Bioprocess Biosyst Eng DOI 10.1007/s00449-015-1375-x

ORIGINAL PAPER

Characterization of salt-tolerant b-glucosidase with increased thermostability under high salinity conditions from Bacillus sp. SJ-10 isolated from jeotgal, a traditional Korean fermented seafood Jong Min Lee • Yu-Ri Kim • Joong Kyun Kim • Gwi-Taek Jeong • Jeong-Chul Ha • In-Soo Kong

Received: 5 November 2014 / Accepted: 8 February 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract The b-glucosidase gene, bglC, was cloned from Bacillus sp. SJ-10 isolated from the squid jeotgal. Recombinant BglC protein overexpression was induced in Escherichia coli. The optimal pH and temperature of the enzyme, using p-nitrophenyl-b-D-glucopyranoside (pNPbGlc) as a substrate, were pH 6 and 40 °C, respectively. Enzymatic activity increased by 3.3- and 3.5-fold in the presence of 15 % NaCl and KCl, respectively. Furthermore, enzyme thermostability improved in the presence of NaCl or KCl. At 45 °C in the presence of salts, the enzyme was stable for 2 h and maintained 80 % activity. In the absence of salts, BglC completely lost activity after 110 min at 45 °C. Comparison of the kinetic parameters at various salt concentrations revealed that BglC had approximately 1.5- and 1.2-fold higher affinity and hydrolyzed pNPbGlc 1.9- and 2.1-fold faster in the presence of 15 % NaCl and KCl, respectively. Additionally, the Gibb’s free energy for denaturation was higher in the presence of 15 % salt than in the absence of salt at 45 and 50 °C. Since enzymatic activity and thermostability were enhanced under high salinity conditions, BglC is an ideal salt-tolerant enzyme for further research and industrial applications. Keywords b-Glucosidase  Bacillus  Salt tolerant  Thermostability

J. M. Lee  J. K. Kim  G.-T. Jeong  I.-S. Kong (&) Department of Biotechnology, Pukyong National University, Busan 608-737, Korea e-mail: [email protected] Y.-R. Kim CBR Defense Research Institute, Seoul, Korea J.-C. Ha Korea Consumer Agency, Seoul 137-700, Korea

Introduction b-Glucosidase (b-D-glucopyranoside glucohydrolases, EC 3.2.1.21) is found in all domains of living organisms. This enzyme is biologically important for hydrolyzing betaglucosidic linkages between carbohydrate residues in arylamino- or alkyl-beta-D-glucosides, cyanogenic-glucosides, short chain oligosaccharides and disaccharides under different physiological conditions [1]. Moreover, under defined conditions, this enzyme can catalyze the synthesis of glycosyl bonds between molecules [2]. b-Glucosidase plays an active role in many biological processes, such as degradation of structural and storage polysaccharides, host–pathogen interactions, cellular signaling and oncogenesis. It has attracted considerable attention due to its roles in various biotechnological processes such as biotransformation for the manufacturing of pharmaceuticals, fine chemicals and food ingredients, as well as the release of aromatic compounds from flavorless precursors [3]. bGlucosidase can be used to generate biofuel and is an important enzyme in the cellulose hydrolysis process by preventing cellobiose-mediated inhibition of other enzymes [4]. Cellulosic biomass is the most abundant renewable resource on earth. Many types of cellulose, including rice straw, corn stalk and wheat stalk, are used as raw materials by biorefineries. A recent study predicted that the use of croplands would increase greenhouse gases because of changes in land use [5]. Therefore, economical conversion of cellulosic biomass is desirable. Differences in the structures of marine biomass and terrestrial cellulosic biomass have been observed. Marine biomass lacks the recalcitrant cell wall structures that are found in cellulosic biomass. Additionally, it is relatively simple to release fermentable sugars from marine biomass compared to

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terrestrial biomass. Thus, marine biomass is applicable as raw material for biorefineries [6, 7]. Moreover, marine glycoside compounds have diverse aglycone and carbohydrate moiety structures compared with their terrestrial origin. Marine glycosides may have several biological functions including antiprotozoal, antifungal and antitumor [8]. However, marine biomass contains a high salt concentration, which is detrimental to the activity and stability of most known b-glucosidases [9]. The use of marine biomasses as a sustainable biorefinery source under high salinity conditions is important for environmental restoration [10]. Thus, salt-tolerant b-glucosidase offers major biotechnological advantages. Bacillus species produce diverse extracellular polysaccharide degrading and industrially important enzymes. bGlucosidases have been characterized from several Bacillus spp. However, the activity and stability of these bglucosidases at a wide range of salinities was low. To identify glucosidase family enzymes with high activity toward b-glucosides (including cellobiose) in the presence of salt, we previously isolated Bacillus sp. SJ-10 (KCCM 90078, JCM 15709) from jeotgal (a traditional Korean salted fermented seafood), which produces a novel b-1,31,4-glucanase with high activity under a range of conditions [11]. We found that this strain produces various tolerant enzymes. In this study, we cloned the gene encoding b-glucosidase (BglC) from Bacillus sp. SJ-10 and overexpressed it in Escherichia coli to investigate its enzymatic properties in the presence of salts. We also investigated the kinetics and thermodynamics of the expressed recombinant enzyme.

