Screening and Comparative Characterization of Microorganisms from Iranian Soil Samples Showing ω -Transaminase Activity toward a Plethora of Substrates

: In this study, soil microorganisms from Iran were screened for ω -transaminase ( ω -TA) activity based on growth on minimal media containing ( rac )- α -methylbenzylamine (rac- α -MBA) as a sole nitrogen source. Then, for the selection of strains with high enzyme activity, a colorimetric o-xylylendiamine assay was conducted. The most promising strains were identiﬁed by 16S rDNA sequencing. Five microorganisms showing high ω -TA activity were subjected to determine optimal conditions for ω -TA activity, including pH, temperature, co-solvent, and the speciﬁcity of the ω -TA toward di ﬀ erent amine donors and acceptors. Among the ﬁve screened microorganisms, Bacillus halotolerans turned out to be the most promising strain: Its cell-free extract showed a highly versatile amino donor spectrum toward aliphatic, aromatic chiral amines and a broad range of pH activity. Transaminase activity also exhibited excellent solvent tolerance, with maximum turnover in the presence of 30% ( v / v ) DMSO.

The significant challenge in industry is to use optically pure amines and amino acids from cost-effective production [1,9]. For the mass production of such compounds, nowadays biocatalysts are frequently used to comply with green chemistry as well be economically feasible [1,5]. Among various enzymatic methods for producing chiral amine compounds, ω-transaminases

Enzymatic Properties and Substrate Specificity
Appropriate temperature, pH, and organic solvents play important roles in ω-TA activity [5,30]. To survey such effects, the optimum reaction condition against these three factors as investigated in the screened bacteria. Several studies have been performed by applying cell and crude extract to convert different substrates [2,18,21,29,[31][32][33], so instead of using high-priced purified protein, we applied cell-free extracts to optimize reaction parameters and to investigate substrate scope.
The relative values of ω-TA activity were calculated based on producing acetophenone as a product of (S)-MBA deamination. The highest activity was considered as 100%.

Effect of Temperature on ω-TA Activity
The cell-free extracts were incubated at defined temperatures (25-65 • C) for one hour. The crude extracts of BaH, RhZ, and BaS shared similar expected mesophilic temperature profiles between 30 and 65 • C, with highest activity at 35 • C, which is reported for other ω-TAs [21,34]. They exhibited reasonable activity of around 70% up to 45 • C. Among all of them, 40-50% of activity remained at 50 • C, which decreased to approximately 10-20% at temperatures above 60 • C (Figure 1a). For ω-TABaE and ω-TABaI, the optimum reaction temperature was 40 °C and 30 °C, respectively. No significant change of activity was observed at 35 °C. Thus, for surveying the effect of pH and solvent on enzyme activity on all crude extracts, the temperature was maintained at 35 °C. Additionally, ω-TABaI showed approximately the same activity at temperatures between 20 and 30 °C (Table S1).

Effect of pH on ω-TA Activity
In contrast to other ω-TAs that usually show their maximum activity at slightly alkaline pH [12,21,31], ω-TABaI and ω-TABaH exhibited an almost constant activity in the pH range of 5 to 9. For ω-TABaE and ω-TABaI, the optimum reaction temperature was 40 • C and 30 • C, respectively. No significant change of activity was observed at 35 • C. Thus, for surveying the effect of pH and solvent on enzyme activity on all crude extracts, the temperature was maintained at 35 • C. Additionally, ω-TABaI showed approximately the same activity at temperatures between 20 and 30 • C (Table S1).

Effect of pH on ω-TA Activity
In contrast to other ω-TAs that usually show their maximum activity at slightly alkaline pH [12,21,31], ω-TABaI and ω-TABaH exhibited an almost constant activity in the pH range of 5 to 9. Surprisingly, for ω-TABaS and ω-TABaE, the highest enzyme activity was observed under acidic conditions (pH 5). Notably, 70% of ω-TABaS activity remained at pH 3, Furthermore, ω-TARhz preferred neutral conditions (Figure 1b). No enzyme activity was observed at pH 12. In the literature data, there is no report concerning ω-TA activity in acidic conditions. ω-TA activity at different pHs was mostly tested in pH range from 6 to 11 [4,17,18].

