GS culture was prepared by adding 0.1 mL of a stock solution obtained from NAMSA G. stearothermophilus cell line 7943 spore suspension (2.4 × 10⁶ spores/0.1 mL, VWR, Atlanta GA, USA) to 10 mL of sterile water. This diluted stock was incubated in a water bath (ISOTEMP 210, Fisher Scientific, Dexter MI, USA) for 10 min at 80˚C. 1 mL of this was added to 25 mL of tryptic soy broth (TSB, VWR, Atlanta GA, USA) media in a sterile 250 mL Erlenmeyer flask. The flask was covered with sterile foil and the sample incubated at 55˚C for 24 h in a shaker at 100 rpm (New Brunswick Scientific Series 25, Incubator Shaker, New Jersey, USA). The optical density (OD) of bacterial growth was measured using a dual beam spectrophotometer (Shimadzu, BioSpec-1601, Kyoto, Japan) at 600 nm [10].

A. flavithermusTNO-09.006 culture was a gift from RemcoKort (Vrije University, Amsterdam, The Netherlands) in the form of slant tubes. One loop from the slant tube was used to make a streak plate on tryptic soy agar (VWR, Atlanta GA, USA) and was incubated at 55˚C for 48 h. A subculture was grown by inoculating 25 mL of TSB with one loop of the culture grown on the streak plate in a sterile 250 mL Erlenmeyer flask covered with sterile foil. Cells were grown with aerobic condition at 55˚C in a shaker at 100 rpm for 16 - 18 h. Growth of AF was confirmed by measuring OD 600 nm using a spectrophotometer [11]. Frozen stocks were prepared by inoculating 2 mL of overnight culture into 20 ml of a 30% glycerol solution (Mallinckrodt Specialty Chemicals Co, St. Louis, MO USA) (glycerol/TSB, w/v) and stored in 2.0 mL cyro-vials at −20˚C. Frozen stocks (0.1 mL) of each organism was added to 25 mL TSB in a sterile 250 mL Erlenmeyer flasks, then covered with sterile foil and incubated at 55˚C for 16 - 18 h in a shaker at 100 rpm [11]. The 25 mL growth of bacteria was added to 1 L of TSB and cells were grown at 55˚C at 100 rpm until the OD at 600 nm was between 0.7 to 0.9.

The bacteria were separated from the medium by centrifuging at 3000 ×g for 10 min at 10˚C. The cells were washed in 10 ml 50 mM Tris-HCl (tris hydrochloride, Fisher Scientific, Waltham, MA USA) buffer pH 7.5 and centrifuged again as described above. After centrifugation, the cells were mixed with 5 - 10 mL Tris-HCl buffer (50 mM Tris-HCl, pH 7.5), and this mixture was sonicated at 40% amplitude for 1 min using a 4.5 mm microtip and a QsonicaSonicator power source (500 W power; QSonica Q500, Newtown, CT). The sonicated solution was centrifuged at 5000 ×g at 0˚C for 20 min and the lysate was filtered using a 1.0 μm microfilter (GlassfiberPrefilter, Tullagreen, Carrigtwohill Co. Ireland).

The cell lysates from GS and AF were adjusted to 0.95 mg/mL with Tris-HCl buffer, then heated at 70˚C for 10 min in a water bath then placed in an ice bath to cool. The protein content was determined using a protein determination kit (BCA kit, Thermo Scientific, Waltham, MA, USA). The samples were centrifuged at 10,000 ×g for 30 min at 4˚C to precipitate denatured proteins [12]. The supernatants were dried using a VIRTIS sentry freeze-dryer overnight.