Materials and methods Chemicals and reagents p-Nitrophenol, p-nitrophenyl-b-D-glucopyranoside (pNPb Glc), p-nitrophenyl-a-D-glucopyranoside (pNPaGlc), o-nitrophenyl-b-D-galactopyranoside (oNPbGal), cellobiose, cellotriose and cellotetraose were purchased from Sigma (St. Louis, MO, USA). All restriction enzymes were purchased from Enzynomics (Seoul, korea). T4 DNA ligase and DNA Polymerase were supplied by Takara Bio Co. (Japan). Isopropyl-b-D-1-thiogalactopyranoside (IPTG) and ampicillin were purchased from USB (Cleveland, USA). Bacterial strains, plasmids, media, and growth conditions Bacillus sp. SJ-10 (KCCM 90078, JCM 15709) isolated from the squid jeotgal was grown in HM (5 % NaCl, 0.5 % yeast extract, 0.5 % proteose peptone, and 0.1 % glucose)

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medium at 37 °C overnight. E. coli DH5a and E. coli BL21 (DE3) were grown in Luria broth (LB) at 37 °C for overexpression. pETDuet-1 (Novagen) was used as an expression vector. Positive transformants were grown in LB supplemented with ampicillin (100 lg/ml) at 37 °C. All strains were mixed with glycerol or DMSO and kept at -70 °C until use. Cloning, expression and purification of recombinant BglC The gene encoding b-glucosidase (bglC) was amplified from Bacillus sp. SJ-10 genomic DNA using PCR. The forward (BS-bglC-UP, 50 -GGCCGGATCCGATGATACATRAAAAACMGGG-30 ) and reverse primers (BSbglC-RP, 50 -GGCCGTCGACTTATAAACTYTTACCG TTTG-30 ) contained BamHI and SalI restriction sites (underlined), respectively. PCR was performed for 25 amplification cycles at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s. PCR products and the pETDuet-1 vector were treated with BamHI and SalI restriction enzymes. The bglC gene from Bacillus sp. SJ-10 and plasmid pETDuet-1 were ligated using T4 ligase to construct the recombinant expression plasmid. The vector was transformed into E. coli DH5a and finally retransformed into E. coli BL21 (DE3). For overexpression and purification of BglC, recombinant E. coli was grown until it reached an OD600 of 0.6. IPTG (0.5 mM) was added to induce expression of the recombinant protein at 25 °C for 6 h. Cells were harvested by centrifugation (6,000 rpm, 4 °C, 10 min) and resuspended in 50 mM Tris–HCl (pH 8.0). After sonication, the homogenates were centrifuged (12,000 rpm, 4 °C, 10 min). Pellets were resuspended in 20 mM Tris–HCl (pH 8.0) containing 6 M urea and dialyzed against 20 mM Tris–HCl (pH 8.0) for application to the column. Recombinant BglC was purified using a Ni–NTA column. The protein concentration was determined according to the Bradford method using bovine serum albumin as a standard. The purity and molecular weight of the protein were assessed using SDS-PAGE. Enzymatic activity assay The b-glucosidase assay was modified from the pNPG method [12]. The activity of recombinant BglC was determined by measuring the release of p-nitrophenol from the chromogenic substrate p-nitrophenyl-b-D-glucopyranoside (pNPbGlc) in microtiter plates. Assay mixtures (100 ll) containing 5 mM pNPbGlc and enzyme solution (1 mg/ml) in 50 mM Tris–HCl buffer pH 6 were incubated for 1 h at 40 °C. Reactions were stopped by adding two volumes of 1 M Na2CO3. The resulting color was read at 405 nm using an ELISA Reader (Bio-Tek; EL800). The

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amount of released p-nitrophenol was quantified using the pNP standard. One unit of enzymatic activity was defined as the amount of enzyme required to produce 1 nmol of pNP per min under the above assay conditions. All assays were performed in triplicate. Characterization of BglC Optimal pH and temperature To determine the optimal pH and temperature for BglC activity, an enzymatic activity assay was performed using the pNPG method described above. The relative activity of the enzyme against pNPbGlc was examined at pH values of 2–10. The substrates were dissolved in either 50 mM potassium chloride–HCl (pH 2), 50 mM Tris–HCl (pH 3–8) or 50 mM glycine–NaOH (pH 9–10). The optimal temperature was determined by examining b-glucosidase activity in 50 mM Tris–HCl buffer (pH 6) at temperatures ranging from 25 to 45 °C.

for 2 h in the presence of 15 % NaCl or KCl. Samples were cooled immediately and harvested at 10-min intervals, and the remaining activity was determined under the standard assay conditions. Kinetic parameters Km, kcat and the Km/kcat ratio for hydrolysis of pNPbGlc in the absence of salt or in 15 % NaCl or KCl by BglC were determined using 0–20 mM substrate and by analyzing the data based on double-reciprocal (Lineweaver–Burk) plots. Thermodynamic parameters The activation energy was calculated from the Arrhenius equation, which is expressed as [13]: lnðkd Þ ¼ ln k0  ðEd =RTÞ

ð1Þ

Stability was determined at pH 3–9 and at temperatures of 25–50 °C. A total of 100 ll of BglC (1 mg/ml) was incubated at various pH values and temperatures. The sample was withdrawn after 1 h and the remaining activity was determined using the pNPG method.

Ed values were estimated from the slope of the plot of ln(kd) versus 1/T. The kd is the first-order deactivation rate constant, R is universal gas constant and T is the absolute temperature in degrees Kelvin. From the plot of ln(Et/E0) versus t, the slope is the value of kd. Et is the enzymatic activity deactivated after specific times, E0 is the initial activity of b-glucosidase and t is the time of deactivation of the enzyme. Other thermodynamic data were calculated using the following equations [14]:

Substrate specificity

DH ¼ Ed  RT

ð2Þ

DG ¼ RTlnfðkd hÞ=kB T g

ð3Þ

DS ¼ ðDH  DGÞ=T

ð4Þ

Stability

Various pNP-glycoside and cello-oligosaccharide substrates were used to evaluate the substrate specificity of BglC. The b-glycosides used include pNPbGlc, pNPaGlc and oNPbGal, and cello-oligosaccharides used include cellobiose, cellotriose and cellotetraose. pNP-glycoside activity was determined by measuring pNP release, as described above. Hydrolysis of cello-oligosaccharides was monitored based on the release of glucose using the same reaction conditions for pNP glycosides. All compounds were used at 5 mM under optimal conditions at pH 6 and 40 °C, and were incubated with 10 lg of enzyme for 1 h. Effects of salts on b-glucosidase activity and thermostability The effects of various salts, including MgCl2, CaCl2, NaCl, KCl and LiCl on the hydrolysis of pNPbGlc by BglC were determined using the pNPG method at 0–30 % salt concentrations. The enzymatic activity assay was performed under the optimal conditions using pNPbGlc as a substrate, as described above. To measure the thermostability of BglC, the enzyme was pre-incubated at 40, 45 and 50 °C

where h, kB, DH, DG and DS are Planck’s constant, Boltzmann’s constant, enthalpy change, free energy and entropy change, respectively. Molecular modeling and calculation of intraprotein interactions The amino acid sequence of BglC was submitted to SWISS-MODEL (http://www.expasy.org/swissmod/) to obtain the predicted 3D structure [15]. For homology modeling, the crystal structure of 6-phospho-beta-glucosidase from Streptococcus mutans UA159 (PDB ID: 4f66) was used as template. The model was viewed using SwissPDB Viewer and its quality was evaluated by local model quality estimation in SWISS-MODEL. Intramolecular interactions for the BglC were calculated using the PIC (Protein Interactions Calculator) server (http://crick.mbu. iisc.ernet.in/*PIC/) [16]. For comparison with the tertiary structure and intra molecular interactions, the bioinformatics analysis was performed with BglC from

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B. amyloliquefaciens Y2 (NCBI accession number; WP_014720704.1) showing the highest sequence homology with BglC. Nucleotide sequence of BglC Nucleotide sequence of BglC was determined by BigDye(R) Terminator v3.1 Cycle Sequencing Kits (Applied Biosystems). The GenBank accession number for the bglC gene sequence of Bacillus sp. SJ-10 is KF732821.

of these residues is a general acid/base while the second acts as a catalytic nucleophile [18, 19]. Despite the very high sequence homology with reported glycoside hydrolase family 1 from B. amyloliquefaciens spp., a noteworthy feature of BglC was the modification of several amino acid residues existing only in BglC. The I96, A282, F295, V298, K338, Y397 and N474 in the amino sequences of B. amyloliquefaciens spp. were modified with T96, P282,V295, L298, E338, C397 and S474 in BglC, respectively. These specific amino acid residues will be discussed more detail in homology modeling and intra molecular interactions section.

Results and discussion Overexpression, solubilization and purification of BglC Gene cloning and sequence analyses of b-glucosidase Bacillus sp. SJ-10 was chosen for this study because it grows efficiently under high salinity conditions with glucan. Thus, this strain efficiently hydrolyzes carbohydrates into glucose in the presence of high salt concentrations, catalyzed by b-glucosidase. Based on these facts, the bglucosidase of Bacillus sp. SJ-10 was thought to be functional under high salt concentrations. b-glucosidase was amplified from the genome of Bacillus sp. SJ-10 by PCR using universal primers designed based on the nucleotide sequences of the genes from related Bacillus species. A 1,437 bp fragment was obtained and cloned into the pETDuet-1 vector. The deduced amino acid sequence consisted of 478 amino acid residues. b-Glucosidases are divided into two subfamilies; the BGA subfamily (b-glucosidases and phospho-b-glucosidases from bacteria to mammals), which are 450 amino acids in length with a molecular mass of about 50 kDa, and the BGB subfamily (b-glucosidases from yeasts, molds, and rumen bacteria), which are approximately 800 amino acids in length with a molecular mass of *80 kDa [17]. Based on the amino acid sequence, BglC could be classified as a member of the BGA subfamily. BglC was compared to known b glucosidases from Bacillus and other species. BglC of Bacillus sp. SJ-10 showed the highest nucleotide sequence similarity (94.7–98.1 %) and amino acid sequence identity (92.9–99.1 %) with the glycoside hydrolase family 1 b-glucosidases of Bacillus amyloliquefaciens strains. Alignment of BglC amino acid sequences from other b-glucosidase in the glycoside hydrolase family 1 revealed conserved NEX and ENG motifs, containing glutamic acid residues Glu170 and Glu378. These two glutamate residues are important for the enzymatic activity of family 1 b-glucosidases. In general, b-glucosidase hydrolyzes b-glycosidic bonds between a monosaccharide and a moiety, forming an enzyme-substrate complex. Two carboxylic acids of the glutamate side-chain residues positioned in the active site are involved in this reaction. One

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The recombinant plasmid pDuet-BSblgC was transformed into E. coli BL21 (DE3) and the enzyme BglC was overexpressed as an N-terminal His6-tagged protein. After IPTG induction, protein expression was confirmed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A significant amount of induced protein was present in the insoluble fraction at 37 °C after induction with 1 mM IPTG, and was found to aggregate during the purification process. This aggregation is disadvantageous since it reduces enzyme yield, lowers specific activity and increases the cost since a refolding process is required for industrial applications. To address this problem, we expressed BglC under mild conditions, such as low temperature (10, 15, 25 and 37 °C), low inducer concentrations (0.1–1 mM IPTG) and expression time (4, 6, 8, 12 and 24 h), to increase protein solubility. At low temperatures and low inducer concentrations, Bacillus sp. SJ-10 BglC was soluble with high expression levels (Fig. 1b). We determined the optimal conditions for expression of active BglC protein as 25 °C for 6 h induction with 0.5 mM IPTG. Recombinant BglC could be effectively purified by Ni–NTA affinity chromatography (His-bind resin, Novagen), which produced a single band on a SDS-PAGE gel with a molecular weight of 52 kDa (Fig. 1a). Optimal pH, temperature and stability Optimal pH and temperature of recombinant BglC were determined using pNPbGlc as substrate in the absence of salts. The maximal hydrolysis by BglC was observed at pH 6 and 40 °C, respectively. Moreover, more than 60 % enzymatic activity was retained at pH 3–7 at 40 °C (Fig. 2a). Additionally, BglC retained at least 80 % activity when pretreated at a range of pHs between 4 and 8 for 1 h at 40 °C. Enzymatic activity of 95 % was maintained up to 40 °C (Fig. 2b). After 1 h at 45 °C, the residual enzymatic activity was 13 %.