Effect of Organic Solvents on ω-TA Activity
Organic solvents are often added to ω-TA reactions to increase substrate solubility. However, this mostly causes negative effects on enzyme activity [10]. Therefore, the examination of the tolerance of the applied enzyme to different solvents is highly required [5]. To analyze whether solvents affect enzyme activity, four different solvents with a 0%-20% with the exception of DMSO which increased up to 40% v/v concentration were tested. The ω-TA activity in aqueous buffer without addition of organic solvents was set as 100%. The results were expressed as relative activity in %.
Dimethyl sulfoxide (DMSO) is a common solvent that was used in concentrations ranging from 5%-20% v/v in different projects [5]. DMSO as co-solvent led to the highest activity, followed by methanol, N,N-Dimethylformamide (DMF), and isopropanol. In some cases (BaI and Rhz), the reactions without any co-solvent showed better activity than methanol. It is remarkable to mention that ω-TABaH activity was enhanced threefold with a concentration of DMSO up to 30%. The results again suggest that DMSO is the most suitable organic solvent for catalytic activation of ω-TAs. However, Catalysts 2019, 9, 874 5 of 13 methanol in some cases also increased enzyme activity (Figure 2). Methanol was mentioned as a second co-solvent after DMSO for enhancing ω-TA activity in Pawer et al.'s experiment [2].    (e) BaI. The value of 100% corresponds to enzyme activity in the no co-solvent condition. 1 mg/mL cell-free extract was added to react under the following conditions: (S)-MBA (10 mM), pyruvate (10 mM), PLP (0.1 mM), and co-solvent (0-30% v/v) in Britton-Robinson buffer (pH 7) at 35 • C. Formation of acetophenone was detected by HPLC at 254 nm. Error bars represent ± standard deviation.

Substrate Specificity and Enantioselectivity
The substrate spectrum of all strains was investigated in order to find the most suitable amino donor and acceptor.
Regarding amino donors, 28 substrates, including aliphatic and aromatic amino compounds were reacted to pyruvate as an amino acceptor to investigate amino donor specificity, and alanine generation was monitored by the HPLC technique. Racemates as well as the pure, (S) and (R) enantiomers (if available) of amino donors were chosen to explore the enantioselectivity of ω-TA. The results of the substrate screening are illustrated in Table 2. HPLC analysis proved that all enzymes show predominant formation of an (S)-configuration. This is in accordance with most previously reported publications in the literature indicating that (R)-stereoselectivity are rather rare in wild-type microorganisms [35]. All cell-free extracts showed high activity against (S)α-methylbenzylamine (S-MBA) 1s.
BaH showed considerable activity toward a wide range of substrates, including (S), (R), and racemates of aromatic and aliphatic amino donors. Although this cell-free extract also showed activity toward some tested (R)-enantiomers (1r, 2r, 4r, 5r, 7r, 8r, 13r), (S)-selectivity appeared to be not so strict in this strain. Notably, it is difficult to conclude that BaH showed (R)-selectivity since in this study, crude extract was applied. There is a possibility that (R) converted to (S)-enantiomer via enzymatic racemization [36], and then the latter was subjected to ω-TA activity. On the other hand, (R)-preference towards 13r in compare to 13s leads to another hypothesis, in which both R and S transaminases are present in crude extract.
While the cell-free extract of BaH revealed varying enantiopreference toward all tested amino donors to different degrees, the substrate specificity was found to resemble what has previously been reported by Mathew et al. [5,30], as a preference to react with aromatic β-amino acids is clearly visible [5]. (S)-MBA 1s is converted with the highest activity, which therefore was set as 100%. Although a few ω-TAs are described with activity toward aromatic β-amino acids (56), it is remarkable that BaH uses β-phenylalanine (13, 13s, 13r) and (rac)-β-homophenylalanine (14) as an amino donor; both of these were inert for crude extraction from other bacteria in this experiment, which implies the presence of a broad binding pocket in the active site of ω-TABaH. ω-TA activity against 14 has been reported by Mathew et al. for ω-TABG, with 50% relative activity in comparison with 13 [30]. A recent study by Buß et al. explained the lack of ω-TA activity against 14 (by an ω-TA that was highly active against 13) by sterice hindrance caused by the additional carbon atom located between the amino group and phenyl group [36]. Several substituted derivatives of β-phenylalanine are converted as well. Table 2. Amino donor screened assays were carried out in Britton-Robinson buffer, pH 7 with amino donor (10 mM), pyruvate (10 mM), PLP (0.1 mM), and 15% (v/v) DMSO. The reaction was initiated by adding crude extract to a final concentration of 1 mg protein per ml. Reactions were performed at 35 • C, 600 rpm for 20 h. Relative activity was measured by HPLC detection of alanine formation at 338 nm. All reactions were conducted in triplicate, and each cell-free extract was separately analyzed for alanine content as a control.

