Next Article in Journal
Indigenous Malolactic Starter Cultures as Innovative Tools to Modify the Sensory Profile of a Wine: An Oenological Challenge
Previous Article in Journal
Immobilized Plant-Based Presumptive Probiotics as Functional Ingredients for Breakfast Cereals
Previous Article in Special Issue
Sustainable Production of Medium-Chain Fatty Acids from Fresh Leachates in the District of Abidjan: Study of the Feasibility of the Process and Environmental Benefits
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Fermentation
Volume 11
Issue 6
10.3390/fermentation11060336
fermentation-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Unlocking the Potential of Pomelo Albedo: A Novel Substrate for Alpha-Amylase Production Using Bacillus licheniformis

by
Thi Ngoc Tran
1,
Si-Chun Chen
2,
Chien Thang Doan
1,* and
San-Lang Wang
2,3,*
1
Faculty of Natural Science and Technology, Tay Nguyen University, Buon Ma Thuot 630000, Vietnam
2
Department of Chemistry, Tamkang University, New Taipei City 25137, Taiwan
3
Life Science Development Center, Tamkang University, New Taipei City 25137, Taiwan
*
Authors to whom correspondence should be addressed.
Fermentation 2025, 11(6), 336; https://doi.org/10.3390/fermentation11060336
Submission received: 5 April 2025 / Revised: 27 May 2025 / Accepted: 9 June 2025 / Published: 11 June 2025
(This article belongs to the Special Issue Fermentation of Organic Waste for High-Value-Added Product Production)
Browse Figures
Versions Notes

Abstract

:
The bioprocessing of agricultural wastes to produce microbial enzymes has become significant due to its benefits in reducing enzyme production costs and improving waste management. In this study, various substrates, including spent coffee grounds, coffee husks, coffee pulp, rice husks, rice bran, pomelo albedo, pomelo flavedo, orange peel, banana peel, sugarcane bagasse, and starch, were used as organic nutrient sources for α-amylase biosynthesis by B. licheniformis TKU004. Among the tested substrates, pomelo albedo (3%, w/v) was the most suitable carbon source for amylase production, with a productivity of 80.645 U/mL. The purification process resulted in a 60 kDa amylase. The protein identification of B. licheniformis TKU004 amylase revealed a coverage rate of 39% with α-amylase from Bacillus subtilis 168. B. licheniformis TKU004 amylase exhibited optimal activity at 60 °C and pH = 7 and showed a high compatibility with EDTA (Ethylenediaminetetraacetic acid). HPLC (high-performance liquid chromatography) analysis demonstrated that B. licheniformis TKU004 amylase is an α-amylase with the final products of maltobiose, maltose, and glucose. Due to its important properties, such as tolerance to EDTA, B. licheniformis TKU 004 amylase may be valuable for industrial applications, especially in detergents and food processing.

1. Introduction

Enzymes have been exploited for industrial applications for a long time due to their excellent properties, such as high specificity and being environmentally friendly [1]. Amylases are among the most widely utilized commercial enzymes due to their critical roles in starch hydrolysis [2,3]. They are extensively used in the food, biofuel, textile, detergent, and paper industries [3]. Amylases account for about 25–33% of the global enzyme market [4]. Notably, the α-amylase market alone is expected to reach USD 382.4 million by 2025 [1]. Microbial sources, particularly bacteria from the Bacillus genus, have dominated industrial amylase production. Bacillus strains include B. cereus [5], B. amyloliquefaciens [6], B. stearothermophilus [7], B. halotolerans [8], B. subtilis [9], and B. licheniformis [10]. Among them, B. licheniformis is the most prominent due to its suitable fermentation characteristics and high production yield. In addition, the non-toxic nature of B. licheniformis makes it a safe choice for industrial applications [11]. Importantly, α-amylases from B. licheniformis exhibit higher thermal stability than those from other bacterial sources and are widely used in industry [12]. However, the economic viability of enzyme production remains challenged by the high costs of conventional fermentation substrates, necessitating the exploration of sustainable, low-cost alternatives [13,14].
Agricultural by-products offer a sustainable and cost-effective carbon source for microbial enzyme production through fermentation, addressing both waste management and production cost challenges [11,15]. Various agro-industrial wastes, including corn cobs, potato peel, rice bran, and wheat bran, have been utilized as substrates for amylase production. Surprisingly, despite its nutritional composition being well suited for microbial use in producing hydrolytic enzymes [16], citrus waste is rarely used for fermentation to produce amylase. Compared to other citrus fruits, pomelo has more peel, thus creating a significant amount of waste [17]. Pomelo peel, a lignocellulosic residue generated in large quantities (around 30–50% of the fruit weight) by food processing, is an underutilized resource rich in polysaccharides and fermentable sugars [18]. Additionally, pomelo peels have more pectin and less lignin than other citrus peels. This allows for a more efficient breakdown of pomelo peels into fermentable sugars [17]. The albedo component accounts for up to 30% of the fruit’s weight, constituting the majority of the mass of pomelo peels [19]. Pomelo albedo comprises approximately 72.62% carbohydrates [20], which makes it an attractive carbon source for microbial fermentation [11]. However, its potential for manufacturing value-added products, such as enzymes, is yet to be fully explored. In particular, limited research has investigated using pomelo peel as a substrate for microbial amylase production, especially using B. licheniformis. Therefore, this study aims to explore the potential of pomelo albedo as a substrate for α-amylase production and to characterize the α-amylase produced by B. licheniformis TKU004.

2. Materials and Methods

2.1. Bacteria Strain and Materials

B. licheniformis TKU004 was isolated and described in an earlier report [21]. Spent coffee grounds, coffee husks, coffee pulp, rice husks, rice bran, pomelo peel, orange peel, banana peel, and sugarcane bagasse were collected from New Taipei (New Taipei, Taiwan). Pomelo peel was separated into flavedo and albedo. All organic materials were then dried and milled into powder. Starch from potato and 3,5-Dinitrosalicylic acid (DNS) were purchased from Merck (Darmstadt, Germany). High Q resin was purchased from Bio-Rad (Hercules, CA, USA). All other chemicals were of the highest possible quality.

