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Article

Production, Purification, and Characterization of a Cellulase from Paenibacillus elgii

by
Chien Thang Doan
1,
Thi Ngoc Tran
1,
Thi Phuong Pham
1,
Thi Thanh Thao Tran
1,
Ba Phong Truong
1,
Thi Tinh Nguyen
1,
The Manh Nguyen
1,
Thi Quynh Hoa Bui
1,
Anh Dzung Nguyen
2 and
San-Lang Wang
3,4,*
1
Faculty of Natural Science and Technology, Tay Nguyen University, Buon Ma Thuot 630000, Vietnam
2
Institute of Biotechnology and Environment, Tay Nguyen University, Buon Ma Thuot 630000, Vietnam
3
Department of Chemistry, Tamkang University, New Taipei City 25137, Taiwan
4
Life Science Development Center, Tamkang University, New Taipei City 25137, Taiwan
*
Author to whom correspondence should be addressed.
Polymers 2024, 16(14), 2037; https://doi.org/10.3390/polym16142037
Submission received: 11 June 2024 / Revised: 14 July 2024 / Accepted: 15 July 2024 / Published: 17 July 2024
(This article belongs to the Special Issue Production and Potential Uses of Biopolymers from Natural Sources and Agro-Industrial Waste II)
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Abstract

:
Cellulases are one of the most essential natural factors for cellulose degradation and, thus, have attracted significant interest for various applications. In this study, a cellulase from Paenibacillus elgii TKU051 was produced, purified, and characterized. The ideal fermentation conditions for cellulase productivity were 2% carboxymethyl cellulose (CMC) as the growth substrate, pH = 8, temperature of 31 °C, and 4 days of culturing. Accordingly, a 45 kDa cellulase (PeCel) was successfully purified in a single step using a High Q column with a recovery yield of 35% and purification of 42.2-fold. PeCel has an optimal activity at pH 6 and a temperature of 60 °C. The activity of cellulase was significantly inhibited by Cu2+ and enhanced by Mn2+. The PeCel-catalyzed products of the CMC hydrolysis were analyzed by high-performance liquid chromatography, which revealed chitobiose and chitotriose as the major products. Finally, the clarity of apple juice was enhanced when treated with PeCel.

1. Introduction

Lignocellulosic biomass primarily comprises cellulose, an essential structural element that gives plant tissues stiffness and strength [1]. Cellulose is an unbranched biopolymer comprising glucose units connected by β-1,4-glycosidic linkages [2]. Cellulose chains interact with each other through hydrogen bonds and van der Waals forces, forming highly ordered crystalline structures. Among allomorphic forms, cellulose I is the most commonly found in nature. The crystal structure and hydrogen-bonding arrangement of cellulose Iα and cellulose Iβ were determined earlier using atomic-resolution synchrotron and neutron diffraction data [3,4]. More recent spectroscopic studies revealed diverse molecular-level structures of cellulose [5,6]. The transformation of cellulose into fermentable sugars plays a crucial role in providing an abundant carbon supply for fermentation. Despite its abundance, cellulose breakdown remains a significant issue mainly because of its complicated and refractory nature [7]. Striving to achieve a ‘green industry’, it is evident that using enzymes in cellulose conversion is a more ecologically friendly alternative than using chemicals (acid and alkali) [8].
Cellulases, which break down cellulose by splitting the β-1,4-D-glycosidic linkages, are categorized into endoglucanases, exoglucanases, and β-glucosidases [9,10,11]. The synergistic action of these three types of cellulases is crucial to the effective breakdown of cellulose. Endoglucanases shorten cellulose chains by hydrolyzing glycosidic linkages in amorphous cellulose. They also act on cellodextrin, which is formed during the hydrolysis of cellulose. The two main types of exoglucanases include 1,4-β-D-glucan cellobiohydrolase I (CHB I), which acts on the reducing ends of cellulose, and 1,4-β-D-glucan cellobiohydrolase II (CBH II), which targets non-reducing ends, producing cellobiose. Then, the β-glucosidases convert this cellobiose into glucose [12]. Microscopic approaches have been used to understand the mechanisms behind cellulase breakdown in plant cell walls, facilitating the cost-effective transformation of cellulosic biomass [13]. Cellulases are widely used across industries, including paper, textiles, detergents, feed, food, and biofuels [12,14], and account for approximately 20% of the enzyme market [15].
Cellulase is produced by microbes, including fungi and bacteria [2,16], with fungi being the leading cellulase producers, for example, Aspergillus niger [17,18] and Trichoderma reesei [19]. However, bacteria have recently gained importance as potential cellulase producers [20]. Paenibacillus, a former member of the genus Bacillus [21], produces various kinds of enzymes [22,23,24,25]. While there is a plethora of published studies on cellulase production by Paenibacillus strains [20,21,22,23,24,25,26], reports concerning P. elgii are notably scarce. Tran et al. (2024) reported a cellulase-producing strain, P. elgii YSY-1.2 [27]. However, the purification and characterization of P. elgii’ cellulase is yet to be fully elucidated.
In this research, we investigated the conditions for cellulase biosynthesis by P. elgii TKU051 strain and attempted to purify the enzyme. In addition, the expense of enzyme purification is still crucial in bringing products to the market. