A Surfactant, Oxidant and Inhibitor Compatible Thermo-solvent-tolerant Amylase From a Novel Extrimophilic Bacillus Subtilis Strain Clb-34 Mk443366: Study on Purication and Characterization

It is the rst time when thermo-tolerant, heavy metal resistance amylase producing strain Bacillus subtilis isolated from soil sample. Amylase was puried 3.8-fold with a specic activity of 11,305.0 U mg -1 . The molecular weight of puried amylase was 67 kDa as revealed by SDS-PAGE and activity gel analysis. The amylase was active in broad pH and temperature range of 4.0-11.0 and 35-110°C, respectively, with maxima at pH 7.0 and 85°C temperature. The amylase has Km and Vmax value of 2.181 mg ml -1 and 909.09 µg ml -1 min -1 , respectively when starch used as substrate. The amylase was not only stable but also its activity enhanced in the presence of n-dodecane, iso-octane, n-decane, xylene, toluene, n-butanol, acetone, and cyclohexane, after prolonged incubation (7 days). Amylase activity and stability was inhibited in the presence of Hg 2+ , benzene, sodium perborate. The unique property of solvent tolerance and heavy metal resistance proves the potential candidature of this isolate not only for starch liquefaction for food application but also for bioremediation strategies involved in environmental cleanup.


Introduction
In modern times, the products of biological origin, particularly enzymes, are attracting the attention of researchers. Their role in several biological and commercial processes has been duly emphasized.
Among all the enzymes, α-amylases constitute a class of industrial enzyme having approximately 30% of the world enzyme production (Van der Maarel Marc et al. 2002) and represent one of the three largest groups of industrial enzymes and account for approximately 25% of total enzymes sales in the world (Rao et al. 1998) and are an important enzyme, particularly in the process of starch or glycogen hydrolysis.
The amylases can be derived from several sources such as plants, animals and microbes. The major advantage of using microorganisms for production of amylases is in economical bulk production capacity and microbes are also easy to manipulate to obtain enzymes of desired characteristics (Aiyer 2005; Vidyalakshmi et al. 2009). Based on their mode of action, they are further classi ed into three categories α-amylases, β-amylases and glucoamylases. All amylases are glycoside hydrolyser and act on α-1,4 glycosidic bonds (Maton et al. 1993). Industrially, α-amylase is used particularly in starch liquefaction, brewing, textile, pharmaceuticals, paper, detergents, drugs, toxic wastes removal and oil drilling (Ajayi and Fagade 2003). Since α-amylases are active over a broad pH (5)(6)(7)(8)(9) and temperature (35- The application of an amylase in industrial reactions depends on its unique characteristics, such as its action pattern, substrate speci city, major reaction products, optimal temperature, and optimal pH (Yun et al. 2004). They are mainly employed for starch liquefaction to reduce their viscosity, production of maltose, oligosaccharide mixtures, high fructose syrup and maltotetraose syrup. In detergents production, they are applied to improve cleaning effect and are also used for starch de-sizing in textile industry (Chengyi et al. 1999). Generally production of this enzyme has been carried out by submerged fermentation (Enhasy 2007) because of the ease of sterilization and process control easier to engineer in these systems. The purpose this studies to isolate a novel thermoloterant amylase producing bacteria in the presence of organic solvent. Puri cation and characterization of amylase by bacteria was also performed in this study.

Materials
All analytical grade reagents and media components were purchased from Hi-Media (Bombay, India) and Merk (India). Column chromatography materials, protein ladder for electrophroresis, and starch were procured from Sigma-Aldrich Pvt. Ltd., USA.

Microorganism
Bacillus subtilis strain CLB-34 MK443366 used in this study was isolated from the soil sample and identi ed on the basis of phenotypic (16S r DNA) and biochemical tests. Analysis of 16S rDNA sequence (799 bp) revealed its 95.0% homology with Bacillus subtilis strains, and was designated as Bacillus subtilis CLB-34. The 16S rDNA sequence of Bacillus subtilis was submitted to Gene bank [MK: 443366] and a link to the dataset is https://www.ncbi.nlm.nih.gov/nuccore/MK443366 (All details are submitted in the form of Supporting Data). The strain CLB-34 was in the same cluster of phylogenetic tree ( Fig. 1) with different strains of Bacillus subtilis. It was maintained on starch nutrient agar slants at 4 °C.

Crude enzyme preparation
One full loop of 24 h grown culture take from nutrient starch slant was transferred in 50 ml basal broth (2.0%, starch; 0.5%, peptone; 0.3%, beef extract; 0.5%, NaCl) and incubated at 55 °C for 24-48. To obtain crude enzyme, culture broth was transferred to micro-centrifuge tubes and centrifuged at 10000 rpm for 10 min. Cells were discarded and resultant supernatant was used as the crude enzyme for various enzyme assay.

Enzyme assay
The activity of α-amylase was assayed by measuring the reducing sugar released by reaction on starch.
Amylase assay was done (Nelson 1944;Somogyi 1952) by using a reaction mixture consisting 500 µl of substrate solution (1.0% soluble starch in 1.0 M phosphate buffer pH 7.0), 100 µl of the enzyme solution and 1 ml volume make up by adding 400 µl distilled water. The reaction mixture was incubated for 10 min at 55 °C. Reaction was stopped by adding 1 ml of alkaline copper tartrate solution and incubated in boiling water bath for 10 min and cooled, then added arsenomolybdate solution for color stabilization.
Optical density of each sample with reaction mixture was taken at 620 nm in a spectrophotometer (Shimadzu, Japan). One unit of enzyme activity was de ned as the amount of enzyme that liberates 1.0 µg of glucose min/ml.

Extraction and Puri cation of Amylase
A three step puri cation method was used to purify the amylase produced by the B. subtilis CLB-34. All the puri cation steps were performed at temperatures between 0 and 4 °C unless otherwise stated.

Enzyme extraction
The crude culture supernatant obtained from 24 h old cultures of B. subtilis CLB-34 grown under optimal conditions was subjected to puri cation. The crude culture ltrate was subjected to a cooling centrifugation at 10,000 rpm to remove the cells and the residual medium. The resulted supernatant was used as crude enzyme extract.

Acetone precipitation
The cell free crude enzyme was saturated by the addition of different volumes (45%, 55%, 65% and 75%) of enzyme grade chilled acetone with gentle stirring on ice-bath. The mixture was left at 4 °C for 4 h and the precipitate was recovered by centrifugation at 10,000 g for 10 min. The supernatant was drained off and precipitate was kept at room temperature for few minutes to remove traces of acetone. The precipitate was dissolved 100 mM phosphate buffer (pH 7.0). The corresponding precipitates were recovered, dissolved individually in fresh buffer and assayed for both total protein content and amylase activity.

Ion exchange chromatography
An anion-exchanger (Q-sepharose) column (Sigma-Aldrich Pvt. Ltd., USA; 15 × 70 mm 2 ), pre-equilibrated with 100 mM phosphate buffer (pH 7.0) was used for further puri cation of the enzyme. The active fraction of acetone precipitates was suitably diluted ( nal volume 3 ml) with 100 Mm phosphate buffer (pH 7.0) prior to loading on column and the ow rate was adjusted to 20 ml/h. Afterthat, the diluted enzyme fraction was allowed to bind with matrix for 2 h at 4 °C. Then the unbound fraction was collected and analyzed for enzyme activity and for protein content. After collecting the loaded sample, column was washed with the same buffer until OD620 of the e uent was zero. The bound fractions were eluted with a linear gradient of NaCl (0.1-0.5 M, 10 ml each) in the same buffer.

Gel ltration chromatography on Sephadex G-75 column
The partially puri ed enzyme was applied to gel-ltration chromatography for puri cation up to homogeneity. The Sephadex-75 column (Sigma Aldrich Pvt. Ltd., USA, 1.5 × 40 cm) was equilibrated with sodium phosphate buffer (100 mM, pH 7.0) and 1 ml of concentrated sample was applied to the column.
The ow rate was adjusted to 5-6 ml/h and fraction of 2 mL each was collected. Amylase activity and estimation of protein content were determined for each individual fraction.

Determination of protein concentrations
Quantitative estimation of protein content was done by the method of Lowry et al. (1951) using Bovine serum albumin (BSA) as standard and expressed as mg/ml. The protein content of individual fraction obtained after different steps of chromatography was monitored by measuring the extinction at 280 nm.