2.2. Amylase Assay

Quantifying reducing sugars from starch hydrolysis was used to determine amylase activity. The reaction setup consisted of 0.1 mL enzyme and 0.9 mL starch (1% in phosphate buffer (pH 7, 100 mM)). Later, the mixture was incubated at 60 °C for 30 min. To stop the reaction, 3 mL of 3,5-Dinitrosalicylic acid reagent (DNS) was added, followed by 10 min of incubation at 100 °C in a hot water bath. The mixture’s color intensity was measured at 540 nm. According to conventional assay settings, one unit (U) of amylase activity is the quantity needed to release 1 μmol of reducing sugar (glucose equivalents) per minute [22]. Control was established using 0.1 mL of denatured enzyme (heated at 100 °C for 20 min) instead of including the enzyme in the reaction setup. Protein concentration was measured by the Lowry method [23].

2.3. Fermentation Conditions

Spent coffee ground powder (SCGP), coffee husk powder (CHP), coffee pulp powder (CPP), rice husk powder (RHP), rice bran powder (RBP), pomelo albedo powder (PAP), pomelo flavedo powder (PFP), orange peel powder (OPP), banana peel powder (BPP), sugarcane bagasse powder (SCBP), and starch were supplemented to the basal medium containing 0.5% NH4NO3, 0.1% K2HPO4, and 0.05% MgSO4 to find the suitable carbon source for amylase production. Each carbon source was added at a concentration of 1%. The initial pH of the medium was set at pH = 7.0. The inoculum size was 2 mL of seed culture to 100 mL of medium. The cultures were incubated in a shaking incubator at 37 °C for 3 days. After incubation, the cultures were tested and screened based on the highest amylase productivity. In the next experiment, different amounts of PAP (0.5–4%) were added to the medium to determine the optimal concentration for amylase production. Finally, cultivation was carried out for 0, 1, 2, 3, 4, and 5 days to investigate the optimal time for maximum amylase activity.

2.4. Enzyme Purification

The liquid supernatant containing amylase was collected after centrifugation at 6000 rpm for 10 min. Ammonium sulfate was added to the liquid supernatant at a concentration of 60% to precipitate amylase [24]. The crude enzyme was dialyzed against 50 mM acetate buffer (pH = 5.8) to remove the salts. The crude enzyme was applied to a High Q column pre-equilibrated with 50 mM acetate buffer (pH = 5.8). The unbound proteins were removed by washing with the same buffer. Proteins bound to the column were eluted with 50 mM acetate buffer (pH = 5.8) containing 0–1 M NaCl. The fraction exhibiting amylase activity was analyzed for purity and mass using the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method [25]. The extracted SDS-PAGE target band was digested in-gel with trypsin and analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) to determine the protein identity [14].

2.5. Enzyme Characterization

The optimal temperature of B. licheniformis TKU004 amylase was determined by assessing its activity from 40 °C to 90 °C. The thermostability of B. licheniformis TKU004 amylase was determined by incubating the enzyme at different temperatures (40–90 °C) for one hour and then measuring its residual activity. The optimal pH was determined by measuring the activity in different buffer systems (50 mM) ranging from pH = 3 to pH = 10.6. The buffer systems were citrate buffer (pH = 3–5), sodium phosphate buffer (pH = 6–8), Tris-HCl buffer (pH = 8–9), and carbonate buffer (pH = 10–10.6). To determine the pH stability, B. licheniformis TKU004 amylase was incubated in the above buffer for one hour, and then its residual activity was measured.
B. licheniformis TKU004 amylase was tested with various chemicals at a final concentration of 5 mM, including KCl, FeCl2, CaCl2, BaCl2, MgCl2, CuCl2, NaCl, ZnCl2, EDTA, Phenylmethanesulfonyl fluoride (PMSF), and β-mercaptoethanol. For surfactants like sodium dodecyl sulfate (SDS), Tween 20, Tween 40, and Triton X-100, a final concentration of 1% was employed. The incubation lasted one hour at 4 °C. Control enzyme activity was tested without chemicals.
The substrate specificity of B. licheniformis TKU004 amylase was confirmed using starch powder, gelatinized starch, carboxymethyl cellulose (CMC), pectin, gum arabic, dextran, glycogen, β-1,3-glucan, stachyose, and raffinose. The activity measured using 1% gelatinized starch served as the control.

2.6. HPLC Analysis

The starch hydrolysis mechanism of B. licheniformis TKU004 amylase was studied at 1% with a reaction time of 0 to 24 h. A KS-803 column, a water mobile phase (with a flow rate of 1 mL/min), and a refractive index detector were used for the analysis. The temperature of the column was set at 80 °C. The standards were glucose and maltose.