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room temperature, it can be considered an acido-tolerant enzyme with high activity and stability at a wide range of pH values. BglC substrate specificity

Fig. 1 SDS-PAGE analysis of the expression and solubility of recombinant b-glucosidase (BglC) induced at 25 and 37 °C. a Expression of BglC. Lane M molecular weight marker. Lane 1 crude E. coli extract without induction. Lane 2 cells harboring pDuetBSblgC that were cultured for 6 h with IPTG. Lane 3 soluble fraction after induction. Lane 4 inclusion bodies. Lane 5 purified b-glucosidase. b Expression of BglC was induced by different IPTG concentrations and temperatures. Soluble (S) and insoluble (I) proteins of induced E. coli BL21(DE3) transformed with pDuet-BSblgC

Most known b-glucosidases from Bacillus strains, including B. subtilis natto [20], B. circulans subsp. Alkalophilus [21], B. polymyxa [22], B. licheniformis (KCTC 1918) [23], B. halodurans [24] and Bacillus sp. GL1 [25] show optimal activity in slightly acidic or neutral pH values at 37–55 °C. The optimal pH for Bacillus sp. SJ-10 bglucosidase was 6, which was similar to the optimal conditions for b-glucosidases purified from Bacillus sp. GL1 (BglA), B. subtilis natto (BglH, YckE) and B. licheniformis KCTC 1918 (BglH). Also, the optimal temperature for recombinant BglC was similar to BglB, an enzyme identified in Bacillus sp. GL1 (a gellan-degrading enzyme). Recombinant BglC from Bacillus sp. SJ-10 showed 61.7 and 73.2 % activity at pH 3 and 7, while the activities of reported b-glucosidases from other Bacillus species such as B. subtilis natto (YckE), B. halodurans (BglA), and B. licheniformis KCTC 1918 (BglH) were less than 35 % under these conditions. Moreover, since BglC retained 73 % of its initial activity at pH 3 after 1 h incubation at

To examine the substrate specificity of purified recombinant BglC, purified protein was incubated for 1 h at the optimal temperature and pH (40 °C, pH 6) in 50 mM Tris– HCl buffer containing 5 mM b-glycosides or cellooligosaccharides (di-, tri-, tetra-) (Table 1). BglC hydrolyzed both pNPbGlc and pNPbGal at about equal rates. BglC showed the highest activity against pNPbGlc, and enzymatic activity on pNPbGal and oNPbGal was 98.3 and 85.7 %, respectively. However, the enzymatic activity was very low towards pNPaGlc. Similar to our results, Kuo and Lee [20] also reported that broad specificity of b-glucosidase from B. subtilis natto with regard to p-nitrophenyl glycoside [20]. BglC from Bacillus sp. SJ-10 was preferentially active against pNPbGlc among the b-glycosides used in this study. In contrast, hydrolysis activity against cello-oligosaccharides was not detected. b-Glucosidases can be classified into three groups according to their substrate specificity. The first group, arylb-glucosidases, has a strong affinity for aryl-b-glucosides. The second group, cellobiases, only hydrolyzes oligosaccharides including cellobiose. Finally, b-glucosidases (the most common b-glucosidases) are active towards both types of substrates [26]. Our results showed that BglC had activity against aryl-glycosides, but not cello-oligosaccharides. Therefore, BglC from Bacillus sp. SJ-10 belongs to the aryl-b-glucosidase group. Effects of salts on the activity and thermostability of BglC The effects of various salts (MgCl2, CaCl2, LiCl, NaCl and KCl) on enzymatic activity were investigated by adding salt to a final concentration of 10 mM. As shown in Table 2, BglC was activated by 147.9, 127.2, 115.8, 117.1 and 116.1 % by 10 mM MgCl2, CaCl2, LiCl, NaCl and KCl, respectively. Enzymatic activity was increased in all tested salts at 10 mM, while its activity differed at high concentrations. BglC showed significantly increased activity in the presence of NaCl or KCl. The highest activity was observed with 15 % NaCl and KCl, in which activity was increased by 3.3- and 3.5-fold, respectively. Enzymatic activity toward pNPbGlc was observed even in the presence of 30 % NaCl or KCl, which is the saturation concentration. In contrast, 15 % MgCl2, CaCl2 and LiCl decreased activity by 21.3, 2.4 and 4.3 %, respectively. The pH and temperature optima were not affected by 15 % NaCl or KCl (Fig. 3a, b).

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Fig. 2 Effect of pH and temperature on purified BglC. a The optimal pH and stability of BglC were measured using 5 mM pNPbGlc as the substrate at various pH values at 40 °C. To examine pH stability, enzymatic activity was measured after incubation at room

Table 1 Substrate specificity of recombinant BglC

temperature at various pH values for 1 h. Activity was then assayed at 40 °C for 1 h. b To examine the optimal temperature and stability of the enzyme, 200 lg/ml BglC was incubated at 25–45 °C in Tris– HCl buffer (pH 6) for 1 h

Substrate

Relative activity (%)

p-Nitrophenyl-b-D-glucopyranoside (pNPbGlc)

100.0

p-Nitrophenyl-b-D-galactopyranoside (pNPbGal)

98.3

o-Nitrophenyl-b-D-galactopyranoside (oNPbGal)

85.7

p-Nitrophenyl-a-D-glucopyranoside (pNPaGlc)

