Bacillus spp. are known for their robust production of extracellular alkaline proteases. In this study, the extracellular alkaline proteases produced from four Bacillus strains were studied. To optimize the production of alkaline protease, the bacteria were cultured under different conditions. Yeast extract was identified as the optimal organic nitrogen source for the production of protease activity in all tested strains (Fig. 1A-D), similar to previous reports on B. stearothermophilus (Karray et al. 2021)d subtilis ATCC 6633 (Chatterjee et al. 2015). In contrast, peptone was a much less effective organic nitrogen source, unlike that reported for production of alkaline protease activity in B. pumilus MP 27 and Bacillus sp. RGR-14 (Puri et al. 2002; Baweja et al. 2016). Other organic nitrogen sources, including skim milk for TBRC 7773 and soytone for TBRC 6663 were comparable to yeast extract for the production of alkaline protease activity, suggesting that the optimal organic nitrogen source is strain-specific. Alternative organic nitrogen sources effective for the production of protease activity in other Bacillus strains include beef extract for B. amyloliquefaciens, B. licheniformis, B. megaterium and B. subtilis (Boominadhan et al. 2009; Sarker et al. 2013), soybean meal for B. cereus MCM B-326 (Nilegaonkar et al. 2007), soybean meal for B. subtilis PCSIR-5 (Qazi et al. 2006), and soya meal mixed with wheat bran for B. mojavensis SA (Hammami et al. 2018).
Regarding the effect of inorganic nitrogen sources on alkaline protease activity, diammonium hydrogen phosphate ((NH4)2HPO4) boosted the protease activity of TBRC 2902 to 20.8% of the activity from the basal medium, whereas potassium nitrate (KNO3) increased the activity of TBRC 1180 by 11.2%, similar to that previously reported for B. licheniformis MTCC NO. 7053 (Lakshmi et al. 2014). However, none of the five tested inorganic nitrogen sources in this study were able to enhance the protease activity of TBRC 6663 and TBRC 7773. The activity of these two strains might be induced by other inorganic nitrogen sources as previously reported. For instance, the protease activity of B. licheniformis P003 in the basal medium was as high as the activity in the basal medium supplemented with ammonium sulphate ((NH4)2SO4) (Sarker et al. 2013). Moreover, ammonium sulphate could optimize the protease activity of B. amyloliquefaciens SH-2 (Boominadhan et al. 2009), B. halodurans (Balachandran et al. 2021)d licheniformis SW2 (Boominadhan et al. 2009), while ammonium carbonate ((NH4)2CO3) was essential for protease activity produced by B. subtilis DL-1 and B. megatherium MW-1 (Boominadhan et al. 2009). B. subtilis EFRL 01 (Qureshi et al. 2011)d subtilis IC-5 (Gul et al. 2015) preferred ammonium nitrate (NH4NO3) and sodium nitrate (NaNO3) as inorganic nitrogen sources, respectively.
In addition to the nitrogen source, the carbon source is important for the production of extracellular alkaline protease. Tapioca starch was identified as the optimal carbon source for enzyme production in TBRC 1180 and TBRC 2902, whereas the optimal carbon sources for TBRC 6663 and TBRC 7773 were soluble starch and sucrose, respectively. The ability of TBRC 1180 and TBRC 2902 to optimally produce protease using tapioca starch, an inexpensive carbon source, makes these strains potentially advantageous for industrial application over other Bacillus strains that require more expensive simple carbon sources, such as glucose for B. luteus H11 (Kalwasińska et al. 2018)d subtilis IC-5 (Gul et al. 2015), and maltose and fructose for B. horikoshii (Joo and Choi 2012). Nonetheless, strain-specific preferences for other inexpensive carbon sources have been reported, including rice flour for B. licheniformis P003 (Sarker et al. 2013), wheat straw for B. pumilus MP 27 (Baweja et al. 2016) and Bacillus sp. BBXS-2 (Qureshi et al. 2016), and molasses for Bacillus sp. BGS (Moorthy and Baskar 2013), B. subtilis (Helal et al. 2012)d pantotheneticus (Shikha et al. 2007).