3. Results and Discussion

3.1. Enzyme Production

In this study, spent coffee ground powder (SCGP), coffee husk powder (CHP), coffee pulp powder (CPP), rice husk powder (RHP), rice bran powder (RBP), pomelo albedo powder (PAP), pomelo flavedo powder (PFP), orange peel powder (OPP), banana peel powder (BPP), sugarcane bagasse powder (SCBP), and starch were used to provide carbohydrate sources for the fermentation of B. licheniformis TKU004. The amylase activity of the culture media was evaluated to find the most suitable by-product source. According to Figure 1a, except for the medium containing RHP, the strain could produce amylase on all the remaining by-product media at different levels. The highest amylase productivity was obtained in the presence of PAP (27.369 ± 1.289 U/mL) and starch (26.425 ± 0.986 U/mL). A one-way ANOVA indicated insignificant differences between the starch and PAP groups (with F-value = 0.550 and p-value = 0.499). This suggests that PAP could be a potential alternative substrate to starch for amylase production. Likewise, Iram et al. (2020) found that B. licheniformis synthesized the highest amount of amylase in a medium containing grapefruit peel as the sole carbon source [26]. The elevated amylase production observed on the PAP-containing medium indicates that B. licheniformis TKU004 may not require starch to produce amylase. In this instance, it is plausible that carbohydrates in PAP affected the amylase production capability of B. licheniformis TKU004. Rajagopalan and Krishnan (2008) discovered that B. subtilis KCC103 produced amylase in a medium containing hydrolysate from sugarcane bagasse, with xylose being the most effective inducer of amylase production [27]. Leloup et al. (1997) reported that sucrose induced the α-amylase production of B. subtilis [28]. The secretion pathway of α-amylase exhibits notable similarities to that of levansucrase, highlighting the significant connections between these enzymes. It is worth noting that B. licheniformis TKU004 produced the highest level of levansucrase in PAP-containing medium [11]. The effect of varying amounts of PAP on the amylase productivity of B. licheniformis TKU004 was investigated at a range of 0.5–4%. Figure 1b showed that at 3% PAP, the highest amylase productivity was achieved (65.814 ± 6.270 U/mL). At a higher amount of PAP (4%), the amylase productivity of B. licheniformis TKU004 (64.291 ± 4.731 U/mL) was almost similar to that observed at 3% PAP. Thus, 3% PAP was chosen for further experiments.
Incubation time is a critical factor in fermentation, dramatically affecting production yield and process efficiency [29]. To explore the optimal incubation period for the amylase production of B. licheniformis TKU004 on the medium containing 3% PAP, enzyme activity was evaluated at 0, 1, 2, 3, 4, and 5 days. Figure 1c reveals that the highest amylase activity (80.645 ± 5.724 U/mL) occurred on the fourth day. Beyond this point (on the fifth day), a decline in activity was observed. Consequently, a 4-day incubation period was chosen for subsequent amylase production experiments.
From the literature, the amylase productivity of Bacillus strains varies depending on strain and fermentation conditions. For example, when using apple peel and potato peel as carbon sources, Bacillus subtilis NAIMCC-B-01934 exhibited the highest amylase productivity of 17468 U/L and 5229 U/L [30]. A new Bacillus strain produced amylase of 18.48 U/mL on a starch-containing medium [31], while Bacillus cereus D3 produced 13.89 ± 0.07 U/mL when using glucose as the carbon source [32]. In addition, Bacillus licheniformis WF67 produced the highest amylase activity (6.633 U/mL) on a soluble starch-containing medium [33], while Bacillus subtilis MK1 produced a higher activity of 145.4 U/mL on the same substrate [34]. Thus, in this study, the amylase productivity of B. licheniformis TKU004 was assessed as relatively good. Notably, using PAP instead of starch can create an economic and environmental advantage, including reducing enzyme production costs and supporting effective waste management. Therefore, choosing B. licheniformis TKU004 combined with PAP substrate is a potential direction in industrial amylase production.

3.2. Enzyme Purification and Identification

Amylase from the liquid supernatant of B. licheniformis TKU004’s medium was purified using (NH4)2SO4 precipitation followed by a High Q column, and the results are summarized in Table 1. The 344.952-fold purification has a specific activity of 622.547 U/mg, as seen in Table 1. According to Figure 2, the molecular weight (MW) of the purified amylase was determined to be 60 kDa by SDS-PAGE. From the literature, the MW of α-amylases ranges from 55 to 70 kDa [35], suggesting that the MW of B. licheniformis TKU004’s α-amylase falls in the range determined by previous studies. In comparison to other strains of B. licheniformis, the MW of B. licheniformis TKU004’s α-amylase is similar to that of B. licheniformis B4-423 (58 kDa) [36] and relatively lower than that of B. licheniformis So-B3 (74 kDa) [37] and B. licheniformis LB04 (130 kDa) [35]. Božić et al. (2011) reported an amylase from B. licheniformis ATCC 9945a with an MW of only 32 kDa [38].
In order to identify the amylase obtained from B. licheniformis TKU004, which appeared as a distinct 60 kDa band on the SDS-PAGE gel, the band was excised and subjected to tryptic digestion. The resulting peptides were analyzed by electrospray tandem mass spectrometry, and their spectra were compared against the NCBI non-redundant protein database. As detailed in Table 2, eleven peptide sequences of the amylase matched those of alpha-amylase from Bacillus subtilis 168, covering approximately 39% of its sequence. This peptide matching suggests that B. licheniformis TKU004 amylase is an α-amylase. Sumrin et al. (2011) reported that the MW of Bacillus subtilis 168 α-amylase was 55 kDa [39], relatively smaller than B. licheniformis TKU004 amylase (60 kDa).

