by moderately

halophilic bacteria. Halophilic microorganisms (Ventosa A,

ed.), pp. 285–295. Springer, Berlin.Miller G (1959) Use of dinitrosalicylic acid reagent for

determination of reducing sugars. Anal Chem 31: 426–428.Ogino H & Ishikawa H (2001) Enzymes which are stable in

the presence of organic solvents. J Biosci Bioeng 91: 109–116.Prakash B, Vidyasagar M, Madhukumar MS, Muralikrishna G

& Sreeramulu K (2009) Production, purification, and

characterization of two extremely halotolerant, thermostable,

and alkali-stable a-amylases from Chromohalobacter sp.

TVSP 101. Process Biochem 44: 210–215.Rao MB, Tanksale AM, Ghatge MS & Deshpande VV (1998)

Molecular and biotechnological aspects of microbial

proteases. Microbiol Mol Biol Rev 62: 597–635.Ruiz DM & De Castro RE (2007) Effect of organic solvents on

the activity and stability of an extracellular protease secreted

by the haloalkaliphilic archaeon Natrialba magadii. J Ind

Microbiol Biotechnol 34: 111–115.Shafiei M, Ziaee AA & Amoozegar MA (2011) Purification and

characterization of an organic-solvent-tolerant halophilic

a-amylase from the moderately halophilic Nesterenkonia sp.

strain F. J Ind Microbiol Biotechnol 38: 275–281.

Shen GJ, Saha BC, Lee YE, Bhatnagar L & Zeikus JG (1988)

Purification and characterization of a novel thermostable

beta-amylase from Clostridium thermosulphurogenes. Biochem

J 254: 835–840.van den Burg B (2003) Extremophiles as a source for novel

enzymes. Curr Opin Microbiol 6: 213–218.Ventosa A, Nieto JJ & Oren A (1998) Biology of moderately

halophilic aerobic bacteria. Microbiol Mol Biol Rev 62: 504–544.

Xiong H, Song L, Xu Y, Tsoi MY, Dobretsov S & Qian PY

(2007) Characterization of proteolytic bacteria from the

Aleutian deep-sea and their proteases. J Ind Microbiol

Biotechnol 34: 63–71.Yoon JH, Kang SJ & Oh TK (2007) Reclassification of

Marinococcus albus Hao et al. 1985 as Salimicrobium album

gen. nov., comb. nov. and Bacillus halophilus Ventosa et al.

1990 as Salimicrobium halophilum comb. nov., and

description of Salimicrobium luteum sp. nov. Int J Syst Evol

Microbiol 57: 2406–2411.Yoon JH, Kang SJ, Oh KH & Oh TK (2009) Salimicrobium

flavidum sp. nov., isolated from a marine solar saltern. Int J

Syst Evol Microbiol 59: 2839–2842.Young MH, Gun LD, Hoon YJ, Ha PY & Jae KY (2001) Rapid

and simple purification of a novel extracellular b-amylase

from Bacillus sp. Biotechnol Lett 23: 1435–1438.Zaks A & Klibanov AM (1988) Enzymatic catalysis in

nonaqueous solvents. J Biol Chem 263: 3194–3201.


8 X. Li & H.-Y. Yu

purification and characterization of novel organic-solvent-tolerant β-amylase and serine protease...

Documents