Amino Donor
Relative Activity%

BaH
BaE BaS BaI RhZ 1 (rac) α-methylbenzylamine small binding pocket. The former can accommodate bulky residues, including aryl groups, whereas the latter is loaded by a small group such as a methyl substituent [14]. So most ω-TAs have a limitation in accepting bulky substrates without applying protein engineering. Voss et al. reported that after double mutation on ω-TA 3FCR from Ruegeria sp. TM1040, this enzyme gained activity toward bulkier substrates such as phenylpropylamine and phenylbutylamine [16]. Strain BaH showed a good ability to convert bulky substrates and showed different substrate spectra with known ω-TAs [15,35]. Purification and further investigation of this enzyme is intended for further studies.  3-amino-3-(4-chlorophenyl) propionic acid small binding pocket. The former can accommodate bulky residues, including aryl groups, whereas the latter is loaded by a small group such as a methyl substituent [14]. So most ω-TAs have a limitation in accepting bulky substrates without applying protein engineering. Voss et al. reported that after double mutation on ω-TA 3FCR from Ruegeria sp. TM1040, this enzyme gained activity toward bulkier substrates such as phenylpropylamine and phenylbutylamine [16]. Strain BaH showed a good ability to convert bulky substrates and showed different substrate spectra with known ω-TAs [15,35]. Purification and further investigation of this enzyme is intended for further studies. 3-amino-3-(4-chlorophenyl) propionic acid propionic acid the latter is loaded by a small group such as a methyl substituent [14]. So most ω-TAs have a limitation in accepting bulky substrates without applying protein engineering. Voss et al. reported that after double mutation on ω-TA 3FCR from Ruegeria sp. TM1040, this enzyme gained activity toward bulkier substrates such as phenylpropylamine and phenylbutylamine [16]. Strain BaH showed a good ability to convert bulky substrates and showed different substrate spectra with known ω-TAs [15,35]. Purification and further investigation of this enzyme is intended for further studies.
propionic acid the latter is loaded by a small group such as a methyl substituent [14]. So most ω-TAs have a limitation in accepting bulky substrates without applying protein engineering. Voss et al. reported that after double mutation on ω-TA 3FCR from Ruegeria sp. TM1040, this enzyme gained activity toward bulkier substrates such as phenylpropylamine and phenylbutylamine [16]. Strain BaH showed a good ability to convert bulky substrates and showed different substrate spectra with known ω-TAs [15,35]. Purification and further investigation of this enzyme is intended for further studies.
limitation in accepting bulky substrates without applying protein engineering. Voss et al. reported that after double mutation on ω-TA 3FCR from Ruegeria sp. TM1040, this enzyme gained activity toward bulkier substrates such as phenylpropylamine and phenylbutylamine [16]. Strain BaH showed a good ability to convert bulky substrates and showed different substrate spectra with known ω-TAs [15,35]. Purification and further investigation of this enzyme is intended for further studies.  The activity against (S)-MBA was set as 100. An amount under 1% was considered as zero activity.  The activity against (S)-MBA was set as 100. An amount under 1% was considered as zero activity.  The activity against (S)-MBA was set as 100. An amount under 1% was considered as zero activity.  The activity against (S)-MBA was set as 100. An amount under 1% was considered as zero activity.  The activity against (S)-MBA was set as 100. An amount under 1% was considered as zero activity.  The activity against (S)-MBA was set as 100. An amount under 1% was considered as zero activity.  The activity against (S)-MBA was set as 100. An amount under 1% was considered as zero activity.  The activity against (S)-MBA was set as 100. An amount under 1% was considered as zero activity.   The activity against (S)-MBA was set as 100. An amount under 1% was considered as zero activity.  The activity against (S)-MBA was set as 100. An amount under 1% was considered as zero activity.  The activity against (S)-MBA was set as 100. An amount under 1% was considered as zero activity. Table 3. Amino acceptor screened assays were carried out in Britton-Robinson buffer, pH 7 with (S)-MBA (10 mM), amino acceptor (10 mM), PLP (0.1 mM), and 15% (v/v) DMSO. The reaction was started by the addition of crude extract to a final concentration of 1 mg/mL in the following conditions: 35 °C, 600 rpm for 20 h. Relative activity was measured by HPLC detection of acetophenone formation at 254 nm. All reactions were conducted in triplicate, and each cell-free extract was separately analyzed as a control.