Cell lysates were assayed using p-nitrophenyl acetate (PNPC2) (Sigma-Aldrich, MO, USA) as the substrate. 5 mL of substrate stock solutions (20 mM of PNPC2) were prepared using an acetonitrile and isopropanol mixture (1:4 v/v) as described by [13]. 1 mL of the substrate stock was diluted with 20 mL of assay buffer (50 mM Tris HCl, pH 7.5 containing 1 mM CaCl₂ (Thermo Fisher Scientific, Waltham, MA, USA) and 0.3% Triton X100 (Mallinckrodt Specialty Chemicals Co, St. Louis, MO, USA) for a final concentration of 0.055 mM substrate solution. For the standard lipase assay, 900 µL of substrate solution was pipetted in 1.5 mL centrifuge tube (VWR, Atlanta GA, USA) and mixed with 100 µL of enzyme solution (50 mM Tris HCl, pH 7.5 containing 1 mM CaCl₂) for a final volume of 1 ml. After 1 min at 20˚C, the sample was transferred to 1.5 mL disposable plastic cuvette (VWR Atlanta GA, USA). Absorbance at 410 nm was recorded every 5 seconds over 1 min using a dual beam spectrophotometer at room temperature. The blank consisted of 900 μl of substrate solution and 100 μl of water. One unit of lipase activity (U) was defined as 1 µmoL substrate hydrolyzed per minute under the assay conditions. This is referred to as the standard lipase assay. Specific activity was determined by calculating the ratio of the lipase activity (U) and protein concentration (mg) and expressed as U/mg. The protein content was determined using a protein determination kit (BCA kit, Thermo Scientific, Waltham, MA, USA).

To determinate the optimal pH, lipase activity was assayed in the range of pH 4 to 12. The buffers were 50 mM sodium acetate buffer (pH 4 to 5), 50 mM Tris-HCl buffer (pH 6 to 8), and 50 mM glycine-NaOH buffer (pH 9 to 12). Standard activity assay using PNPC2 as the substrate in the reactions for each pH solution buffer and reading the absorbance at 410 nm at 20˚C.

The pH stability was tested after incubation of 50 µL of the lipase sample with 100 µL of various pH buffers at pH values of 5 to 12 for 24 h at 4˚C. After incubation, 100 µL of samples were diluted into 900 µL of assay buffer. Lipase activity was determined using the standard lipase assay. The data were graphed using the relationship of pH and incubation time with the relative activity of lipases. The relative activity (%) was calculated using the highest activity obtained and set at 100% using the standard enzyme assay. Relative activity (%) is the ratio between the highest enzyme activity and activity of lipase at various pH values and therefore expressed a percentage.

Thermostability of lipases was determined by measuring the residual activity after incubating the enzyme solutions (50 mM Tris HCl, pH 7.5) at 20˚C to 90˚C for 24 h in 1 mL microcentrifuge tubes. At the end of incubation, the tubes were cooled down in an ice bath and were assayed by added 100 µL of enzyme solution to 900 µL of the assay buffer solution using the standard lipase assay at room temperature. The data were graphed using the relationship of various temperatures and incubation times with the relative activity of the lipases. The relative activity (%) was calculated in relation to values obtained with the highest activity of the enzyme. Relative activity (%) is the ratio between the highest enzyme activity and activity of lipase for various temperatures and therefore expressed as a percentage.

The kinetic parameters including maximum reaction rate (V_max) and Michaelis-Menten constant (K_m) were calculated using different p-nitrophenyl esters (p-nitrophenyl acetate (PNPC2), p-nitrophenyl octanoate (PNPC8), p-nitrophenyl laurate (PNPC12), p-nitrophenyl myristate (PNPC14), and p-nitrophenyl palmitate (PNPC16) (Sigma-Aldrich, MO, USA)). Initial rate measurements with lipases at a constant temperature of 20˚C and 50 mM Tris HCl, pH 7.5 with increasing substrate concentrations (0.06 - 1.2 mM), were performed to determine the kinetic parameters. Substrate solutions containing PNPC12, PNPC14, and PNPC16 were placed in a 60˚C water bath (ISOTEMP 210, Fisher Scientific, Waltham, MA, USA) until substrate solutions were clear before use. Substrate solutions containing PNPC2 and PNPC8 did not need heating to form a clear solution. Absorbance at 410 nm for 1 min reaction times was used to determine the initial enzyme activity. Lineweaver-Burk plots were used to determine the maximum velocity (V_max) and Michaelis-Menten constants (V_max) by linear regression analysis for each experiment using Microsoft Excel (2016 Office 365). The reciprocal of velocity (v) was graphed vs. the reciprocal of substrate concentration [S] and the best-fit line was obtained. Values of V_max and K_m were determined using the equation:

1 v = K m V max [ S ] + 1 V max .