Polyacrylamide gel electrophoresis
The active fraction, with maximum speci c activity, obtained after gel ltration chromatography along with crude, acetone precipitate and anion-exchange chromatography was electrophorezed by Sodium Dodecyl Sulphate-Poly Acrylamide Gel Electrophoresis in a 12.5% polyacrylamide gel according to the method of Laemmli (1970). Approximate molecular weight of the amylase was estimated by SDS-PAGE against the molecular mass markers i.e. lysozyme (14. The in uence of temperature on activity of amylase was studied by incubating the reaction mixture at different temperatures (35-110 °C). The enzyme was incubated at different temperatures 35-110 °C for 1 h to study the stability of the enzyme. The residual amylase activity was measured by conducting the reaction at temperature 55 °C and pH 7.0. The activity of the enzyme was considered as 100% under standard assay conditions.

Effect of pH on enzyme activity and stability:
The effect of pH on amylase activity was measured in the pH range of 4 to 11, using the appropriate buffers at concentration of 100 mM (4.0-6.0, sodium acetate; 6.0-8.0, sodium phosphate; 8.0-10.0, Tris-HCl; 9.0-11.0, glycine-NaOH) under standard assay conditions. To study stability as a function of pH, 100 µl of the puri ed enzyme was mixed with 100 µl of the buffer solutions and incubated at 55 °C for 1 h then aliquots of the mixture were taken to measure the residual amylase activity (%) under standard assay conditions.

Effect of metal ions on activity and stability
The effect of various metal ions (5 mM and 10 mM) on enzyme activity was investigated using FeSO 4, CaCl 2, KCl, NaCl, MgCl 2, MnCl 2, ZnSO 4, CuSO 4, HgCl 2 and NiCl 2 . The enzyme was incubated with different metals at 55 °C for 1 h to study metal ion stability and assayed under standard assay conditions.

Effect of organic solvent on amylase stability
Cell free supernatant having maximum amylase activity was ltered with nitrocellulose membrane (pore size 0.22 µm) and incubated with 30% (v/v) of different organic solvent viz., n-dodecane, n-decane, isooctane, xylene, n-hexane, n-butanol, cyclohexane, acetone, toluene, benzene, ethanol, methanol and propanol for 1 week in screw crapped tubes at 55 °C and 120 rpm. The residual amylase activity was estimated against the control, in which solvent was not present.

Substrate speci city
Substrate speci city of the puri ed enzyme was determined by assaying with different substrates (Soluble starch, Dextrin, Pullulan, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, wheat starch, Potato starch, Rice starch) using 1% (w/v) concentration at pH 7.0 and 55 °C. The enzyme activity on soluble starch was de ned as 100%, and the enzyme activities on other substrates were calculated as relative activities.

Kinetic analysis
The in uence of substrate concentration on the reaction velocity of the puri ed amylase was studied with starch. The puri ed amylase was incubated with various concentration of starch. The nal concentration ranged from 0.25-4.0 mg/ml. In all cases, the enzymatic activity was assayed under standard conditions. The Michaelis constant (K m ) and maximum velocity (V max ) was determined from Lineweaver-Burk plots

Enzymatic reaction product analysis
The puri ed enzyme was used at a dose of 0.5 U/mg soluble starch in 100 mM phosphate buffer (pH 7.0) at 55 °C and at different time intervals (2 h, 8 h and 12 h). Hydrolysis products were subjected to analyzed by High-performance liquid chromatography (HPLC) using a Hypersil NH 2 column at 50 °C using a 75% acetonitrile (V/V) was used as mobile phase at a ow rate of 1.0 ml/min and the hydrolysis products were detected using a RID-10A SHIMADZU refractive detector. Authentic chromatographic grade glucose, maltose, maltotriose, maltotetrose, and maltopentose were used as standards for identi cation of the hydrolysis products in the reaction mixture.

Statistical analysis
Each experiment with the required controls was performed in triplicate and the data are presented as the mean ± one standard deviation (SD). Signi cance of the differences between means was tested for by analysis of variance (ANOVA) and Duncan's multiple means tests (DMMT) on the parametric or arc-sine square root transformed data using the SPSS software, where a value of less than 0.05 was considered as signi cant.

Culture identi cation
The 16S rRNA gene sequencing has been by far the most common housekeeping genetic marker to study bacterial phylogeny and taxonomy attributed to (i) its presence in almost all bacteria, often existing as a multigene family, or operons; (ii) the function of the 16S rRNA gene over time has not changed, suggesting that random sequence changes are a more accurate measure of time (evolution); and (iii) the 16S rRNA gene (1,500 bp) is large enough for informatics purposes (Patel 2001). In the present investigation, the 16S rDNA sequence analysis revealed its maximum closeness with Bacillus subtilis hence the isolate was designated as Bacillus subtilis CLB-34. However, the 16S rDNA sequence analysis indicates that it is a different and novel strain of Bacillus subtilis (Fig. 1).

