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Proceeding Paper

Partial Purification of Bacillus cereus Enzyme Expression for Bio-Pulping of Lignin Degraders Isolated from Coptotermus curvignathus †

by
Sharfina Mutia Syarifah
1,
Ashuvila Mohd Aripin
2,
Nadiah Ishak
3,
Nosa Septiana Anindita
1,
Mohd Firdaus Abdul-Wahab
4 and
Angzzas Sari Mohd Kassim
2,*
1
Biotechnology Study Programme, Faculty of Science and Technology, Universitas ‘Aisyiyah Yogyakarta, Yogyakarta 55292, Indonesia
2
Department of Chemical Engineering Technology, Faculty of Engineering Technology, Universiti Tun Hussein Onn Malaysia, Pagoh 84300, Malaysia
3
Faculty of Business, Hospitality and Technology, Universiti Islam Melaka, Kuala Sungai Baru 75400, Malaysia
4
Department of Biosciences, Faculty of Science, Universiti Teknologi Malaysia, Skudai 81310, Malaysia
*
Author to whom correspondence should be addressed.
†
Presented at the 8th Mechanical Engineering, Science and Technology International Conference, Padang Besar, Perlis, Malaysia, 11–12 December 2024.
Eng. Proc. 2025, 84(1), 41; https://doi.org/10.3390/engproc2025084041
Published: 7 February 2025
(This article belongs to the Proceedings of The 8th Mechanical Engineering, Science and Technology International Conference)
Browse Figures
Figure 1
<p>The linear graph of the Bradford assay standard curve. The assay was performed using bovine serum albumin (BSA) as a standard. The straight line represents a linear fit of the measured data.</p> "> Figure 2
<p>SDS-PAGE gel electrophoresis image. Indicated are the protein marker (Lane 1), the crude enzyme (Lanes 2 and 3), and the ultrafiltered enzyme (Lanes 4 and 5). Lane 2 indicates presence of proteins with 11 different molecular weight size of proteins. The red labels show the chosen bands for protein sequencing.</p> ">
Versions Notes

Abstract

:
Despite extensive research on Bacillus sp. as lignin degraders, the enzyme mechanisms involved, particularly in Bacillus cereus isolated from termite guts, remain unclear. In this study, the selected Bacillus cereus was fermented to extract the lignin-degrading enzymes to identify the enzymes responsible for lignin degradation using the sample substrate empty fruit bunch (EFB) as their sole carbon source. After 7 days of submerged fermentation (SmF), the crude enzyme was extracted, and SDS-PAGE gel was used to determine the weight of the proteins, and bands with sizes of 20 kDa–97 kDa were extracted for further analysis. The extracted proteins were partially characterized and sequenced using liquid chromatography–mass spectrometry (LC–MS/MS). The results identified 11 enzymes that are responsible for lignin degradation, such as 4-aminobutyrate aminotransferase (GABA), amidohydrolase, chemotaxis protein, serine hydrolase, GMC family protein, glycosyltransferase, phosphate binding protein PstS, ABC transporter ATP-binding protein, heme peroxidase, nitrate reductase, and nitrite reductase. The value of the mutual relationships between all the enzymes in Bacillus cereus indicates the synergistic mechanism under carbon scrutinization. Also, the peptides sequenced in this study identified various uncharacterized proteins and hypothetical proteins that might not be discovered for their protein functions. Further analysis is essential to uncover more lignin degradation enzymes that can work synergically for paper and pulp bioprocessing.