The catalytic constant (K_cat) was calculated using V_max, molecular weight, and concentration of the enzyme.

Lipase activity was measured spectrophotometrically at 410 nm by mixing 100 µL of the enzyme with 900 µL substrate buffer for a 1 min reaction time. The highest activity values for p-nitrophenyl esters are reported as 100%. Relative activity (reported in %) was expressed as the ratio of enzyme activity for different p-nitrophenyl ester substrates compared to the highest activity value recorded amongst the p-nitrophenyl ester substrates. For the lipase from GS, PNPC2 was the control substrate to calculate the relative activity, while PNPC14 was the control substrate to calculate the relative activity of lipases from AF.

Three replicates were used to grow the GS and AF cultures and produce heat treated lysates. Analysis was done in triplicate for each replicate. The values are presented as means and standard deviation (SD) as calculated in Microsoft Excel 2016, Office 365.

LGS and LAF were assayed using PNPC2 as a substrate to determine the optimum pH and the pH-stability at 20˚C with a pH range of 4 to 12. Relative activity (%) is the ratio between the activity at the optimum pH and the activity at various pH values and is therefore expressed as a percentage. The relative activity (%) was calculated as a percentage of the enzyme activity measured at pH 7.5 for GS and pH 8.0 for AF. The results show (Figure 1(a)) that the optimum pH of LGS was 7.5; however, the enzyme still had up to 60% activity at pH 9. Also, the result show the highest activity for LAF was between pH 8 to 9 and at pH 10, 40% of the activity remained (Figure 1(b)). The optimum pH for both lipases was observed in alkaline conditions when the pH increased from 7 to 9. The lipase activity decreased in acidic conditions with the enzyme activity decreasing to 20% at pH 6 and lower for GS and less than 10% activity for AF at pH values of 7.5 and less (Figure 1(a) and Figure 1(b)). Previous studies reported that G. stearothermophilu est 30 and est 55 have optimal activity between pH 8 to 9 [8], and G. sterarothermophilus JC has optimal activity at pH of 9 [15]. Anoxybacillusgonensis G2 and Anoxybacillus sp. PDF1 reported a similar observation of optimum activity at pH 7.5 and 8 [16] [17].

The pH stability profiles of LGS and LAF are shown in Figure 2(a) and Figure 2(b). The pH-stability profile shows high enzyme activity between pH 6 to 11, but less stability at pH 5. Also, the enzyme activity was relatively stable at all

pH values except at pH 5 for 24 hours. The result show that more than 80% of LGS activity could be retained at pH values between 6 and 11, but the enzyme activity decreased at pH 5 (Figure 2(a)). LAF showed the highest stability at pH 9 and the activity was 70% at pH 6 to 8 and 90% at pH 10 to 11. At pH 5, only 60% activity was seen for LAF compared to pH 9 (Figure 2(b)). Both lipases showed higher stability with a pH range of 6 to 11 which indicates the ability of both enzymes to work in alkaline conditions compared to acidic. Previous reports stated similar stability profiles of lipases from different strains of Geobacillus and Anoxybacillus. For example, a lipase from Geobacillus thermodenitrificans PS01 had stability at pH 6 to 9 over 30 days at 4˚C [18], Geobacillus stearothermophilus AH22 lipase was stable in the pH range of 4 to 10 at 4˚C [2], Anoxybacillus gonensis A4lipase had stability at pH 6 at 4˚C for 24 h [19], and A. flavithermusHBB 134 lipase was stabilize at pH 6 to pH 11 at 25˚C, 40˚C, and 50˚C for 24 h [9].