2.6

Table 2 The effects of salts on BglC Salts

Relative activity (%) 10 mM

5%

10 %

15 %

20 %

MgCl2

147.9 ± 3.5

31.6 ± 1.2

55.4 ± 1.8

21.3 ± 0.9

0.4 ± 0.1

CaCl2

127.2 ± 2.4

2.0 ± 0.1

1.9 ± 0.1

2.4 ± 0.1

1.5 ± 0.1

NaCl

117.1 ± 3.1

282.2 ± 3.8

325.8 ± 5.9

335.6 ± 7.8

158.9 ± 3.8

KCl

116.0 ± 2.8

242.9 ± 4.9

331.2 ± 6.8

353.4 ± 6.2

263.8 ± 4.5

LiCl

115.8 ± 3.8

38.4 ± 1.1

9.6 ± 0.3

4.3 ± 0.2

0.4 ± 0.1

The effect of salts on BglC was tested for b-glucosidase activity using the pNPbGlc method. Relative activity (%) based on the enzymatic activity in Tris–HCl buffer (pH 6) at 40 °C in the absence of salts

To evaluate the thermostability of BglC depending on the presence or absence of salts, the enzyme was incubated at 40–50 °C for 2 h. Regardless of the presence of salt, the residual enzymatic activity was greater than 90 % at 40 °C after 2 h (Fig. 4a, b). However, the results differed at temperatures above 40 °C. In the absence of salts, the activity slowly decreased at 45 °C. The enzyme showed residual activities of 50 % after 30 min at 45 °C, and activity was lost after 110 min. However, in the presence of salts, at 45 °C the enzyme was stable for 2 h with more than 80 % activity. At 50 °C in the absence of salts, BglC

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lost activity within 10 min. In the presence of NaCl, BglC maintained activity after 60 min, and BglC activity was prolonged compared with that in 15 % KCl. These results indicated that NaCl and KCl play an important role in enzymatic activity and thermostability. The salt tolerance of b-glucosidase has been investigated because of its potential use in industrial processes. Although a number of cellulolytic halophiles have been isolated, salt tolerant or halophilic b-glucosidases are rare. To our knowledge, the effect of salt on the activity of only two b-glucosidases has been investigated. The thermostable

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Fig. 3 Effect of a pH and b temperature on purified BglC in the presence of 15 % NaCl or KCl. Enzymatic activity of BglC toward pNPbGlc was measured using the same method and expressed as a percentage of the activity obtained in the absence of salts

Fig. 4 BglC thermal stability at 40, 45 and 50 °C in the presence of a NaCl and b KCl. The enzyme was incubated with 15 % NaCl or KCl at 40, 45, and 50 °C for 2 h, after which residual activity was measured

b-glucosidases from a marine Aspergillus niger showed the highest activity in 6 % (1 M) NaCl, which was 1.4-fold higher than in the absence of salt [10]. The glucose-enhanced b-glucosidase from marine Streptomycete isolated from deep sea sediment had the highest activity in 0.5 M (2.9 %) NaCl, which was 1.6-fold higher than in the absence of salt [27]. Compared with these two b-glucosidases, BglC had considerable enzymatic activity at high NaCl concentrations. Interestingly, enzymatic activity in the presence of KCl was slightly higher than in NaCl. The effect of salts on enzymatic activity may be dependent on both cations and anions. There was no obvious correlation between the inhibitory or activator effects with any single salt property, such as charge density, ionic radii, hydration energies, hydrophobicity or chaotropes (attachment to water molecules and adsorption to non-polar

surfaces) [28, 29]. It is known that family 1 glycosyl hydrolases, including b-glucosidase and other GH families, catalyze glycoside hydrolysis via a double-displacement mechanism involving two carboxylic acid-containing side chains in the active site. One of these groups (a carboxylate) functions as a nucleophile, leading to a glucosyl-enzyme intermediate (an acylal). The other group (a carboxylic acid) acts as a general acid catalyst during the formation of this intermediate and a general base catalyst after its breakdown [18, 30]. The synergistic effect of BglC in the presence of salts (NaCl and KCl) may increase the ionic interactions between charged substrates and the catalytic residues, allowing for a conformational change in the protein and resulting in an enhanced interaction between catalytic residues. However, the mechanism underlying the salt tolerance of BglC remains unclear.

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A significant increase in the activity and thermostability of b-glucosidase from Bacillus sp. strains under high salt conditions has not been reported. BglC, which exhibited increased activity and thermostability in the presence of salts (NaCl and KCl), can be used to hydrolyze and synthesize glycoside substrates under high salinity conditions. BglC may be able to hydrolyze marine biomass, including algae and sponge, as raw materials since it converts glycosides to aglycone and glucose directly using ionic liquids and seawater without a desalting step. It could also be applied to environmental restoration under high salinity conditions, such as food and marine waste. In this regard, salt-tolerant b-glucosidase BglC has significant advantages for the recycling of marine biomass. Kinetic parameters To determine its kinetic properties, the enzymatic activity of BglC was assayed by monitoring the hydrolysis of pNPbGlc at a wide range of substrate concentrations. The specific activity of recombinant BglC showed 3.2 nmol/ min mg in the absence of salts (Table 3). It was about 2.4 times higher compared with the YckE which the corresponding gene product in Bacillus subtilis 168 [31]. YckE was about 0.4 and 1.35 nmol/min mg on 4-methylumbelliferyl-b-D-glucopyranoside-6-phosphate and 4-methylumbelliferyl-b-D-galactopyranoside (fluorogenic substrates for b-glucosidase and b-galactosidase), respectively. Meanwhile, BglC showed a lower Michaelis constant (Km) and higher kcat for pNPbGlc in the presence of 15 % NaCl or KCl than in the absence of salts. Comparison of the Km and kcat values revealed that BglC showed approximately 1.5and 1.2-fold higher affinity and hydrolyzed pNPbGlc approximately 1.9- and 2.1-fold faster in the presence of 15 % NaCl and KCl, respectively. The specificity constants of BglC for pNPbGlc in the presence of 15 % NaCl and KCl were higher than for pNPbGlc alone. The catalytic efficiency (kcat/Km) of recombinant BglC in the absence of salt, and with 15 % NaCl and KCl were 3.2, 10.3 and 11.0, respectively. Thus, recombinant BglC showed the highest catalytic efficiency in the presence of 15 % KCl.

To the best of our knowledge, it has been first reported by the present authors that the catalytic efficiency of BglC is increased by salts. So far, bglC gene has been tentatively identified as a b-glucosidase, but the activity of the corresponding gene product has not been demonstrated. Certainly not only the precise function of BglC is not yet fully elucidated, but also the study on the BglC from Bacillus spp. was extremely rare. Setlow et al. [31] reported that YckE expression is not induced by aryl-b-D-glucosides nor does it vary notably with the stage of growth, However, YckE comprises significantly more of the total glucosidase activity on 4-methylumbelliferyl-b-D-glucopyranoside-6phosphate in dormant spores than in late-exponential phase or stationary-phase cells [31]. Additionally, according to this report, it was mentioned that although the specificity of this hydrolase has not yet been explored in detail, perhaps YckE has very high activity on some unusual b-glucoside. In the current work, we found that Bacillus sp. SJ-10 grow up using indican (indoxyl-b-D-glucopyranoside) as the sole carbon source, and it can be hydrolysed by BglC. Therefore, taken together, these results suggest that BglC may play an important role in the hydrolysis of other b-glucosides. Additionally, BglC may not the major b-glucosidase in natural environments, but there is a possibility that can be major in harsh conditions including high salinity unable to act other b-glucosidases. Thermodynamic parameters Thermal denaturation of enzymes is a two-step process [32]: N $ U!I where N is the native enzyme, U is the unfolded inactive enzyme that could be reversibly refolded upon cooling, and I is the inactivated enzyme formed after prolonged exposure to heat and therefore cannot be refolded upon cooling [33]. The thermal denaturation of enzymes results in the breakage of non-covalent linkages, including hydrophobic interactions, with concomitant increases in the enthalpy of activation (DH). The opening of enzyme structures is

Table 3 Kinetic properties of recombinant BglC Substrate

Km (mM)

105 9 kcat (per min)

105 9 kcat/Km (per min mM)

Specific activity (per nmol/ min mg)

Relative activity (%)

pNPbGlc

5.4 ± 0.15

5.8 ± 0.09

1.1 ± 0.02

3.2 ± 0.07

100 ± 1.3

pNPbGlc ? 15 % NaCl

3.6 ± 0.08

11.2 ± 0.20

3.1 ± 0.10

10.3 ± 0.25

323 ± 4.1

pNPbGlc ? 15 % KCl

4.6 ± 0.04

13.6 ± 0.31

3.0 ± 0.09

11.0 ± 0.25

344 ± 3.3

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accompanied by increases in the disorder or entropy of activation (DS) [34]. Enzymes can increase thermostability by either stabilizing the native form through non-covalent bonds including hydrogen bonds, salt bridges and hydrophobic interactions or by decreasing the entropy of unfolding [35, 36]. Thermal inactivation of the BglC isolated from Bacillus sp. SJ-10 was investigated at three temperatures (40, 45 and 50 °C), and first-order deactivation rate constants were determined in 0 % salt, 15 % NaCl and 15 % KCl solution (Fig. 5a–c). We found that b-glucosidase was very stable at 40 °C, and an increase in temperature to 50 °C resulted in a progressive loss of activity. The thermodynamics of enzyme thermo-inactivation was also calculated (Table 4). The energy of activation (Ea) for denaturation was 729.52 kJ/mol in the absence of salt, 377.53 kJ/mol in 15 % NaCl and 604.11 kJ/mol in 15 % KCl. The Ed values

were calculated based on the plots of ln kd versus 1/T (Fig. 5d). The thermodynamic parameters, such as DH and DS, of b-glucosidase in 15 % NaCl and KCl were lower than those under salt-free conditions. The thermostabilization of enzymes is mostly accompanied by decreases in DH and DS, as shown by the carboxyl group-modified glucoamylase from A. niger [37], acetylated a-amylase [38] and protein-engineered T4 lysozyme [36]. Both the DH and DG of BglC in the absence of salt, and with 15 % NaCl and KCl decreased with increasing temperature, whereas DS did not show a linear decrease with an increase in temperature from 45 to 50 °C. This showed that the increased thermostability of BglC in 15 % NaCl and KCl is due to conformational changes at high temperatures, as observed for an acetylated a-amylase from B. subtilis. Further decreases in DS in the presence of 15 % salt compared to salt-free conditions could be due to reduced

Fig. 5 First-order plots of the effect of thermal denaturation of BglC in a 0 % salt, b 15 % NaCl and c 15 % KCl solution. Samples were incubated at 40 °C (open square), 45 °C (closed triangle) and 50 °C (open circle) in 50 mM Tris–HCl buffer, pH 6, and aliquots

withdrawn at different time points were cooled on ice before assaying for residual enzymatic activity at 40 °C. d Arrhenius plot for the determination of the activation energy (Ed) of denaturation of BglC

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Bioprocess Biosyst Eng Table 4 Thermodynamic parameters of the BglC at different salinities

Salt (%) 0 % Salt

15 % NaCl Ed = 729.52 kJ/mol in 0 % salt buffer; Ed = 377.53 kJ/mol in 15 % NaCl buffer; Ed = 604.11 kJ/mol KCl buffer. The values of Ed were calculated by the plots of ln kd versus 1/T

15 % KCl

Temperature (K)

DH (kJ/mol)

DG (kJ/mol)

DS [J/(mol K)]

313.15

726.93

69.07

2,100.77

318.15

726.88

56.21

2,108.04

323.15

726.84

47.92

2,100.95

313.15

374.94

66.21

985.87

318.15

374.89

64.32

976.19

323.15

374.85

56.15

986.24

313.15

601.51

65.46

1,711.79

318.15

601.47

64.80

1,686.84

323.15

601.43

48.09

1,712.32

Fig. 6 Prediction of three-dimensional structure of BglC. a Representation of the structure, showing the (a/b)8 barrel form of BglC. b Difference of BglC compared with corresponding b-glucosidase from B. amyloliquefaciens Y2. c Formation of putative ionic bonds

with Glu307 and Lys341, and with Lys318 and Glu338 in BglC are shown. Structure colors: green BglC from Bacillus sp. SJ-10 and blue BglC from B. amyloliquefaciens Y2

repulsion between negatively charged carboxyl groups, thus decreasing the flexibility of an external loop and stabilizing BglC in salt solution. Gibb’s free energy of activation of denaturation (DG), enthalpy for denaturation (DH), and entropy change (DS) are reliable markers of protein stability [14]. A smaller DG is indicative of lower protein stability. As shown in Table 4, the DG values in 15 % NaCl and KCl were higher than that under salt-free conditions at 45 and 50 °C. The thermodynamic parameters suggested that BglC was more stable under high salt conditions than in the absence of salt.

(Fig. 6a). It was composed of an inner core of eight parallel b-strands surrounded by eight a-helices with additional units of secondary structure inserted between the a/b units. The two conserved glutamate residues participating catalysis of BlgC, Glu170 and Glu378, are located at the loop (which links a b-strand with an a-helix) and inner core b-strand, respectively. According to the overall model, superimposition of the BglC structure on a reported family 1 glycosyl hydrolase (including family 1 glycosyl hydrolase of B. amyloliquefaciens Y2) ortholog indicated a very similar three-dimensional arrangement. However, it was found that a slightly different structure in extra regions of secondary structure. Unlike the helix form that appears in a typical family 1 glycosyl hydrolases, BglC forms a long loop at the amino acid residues 315–341 which the surrounding a deep cavity as a active site channel (Fig. 6b). Calculated intramolecular interactions results showed that specific amino acid residues in BglC could induce result in significant change of intra molecular interactions when compared with B. amyloliquefaciens Y2. Glu338 enables the formation of putative

Homology modeling and intra molecular interactions We performed bioinformatics analysis to examine the differences of protein structure and intra molecular interactions based on the specific amino acid residues in BglC compared with family 1 glycosyl hydrolases of B. amyloliquefaciens spp. The predicted three-dimensional structure model shows that BglC folds into an (a/b)8 barrel which structure was well-known up to the present

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ionic bonds with the Glu307 and Lys341, and with the Lys318 and Glu338 (Fig. 6c). The occurrence of ionic bond was the cause of the loop formation at the amino acid residues 315–341. The increased number of putative ionic bonds at these positions strengthens the interaction between surface residues, which is believed to be important for the maintenance of the backbone rigidity and protein stability. In addition, aromatic sulfur interactions could lead to improved packing efficiency and protein thermostability. The Thr96 and Ser474 also indicate that serine and threonine are the best residues for interacting with water molecules surrounding protein structures [39]. Therefore, two ionic bonds of BglC probably make the main contribution to the salt tolerant. Additionally, it is likewise possible on BglC that specific amino acids may induce change of electrostatic potential energy of protein surface, and exhibit improved salt tolerance and salt-dependent thermal stability.

Conclusion The biochemical properties of b-glucosidase (BglC) from Bacillus sp. SJ-10 were investigated in the presence of different salts at various concentrations. BglC showed higher activity and better thermostability under high salinity conditions. Enzymatic activity was retained even in the presence of 30 % NaCl or KCl (the saturation concentration), indicating that this enzyme is active and stable at both high and low salt concentrations. The kinetic and thermodynamic properties of BglC also showed that BglC is a salt-tolerant b-glucosidase. The synergistic effects of multiple salts on b-glucosidase activity have not yet been reported from other Bacillus spp. Based on the results in this study, BlgC may be a suitable enzyme for the manufacture of pharmaceuticals, fine chemicals and food ingredients. Also, it is a promising candidate for recycling of marine wastes containing b-glycosidic bonds under high salt conditions, and provides a new model to investigate the effects of salt on protein structure. Acknowledgments This research was supported by iPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

References 1. Ketudat Cairns JR, Esen A (2010) b-Glucosidases. Cell Mol Life Sci 67:3389–3405 2. Singhania RR, Patel AK, Sukumaran RK, Larroche C, Pandey A (2012) Role and significance of beta-glucosidases in the hydrolysis of cellulose for bioethanol production. Bioresource Technol 127:500–507

3. Bhatia Y, Mishra S, Bisaria VS (2002) Microbial b-glucosidases: cloning, properties, and applications. Crit Rev Biotechnol 22:375–407 4. Beguin P, Aubert JP (1994) The biological degradation of cellulose. FEMS Microbiol Lett 13:25–58 5. Searchinger T, Heimlich R, Houghton RA, Dong F, Elobeid A, Fabiosa J, Tokgoz S, Hayes D, Yu TH (2008) Use of U.S. croplands for biofuels increases greenhouse gases through emissions from land-use change. Science 319:1238–1240 6. Chisti Y (2007) Biodiesel from microalgae. Biotechnol Adv 25:294–306 7. Hsieh CH, Wu WT (2009) Cultivation of microalgae for oil production with a cultivation strategy of urea limitation. Bioresource Technol 100:3921–3926 8. Kalinin VI, Ivanchina NV, Krasokhin VB, Makarieva TN, Stonik VA (2012) Glycosides from marine sponges (Porifera, Demospongiae): structures, taxonomical distribution, biological activities and biological roles. Mar Drugs 10:1671–1710 9. Fu CC, Hung TC, Chen JY, Su CH, Wu WT (2010) Hydrolysis of microalgae cell walls for production of reducing sugar and lipid extraction. Bioresource Technol 101:8750–8754 10. Xue DS, Chen HY, Ren YR, Yao SJ (2012) Enhancing the activity and thermostability of thermostable b-glucosidase from a marine Aspergillus niger at high salinity. Process Biochem 47:606–611 11. Kim YR, Kim EY, Lee JM, Kim JK, Kong IS (2013) Characterization of a novel Bacillus sp SJ-10 b-1,3-1,4-glucanase isolated from jeotgal, a traditional Korean fermented fish. Bioprocess Biosyst Eng 36:721–727 12. Neil JP, David EB, Emyr O, Isabel V, Jozef VB, Mahalingeshwara KB (2001) Biochemical characterization and mechanism of action of a thermostable b-glucosidase purified from Thermoascus aurantiacus. Biochem J 353:117–127 13. Gummadi SN (2003) What is the role of thermodynamics on protein stability? Biotechnol Bioproc E 8:9–18 14. Marangoni AG (2003) Enzyme kinetics: a modern approach. Wiley 146–150 15. Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISSMODEL: an automated protein homology-modeling server. Nucleic Acids Res 31:3381–3385 16. Tina KG, Bhadra R, Srinivasan N (2007) PIC: protein interactions calculator. Nucl Acids Res 35:473–476 17. Gra¨bnitz F, Seiss M, Ru¨cknagel KP, Staudenbauer WL (1991) Structure of the beta-glucosidase gene bglA of Clostridium thermocellum. Sequence analysis reveals a superfamily of cellulases and beta-glycosidases including human lactase/phlorizin hydrolase. Eur J Biochem 200:301–309 18. Sinnott ML (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem Rev 90:1171–1202 19. McCarter JD, Withers SG (1994) Mechanisms of enzymatic glycoside hydrolysis. Curr Opin Struc Biol 4:885–892 20. Kuo LC, Lee KT (2008) Cloning, expression, and characterization of two b-glucosidases from Isoflavone glycoside-hydrolyzing Bacillus subtilis natto. J Agr Food Chem 56:119–125 21. Paavilainen S, Hellman J, Korpela T (1993) Purification, characterization, gene cloning, and sequencing of a new b-glucosidase from Bacillus circulans subsp. Alkalophilus. Appl Environ Microb 59:927–932 22. Gonza´lez-Candelas L, Aristoy MC, Polaina J, Flors A (1989) Cloning and characterization of two genes from Bacillus polymyxa expressing b-glucosidase activity in Escherichia coli. Appl Environ Microb 55:3173–3177 23. Choi IS, Wi SG, Jung SR, Patel DH, Bae HJ (2009) Characterization and application of recombinant b-glucosidase (BglH) from Bacillus licheniformis KCTC 1918. J Wood Sci 55:329–334

123

Bioprocess Biosyst Eng 24. Naz S, Ikram N, Rajoka MI, Sadaf S, Akhtar, MW (2010) Enhanced production and characterization of a b-glucosidase from Bacillus halodurans expressed in Escherichia coli. BiochemistryMoscow 75:513–513 25. Hashimoto W, Miki H, Nankai H, Sato N, Kawai S, Murata K (1998) Molecular cloning of two genes for b-D-glucosidase in Bacillus sp. GL1 and identification of one as a gellan-degrading enzyme. Arch Biochem Biophys 360:1–9 26. Enari TM, Niku-paavola ML (1987) Enzymatic hydrolysis of cellulose: is the current theory of the mechanism of hydrolysis valid? Crit Rev Biotechnol 5:67–87 27. Mai Z, Yang J, Tian X, Li J, Zhang S (2013) Gene cloning and characterization of a novel salt-tolerant and glucose-enhanced bglucosidase from a marine Streptomycete. Appl Biochem Biotech. 169:1512–1522 28. Zhao YH, Abraham MH (2005) Octanol/water partition of ionic species, including 544 cations. J Org Chem 70:2633–2640 29. Collins KD (1995) Sticky ions in biological systems. P Natl Acad Sci USA 92:5553–5557 30. McCarter JD, Withers SG (1994) Mechanisms of enzymatic glycoside hydrolysis. Curr Opin Struc Bio l4:885–892 31. Setlow B, Cabrera-Hernandez A, Cabrera-Martinez RM, Setlow P (2004) Identification of aryl-phospho-b-D-glucosidases in Bacillus subtilis. Arch Microbiol 181:60–67

123

32. Montes FJ, Battaner E, Catalan J, Galan MA (1995) Kinetics and heat-inactivation mechanism of D-amino acid oxidase. Process Biochem 30:217–224 33. Zale SE, Klibanov AM (1986) Why does ribonuclease irreversibly inactivate at high temperatures? Biochemistry 25:5432–5444 34. Vieille C, Zeikus JG (1996) Thermozymes: identifying molecular determinants of protein structural and functional stability. Trends Biotechnol 14:183–190 35. Daniel RM (1996) The upper limits of enzyme thermal stability. Enzyme Microb Tech 19:74–79 36. Matthews BW, Nicholson H, Becktel WJ (1987) Enhanced protein thermostability from site directed mutations that decrease the entropy of unfolding. P Natl Acad Sci USA 84:6663–6667 37. Munch O, Tritsch D (1990) Irreversible thermoinactivation of glucoamylase from Aspergillus niger and thermostabilization by chemical modification of carboxyl groups. Biochim Biophys Acta 1041:111–116 38. Urabe I, Nanjo H, Okada H (1973) Effect of acetylation of Bacillus subtilis a-amylase on the kinetics of heat inactivation. Biochim Biophys Acta 302:73–79 39. Pei XQ, Yi ZL, Tang CG, Wu ZL (2011) Three amino acid changes contribute markedly to the thermostability of b-glucosidase BglC from Thermobifida fusca. Bioresource Technol 102:3337–3342