1. Introduction

Prior to mechanical or chemical pulping, the process of bio-pulping uses natural degraders like white-rot fungi and bacteria to produce enzymes that can break down lignocellulosic materials [1,2]. The intention is to reduce cellulose degradation during the pretreatment stage and eliminate lignin, and so bio-pulping offers tremendous potential to improve the current pulping process. [2]. Bacteria lignin degraders can be classified into three classes: α-proteobacteria, γ-Proteobacteria, and actinomycetes [3].
Previous research has demonstrated the existence of bacterial delignification enzymology. There is evidence, however, that bacteria not only secrete similar extracellular ligninolytic enzymes as fungi, but also possess some unique and special enzymes, such as laccase, glutathione s-transferases, cleaving dioxygenases, monooxygenases, and phenol oxidases [4]. Bacterial enzymes may have advantages over fungal enzymes in terms of thermostability, halotolerance, specificity, and mediator dependency. A hyperthermophilic laccase produced by Thermos thermophilus HB27 bacteria with the optimal temperature requirement of approximately 92 °C and a half-life of thermal inactivation at 80 °C was successfully isolated by Miyasaki [5,6]. This demonstrated the possibility of using bacteria with lower sensitivities, which attracted the interests of industries.
Lignin degradation on palm oil empty fruit bunch (EFB) by Bacillus cereus has previously been proven to successfully remove 21.7% of lignin [7]. The advantages given by Bacillus sp. include their rapid growth rates which leads to short fermentation periods, and their ability to secrete proteins into the extracellular medium. The use of Bacillus sp. in lignin degradation has shown great prospects. Bacillus sp. have the ability to degrade Kraft lignin, with a preference for recalcitrant phenothiazine dye groups such as Azure B, Methylene Blue, and Toluidene Blue O dyes. Nonetheless, the addition of copper sulphate and yeast extract to the growth medium was necessary for decolorizing Kraft lignin after 6 days of incubation [8].
Other lignin-degrading enzymes produced by Bacillus sp. have been reported. Other Bacillus sp. such as Bacillus aryabhattai and Bacillus cereus ATCC11778 are known to secrete two types of lignolytic enzymes, which are lignin peroxidase and laccase [9]. Moreover, Bacillus tequilensis SN4 is identified to produce only laccase [10]. Bacillus cereus ATCC11778 is reported to have 12 U/mg lignin peroxidase activity and 0.027 U/mg laccase activity [8]. Other than that, Bacillus ligniniphilus L1 is chosen for lignin degradation because of its ability to use alkaline lignin as a single carbon or energy source, as well as its exceptional ability to survive in extreme environments. Gentisate and benzoic acid pathways are two of three lignin degradation pathways in strain L1, and the β-ketoadipate pathway includes two divisions of catechuate and protocatechuate. [11]. Bacillus sp. LG7 shows the capability to produce three promising enzymes, such as laccase activity (274.03 ± 2.45 U/L), and LiP activity (265.48 ± 1.24 U/L). Bacillus sp. LG7 is one of the promising sources of ligninolytic enzymes for industrial applications [12].
With advanced methods such as protein extraction and protein sequencing analysis, researchers can determine the type of enzymes involved during the mechanism of lignin degradation by Bacillus sp. Therefore, this article aims to discover the enzymes that are associated with lignin degradation in Bacillus cereus using EFB as the sole carbon source for lignocellulose material.

2. Materials and Methods

The enzyme expression was cultivated via submerged fermentation (SmF) [13]. The selected Bacillus cereus was fermented with empty fruit bunch (EFB) that act as the sole source of carbon to trigger the expression of enzymes that are responsible for the lignin degradation. Bacillus cereus was grown on an agar plate for 24 h at 37 °C, then placed into an inoculum of 10 mL of LB broth and grown there for another 24 h until the solution turned cloudy. The overnight suspension was diluted to a fixed density of 0.5 McFarland standard [MF] which equivalent to 1.5 × 108 CFU/mL and evaluated using UV-VIS spectrophotometry at a minimum value of 0.7–0.8 OD with roughly 1 × 108 cells. A 1 mL suspension of Bacillus cereus was transferred into a 500 mL conical flask containing the medium and substrates for continuous expression. The bacterium strain was cultured at 250 mL of LB broth supplemented with 5 g of fully submerged empty fruit bunch (EFB). The pH of the SmF culture conditions was adjusted to 6.5 at 37 °C, 120 rpm, and incubated for 7 days in an incubator shaker [14].

2.1. Protein Extraction

The crude enzyme was extracted after 7 days of submerged fermentation (SmF) by centrifugation at 10,000× g rpm for 15 min at 4 °C. The supernatant was then precipitated using 60% ammonium sulphate ((NH4)2SO4). After 24 hours, the precipitated enzyme was recovered by centrifugation at 10,000× g rpm for 10 min [14]. The centrifuged pellets were dissolved in a 0.3 M sodium phosphate buffer at pH 7. The solution was placed in a dialysis bag and dialyzed for 24 h against the same buffer. The dialysate protein was collected and transferred to a centrifuge ultrafiltration vivaspin 6 kDa tube, and the solvent was centrifuged for 15 min at 4 °C at an adjusted 4500 rpm [15]. A Bradford assay test was carried out at each step of the purification procedure to determine the total protein level.