3.3. Enzyme Characterization

Temperature is a critical parameter that significantly influences amylase activity. Understanding the optimum temperature of an enzyme is important for its applications. According to Figure 3a, B. licheniformis TKU004 amylase has an optimum temperature of 50 °C. Studies have shown that various optimum temperatures could be observed from different Bacillus strains. For example, B. halotolerans RFP74 amylase has an optimum temperature of 37 °C [8], while B. licheniformis LB04 works best at 65 °C [35]. Similarly, B. cereus shows optimum activity at 45 °C [40]. Amylase from B. subtilis J12 works well between 40 and 70 °C, with an optimum pH of 6.0 [41]. Amylase from Bacillus sp. H7 shows maximum enzymatic activity at 40 °C [42]. Purified BH072 α-amylase from B. amyloliquefaciens BH072 was most active at 60 °C [43]. An extraordinarily thermostable and acidophilic amylase showed maximum enzymatic activity at 100 °C [36]. These differences emphasize that the optimum temperature is highly dependent on the specific enzyme and its source. High temperatures can lead to enzyme denaturation and reduced activity, while low temperatures can significantly reduce amylase activity. To investigate the thermal stability of B. licheniformis TKU004 amylase, the enzyme underwent preincubation for one hour at temperatures ranging from 40 °C to 100 °C. The residual activity of B. licheniformis TKU004 amylase was subsequently assessed. The activity of B. licheniformis TKU004 amylase was observed to decrease non-significantly at 50 °C. At higher temperatures, the enzyme activity was sharply decreased.
The optimal pH and pH stability of enzymes are critical in industrial applications. According to Figure 3b, B. licheniformis TKU004 amylase exhibited peak activity at pH = 7.0, suggesting that the optimal pH for this enzyme’s activity is 7.0. Likewise, amylases from B. amyloliquifaciens TSWK1-1 [44], B. amyloliquefaciens BH072 [43], and B. methylotrophicus P11-2 have optimal pH at pH = 7.0 [22]. However, some amylases from B. licheniformis show optimal activity in acidic conditions [35,36,37]. B. licheniformis TKU004 amylase demonstrated significant pH stability within the range from 5.0 to 9.0, maintaining over 80% of its initial activity.
Table 3 shows how various chemicals affect the amylolytic activity of B. licheniformis TKU004 amylase differently. Among metal salts, KCl and FeCl2 did not significantly affect the enzyme activity; CaCl2, BaCl2, MgCl2, and NaCl mildly decreased the enzyme activity; CuCl2 and ZnCl2 dramatically inhibited the enzyme activity. The inhibitory effect of Cu2+ and Zn2+ could be observed in the reports of Xie et al. (2014) [22] and Mabrouk et al. (2025) [31]. Interestingly, the chelating agent EDTA did not inhibit the amylolytic activity of B. licheniformis TKU004 amylase. EDTA could drastically inactivate amylase activity in many cases, for example, amylases from B. atrophaeus NRC1 [45], B. methylotrophicus strain P11-2 [22], and B. licheniformis strain LB04 [35]. Since many α-amylases belong to metalloenzymes, they are inhibited by EDTA [46]. The resistance of B. licheniformis TKU004 amylase to EDTA may arise from its minimal reliance on metal ions for structural integrity and activity. An amylase from Bacillus strain KSM-38 was highly resistant to EDTA due to the weak binding affinity of Ca2+ for the enzyme [47]. The EDTA resistance of amylases may be important for their application in the detergent industry [36]. Thus, it is worth noting that B. licheniformis TKU004 amylase could be used as a detergent component.
Table 3 shows that surfactants (SDS, Tween 20, Tween 40, and Triton X-100) significantly inhibited B. licheniformis TKU004 amylase. In the presence of SDS, Tween 20, Tween 40, and Triton X-100, the enzyme retained 68.313 ± 2.650%, 76.533 ± 0.900%, 40.684 ± 3.650%, and 44.414 ± 3.850% (respectively) of its initial activity. Indeed, surfactants can have an inhibitory effect on amylase activity [36]. Their inhibitory effect was mainly due to the changes in enzyme structure, which decreased enzyme activity [48]. PMSF and β-mercaptoethanol had no significant effect on B. licheniformis TKU004 amylase activity. Likewise, PMSF did not significantly affect the amylase of B. amyloliquefaciens BH072; however, this enzyme was boosted by β-mercaptoethanol [43]. In other reports, PMSF and β-mercaptoethanol moderately inhibited amylase activity [36,49].
According to Table 4, B. licheniformis TKU004 amylase exhibits its highest specificity toward gelatinized starch (100.000 ± 1.194%), followed by glycogen (79.055 ± 3.270%). However, the enzyme exhibited relatively low or no activity on non-starch substrates. This indicates the high substrate specificity of B. licheniformis TKU004 amylase, suggesting that it primarily targets starch for hydrolysis. This phenomenon could be easily observed in other amylases [50,51,52]. In addition, B. licheniformis TKU004 amylase shows low activity on starch powder (3.266 ± 1.755 %), indicating that the physical form of starch is also a key parameter for enzymatic hydrolysis. Likewise, Alonazi et al. (2021) confirmed that α-amylase from Bacillus pacificus could not hydrolyze raw starch [53].

3.4. HPLC Analysis of the Hydrolysis Products

Hydrolysis products of starch catalyzed by B. licheniformis TKU004 were analyzed by HPLC method. Figure 4 indicates that three product peaks were observed at the early stage (0.5 h), including 11.8 min, 12.3 min, and 13.2 min. Of those, the peak at 13.2 min refers to maltose; the peaks at 12.3 min and 11.8 min could be maltotriose and maltotetraose (respectively). This result reveals that B. licheniformis TKU004 amylase belongs to α-amylase, which can hydrolyze starch to different sizes of oligosaccharides. After one hour, a trace peak of glucose (14.9 min) could also be observed. As the incubation time was prolonged, the intensity of maltotriose, maltose, and glucose peaks increased, but the maltotetraose peak decreased. This indicates that the enzyme could further hydrolyze maltotetraose to release smaller products. Finally, at 24 h, only glucose, maltose, and maltotriose peaks were observed. Of those, maltose is the predominant product. Likewise, amylase from B. methylotrophicus strain P11-2 produces the final products of maltotriose, maltose, and glucose [22]. However, the α-amylase from B. licheniformis NCIB 6346 releases the predominant product of maltopentaose [54].

4. Conclusions

The utilization of agricultural wastes for the production of high-quality products via microbial fermentation is progressively rising. In this work, B. licheniformis TKU004 revealed its ability to produce amylase with the highest productivity of 80.645 U/mL in a medium containing 3% PAP. The extracted amylase had a MW of 60 kDa on SDS-PAGE gel and belongs to α-amylase, according to protein identification analysis. B. licheniformis TKU004 amylase worked best at 60 °C and pH = 7 and had high compatibility with EDTA. The amylase can convert starch into maltotriose, maltose, and glucose as the final products. The beneficial properties of B. licheniformis TKU004 amylase show great potential for the detergent and food industries. Moreover, since the enzyme is produced using agricultural waste, this bioprocess aligns with current trends toward sustainable and circular bioeconomy practices. Future work should focus on optimization and scale-up studies to evaluate its economic and operational feasibility on a commercial scale.

Author Contributions

Conceptualization and methodology, S.-L.W. and C.T.D.; software, validation, formal analysis, investigation, resources, and data curation, C.T.D., T.N.T., S.-C.C. and S.-L.W.; writing—original draft preparation, writing—review and editing and visualization, C.T.D., S.-L.W. and T.N.T.; supervision, project administration, and funding acquisition, S.-L.W. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported in part by a grant from the National Science and Technology Council, Taiwan (NSTC-112-2320-B-032-001).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Mondal, S.; Mondal, K.; Halder, S.K.; Thakur, N.; Mondal, K.C. Microbial amylase: Old but still at the forefront of all major industrial enzymes. Biocatal. Agric. Biotechnol. 2022, 45, 102509. [Google Scholar] [CrossRef]
  2. Zinck, S.S.; Christensen, S.J.; Sørensen, O.B.; Svensson, B.; Meyer, A.S. Importance of inactivation methodology in enzymatic processing of raw potato starch: NaOCl as efficient α-amylase inactivation agent. Molecules 2023, 28, 2947. [Google Scholar] [CrossRef] [PubMed]
  3. De Souza, P.M.; De Magalhães, P.O. Application of microbial α-amylase in industry—A review. Braz. J. Microbiol. 2010, 41, 850–861. [Google Scholar] [CrossRef] [PubMed]
  4. Bhatt, B.; Prajapati, V.; Patel, K.; Trivedi, U. Kitchen waste for economical amylase production using Bacillus amyloliquefaciens KCP2. Biocatal. Agric. Biotechnol. 2020, 26, 101654. [Google Scholar] [CrossRef]
  5. Vaikundamoorthy, R.; Rajendran, R.; Selvaraju, A.; Moorthy, K.; Perumal, S. Development of thermostable amylase enzyme from Bacillus cereus for potential antibiofilm activity. Bioorg. Chem. 2018, 77, 494–506. [Google Scholar] [CrossRef]
  6. Zalila-Kolsi, I.; Ben-Mahmoud, A.; Al-Barazie, R. Bacillus amyloliquefaciens: Harnessing its potential for industrial, medical, and agricultural applications—A comprehensive review. Microorganisms 2023, 11, 2215. [Google Scholar] [CrossRef]
  7. Yao, D.; Su, L.; Li, N.; Wu, J. Enhanced extracellular expression of Bacillus stearothermophilus α-amylase in Bacillus subtilis through signal peptide optimization, chaperone overexpression and α-amylase mutant selection. Microb. Cell Fact. 2019, 18, 69. [Google Scholar] [CrossRef]
  8. Rafanomezantsoa, P.; Gharbi, S.; Karkachi, N.; Kihal, M. Optimization of amylase production by the biological control agent Bacillus halotolerans RFP74 using response surface methodology. J. Genet. Eng. Biotechnol. 2023, 21, 63. [Google Scholar] [CrossRef]
  9. Wang, M.; Zhao, W.; Zhou, D.; Huang, J. Bacillus subtilis contributes to amylase production in the honey sac of Apis mellifera. Insects 2025, 16, 221. [Google Scholar] [CrossRef]
  10. Zhang, A.; Ma, Y.; Deng, Y.; Zhou, Z.; Cao, Y.; Yang, B.; Bai, J.; Sun, Q. Enhancing protease and amylase activities in Bacillus licheniformis XS-4 for traditional soy sauce fermentation using ARTP mutagenesis. Foods 2023, 12, 2381. [Google Scholar] [CrossRef]
  11. Doan, C.T.; Tran, T.N.; Nguyen, T.T.; Tran, T.P.H.; Nguyen, V.B.; Tran, T.D.; Nguyen, A.D.; Wang, S.-L. Production of sucrolytic enzyme by Bacillus licheniformis by the bioconversion of pomelo albedo as a carbon source. Polymers 2021, 13, 1959. [Google Scholar] [CrossRef] [PubMed]
  12. Lee, J.; Xiang, L.; Byambabaatar, S.; Kim, H.; Jin, K.S.; Ree, M. Bacillus licheniformis α-amylase: Structural feature in a biomimetic solution and structural changes in extrinsic conditions. Int. J. Biol. Macromol. 2019, 127, 286–296. [Google Scholar] [CrossRef] [PubMed]
  13. Mamo, J.; Kangwa, M.; Fernandez-Lahore, H.M.; Assefa, F. Optimization of media composition and growth conditions for production of milk-clotting protease (MCP) from Aspergillus oryzae DRDFS13 under solid-state fermentation. Braz. J. Microbiol. 2020, 51, 571–584. [Google Scholar] [CrossRef] [PubMed]
  14. Doan, C.T.; Tran, T.N.; Tran, T.P.H.; Nguyen, T.T.; Nguyen, H.K.; Tran, T.K.T.; Vu, B.T.; Trinh, T.H.T.; Nguyen, A.D.; Wang, S.-L. Chitosanase production from the liquid fermentation of squid pens waste by Paenibacillus elgii. Polymers 2023, 15, 3724. [Google Scholar] [CrossRef]
  15. Khaswal, A.; Mishra, S.K.; Chaturvedi, N.; Saini, S.; Pletschke, B.; Kuhad, R.C. Microbial enzyme production: Unlocking the potential of agricultural and food waste through solid-state fermentation. Bioresour. Technol. Rep. 2024, 27, 101880. [Google Scholar] [CrossRef]
  16. Lima, C.A.; Contato, A.G.; de Oliveira, F.; da Silva, S.S.; Hidalgo, V.B.; Irfan, M.; Gambarato, B.C.; Carvalho, A.K.F.; Bento, H.B.S. Trends in enzyme production from Citrus by-products. Processes 2025, 13, 766. [Google Scholar] [CrossRef]
  17. Huang, R.L.; Cao, M.; Guo, H.; Qi, W.; Su, R.X.; He, Z.M. Enhanced ethanol production from pomelo peel waste by integrated hydrothermal treatment, multienzyme formulation, and fed-batch operation. J. Agric. Food Chem. 2014, 62, 4643–4651. [Google Scholar] [CrossRef]
  18. Xiao, L.; Ye, F.Y.; Zhou, Y.; Zhao, G.H. Utilization of pomelo peels to manufacture value-added products: A review. Food Chem. 2021, 351, 129247. [Google Scholar] [CrossRef]
  19. Wang, X.Y.; Xu, R.; Wang, Y.X.; Ma, L.Y.; Nie, S.P.; Xie, M.Y.; Yin, J.Y. Physicochemical and rheological properties of pomelo albedo pectin and its interaction with konjac glucomannan. Int. J. Biol. Macromol. 2020, 151, 1205–1212. [Google Scholar] [CrossRef]
  20. Zain, N.F.M.; Yusop, S.M.; Ahmad, I. Preparation and characterization of cellulose and nanocellulose from pomelo (Citrus grandis) albedo. J. Nutr. Food Sci. 2014, 5, 1000334. [Google Scholar]
  21. Wang, S.L.; Kao, T.Y.; Wang, C.L.; Yen, Y.H.; Chern, M.K.; Chen, Y.H. A solvent stable metalloprotease produced by Bacillus sp. TKU004 and its application in the deproteinization of squid pen for β-chitin preparation. Enzym. Microb. Technol. 2006, 39, 724–731. [Google Scholar] [CrossRef]
  22. Xie, F.; Quan, S.; Liu, D.; Ma, H.; Li, F.; Zhou, F.; Chen, G. Purification and characterization of a novel α-amylase from a newly isolated Bacillus methylotrophicus strain P11-2. Process Biochem. 2014, 49, 47–53. [Google Scholar] [CrossRef]
  23. Lowry, O.; Rosebrough, N.; Farr, A.L.; Randall, R. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951, 193, 265–275. [Google Scholar] [CrossRef] [PubMed]
  24. Abo-Kamer, A.M.; Abd-El-Salam, I.S.; Mostafa, F.A.; Mustafa, A.-E.A.; Al-Madboly, L.A. A promising microbial α-amylase production, and purification from Bacillus cereus and its assessment as antibiofilm agent against Pseudomonas aeruginosa pathogen. Microb. Cell Factories 2023, 22, 141. [Google Scholar] [CrossRef]
  25. Tran, T.N.; Doan, C.T.; Dinh, T.K.L.; Duong, T.H.N.; Phan, T.T.U.; Le, T.T.L.; Tran, T.D.; Hoang, P.H.Q.; Nguyen, A.D.; Wang, S.-L. Optimization production of an endo-β-1,4-xylanase from Streptomyces thermocarboxydus using wheat bran as sole carbon source. Recycling 2024, 9, 50. [Google Scholar] [CrossRef]
  26. Iram, N.; Shakir, H.A.; Irfan, M.; Khan, M.; Ali, S.; Anwer, A.; Saeed, S.; Qazi, J.I. Statistical optimization of amylase production using grape fruit peels in submerged fermentation. Acta Sci. Technol. 2020, 43, e50538. [Google Scholar] [CrossRef]
  27. Rajagopalan, G.; Krishnan, C. α-Amylase production from catabolite derepressed Bacillus subtilis KCC103 utilizing sugarcane bagasse hydrolysate. Bioresour. Technol. 2008, 99, 3044–3050. [Google Scholar] [CrossRef]
  28. Leloup, L.; Haddaoui, E.A.; Chambert, R.; Petit-Glatron, M.F. Characterization of the rate-limiting step of the secretion of Bacillus subtilis alpha-amylase overproduced during the exponential phase of growth. Microbiology 1997, 143, 3295–3303. [Google Scholar] [CrossRef]
  29. Özdemir, S.; Matpan, F.; Güven, K.; Baysal, Z. Production and characterization of partially purified extracellular thermostable α-amylase by Bacillus subtilis in submerged fermentation (SmF). Prep. Biochem. Biotechnol. 2011, 41, 365–381. [Google Scholar] [CrossRef]
  30. Singh, R.; Langyan, S.; Sangwan, S.; Gaur, P.; Khan, F.N.; Yadava, P.; Rohatgi, B.; Shrivastava, M.; Khandelwal, A.; Darjee, S.; et al. Optimization and production of alpha-amylase using Bacillus subtilis from apple peel: Comparison with alternate feedstock. Food Biosci. 2022, 49, 101978. [Google Scholar] [CrossRef]
  31. Mabrouk, S.B.; Hmida, B.B.H.; Sebii, H.; Fendri, A.; Sayari, A. Production of an amylase from newly Bacillus strain: Optimization by response-surface methodology, characterization and application with a fungal lipase in bread making. Int. J. Biol. Macromol. 2025, 285, 138147. [Google Scholar] [CrossRef] [PubMed]
  32. Wang, S.; Bai, H.; Wang, Z.; Peng, L.; Li, L.; Yan, H.; Zhu, L.; Wang, Y.; Shao, J.; Liu, J. Characterization of newly isolated Bacillus cereus D3 co-producing α-amylase and protease and its application in food raw materials. Food Biosci. 2024, 62, 105255. [Google Scholar] [CrossRef]
  33. Saad, W.F.; Othman, A.M.; Abdel-Fattah, M.; Ahmad, M.S. Response surface methodology as an approach for optimization of α-amylase production by the newly isolated thermotolerant Bacillus licheniformis WF67 strain in submerged fermentation. Biocatal. Agric. Biotechnol. 2021, 32, 101944. [Google Scholar] [CrossRef]
  34. Ahmed, S.A.; Abdella, M.A.; El-Sherbiny, G.M.; Ibrahim, A.M.; El-Shamy, A.R.; Atalla, S.M. Application of one-factor-at-a-time and statistical designs to enhance α-amylase production by a newly isolate Bacillus subtilis strain-MK1. Biocatal. Agric. Biotechnol. 2019, 22, 101397. [Google Scholar] [CrossRef]
  35. Silva-Salinas, A.; Rodríguez-Delgado, M.; Gómez-Treviño, J.; López-Chuken, U.; Olvera-Carranza, C.; Blanco-Gámez, E.A. Novel thermotolerant amylase from Bacillus licheniformis strain LB04: Purification, characterization and agar-Agarose. Microorganisms 2021, 9, 1857. [Google Scholar] [CrossRef]
