Optimization of Process Parameters for the Enhanced Biosynthesis of ⍺-Amylase by Bacillus subtilis

Starch is an important macromolecule and the main source of carbohydrates in food. Chemically, it is a glucose polymer with amylose and amylopectin as structural components. Amylose, which makes up approximately 20% of starch, is made up of linear alpha-1, 4 linked glucose units. Whereas, amylopectin is a highly branched glucose polymer with alpha-1, 4 linked residues and alpha-1, 6 linkage at the branching point. It makes up about 80% of starch. Starch hydrolysis is a key process in many industries that is carried out preferably by amylases (Wang et al., 2022; Chorfa et al., 2022; Gan and Evers, 2022; Fuentes-Zaragoza et al., 2010). Amylases are categorized into beta amylases with EC (Enzyme Commission number) 3.2.1.2, glucoamylases (EC: 3.2.1.3) and alpha amylases with (EC: 3.2.1.1). Alpha amylases break down alpha-1, 4-glycosidic linkages, thus, resulting in the conversion of glycogen and starch and related polysaccharides into a range of products from monosaccharides (glucose) to low molecular weight oligosaccharides including maltose and maltotriose units, maltodextrin derivatives or cyclodextrins (Gupta et al., 2003; Rajagopalan et al., 2008).

Starch hydrolyzing enzymes i.e., amylases are considered to be quite versatile and have numerous biotechnological applications. Alpha-amylases are the major shareholder in the world market occupying 25-30% of the market (Chavez-Camarill et al., 2022; Hallol et al., 2022). It has extensive applications in textile, paper, animal feeds, environment, biofuel, detergent and clinical., health care, pharmaceutical and most importantly the food processing industry (Gavrilescu and Chishti, 2005; Couto and Sanroman, 2006; Ghorai et al., 2009; Al-Dhabi et al., 2020; Chavez-Camarill et al., 2022; Balakrishnan et al., 2021; Hallol et al., 2022).

Alpha-amylases are produced naturally in plants, insects, animals, and microorganisms (Balakrishnan et al., 2021). However, enzymes obtained from microbes (bacterial sources) have significant industrial applications (Kandra, 2003; Gupta et al., 2003; Reddy et al., 2003; Al-Dhabi et al., 2020). Members of genus Bacillus such as B. subtilis, B. circulans, B. amyloliquefaciens, B. megaterium, B. stearothermophilus, and B. licheniformis are the leading producer of commercial alpha-amylases (Konsoula and Liakopouloukyriakides, 2007; Prakash and Jaiswal, 2009; Luang-In et al., 2019). The rod-shaped B. subtilis (hay bacillus) is a catalase-positive and gram-positive soil bacterium. It is capable of producing a rigid, protective endospore which allows it to tolerate unusual environmental conditions. It produces thermostable enzymes that decrease the risk of contamination and exhibit increased the diffusion rate in reaction (Peixoto et al., 2003; Madigan and Martinko, 2005; Pant et al., 2015).

Two main fermentation setups are SmF (Submerged fermentation) and SSF (solid-state fermentation) which are utilized for alpha-amylase production. SmF has long been used for enzymes production because the different reaction parameters such as pH, temperature, aeration, moisture and oxygen transfer are easily controlled in SmF (Couto et al., 2006; Gangadharan et al., 2008; Balakrishnan et al., 2021; Hallol et al., 2022). Yet, many researchers prefer SSF for enzyme production (Tanyildizi et al., 2007; Couto et al., 2006). SSF has the natural advantages of high volumetric productivity, better product recovery, reduced level of catabolite repression, low capital investment, less usage of water, and the utilization of agricultural industrial wastes which reduces pollution problems and less effluent generation. Moreover, SSF also resembles the native habitat of microorganisms and thus, is a preferable choice for the growth of fungal, yeast and bacteria as well as the biosynthesis of valuable products (Pandey et al., 1999, 2000; Sivaramakrishnan et al., 2006; Singhania et al., 2009; Chimata et al., 2010).

The development of a successful fermentation process is directly dependent on the optimization of fermentation conditions. Thus, much attention has been shifted towards the optimization of culture medium as it is one of the main strategies for achieving maximum enzyme yield. Enzyme yield is also dependent on microbial strain, incubation time, cultivation technique, optimal pH and temperature, nutrients and cultivation temperature (Al-Dhabi et al., 2020). As these conditions and requirements vary from strain to strain so, it is crucial to optimize these process parameters to evaluate the amylase-producing potential of the individual strains.

Pakistan has a wide range of agricultural resources such as rice straw, rice husk, soybean meal, wheat straw and wheat bran, etc. which have tremendous potential as a solid substrate for commercially significant enzymes bioproduction. Therefore, the present project is focused on optimizing batch fermentation parameters of B. subtilis for the utilization of the locally available and cheap substrate to synthesize alpha-amylase via SSF.

Bacillus subtilis was obtained from stock culture of PCSIR labs, Lahore and was maintained on the nutrient agar medium.

Overnight grown inoculum of B. subtilis (at 37°C and 200 rpms) in nutrient broth (pH 6.8) was used.

The batch fermentation was carried out in a 250 ml flask for the bioproduction of alpha-amylase. Diluent (10 ml) was transferred into the flask containing wheat bran (10 g), was mixed well and sterilized using an autoclave at 121 ºC and lbs/in2 for 15 minutes. Inoculated the flasks aseptically with 1.0 ml of vegetative inoculum after cooling to 30 ºC and incubated at 37+1 ºC for two days. All the Experiments were run in duplicate.

Diluent composition: (%, w/v) glucose 0.5, (NH­)2­SO4 0.25, Urea 0.25 and KH­­2­PO4 0.5 pH 6.5.

Distilled water (50 ml) was added to each flask after an incubation period of 48 h followed by rotation on a rotary shaker for 2 h set at 150 rpm and 37+1ºC. The slurry was filtered and centrifuged at 4ºC at 8000 rpm for 10 minutes to separate fermented substrate, spores, cell mass, and small particles. The supernatant obtained was for enzyme assay.

For analysis of alpha-amylase was performed by Rick and Stegbauer’s Method (1974). The reaction mixture containing 0.5 ml of extracted enzyme and 0.5 ml of starch solution (1.0% solution prepared in acetate buffer of pH 5.0) was incubated for 30 minutes at 50°C. Reducing sugar estimation was carried out by the dinitrosalicylic acid (DNS) method after Miller (1959) using a standard curve of maltose.

One enzyme unit is the protein amount which in 10 minutes hydrolyses 1.0 μM reducing sugar (maltose) from 1.0% Lintner’s soluble starch per min per ml under standard assay conditions.

Computer software Costat was used for statistical analysis of the results of all the experiments that were run in duplicate.

Optimizing incubation time is quite critical for the optimal growth of bacterial culture (Lulko et al., 2007). The alpha-amylase production takes place only in cells in which normal cellular multiplication no longer occurs (Yamaguchi et al., 1974; Nomura et al., 1956). Therefore, a research study consisting of 24-96 hours was carried out for alpha-amylase production by B. subtilis. As shown in Figure 1, enzyme productivity increased with increasing incubation time while the maximum productivity (2.1±0.15 U/ml) was obtained at an incubation time of 48 hours after inoculation. This is because B. subtilis enters the stationary phase after 48 hours and produce maximum alpha-amylase. After 48h of incubation, a rapid decrease in alpha-amylase production was observed due to nutrient depletion and accumulation of metabolites and toxic substances in the fermented bran (Chamber et al., 1999; Gangadharan et al., 2006). These findings are similar to the work of other researchers (Riaz et al., 2003; Haq et al., 2005). Hence, the incubation period of 48 hours was considered optimal for alpha-amylase production by B. subtilis.

Figure 2 shows the effect of the diluent pH (5.5-8.5) on alpha-amylase bioproduction by B. subtilis. It was noted that with increasing pH, alpha-amylase production increased gradually. The maximum enzyme production (4.5±0.31 U/ml) was obtained at neutral pH. Further increase in pH declined the enzyme production by B. subtilis. It is because the optimum pH requirement for the growth of bacteria is neutral pH (7). At acidic pH, enzyme activity was less because it resulted in poor bacterial growth. Similar findings are reported by others as well (Haq et al., 2005; Syu and Chen, 1997; Tanyildizi et al., 2005).

In addition to the cultivation method, alpha-amylase production is greatly affected by essential nutrients among which carbon source is most noticeable. In the present work different mono and polysaccharides; glucose, soluble starch, maltose, sucrose and arabinose were used for optimal alpha-amylase production by B. subtilis (Figure 3) and the maximum production of alpha-amylase (9.5±0.43 U/ml) was obtained with soluble starch (0.25%, w/v). It is because starch as the substrate of alpha-amylase is the best inducer. Similar results are reported by Rajagopalan et al. (2010). However, glucose and arabinose gave the least production i.e., 3.5±0.6 and 3.1±0.23 U/ml, respectively. While contradictory results are reported by Anto et al. (2006) who discussed maximum amylase production in the glucose-supplemented culture medium.

Nitrogen sources (organic and inorganic) are known to enhance alpha-amylase production in SSF system (Pederson and Nielson, 2000). Figure 4 depicts the effect of various inorganic nitrogen sources i.e., ammonium citrate, (NH4)2SO4, NH4SO4, NH4NO3, NH4Cl2 and NaNO3 on the production of alpha-amylase (Figure 4). The maximum enzyme production (15.4±0.92 U/ml) was achieved when the diluent was supplemented with NaNO3. On the contrary, Aiyer (2004) reported different findings; optimal alpha-amylase production using NH4Cl2 as an inorganic nitrogen source. Five organic nitrogen sources (Tryptone, peptone, urea, casein and skimmed milk) were also tested for alpha-amylase fermentation (Figure 5). Among all of them, peptone gave maximum enzyme production (19.7±1.2 U/ml). On the contrary, another study reported a marginal rise in the bioproduction of alpha-amylase with the addition of peptone (Gangadharan et al., 2008).

The effect of four different metal ions on alpha-amylase production was studied (Figure 6). In this context, diluent was supplemented with ZnCl2, CaCl2, MgSO4 and MnCl2 individually. The enzyme production was maximum (25.4± 0.73 U/ml) when CaCl2 (0.5 %) was added to the diluent. It is due to the reason that Ca2+ ions are essential for the activity and stability of alpha-amylases (Hashemi et al., 2010). Similar findings are also described by others (Kokab et al., 2003; Hashemi et al., 2010).

The cultural conditions and nutritional necessities of Bacillus subtilis for the optimal biosynthesis of alpha-amylase via SSF are optimized successfully. The optimal amylase bioproduction was attained when the fermentation was carried out for 48 h at 37ºC using 10 g wheat bran. Alpha amylase production was improved by using the diluent having pH 7. Maximum alpha-amylase production (25.4±0.73 U/ml) was achieved when diluent was supplemented (%, w/v) with soluble starch 0.25, NH4NO3, peptone 0.25 and CaCl2 0.5. However, the production of alpha-amylase can be further improved by mutation prior to scale-up studies.

We acknowledge PCSIR, Laboratories, Lahore for providing the bacterial strain and facilities.

we have reported the optimized conditions for the alpha-amylase biosynthesis by B. subtilis that resulted in a 12.1-fold (1109.5% increase) in enzyme productivity.

UH, AL, MAK: Design of design, experimental work, data analysis and manuscript writing.

MA: Manuscript preparation.

IH: Approval of the experiment design.

The authors have declared no conflict of interest.