2.2. Total Protein Quantification

The protein contents were measured using the technique reported by Bradford in 1976 [16]. Coomassie Brilliant Blue G-250, a blue colour indicator, was dissolved in 12.5 mL 95% ethanol. The Bradford reagent was prepared by diluting the blue indicator solution with 25 mL of 85% (w/v) phosphoric acid to a final volume of 250 mL. Each dilution of 0.0 to 1.8 mg/mL of bovine serum albumin (BSA) solution was mixed in a glass test tube with 3 mL of the Bradford reagent. After 5 min of incubation, the diluted samples were pipetted into 1.5 mL cuvettes and their absorbance was measured using UV-VIS spectrophotometry (Spectro UV-2650, Labomed® Inc., Los Angeles, CA, USA) on a wavelength of 595 nm.
The total protein concentration was determined using the method described by Bradford in 1976. The BSA dilution was used for standard curve determination. Samples were prepared with 0.1 mL protein solutions in glass test tubes. The 3 mL of Bradford reagent was mixed by circular shaking before being incubated in a dark room for 5 min. Continuously, 1.5 mL of the solution was pipetted into cuvettes and a measurement was taken using UV-VIS spectrophotometry.

2.3. SDS-PAGE Electrophoresis Preparation

SDS-PAGE electrophoresis was carried out to determine the homogeneity and apparent molecular mass of the partially purified bacteria sample from the EFB substrate. The gel for electrophoresis was prepared beforehand for the stock solution preparation followed by the working solution in accordance with the Laemmli standard 1970 [17]. SDS-PAGE preparation involved casting two layers of gel as stacking and separating gel between glass plates provided by the Bio-Rad SDS page system, and was carried out using current Bio-Rad methods as well as the working solutions specified in Table 1 [18].
The separating gels were assembled accordingly using the working solutions in Table 1 with 4 mL of Solution A, 2.5 mL of Solution B, and 3.5 mL of distilled water mixed, respectively. Then, 100 µL of 10% (w/v) APS and 5 µL of TEMED were added and mixed well. The mixed solution was then quickly transferred into the gel sandwich equipment by carefully using a micropipette. Further, the solution was levelled out on top with distilled water to create a flat gel surface and to avoid the development of bubbles on the polymerized separating solution. The gel was left to polymerize for around 60 min.
After the gel suspension was polymerized, the distilled water was taken out and the stacking gel solution was then added. The formulae for the stacking gel were mixed accordingly. A total of 0.67 mL of Solution A, 1 mL of Solution C, and 2.3 mL of distilled water were mixed and suspended, respectively. Then, 50 µL of 10% (w/v) APS and 5 µL of TEMED were added and mixed afterwards. Using a micropipette, the stacking solution was swiftly placed onto the separating gel until it reached the top of the plate. The comb was then carefully placed on top of the gel solution and left there for 60 min to polymerize. It was calculated that both solutions yielded two different gels.
To prepare the protein samples, 5 µL of the 5x sample buffer was added to 20 µL of the crude and dialyzed protein samples. The samples were denatured by heating at 90 °C for 5 min and then injected into each well (20 µL per well). The SDS gel electrophoresis was run on a Mini-PROTEAN® Tetra cell (Cat log no. #1658006; Bio-Rad Laboratories Inc., Hercules, California, USA ) in accordance with the Bio-Rad protocol [18]. A total of 10 µL of a protein marker (Sigma Aldrich, Darmstadt, Germany) was used as protein ladder with molecular weight range between 14kb to 200 kb bands. The crude and dialyzed protein samples were loaded into two wells with two replicates for each sample. The electrophoresis was run for 60 min at 150 V until the dye in the sample buffer reached the bottom of the gel [17,18].

2.4. Protein Band Determination

After collection, the gels were stained to determine the bands. In order to make the staining buffer, 0.25 g of Coomassie blue G-250 (0.25%) was dissolved in 100 mL of water, then 100 mL of 40% ethanol was added, and the mixture was stirred until the dye was completely dissolved. After 5 min, 25 mL of 10% glacial acetic acid was added, and 250 mL of distilled water was then added. The staining solution was stored in the dark. A 1 L batch of another de-staining buffer was composed of 40% ethanol and 10% acetic acid [19]. The gel was then transferred to a different container that had been sufficiently filled with staining buffer to saturate the gel. The container was heated in a microwave at high power for 2 min with a loose lid, then placed on a rocking shaker for 1 h. The gel was then transferred to the de-staining gel container, cleaned with distilled water, and set on a rocking shaker for 24 h. The final image of the gel was captured and stored in distilled water for further analysis. A Gel Doc XR+ System was used to capture the gel picture (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

2.5. Protein Sequencing Analysis

The protein sequencing was carried out at the Proteomics International Facility at the Harry Perkins Institute of Medical Research, QEII Medical Centre, Perth, Australia. All protocols were currently accredited for compliance with ISO/IEC 17025 by National Association of Testing Authorities (NATA), accreditation number (16838). The gel pieces of the protein samples were cut and digested with trypsin, then the peptides were extracted [20]. The samples were analyzed using an Agilent 1260 Infinity HPLC system coupled to an Agilent 6540 mass spectrometer with an Agilent 1260 Chipcube Nanospray interface. Peptides were then loaded onto a ProtID-Chip-150 C18 column and isolated using a water/acetonitrile/0.1% formic acid (v/v) gradient. Using Mascot sequence matching software [Matrix Science] and the MSPnr100 database, the resulting spectra were used to identify proteins of interest. The search parameters for the LC-MS/MS analysis on the Agilent 6540 mass spectrometer were as follows: peptide tolerance (Peptide tol): ±0.2; MS/MS tol: ±0.2; peptide charge: 2+, 3+, and 4+; mass; monoisotopic enzyme: trypsin; and miss cleavage: 1. The protein was identified using a statistically significant score (p < 0.05) [21]. Using Mascot sequence matching software [Matrix Science] and the UniProt and SwissProt databases, the spectra results were analyzed to identify proteins of interest. The peptide sequences that could not be identified were obtained from the NCBI protein database using protein–protein BLAST.

3. Results and Discussion

As shown in Figure 1, the standard curve was generated and plotted using the Bacillus cereus absorbance value of BSA serial dilution against the concentration of the BSA serial dilution. The y = f(x) equation of the calibration curve was reported as y = 0.9698x + 0.0925, where the determined R2 value was 0.9904, which fitted with the linear regression range of R2 (0.96–0.99) [22].
According to the equation set by the standard curve in Figure 1, the x value, or the protein quantification, could be determined by measuring the y value, or the absorbance value of Bacillus cereus protein concentration. As the y values were recorded and determined, the concentrations of the enzyme expressed through the SmF of the EFB substrate were tabulated in Table 2.
Based on the results in Table 2, the enzyme concentration was determined at approximately 0.85 mg/mL. As the proteins were concentrated through dialysis and ultrafiltration, the protein volume of the concentrated protein increased to around 1.62 mg/mL. This study shows a higher concentration of protein compared to the study performed by Sahadevan et al. (2016) on the lignin-degrading fungi MVI.2011, which measured approximately 0.69 mg/mL of the crude enzyme. A higher amount of total protein concentration collected would possibly consist higher amount of ligninase, in order to supports lignin degradation activity [23]. A study by Lai et al. (2017) also supported that Bacillus sp. have great potential in producing overall lignin-degrading enzymes in its crude enzyme for synergistic activity [24].

3.1. SDS PAGE Electrophoresis (Molecular Weight Determination)

The SDS-PAGE gel was used to determine the weight of the proteins and for further analysis of their identity. The extracted crude and ultrafiltered enzyme supernatants were analyzed and the scanned image by Gel Doc XR+ System shown in Figure 2.
Based on Figure 2, there were 11 visible bands of the enzyme in the crude protein sample. The result of the crude enzyme sample was comparable with another LDE producer, dimorphic novel fungus MVI.2011 [22]; around 12 different molecular weight size of proteins were discovered in its crude enzyme sample. However, the ultrafiltered protein sample revealed only six visible bands with masses of 95–97 kDa, 66–68 kDa, 43–45 kDa, 38–40 kDa, 25–27 kDa, and 20 kDa. The LDE research discovered that the lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase (Lac) single bands weighed 42, 45, and 62 kDa, respectively. The molecular weight of ligninase varied depending on the organism type. It has also been indicated that the laccases ranged from 20 to 90 kDa [24,25]. Several types of heme peroxidase protein were usually found in the range of 40 to 50 kDa secreted into their microenvironment by ligninolytic fungi [26]. These types of enzymes were also found in bacteria species such as Rhodococcus opacus’s Dyp-type peroxidase, a heme-dependent enzyme that were reported to be observed as single band with MW size at 40 kDa to 50 kDa [27]. Moreover, the heme peroxidase (LiP, MnP, veratyrl peroxidase (VP)) molecular weight of Bacillus subtilis and Bacillus albus MW407057 was reported to be a single monomer of 48 kDA [28,29]. A Bacillus cereus analysis on lignin-degrading enzymes for molecular weight determination has not been reported yet; however, the analysis for cellulose has been reported [30]. Thus, a range of protein from 20 kDa to around 90 kDa is suspected of possible lignin degradation enzymes, and the specific heme peroxidase enzyme molecular weight is expected to be around 40 kDA to 50 kDA.