Amino Acceptors
Relative Activity% The activity against (S)-MBA was set as 100. An amount under 1% was considered as zero activity. The activity against (S)-MBA was set as 100. An amount under 1% was considered as zero activity. BaE presented only (S)-selectivity in relation to tested substrates. Similar to BaH, the strain's crude extract exhibited activity toward 1s, almost double that of 1. BaE and BaH share the same activity toward 12, while BaI showed almost double the activity in comparison with those. The relative activity of ω-TA of BaI toward (rac)-MBA 1 exceeded 60%, which might be related to the presence of racemase in the crude extract of bacteria [37,38].
Crude extracts of RhZ and BaS displayed (S)-selectivity toward the tested amino donors with similar preference profiles. RhZ showed more activity toward 17s as an aliphatic substrate than MBA 1s, which was not observed for other screened strains.
Amino acceptors play a critical role in ω-TA activity [30]. Eight ketones were reacted with (S)-MBA to explore the amino acceptor specificities by HPLC detection of acetophenone production ( Table 3). Aliphatic ketones such as sodium pyruvate 7b and α-ketoglutaric acid 8b showed high conversion, whereas most aromatic ketones were not accepted by the investigated enzymes. The activity against (S)-MBA was set as 100. An amount under 1% was considered as zero activity.  In each column, the highest activity was defined as 100%. Activity less than 1% was considered zero.

Chemicals
All solvents and chemicals used in this study were purchased from Sigma-Aldrich (St Louis, MO, USA), Carl Roth GmbH (Karlsruhe, Germany), and Peptech (Burlington, MA, USA).

Screening and Identification
3.2.1. Enrichment of Microorganisms on (rac)-α-MBA as Sole Nitrogen Source Due to the role of transaminase in nitrogen metabolism [29], areas enriched with nitrogen compounds were chosen as potential sites for screening novel ω-transaminase. Soil samples (2 g) from a petrochemical site, oil well, and an agriculture field in Iran were separately suspended in 200 In each column, the highest activity was defined as 100%. Activity less than 1% was considered zero.

Chemicals
All solvents and chemicals used in this study were purchased from Sigma-Aldrich (St Louis, MO, USA), Carl Roth GmbH (Karlsruhe, Germany), and Peptech (Burlington, MA, USA).

Screening and Identification
3.2.1. Enrichment of Microorganisms on (rac)-α-MBA as Sole Nitrogen Source Due to the role of transaminase in nitrogen metabolism [29], areas enriched with nitrogen compounds were chosen as potential sites for screening novel ω-transaminase. Soil samples (2 g) from a petrochemical site, oil well, and an agriculture field in Iran were separately suspended in 200 mL sterile minimal medium (MIM) in 500 mL Erlenmeyer flasks containing 10 mM (rac)-α-MBA, 100 In each column, the highest activity was defined as 100%. Activity less than 1% was considered zero.

Chemicals
All solvents and chemicals used in this study were purchased from Sigma-Aldrich (St Louis, MO, USA), Carl Roth GmbH (Karlsruhe, Germany), and Peptech (Burlington, MA, USA).

Enrichment of Microorganisms on (rac)-α-MBA as Sole Nitrogen Source
Due to the role of transaminase in nitrogen metabolism [29], areas enriched with nitrogen compounds were chosen as potential sites for screening novel ω-transaminase. Soil samples (2 g In each column, the highest activity was defined as 100%. Activity less than 1% was considered zero.

Chemicals
All solvents and chemicals used in this study were purchased from Sigma-Aldrich (St Louis, MO, USA), Carl Roth GmbH (Karlsruhe, Germany), and Peptech (Burlington, MA, USA).

Enrichment of Microorganisms on (rac)-α-MBA as Sole Nitrogen Source
Due to the role of transaminase in nitrogen metabolism [29], areas enriched with nitrogen compounds were chosen as potential sites for screening novel ω-transaminase. Soil samples (2 g In each column, the highest activity was defined as 100%. Activity less than 1% was considered zero.

Chemicals
All solvents and chemicals used in this study were purchased from Sigma-Aldrich (St Louis, MO, USA), Carl Roth GmbH (Karlsruhe, Germany), and Peptech (Burlington, MA, USA).

Enrichment of Microorganisms on (rac)-α-MBA as Sole Nitrogen Source
Due to the role of transaminase in nitrogen metabolism [29], areas enriched with nitrogen compounds were chosen as potential sites for screening novel ω-transaminase. Soil samples (2 g In each column, the highest activity was defined as 100%. Activity less than 1% was considered zero.

Chemicals
All solvents and chemicals used in this study were purchased from Sigma-Aldrich (St Louis, MO, USA), Carl Roth GmbH (Karlsruhe, Germany), and Peptech (Burlington, MA, USA).

Enrichment of Microorganisms on (rac)-α-MBA as Sole Nitrogen Source
Due to the role of transaminase in nitrogen metabolism [29], areas enriched with nitrogen compounds were chosen as potential sites for screening novel ω-transaminase. Soil samples (2 g In each column, the highest activity was defined as 100%. Activity less than 1% was considered zero.

Chemicals
All solvents and chemicals used in this study were purchased from Sigma-Aldrich (St Louis, MO, USA), Carl Roth GmbH (Karlsruhe, Germany), and Peptech (Burlington, MA, USA).

Enrichment of Microorganisms on (rac)-α-MBA as Sole Nitrogen Source
Due to the role of transaminase in nitrogen metabolism [29], areas enriched with nitrogen compounds were chosen as potential sites for screening novel ω-transaminase. Soil samples (2 g In each column, the highest activity was defined as 100%. Activity less than 1% was considered zero.
Cell-free extract of BaH, BaS, and BaI exhibited some activity toward bulkier substrates, including 1b or 2b. It is supposed that β-amino acid esters were produced by using these acceptors, but this needs to be investigated in further experiments. BaH, BaS, and BaI showed activity toward 1b as amino acceptor. BaI was the only extract to show activity toward acceptor 2b.
The substrate scope of ω-TA is determined by the active site, which consists of a large and a small binding pocket. The former can accommodate bulky residues, including aryl groups, whereas the latter is loaded by a small group such as a methyl substituent [14]. So most ω-TAs have a limitation in accepting bulky substrates without applying protein engineering. Voss et al. reported that after double mutation on ω-TA 3FCR from Ruegeria sp. TM1040, this enzyme gained activity toward bulkier substrates such as phenylpropylamine and phenylbutylamine [16]. Strain BaH showed a good ability to convert bulky substrates and showed different substrate spectra with known ω-TAs [15,35]. Purification and further investigation of this enzyme is intended for further studies.

Chemicals
All solvents and chemicals used in this study were purchased from Sigma-Aldrich (St Louis, MO, USA), Carl Roth GmbH (Karlsruhe, Germany), and Peptech (Burlington, MA, USA).

Enrichment of Microorganisms on (rac)-α-MBA as Sole Nitrogen Source
Due to the role of transaminase in nitrogen metabolism [29], areas enriched with nitrogen compounds were chosen as potential sites for screening novel ω-transaminase. Soil samples (2 g) from a petrochemical site, oil well, and an agriculture field in Iran were separately suspended in  [39]. FeSO 4 and (rac)-α-MBA were separately sterile filtrated and added to the medium. Autoclaving causes precipitation of ferric hydrate [40]. Each sample was prepared in duplicate. Enrichment went through two strategies: In the first method, Erlenmeyer flasks were incubated for 24 h at 180 rpm at 35 • C (depending on the original temperature of the soil samples). After 24 h of incubation, a 100 µL sample was transferred to 20 mL of fresh minimal medium and incubated for 24 h under the same conditions as described before. This serial dilution was repeated 3 times. After that, the culture was diluted with PBS buffer (10: 90 µL) and poured to MIM agar plates (mineral medium, 10 mM (rac)-α-MBA with 1.5% agar) and incubated at 35 • C. As soon as colonies were visible, single colonies were streaked on new MIM agar plates to obtain uniform colonies under the same temperature.
In the second method, Erlenmeyer flasks were transferred to a dark place, without shaking, at 35 • C. During incubation, 1 mL of concentrated MIM (5X), 0.5% v/v glycerol, and 10 mM (rac)-α-MBA were added to the flasks every week. After 3 months, the culture broth was centrifuged (1000 g, 2 min) and 100 µL of supernatant was spread on MIM agar plates. The colonies were isolated after 3 days of incubation at 35 • C. This was continued until the isolation of single colonies was achieved.

Selection of the Most Promising Strains
To identify whether isolated strains use (rac)-α-MBA as a sole nitrogen source or atmospheric nitrogen, o-Xylylenediamine was applied as a smart amino donor. Each colony was cultivated in a shake flask containing 20 mL MIM medium with 12 mM (rac)-α-MBA for three days at 35 • C at 120 rpm. The cells were harvested by centrifugation at 6000× g for 10 min at 4 • C and resuspended in HEPES buffer (50 mM, pH 7.5). The cell concentration was adjusted to around 20 mg/mL by measuring dry cell mass based on Buß et al. [36]. The cells were frozen at −80 • C and thawed at room temperature for cell disruption.
Reaction was started by adding 50 µL of whole cells to 150 µL reaction solution containing 7.5 mM o-Xylylenediamine, 5 mM pyruvate, 1 mM PLP, and 10% DMSO in HEPES buffer (50 mM, pH 7.5) [41] in a 96-well plate. Each reaction was conducted in triplicate. The plate was incubated overnight at 35 • C, 150 rpm.

Identification of Bacteria
The positive strains were grown separately overnight in LB broth at 35 • C, 150 rpm. DNA was extracted by ZR soil Microbe DNA Kit TM . PCR was done with a Q5 high-fidelity PCR kit (NEB, Germany). Universal Primers (27F and 1492R) were applied to amplify 1.5 kb 16S rDNA fragments. PCR was conducted following the manufacturer's instructions for Q5 polymerase. Gene sequencing was performed by Eurofins Company (Ebersberg, Germany). The resulting 16S rRNA gene sequences were compared with available gene sequences in the EZ bioCloud database [42].

Preparation of Crude Extract
The inoculum (3 mL) was prepared by picking a single colony and cultivating it for three days in MIM with 12 mM (rac)-α-MBA. The medium (400 mL) was inoculated by the addition of 1% (v/v) of the latter culture into 1 L shaking flasks containing MIM with 0.5 g/L yeast extract and 12 mM (rac)-α-MBA overnight at 150 rpm and 35 • C. Cells were harvested before reaching stationary phase by centrifugation at 6000× g for 10 min at 4 • C. After washing the pellet with sodium phosphate buffer (50 mM, pH 7), cells were resuspended in 10 mL lysis buffer (sodium phosphate (50 mM, pH 7), 0.1 µM PLP, 100µg/mL lysozyme]. Afterwards, they were incubated for 1 h at room temperature, followed by sonification (3 cycles: 30 s pulse, 20 s pause; 60% amplitude) on ice. Subsequently, the cell debris was removed by centrifugation (20,000 rpm, 4 • C, 15 min) in a JA-30-50 rotor (Coulter-Beckman centrifuge). The protein concentration of crude extract was determined by using the Roti ® -Quant universal kit (Carl Roth, Karlsruhe, Germany) following the manufacturer's instructions. Eventually, they were mixed with 15% glycerol and preserved in −80 • C for further analysis.

Effect of Temperature, pH, and Co-Solvent
The effects of temperature and pH on enzyme activity were examined at different temperatures (25-65 • C at pH7) and various pHs (3-12 at 35 • C) by using Britton-Robinson buffer (0.04 M H 3 BO 3 , 0.04 M H 3 PO 4 , 0.04 M CH 3 COOH) containing an amino donor (10 mM), amino acceptor (10 mM), PLP (0.1 mM), and DMSO (10%). The total reaction volume was set to 0.25 mL. The reactions were initiated by adding crude extract of enzyme (~1 mg/mL).
To study the effect of solvent on ω-TA activity, reactions were carried out applying various organic solvents with 0-20% (v/v) (for DMSO 0-40%). (rac)-α-MBA (10 mM) was used as amino donor and pyruvate (10 mM) as amino acceptor in the presence of PLP (0.1 mM) and cell-free extract (~1 mg/mL). The reactions were carried out in Britton-Robinson buffer (pH 7) at 35 • C as described above.
Each reaction was conducted in triplicate and incubated (ThermoMixer, Eppendorf) at 600 rpm for 1 h. As a control, the reaction was conducted without adding enzyme. Furthermore, the amount of acetophenone in crude extract of enzyme was also evaluated. Reactions were stopped by heating to 95 • C for 5 min. After centrifugation, the supernatant was analyzed by HPLC to detect produced acetophenone according to Section 3.4. Results are shown as relative activity.

Substrate Specificity and Enantioselectivity
The reactions were carried out according to Section 3.3.2. Various amino donors (10 mM) listed in Table 1 were tested using sodium pyruvate (10 mM) as amino acceptor in the Britton-Robinson buffer (pH 7) and 15% (v/v) DMSO. The reaction was carried out at 35 • C, 600 rpm, for 20 h (ThermoMixer, Eppendorf). The produced alanine was analyzed by HPLC according to Section 3.4.
Different amino acceptors (10 mM) were tested with (S)-α-MBA as amino donor (10 mM) in Britton-Robinson buffer, pH 7, at 35 • C, 600 rpm, for 20 h. The production of acetophenone was analyzed by HPLC according to Section 3.4.
Each reaction was conducted in triplicate, and the average of three independent reactions was used for evaluation. Enzyme inactivation was applied by heating to 95 • C for 5 min. The supernatant was used for analysis after centrifugation. In addition, the amounts of alanine and acetophenone were measured in enzyme crude extracts as blanks.

HPLC Analytics
All samples were analyzed by Agilent 1100 series HPLC system (Santa Clara, CA, USA). For the analysis of alanine, derivatization by using ortho-phthalaldehyde was carried out according to Brucher et al. [43] and Buß et al. [44], with an automated precolumn derivatization. A reversed-phase C 18 column (150 × 4.6 mm HyperClone 5 µm ODS, Phenomenex, Germany) with isocratic elution with 35% (v/v) methanol and 65% (v/v) sodium phosphate buffer (40 mM, pH 6.5) at a flow rate of 0.8 mL min −1 and detection at 338 nm at 25 • C was used.

Conclusions
Our motivation for screening novel microorganisms exhibiting transaminase activity resulted from several challenges for the industrial application of ω-TAs, e.g., lacking activity at acidic pH, high temperature, unnatural substrates, (R)-configured molecules, and elevated concentrations of organic solvents (needed due to low substrate solubility in aqueous systems). We succeeded in finding promising new strains from a high variety of Iranian soil samples by enrichment culture using (rac)-α-methylbenzylamine (α-MBA) as a sole nitrogen source.
In particular, a Bacillus halotolerans (BaH) strain was isolated from a petroleum refinery, exhibiting ω-TA activity using a broad spectrum of amino donors over a pH range of 5-9 at elevated concentrations of DMSO and other organic solvents up to 30% (v/v). Two other strains isolated from an agricultural field (Bacillus endophyticus, BaE) and an oilfield (Bacillus subtilis, BaS) showed the highest ω-TA activity against α-MBA at pH 5 with 70% remaining activity at pH 3 (BaS), whereas ω-TAs are usually described to prefer slightly alkaline conditions. The enzymes dedicated to these extraordinary activities will be purified and subjected to in-depth studies for application-technical characterization.
Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4344/9/10/874/s1, Table S1: The Effect of temperature on the amination of pyruvate and (S)-MBA as amino donor using 1 mg/mL cell-free extracts of BaI strain. Reaction conditions are given in the method section. Formation of acetophenone was detected by HPLC at 254 nm. Every reaction was conducted in three replicates.