Regarding the effect of culture incubation time on the production of protease activity, TBRC 1180 and TBRC 6663 produced protease in a short time, with peak activity observed after 24 hours of culture, similar to B. stearothermophilus (Karray et al. 2021). However, TBRC 2902 and TBRC 7773 required a longer culture period (48 hours) to reach peak production, similar to B. licheniformis P003 (Sarker et al. 2013), B. pantotheneticus (Shikha et al. 2007), B. pumilus MP 27 (Baweja et al. 2016), B. subtilis GA CAS8 (Sathishkumar et al. 2015), B. subtilis (Helal et al. 2012), and Bacillus sp. BGS (Moorthy and Baskar 2013). Prolonging the incubation time beyond 48 hours can lead to a decrease in protease activity (Karray et al. 2021).
When used as an additive in detergent, proteases with optimal activity and stability under alkaline conditions are desirable. In this study, the crude enzymes from all tested strains showed protease activity with optimal activity in alkaline conditions. B. amyloliquefaciens TBRC 2902, B. subtilis TBRC 6663, and B. velezensis TBRC 7773 showed optimal activity at pH 8.0, similar to the crude enzyme produced from B. cereus VITSN04 (Sundararajan et al. 2011), and the purified enzymes from B. caseinilyticus (Mothe and Sultanpuram 2016), B. megaterium (Manavalan et al. 2020), B. subtilis DR8806 (Farhadian et al. 2015), B. thuringiensis (Agasthya et al. 2013)d velezensis SW5 (Yang et al. 2020a). By contrast, B. siamensis TBRC 1180 exhibited optimal activity at pH 9.0, similar to the purified enzymes produced from B. alveavuensis CAS 5 (Annamalai et al. 2014), B. koreensis BKP21A (Anbu 2013), B. licheniformis ALW (Emran et al. 2020)d subtilis BP-36 (Mashayekhi et al. 2012) (Table 2).
Proteases generally exhibit optimal activity in the range 50 to 70°C (Haddar et al. 2009b, a). The crude enzymes from TBRC 2902, TBRC 6663, and TBRC 7773, showed optimal activity at 50°C, similar to that of the crude enzyme from B. licheniformis P003 (Sarker et al. 2013), and the purified enzyme from B. altitudinis W3 (Yang et al. 2020b), B. alveayuensis CAS 5 (Annamalai et al. 2014), B. amyloliquefaciens SYB-001 (Wang et al. 2013), B. megaterium (Asker et al. 2013)d subtilis GA CAS8 (Sathishkumar et al. 2015). By contrast, the TBRC 1180 crude enzyme exhibited optimal activity at 60°C, similar to the crude enzymes of B. gibsonii 6BS15-4 (Mahakhan et al. 2023)d invictae AH1 (Hammami et al. 2017), and the purified enzymes of B. caseinilyticus (Mothe and Sultanpuram 2016), B. koreensis BK-P21A (Anbu 2013), B. safensis S406 (Mhamdi et al. 2017)d subtilis BP-36 (Mashayekhi et al. 2012) (Table 2).
Table 2
Comparison of physicochemical properties of proteases from various Bacillus spp. with the crude proteases from four Bacillus strains in this study
Organism
|
Type
|
MW (kDa)
|
Optimal pH
|
Optimal temperature (°C)
|
Reference
|
B. altitudinis W3
|
PE
|
34.9
|
10.5
|
45
|
(Yang et al. 2020b)
|
B. altitudinis W3
|
PE
|
37.3
|
8.5
|
50
|
(Yang et al. 2020b)
|
B. altitudinis W3
|
PE
|
37.9
|
9.5
|
55
|
(Yang et al. 2020b)
|
B. alveayuensis CAS 5
|
PE
|
33.0
|
9.0
|
50
|
(Annamalai et al. 2014)
|
B. amyloliquefaciens TBRC 2902
|
CE
|
n.d.
|
8.0
|
50
|
This study
|
B. amyloliquefaciens SYB-001
|
PE
|
36.8
|
7.0
|
50
|
(Wang et al. 2013)
|
B. caseinilyticus
|
PE
|
66.0
|
8.0
|
60
|
(Mothe and Sultanpuram 2016)
|
B. cereus VITSN0
|
CE
|
32.0
|
8.0
|
30
|
(Sundararajan et al. 2011)
|
B. gibsonii 6BS15-4
|
CE
|
n.d.
|
12.0
|
60
|
(Mahakhan et al. 2023)
|
B. invictae AH1
|
CE
|
n.d.
|
9.0–11.0
|
60
|
(Hammami et al. 2017)
|
B. koreensis BK-P21A
|
PE
|
48.0
|
9.0
|
60
|
(Anbu 2013)
|
B. licheniformis ALW
|
PE
|
n.d.