Lipases which have high stability in neutral to acidic ranges can be used in the oleochemicals sector where the hydrolysis of FFA occurs and requires acidification of the formed soaps to obtain fatty acids [20]. Interesterification of oil by enzymatic reactions utilizes lipases that have high activity in the pH range of 6 to 7 [21]. Lipases which have higher stability in alkaline pH conditions can be used in laundry and household detergents [2].

The thermostability of LGS and LAF was determined by incubation of enzymes at 20˚C to 90˚C, at pH 7.5 without substrate for a time course up to 24 h, and the results are shown in Figure 3(a) for LGS and Figure 3(b) for LAF. The relative activity (%) was calculated in relation to values obtained upon incubation at 24 h and 20˚C. The activity of LGS was relatively stable between 20˚C to 60˚C with the relative activity being between 75% to 100% after 24 h of incubation. A decrease in the relative activity of approximately 55% was observed with an increase in temperature to 70˚C and incubation time of 12 h. LGS activity was significantly reduced at 70˚C after 6 h, and after 12 h the activity was 46%. A dropin activity was induced by incubation at temperatures above 70˚C. Interestingly,

LGS activity was higher at 60˚C that at 50˚C. Using the data from this study, LGS can retain stable activity at 70˚C for 6 hand would operate well at 50˚C. LGS activity was below 40% after 6 h at 90˚C.

LAF showed 60% activity between approximately 20˚C to 80˚C for 24 h (Figure 3(b)). At 90˚C, LAF activity was retained for 6 h. LAF showed remarkable thermal stability at 80˚C compared to LGS and would operate well at this temperature. Results showed that LGS and LAF activity was stable at 20˚C to 60˚C with LAF being more thermostable at temperatures greater than 60˚C. Also, the lipases have the ability to be active at 70˚C for 6 h and LAF can retain 50% of its activity at 80˚C after 24 hrs.

The thermostability data for both enzymes suggests that LGS was more stable at temperatures less than 70˚C while LAF is more stable at 70˚C and 80˚C. In this study, the thermostability of LGS and LAF were within the industry application range (50˚C to 60˚C) for dairy processing such as cheese flavor production [1]. LAF would be suitable for the interesterification of oils which required lipases to have activity at 50˚C to 70˚C [7] [22]. These results are similar to a lipase from A. flavithermusHBB 134 which was thermostable at 25˚C to 50˚C after 24 h [9] and a lipase from Anoxybacillus sp. PDF1 was stable after a 30 min incubation at 60˚C [16]. Based on the data, LGS was stable at 25˚C to 60˚C after a 24 h incubation and retained 50% of initial activity at 70˚C after 12 h. When comparing to previous studies, LGS was more stable than a lipase from Geobacillus sp. strain ARM, which was stable after 150 min at 50˚C [23]. A lipase from Geobacillus sp. SBS-4S was active at 50˚C after 80 min of incubation [24]. When comparing to commercial lipases, the thermostability of LGS and LAF was similar to lipase produced from Thermus thermophilus, which is active in the temperature range of 65˚C to 70˚C [25].

Lipases have different types of specificity including substrate chain length and positional which can be triacylglycerol stereospecific or non-specific. This research investigated the substrate chain length specificity of LGS and LAF. LGS and LAF kinetic constants were determined with different concentrations of various p-nitrophenyl esters (Table 1). With the standard lipase activity conditions (pH 7.5, at 20˚C, and 1 min reaction time), the Michaelis-Menten kinetic constants of K_m and V_max were determined by Lineweaver-Burk plots. Turnover numbers (k_cat) and the catalytic efficiency (k_cat/K_m) were used to compare the differences between LGS and LAF (Table 1). LGS showed the highest V_max with PNPC2, corresponding to 22 mM/min/mg followed by 2.6 mM/min/mg with PNPC12. The lowest K_m of 0.8 mM was obtained using PNPC12 and this substrate also showed the highest K_cat/K_m value. The highest K_cat was with PNPC2, corresponding to 9.3 × 10⁸ s⁻¹ and K_cat/K_m was 1.5 × 10¹⁰ mM⁻¹·s⁻¹ (Table 1).

For LAF, the kinetic parameters V_max, K_m, K_cat, and K_cat/K_m of LAF varied between various chain length esters, but the highest V_max was with PNPC14 and

PNPC2 is p-nitrophenyl acetate (C2), PNPC8 is p-nitrophenyl octanoate (C8), PNPC12 is p-nitrophenyl laurate (C12), PNPC14 is p-nitrophenyl myristate (C14), and PNPC16 is p-nitrophenyl palmitate (C16). Samples were assayed at 410 nm with 20˚C and 7.5 pH value. The values are presented as mean ± S.D.

PNPC8 and the lowest K_m was with PNPC8 which also resulted in the highest K_cat/K_m value (Table 1). The kinetics results showed that the K_m value was low for both enzymes when the substrates had chain length esters between C8 to C16 which indicated that both enzymes had an excellent affinity for p-nitrophenyl esters [26]. The results showed that the kinetic parameter values were close to many lipases for the various substrates from different sources. Lipases from G. stearothermophilus (Est55 and Est 30) showed affinity towards a PNPC8 substrate, and with this substrate Est55 showed a K_m of 0.5 µM and K_cat 39,758 s⁻¹, while Est30 showed a K_m of 2.16 µM and K_cat 38 s⁻¹ respectively [8]. The V_max and K_m of a lipase from Geobacillus sp. SBS-4S using a PNPC2 substrate was 2273 µmoL·min⁻¹·mg⁻¹ and 3.8 mM respectively [24]. Previous research showed a lipase from A. gonensis A4 had activities with a PNPC10 substrate and the K_m and V_max were 176.5 µM and 800 U/L [19].

The substrate specificities of LGS and LAF were examined with different chain length esters (Figure 4). Relative activity (%) of lipases was evaluated between the highest activity values of the enzyme with other activities using different substrates with the same conditions. The control substrate used to calculate relative activity for LGS was PNPC2 and for LAF was PNPC14 which were set at 100% relative activity. The enzyme specificity of LGS was higher towards the short chain fatty acid esters PNPC2 and PNPC8 compared to the longer chain PNPC14 and PNPC16. The results revealed that LGS had the highest activity of 100% with PNPC2, 61% with PNPC8, and 36%, 14%, and 7% with PNPC12, PNPC14, and PNPC16 substrates, respectively (Figure 4). The results were different with LAF, and the enzyme activity was highest with PNPC14 while the lowest enzyme activity was PNPC16. The relative activity of LAF was 12%, 100%,

27%, 35%, and 59% for PNPC16, PNPC14, PNPC12, PNPC8, and PNPC2, respectively (Figure 4). Geobacillus sp. SBS-4S lipases showed higher specificity toward a PNPC12 substrate than PNPC16 and PNPC20 substrates [24].

Lipases from G. stearothermophilus (Est55 and Est 30) more efficiently catalyzed the hydrolysis of esters of PNPC4 and PNPC6, with relative hydrolysis rates of 100%, 97% respectively compared to PNPC2 [8]. A lipase from Anoxybacillus sp. PDF1 had a higher catalytic efficiency with PNPC4 with a relative activity of 100% [16]. The reported substrate specificity observed in lipases from GS and AF agrees with the kinetic parameters. LGS has a specificity towards short chain fatty acid esters, while LAF has a high affinity to a 14 carbon chain fatty acid ester. Both enzymes, with their different specificity can be used in the food industry especially in the hydrolysis of milk fat, cheese ripening, modification of butter, and flavor development in meat and fish. Also, oils can be modified by enzymatic interesterification through lipases which catalyze medium and long chain fatty acid reactions [9] [18] [27].