3.2. Enzyme Sequencing Analysis LC-MS/MS (Enzyme Identification)

The enzyme sequencing results validated by the LC-MS/MS detection were identified using MASCOT sequence matching software and NCBI BLAST and are listed in Table 3. Bacillus cereus in this study was shown to secrete various oxidative enzymes that assist in lignin modifications. The extracellular oxidative enzymes which degrade lignin employ heme-containing peroxidases have been identified in this study and validate the lignin-degrading capability [31]. On the other hand, some other oxidizing mediators or small oxidizing agents, such as up-regulated proteins, which might be related to lignin degradation, including oxidoreductase, transferases, hydroxylases, reductases, dehydrogenases, and ABC transporters have been discovered as well.
Based on the results, gamma-aminobutyrate transaminase (GABA-T), also known as 4-aminobutyrate aminotransferase, is involved in the 4-aminobutyrate and glutamate degradation pathways. The accumulation of GABA is influenced by the feeding habit of insects that consume lignocellulose, which, in turn, harbour gut bacteria capable of producing enzymes related to GABA production [32]. Recently, it was found that there was a reaction in empty fruit bunch oil palm through high glutamate-producing Corynebacterium glutamicum strains for the co-utilization of glucose and xylose as carbon sources to enhance the production of GABA [33]. The GABA activity observed was analyzed in a batch fermentation with a pH of 6 and 7, which is a suitable pH to exhibit high amount of GABA. This in accordance with the pH value designed in this study, which were set to pH 6 for the empty fruit bunch Bacillus cereus treatment. The production of GABA could be related with the lack of nitrogen as well as the delignification process continuing since no carbon source other than lignin was fed to the bacteria. This was shown in another study, where the presence of exogenous GABA contributed towards the decrease in the lignin and hemicellulose structure due to the decrease depending on the nitrogen source [34].
Hydrolases were present due to the biochemical catalyze activity, which used water to cleave chemical bonds paralleled with the liquid used in the submerged fermentation (SmF) which was used in this study. Serine hydrolases (SHs) catalyzed one of the main biochemical reactions: the cleavage of the amide and ester bond. SH is known as one of the largest and most complex enzyme families, as well as being important to the catabolism of aromatic compounds [35,36]. SHs from various species have been involved in aromatic compound cleavage pathways and have a high catalytic activity on some hydrolysis of C-C bonds [36]. It was also stated that the methyl-acceptant chemotaxis protein and the methyl-acceptant sensory chemotaxis transducer were substantially upregulated when lignin was the sole carbon source [36].
Moreover, in a study it was discovered that Bacillus cereus protease enzymes detected on a molecular mass of 45.6 kDa were able to degrade wool fibre, and this was also recorded in this study, which shows an enzyme activity at a similar molecular weight detected at 45 kDa [37]. The GMC oxidoreductase family enzymes have vital roles in the oxidation of alcohol group-type lignin-derived compounds in the Sphingobium sp. SYK-6 [38]. It was suspected that Bacillus cereus yielded a similar reaction of the enzyme. Additionally, glycosyltransferases (GT) were reported to be involved in the sequence of carbohydrate-active enzymes (CAZymes). The GT family was shown to be encoded in the Ganoderma lucidum G0119 genome for lignocellulolytic enzymes [39]. However, a glycosyltransferases (GT) from family 2 (a repertoire of Carbohydrate Active Enzymes (CAZymes)) showed activity towards the synthesis of various oligo- and polysaccharides, including cellulose, hemicellulose, chitin, and peptidoglycan, which possibly increased the amount of lignocellulose content instead [40].
Bacillus cereus secretes various oxidative enzymes of different types of heme-containing peroxidases, which include the so-called LiP, MnP, and versatile peroxidases (VP) [41]. While some of those peroxidases can attack fragments of lignin, peroxidases also attack lignin from a distance. Small oxidizing agents are generated in the presence of oxidizing mediators that penetrate the branched lignin polymer to trigger depolymerization via radical chemistry [42]. For this reason, the sequence was detected as heme peroxidase instead of a specific protein identification. These heme peroxidases enzymes act play important role in lignin degradation, thus Bacillus cereus potentially could be an excellent lignin degrader.
Lignin degradation was primarily moderated by initial macronutrient levels, such as nitrogen and phosphate ratio concentrations, which are the basic nutrients for microbial activity [43]. For example, phosphate-binding protein (PBP) were induced under phosphate starvation particularly PstS, a periplasmic binding protein that plays a crucial role in phosphate uptake; to facilitate organic and inorganic degradation process in order to meet the metabolic demands of the cell [44]. The movement of aromatic compounds across the cell, including decarboxylase benzoyl for benzene, xylene, and toluene degradation, could be regulated by ABC transporters [45]. It is also demonstrated by Alphaproteobacteria, which possess a diverse set of transport capabilities for lignin-derived compounds [46]. DeAngelis et al. (2013) proved that the increments of NADH–quinone oxidoreductase, ATP synthase, ATP-binding cassette (ABC) transporters, and other electron transport chain proteins in their studies had increased their degradation of lignin in Enterobacter lignolyticus under starvation of carbon source [47]; hence, similar speculation can be made for Bacillus cereus expression in this experiment. Thus, the overall findings in this study support that the synergistic between lignin degrader enzymes enhances lignin degradation process in gut microbes.

4. Conclusions

In this study, the protein quantification was sufficient to be used to further the analysis and to characterize and identified enzymes associated with lignin degradation process. The identification of several known enzyme degraders, such as the hydrolases (serine, amidohydrolase), oxidoreductases (GMC family protein), heme peroxidase, and chemotaxis protein, are supported by accessory enzymes such as the PstS in combination with ABC transporters, glycosyltransferases, and nitrogen fixation reductases that have an essential role in synergistic lignin degradation. The synergistic of Bacillus cereus’s enzyme combination is pivotal to degrade a complex aromatic compound such as lignin, especially under the state of carbon starvation. Bacillus cereus was shown to secrete various oxidative enzymes that assist in lignin modifications. The vastness of nature’s potential for biodelignification has not yet been fully explored, but it has become crucial for understanding the development of a future more environmentally friendly industry. Optimization of the bacterial enzyme is important to produce scale-up conditions for Bacillus cereus to be produced for the bioprocessing of pulp and in paper production.

Author Contributions

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

Funding

This work was supported and funded by the Ministry of Science and Technology Innovation (MOSTI), ScienceFund (S027). Thanks to the Universiti Tun Hussein Onn Malaysia and Universiti Teknologi Malaysia for the provided facilities as well as for the expertise.

Institutional Review Board Statement

Institutional Review Board approval was not required for this study, as it did not involve human or animal subjects.

Informed Consent Statement

Informed consent does not apply to this research, as it did not involve human subjects, identifiable personal data, or biological material.

Data Availability Statement

The data used in this study are not publicly available due to confidentiality. However, the data presented in this study are available upon reasonable request and appropriate permissions from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Engproc 84 00041 g001
Figure 1. The linear graph of the Bradford assay standard curve. The assay was performed using bovine serum albumin (BSA) as a standard. The straight line represents a linear fit of the measured data.
Figure 1. The linear graph of the Bradford assay standard curve. The assay was performed using bovine serum albumin (BSA) as a standard. The straight line represents a linear fit of the measured data.
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Engproc 84 00041 g002
Figure 2. SDS-PAGE gel electrophoresis image. Indicated are the protein marker (Lane 1), the crude enzyme (Lanes 2 and 3), and the ultrafiltered enzyme (Lanes 4 and 5). Lane 2 indicates presence of proteins with 11 different molecular weight size of proteins. The red labels show the chosen bands for protein sequencing.
Figure 2. SDS-PAGE gel electrophoresis image. Indicated are the protein marker (Lane 1), the crude enzyme (Lanes 2 and 3), and the ultrafiltered enzyme (Lanes 4 and 5). Lane 2 indicates presence of proteins with 11 different molecular weight size of proteins. The red labels show the chosen bands for protein sequencing.
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Table 1. List of working solutions for SDS-PAGE.
Table 1. List of working solutions for SDS-PAGE.
Type of SolutionQuantity/Molarity
Solution A30% (v/v) of acrylamide stock solution
Solution B (4× Separating Gel Buffer)2 M of Tris-HCl (pH 8.8), 10% (w/v) of SDS
Solution C (4× Stacking Gel Buffer)1 M of Tris-HCl (pH 6.8), 10% (w/v) of SDS
Electrophoresis Buffer (1 L)0.6 g of Tris Base, 14.4 g of Glycine, 1 g of SDS, and added up to 1 L of distilled water.
10% (w/v) Ammonium persulfate (APS) (1 mL)0.1 g of APS and added up to 1 mL of distilled water.
5× Sample Buffer (10 mL)0.6 mL of 1M Tris-HCl (pH 6.8), 5 mL of 50% (v/v) glycerol, 2 mL of 10% (w/v) SDS, 0.5 mL of β-mercaptoethanol, and 1 mL of 1% (w/v) Bromophenol Blue
Table 2. The absorbance and enzyme concentration values produced by Bacillus cereus on the EFB substrate.
Table 2. The absorbance and enzyme concentration values produced by Bacillus cereus on the EFB substrate.
Sample of ProteinAbsorbance
(595 nm)		Protein
Concentration (mg/mL)
Negative control0.0000.00
Blank0.2120.12
Crude protein0.9210.85
Ultrafiltered protein1.6681.62
Table 3. Types of identified enzymes and its functions.
Table 3. Types of identified enzymes and its functions.
No.Band SizesEnzymesFunction
1.95–97 kDa
▪
4-aminobutyrate aminotransferase (GABA)
GABA involved in degradation pathways producing succinic acid (lignin degradation product).
2.66–68 kDa
▪
Serine hydrolase
▪
Amidohydrolase family protein
▪
Chemotaxis protein
Hydrolases involved in cleavage aromatic compound degradation as well as playing a chemotaxis role in flagellar rotation for regulating the degradation.
3.43–45 kDa
▪
GMC family oxidoreductase
▪
Glycosyltransferase group 2 family
Oxidoreductases act as complimentary enzymes and responsible for the transferase activity involved in transferring glycosyl groups.
4.38–40 kDa
▪
Heme peroxidase
A group of peroxidases which acts on prime delignification.
5.25–27 kDa
▪
Phosphate-binding protein/PstS
▪
ABC transporter ATP-binding protein
A delignification product alongside the ABC transporter for binding proteins.
6.20 kDa
▪
Assimilatory nitrate reductase (NADH)
▪
Nitrite reductase
Nitrite promotes the growth and decreases the lignin content.
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MDPI and ACS Style

Syarifah, S.M.; Aripin, A.M.; Ishak, N.; Anindita, N.S.; Abdul-Wahab, M.F.; Kassim, A.S.M. Partial Purification of Bacillus cereus Enzyme Expression for Bio-Pulping of Lignin Degraders Isolated from Coptotermus curvignathus. Eng. Proc. 2025, 84, 41. https://doi.org/10.3390/engproc2025084041

AMA Style

Syarifah SM, Aripin AM, Ishak N, Anindita NS, Abdul-Wahab MF, Kassim ASM. Partial Purification of Bacillus cereus Enzyme Expression for Bio-Pulping of Lignin Degraders Isolated from Coptotermus curvignathus. Engineering Proceedings. 2025; 84(1):41. https://doi.org/10.3390/engproc2025084041

Chicago/Turabian Style

Syarifah, Sharfina Mutia, Ashuvila Mohd Aripin, Nadiah Ishak, Nosa Septiana Anindita, Mohd Firdaus Abdul-Wahab, and Angzzas Sari Mohd Kassim. 2025. "Partial Purification of Bacillus cereus Enzyme Expression for Bio-Pulping of Lignin Degraders Isolated from Coptotermus curvignathus" Engineering Proceedings 84, no. 1: 41. https://doi.org/10.3390/engproc2025084041

APA Style

Syarifah, S. M., Aripin, A. M., Ishak, N., Anindita, N. S., Abdul-Wahab, M. F., & Kassim, A. S. M. (2025). Partial Purification of Bacillus cereus Enzyme Expression for Bio-Pulping of Lignin Degraders Isolated from Coptotermus curvignathus. Engineering Proceedings, 84(1), 41. https://doi.org/10.3390/engproc2025084041

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