|
9.0
|
70
|
(Emran et al. 2020)
|
B. licheniformis P003
|
CE
|
n.d.
|
10.0
|
50
|
(Sarker et al. 2013)
|
B. licheniformis RSP-09-37
|
PE
|
55
|
10.0
|
55
|
(Sareen and Mishra 2008)
|
B. luteus H11
|
PE
|
37.0
|
10.5
|
45
|
(Kalwasińska et al. 2018)
|
B. megaterium
|
PE
|
25.0
|
7.5
|
50
|
(Asker et al. 2013)
|
B. megaterium
|
PE
|
28.0
|
7.5
|
50
|
(Asker et al. 2013)
|
B. megaterium
|
PE
|
33.0
|
8.0
|
70
|
(Manavalan et al. 2020)
|
B. safensis S406
|
PE
|
29.0
|
11.0
|
60
|
(Mhamdi et al. 2017)
|
B. siamensis TBRC 1180
|
CE
|
n.d.
|
9.0
|
60
|
This study
|
Bacillus sp.SM2014
|
PE
|
71.0
|
10.0
|
60
|
(Jain et al. 2012)
|
B. subtilis TBRC 6663
|
CE
|
n.d.
|
8.0
|
50
|
This study
|
B. subtilis BP-36
|
PE
|
40.0
|
9.0
|
60
|
(Mashayekhi et al. 2012)
|
B. subtilis DR8806
|
PE
|
37.0
|
8.0
|
45
|
(Farhadian et al. 2015)
|
B. subtilis GA CAS
|
PE
|
41.0
|
9.0
|
50
|
(Sathishkumar et al. 2015)
|
B. thuringiensis
|
PE
|
n.d.
|
8.0
|
47
|
(Agasthya et al. 2013)
|
B. velezensis TBRC 7773
|
CE
|
n.d.
|
8.0
|
50
|
This study
|
B. velezensis SW5
|
PE
|
34.0
|
8.0
|
40
|
(Yang et al. 2020a)
|
Remark CE, crude enzyme; PE, purified enzyme; n.d. not determined.
The stability of alkaline proteases to surfactants is important for industrial application in detergent formulations, which was investigated using non-ionic and ionic surfactants for the crude enzymes. Regarding the non-ionic surfactants, the crude enzymes from TBRC 1180 and TBRC 6663 exhibited moderate stability in 5% (v/v) Triton X-100, showing relative activities of 63.41 ± 5.18% and 56.90 ± 3.47%, respectively, after 24 hours of incubation. These values were higher than the relative activity of the crude enzymes from B. licheniformis A10, B. invictae AH1, and B. mojavensis SA, which was 44.38%, 46.83%, and 48.54%, respectively, after 1 hour of incubation (Yilmaz et al. 2016; Hammami et al. 2017, 2018). The relative activities of the crude enzymes from all tested strains against 5% (v/v) Tween 20 were above 45%, surpassing the residual activity of the partially purified enzymes from B. licheniformis A10, and Bacillus sp. RGR-14, which were 34.29% and 35.00% after 1 of incubation at 5% and 1% (v/v) of the surfactant, respectively (Oberoi et al. 2001; Yilmaz et al. 2016). Furthermore, the crude enzyme of B. subtilis TBRC 6663 exhibited the highest relative activity against 5% (v/v) Tween 80, reaching 61.35 ± 3.39%. This activity level was comparable to the relative activity of the purified enzymes from Bacillus sp. MPTK 712 and B. licheniformis A10, which were recorded at 67.50% and 27.84%, respectively (Kumar et al. 2012; Yilmaz et al. 2016).
In terms of activities on 1% (w/v) CTAB at 24 hours of incubation, TBRC 2902, TBRC 6663, and TBRC 7773 showed the relative activity of 30.64 ± 0.62%, 32.25 ± 1.13%, and 27.15 ± 1.48%, respectively. These activities surpassed the relative activity after 1 hour of the purified enzyme from B. subtilis DR8806 at the same concentration, which was recorded at 25% (Farhadian et al. 2015). Among the tested enzymes, only the crude enzyme from TBRC 1180 maintained its activity in 0.5% (w/v) SDS after 24 hours, exhibiting a relative activity of 7.81 ± 0.89%. This relative activity is comparable to the residual activity of B. invictae AH1, which was 20.54 ± 0.83% after 1 hour of incubation with the same concentration of SDS (Hammami et al. 2017).