Puri cation of extra-cellular α-amylase
The crude enzyme extract was rst concentrated by acetone precipitation. Maximum activity was observed in the fraction obtained by the addition of acetone in 50% with protein content of 25.67 mg/ml. This fraction had 11,305.0 U/mg of speci c activity with recovery of 79.7% and with regard to puri cation it showed 3.8-fold puri cation (Table 1). The active fraction of acetone precipitation method was used for further puri cation by using ion exchange chromatography. Sample (1 ml) was loaded into the Q-Sepharose column pre-equilibrated with sodium phosphate buffer (100 mM, pH 7.0) and allowed to pass through the column. The unbound fraction was collected and analyzed for amylase activity and protein content. There was no amylase activity in the fraction, while 2.1 mg/ml of protein was estimated. The absence of enzyme in unbound fraction suggested that total amylase was bound to matrix. The bound enzyme was eluted by sodium phosphate buffer (100 mM, pH 7.0) having NaCl with increasing concentration at gradient of 0.1 M. Ten per ml solution of each concentration of NaCl was used to evade the bound enzyme. The amylase activity was detected in the fraction released by the addition of 0.5 M NaCl Anion-exchange chromatography of amylase on column resulted in one prominent peak at the 24th fraction (Fig. 2).
The active fraction was applied on Sephadex G-75 column. Figure 3 shows the fractionation pattern of amylase on Sephadex G-75 column. One distinctive protein peak was appeared that overlapped with the amylase activity. The puri cation process resulted in 33.8-fold puri cation factor and a nal recovery of 27.1% of the enzyme with speci c activity of 99,491.4 U/mg (Table 1). However, Mesbaha and Wiegelb (2014) reported the amylase was puri ed by a combination of 80% ethanol precipitation, ion exchange chromatography with Q sepharose and Superdex™ 75 gel ltration chromatography. The enzyme was puri ed 4.5 fold with 15.4% recovery and a speci c activity of 250 units/mg protein

Electrophoretic analysis
The purity of the enzyme was con rmed by the presence of a single band on SDS-PAGE and its molecular weight was approximately 67 kDa (Fig. 4) In this experiment the amylase of B. subtilis CLB-34 was absolutely stable in the wide temperature range of 35-95 o C during 1 h incubation. The enzyme retained 98% activity even after treatment at 100 o C (Fig. 5). Similarly 100% activity at 90 ºC for 1 h for amylase from Bacillus sp. has been reported by Teodoro and Martins (2000). However, with further increase in every 5 o C temperature, there was a gradual decrease in enzyme stability ranging between 10-15% upto 110 o C. The amylase of B. subtilis CLB-34 retained 98, 88 and 75% activity even after treatment at 100, 105 and 110 o C, respectively (Fig. 5).
The amylase of Bacillus subtilis CLB-34 is more thermostable than amylase studied by several other researchers. Arikan (2007) have reported a thermostable amylase stable up to 60-100 o C but retained only ~ 96% activity at 100 o C. Most other thermophile Bacillus amylases reported to so far, amylases exhibited higher temperature optimum for activity and showed good thermal stability (Dong et al. 1997;Horikoshi 1999). These are the properties considered to be very important for industrial starch liquefaction. Hence it is evident that the amylase of Bacillus subtilis CLB-34 is more thermostable, and may be applied to several biotechnological and industrial purposes.

Effect of pH on enzyme activity and stability
The pH stability on the puri ed amylase of B. subtilis CLB-34 was determined by measuring the enzyme activity at varying pH values ranging from 4.0-11.0 using different suitable buffers. Figure 5b showed that maximum amylase activity was established at pH 8.0, however it was found to be most stable at pH 7.0 (Fig. 6).

Effect of metal ions on activity and stability
Results suggest that α-amylase of B. subtilis CLB-34 showed maximum relative activity (194%) and stability (160%) in the presence of Calcium ion (10 mM  Enzyme activity was determined at 55 °C in the presence of metal ions in the reaction mixture directly and for stability enzyme was pre-incubated with different metal ions at 55 °C for 1 h and assayed as standard assay method. The enzyme activity without incubation with metal ions was taken as 100%.
Mean standard deviation for all the values is < ± 5.0%.

Effect of organic solvents on amylase stability
In another experiment, the effect of various organic solvents (30%, v/v) on amylase stability was also investigated for one week, and the results are depicted in Table 3. The amylase of Bacillus subtilis is extraordinarily stable in the presence of all organic solvents under study. It was observed that except benzene, propanol and ethanol, presence of other solvents (n-dodecane, iso-octane, n-decane, xylene, toluene, n-haxane, n-butanol, acetone, methanol and cyclohexane) enhanced the amylase activity (Table 3). After incubation with n-dodecane, iso-octane, n-decane, xylene, Toluene, n-haxane, n-butanol, Acetone, Methanol, and cyclohexane the amylase activity increased to 252. 3

Effect of inhibitors on enzyme stability
When the Bacillus subtilis amylase enzyme was incubated with EDTA, PMSF, Urea and βmercaptoethanol, the enzyme activity was retained at 96.3%, 94%, 99.4%, and 95.6% of the original activity at 10 mM (Table 4) 28% activity with 5 mM. EDTA generally shows non-competitive inhibition of amylase activity and a slight inhibition showed us it is a metallo-enzyme. Bacillus subtilis CLB-34 α-amylase has also slight inhibition by 3.3% with 10 mM EDTA. It was reported that amylases from alkaliphilic Bacillus strains were not inhibited by 10 mM EDTA at 40 ºC but was completely inactivated by 8 M urea (Horikoshi 1999   Enzyme was pre-incubated with different inhibitors, surfactants, commercial detergents and oxidizing agents at 55 °C for 1 h and assayed as standard assay method. The enzyme activity without incubation with inhibitor, surfactants, commercial detergents and oxidizing agents was taken as 100%. Mean standard deviation for all the values is < ± 5.0%. Enzyme was pre-incubated with different inhibitors, surfactants, commercial detergents and oxidizing agents at 55 °C for 1 h and assayed as standard assay method. The enzyme activity without incubation with inhibitor, surfactants, commercial detergents and oxidizing agents was taken as 100%. Mean standard deviation for all the values is < ± 5.0%.

Effect of surfactant on enzyme stability
In order to have applications in detergent industries, amylase must be stable to various detergent ingredients, such as surfactants. As shown in Table 4

HPLC analysis of hydrolysis products of α-amylase
The end products of starch hydrolysis by the amylase of Bacillus subtilis CLB-34 were analyzed by HPLC. At an early stage (2 h), the hydrolysis products were maltose, maltotriose and maltotetraose, maltopentose and with a trace amount of glucose. As the incubation time prolonged, the amount of glucose, maltose and maltotriose increased, but the amount of maltotetraose and maltopentose decreased. After 8 hours of incubation, the amount of maltotetraose was hard to be detected, which was hydrolyzed to smaller one by the amylase of Bacillus subtilis CLB-34. The main products were glucose, maltose and maltotriose, the contents of the three were about 16.45%, 58.99% and 27.41%, respectively, at the time of 14 hours of incubation. However, during the whole process of cultivation, the soluble starch could not be completely hydrolyzed to the three main products by the α-amylase of Bacillus subtilis CLB-34, at the time of 14 hours of incubation, the degree of hydrolysis was about 85.67%.

Conclusion
It is the rst instance when a thermo-tolerant amylase being reported from a thermo-tolerant solvent tolerant Bacillus subtilis isolate. The strain is unique with respect to several solvents tolerance, heavy metals, surfactant and inhibitor resistance, makes the isolate applicable under stressed conditions.
Outstanding solvent stability of the amylase proves its possible application under anhydrous conditions and amylase liquefaction. The amylase activity in broad pH and temperature range of 4.0-11.0 and 35-110 °C clearly indicate the thermo-alkaline nature of this enzyme. This enzyme, which possesses unique properties, could be widely used in different types of industries, especially in food and biotechnological applications.

Declarations
Ethics approval and consent to participate This article does not contain any studies with human participants or animals performed by any of the authors.

Consent for publication
All authors consent to publish this manuscript.

Availability of data and materials
Name of the repository is NCBI (National Center for Biotechnology Information) where our data's were deposited and a link to the dataset DOI are https://www.ncbi.nlm.nih.gov/nucleotide/MK443365.1 and https://www.ncbi.nlm.nih.gov/nuccore/MN370035.1.

Competing interests
The author(s) declare that they have no competing interests. homology to other Bacillus subtilis., so it could be stated that therefore it is different from reported Bacillus subtilis. The phylogenetic tree was drawn by MEGA 6 software using Neighbour-joining method and the signi cance of junctions was established using bootstrap method (1000 replicates).