  36. Wu, X.; Wang, Y.; Tong, B.; Chen, X.; Chen, J. Purification and biochemical characterization of a thermostable and acid-stable alpha-amylase from Bacillus licheniformis B4-423. Int. J. Biol. Macromol. 2018, 109, 329–337. [Google Scholar] [CrossRef]
  37. Fincan, S.A.; Özdemir, S.; Karakaya, A.; Enez, B.; Mustafov, S.D.; Ulutaş, M.S.; Şen, F. Purification and characterization of thermostable α-amylase produced from Bacillus licheniformis So-B3 and its potential in hydrolyzing raw starch. Life Sci. 2021, 264, 118639. [Google Scholar] [CrossRef]
  38. Božić, N.; Ruiz, J.; López-Santín, J.; Vujčić, Z. Production and properties of the highly efficient raw starch digesting α-amylase from a Bacillus licheniformis ATCC 9945a. Biochem. Eng. J. 2011, 53, 203–209. [Google Scholar] [CrossRef]
  39. Sumrin, A.; Ahmad, W.; Ijaz, B.; Sarwar, M.T.; Gull, S.; Kausar, H.; Shahid, I.; Jahan, S.; Asad, S.; Hussain, M. Purification and medium optimization of α-amylase from Bacillus subtilis 168. Afr. J. Biotechnol. 2011, 10, 2119–2129. [Google Scholar]
  40. Rakaz, M.A.; Hussien, M.O.; Ibrahim, H.M. Isolation, extraction, purification, and molecular characterization for thermostable α-Amylase from locally isolated Bacillus Species in Sudan. Biochem. Res. Int. 2021, 2021, 6670380. [Google Scholar] [CrossRef]
  41. Mahfudz, M.K.; Jaikhan, S.; Phirom-on, K.; Apiraksakorn, J. Cost-effective strategy and feasibility for amylase production from okara by Bacillus subtilis J12. Fermentation 2024, 10, 561. [Google Scholar] [CrossRef]
  42. Bandal, J.N.; Tile, V.A.; Sayyed, R.Z.; Jadhav, H.P.; Azelee, N.I.W.; Danish, S.; Datta, R. statistical based bioprocess design for improved production of amylase from halophilic Bacillus sp. H7 isolated from marine water. Molecules 2021, 26, 2833. [Google Scholar] [CrossRef] [PubMed]
  43. Du, R.; Song, Q.; Zhang, Q.; Zhao, F.; Kim, R.C.; Zhou, Z.; Han, Y. Purification and characterization of novel thermostable and Ca-independent α-amylase produced by Bacillus amyloliquefaciens BH072. Int. J. Biol. Macromol. 2018, 115, 1151–1156. [Google Scholar] [CrossRef]
  44. Kikani, B.; Singh, S. Single step purification and characterization of a thermostable and calcium independent α-amylase from Bacillus amyloliquifaciens TSWK1-1 isolated from Tulsi Shyam hot spring reservoir, Gujarat (India). Int. J. Biol. Macromol. 2011, 48, 676–681. [Google Scholar] [CrossRef]
  45. Abd-Elaziz, A.M.; Karam, E.A.; Ghanem, M.M.; Moharam, M.E.; Kansoh, A.L. Production of a novel α-Amylase by Bacillus atrophaeus NRC1 isolated from honey: Purification and characterization. Int. J. Biol. Macromol. 2020, 148, 292–301. [Google Scholar] [CrossRef]
  46. Gupta, R.; Gigras, P.; Mohapatra, H.; Goswami, V.K.; Chauhan, B. Microbial alpha-amylases: A biotechnological perspective. Process Biochem. 2003, 38, 1599–1616. [Google Scholar] [CrossRef]
  47. Hagihara, H.; Igarashi, K.; Hayashi, Y.; Endo, K.; Ikawa-Kitayama, K.; Ozaki, K.; Kawai, S.; Ito, S. Novel α-amylase that is highly resistant to chelating reagents and chemical oxidants from the Alkaliphilic Bacillus isolate KSM-K38. Appl. Environ. Microbiol. 2001, 67, 1744–1750. [Google Scholar] [CrossRef]
  48. Kumar, N.M.; Karthikeyan, S.; Jayaraman, G. Thermostable alpha-amylase enzyme production from Bacillus laterosporus: Statistical optimization, purification, and characterization. Biocatal. Agric. Biotechnol. 2013, 2, 38–44. [Google Scholar] [CrossRef]
  49. Sharma, A.; Satyanarayana, T. Cloning and expression of acid-stable, high maltose-forming, Ca2+-independent α-amylase from an acidophile Bacillus acidicola and its applicability in starch hydrolysis. Extremophiles 2012, 16, 515–522. [Google Scholar] [CrossRef]
  50. Sharma, A.; Satyanarayana, T. Microbial acid-stable α-amylases: Characteristics, genetic engineering and applications. Process Biochem. 2013, 48, 201–211. [Google Scholar] [CrossRef]
  51. Ben Hadj Hmida, B.; Ben Mabrouk, S.; Fendri, A.; Hmida-Sayari, A.; Sayari, A. Optimization of newly isolated Bacillus cereus α-amylase production using orange peels and crab shells and application in wastewater treatment. 3 Biotech 2024, 14, 119. [Google Scholar] [CrossRef] [PubMed]
  52. Murakami, S.; Nagasaki, K.; Nishimoto, H.; Shigematu, R.; Umesaki, J.; Takenaka, S.; Kaulpiboon, J.; Prousoontorn, M.; Limpaseni, T.; Pongsawasdi, P.; et al. Purification and characterization of five alkaline, thermotolerant, and maltotetraose-producing α-amylases from Bacillus halodurans MS-2-5, and production of recombinant enzymes in Escherichia coli. Enzym. Microb. Technol. 2008, 43, 321–328. [Google Scholar] [CrossRef]
  53. Alonazi, M.; Karray, A.; Badjah-Hadj-Ahmed, A.Y.; Ben Bacha, A. Alpha amylase from Bacillus pacificus associated with brown algae Turbinaria ornata: Cultural conditions, purification, and biochemical characterization. Processes 2021, 9, 16. [Google Scholar] [CrossRef]
  54. Morgan, F.J.; Priest, F.G. Characterization of a thermostable α-amylase from Bacillus licheniformis NCIB 6346. J. Appl. Bacteriol. 1981, 50, 107–114. [Google Scholar] [CrossRef]
Fermentation 11 00336 g001
Figure 1. Effect of carbon source (a), PAP ratio (b), and incubation time (c) on amylase productivity by B. licheniformis TKU004. Data represent the mean of three replicates, and error bars indicate the standard deviation (SD).
Figure 1. Effect of carbon source (a), PAP ratio (b), and incubation time (c) on amylase productivity by B. licheniformis TKU004. Data represent the mean of three replicates, and error bars indicate the standard deviation (SD).
Fermentation 11 00336 g001
Fermentation 11 00336 g002
Figure 2. SDS-PAGE analysis of the amylase produced by B. licheniformis TKU004. M, protein marker; 1, crude enzyme; 2, High Q chromatography.
Figure 2. SDS-PAGE analysis of the amylase produced by B. licheniformis TKU004. M, protein marker; 1, crude enzyme; 2, High Q chromatography.
Fermentation 11 00336 g002
Fermentation 11 00336 g003
Figure 3. Effect of temperature (a) and pH (b) on activity and stability of B. licheniformis TKU004 amylase. Solid line, effect of pH on activity; dash line, effect of pH on stability. Data represent means of three replicates, and error bars indicate SD.
Figure 3. Effect of temperature (a) and pH (b) on activity and stability of B. licheniformis TKU004 amylase. Solid line, effect of pH on activity; dash line, effect of pH on stability. Data represent means of three replicates, and error bars indicate SD.
Fermentation 11 00336 g003
Fermentation 11 00336 g004
Figure 4. Hydrolysis pattern of B. licheniformis TKU004 amylase toward starch.
Figure 4. Hydrolysis pattern of B. licheniformis TKU004 amylase toward starch.
Fermentation 11 00336 g004
Table 1. Purification of amylase produced by B. licheniformis TKU004.
Table 1. Purification of amylase produced by B. licheniformis TKU004.
Purification StepTotal Protein
(mg)		Total Activity
(U)		Specific Activity (U/mg)Purification FoldRecovery (%)
Liquid supernatant11361.58420558.2711.8091100
(NH4)2SO4 precipitation866.58216552.18019.10110.55680.513
High Q column5.5283441.206622.547344.05216.739
Table 2. Identification of B. licheniformis TKU004 amylase by LC-MS/MS.
Table 2. Identification of B. licheniformis TKU004 amylase by LC-MS/MS.
Peptide SequenceIdentified Protein and Coverage RateStrain
69DIHDAGYTAIQTSPINQVK87
114YLGTEQEFKEMCAAAEEYGIK134
157SIPNWTHGNTQIK169
206ALNDGADGFR215
284NLGVSNISHYASDVSADK301
329LGWAVIASRSGSTPLFFSRPEGGGNGVR356
367GSALFEDQAITAVNRFHNVMAGQPEELSNPNGNNQIFMNQR407
439AGAGSFQVNDGKLTGTINARSVAVLYPDDIAKAPHVFLENYKTGVTHSFNDQLTITLR496
504AVYQINNGPETAFKDGDQFTIGK526
533TYTIMLK539
603NADGIYTLTLPADTDTTNAK622		α-amylase 39%B. subtilis strain 168
Table 3. Effect of chemicals on the activity of B. licheniformis TKU004 amylase.
Table 3. Effect of chemicals on the activity of B. licheniformis TKU004 amylase.
Relative Activity (%)
(Mean ± SD)
Control100.000 ± 3.016
KCl99.465 ± 3.000
FeCl297.600 ± 6.350
CaCl279.158 ± 0.400
BaCl280.263 ± 0.900
MgCl278.260 ± 1.150
CuCl28.634 ± 0.950
NaCl91.176 ± 3.000
ZnCl22.141± 0.350
SDS68.313 ± 2.650
Tween 2076.533 ± 0.900
Tween 4040.684 ± 3.650
Triton X-10044.414 ± 3.850
EDTA123.986 ± 0.850
PMSF97.048 ± 5.150
β-mercaptoethanol96.495 ± 3.350
Table 4. Substrate specificity of B. licheniformis TKU004 amylase.
Table 4. Substrate specificity of B. licheniformis TKU004 amylase.
Relative Activity (%)
(Mean ± SD)
Starch powder3.266 ± 1.755
Gelatinized starch 100.000 ± 1.194
CMCN.A.
Pectin16.439 ± 1.849
Gum arabic22.061 ± 2.285
Dextran4.120 ± 1.587
Glycogen79.055 ± 3.270
β-1,3-glucan14.675 ± 1.972
StachyoseN.A.
RaffinoseN.A.
N.A., no activity.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Tran, T.N.; Chen, S.-C.; Doan, C.T.; Wang, S.-L. Unlocking the Potential of Pomelo Albedo: A Novel Substrate for Alpha-Amylase Production Using Bacillus licheniformis. Fermentation 2025, 11, 336. https://doi.org/10.3390/fermentation11060336

AMA Style

Tran TN, Chen S-C, Doan CT, Wang S-L. Unlocking the Potential of Pomelo Albedo: A Novel Substrate for Alpha-Amylase Production Using Bacillus licheniformis. Fermentation. 2025; 11(6):336. https://doi.org/10.3390/fermentation11060336

Chicago/Turabian Style

Tran, Thi Ngoc, Si-Chun Chen, Chien Thang Doan, and San-Lang Wang. 2025. "Unlocking the Potential of Pomelo Albedo: A Novel Substrate for Alpha-Amylase Production Using Bacillus licheniformis" Fermentation 11, no. 6: 336. https://doi.org/10.3390/fermentation11060336

APA Style

Tran, T. N., Chen, S.-C., Doan, C. T., & Wang, S.-L. (2025). Unlocking the Potential of Pomelo Albedo: A Novel Substrate for Alpha-Amylase Production Using Bacillus licheniformis. Fermentation, 11(6), 336. https://doi.org/10.3390/fermentation11060336

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop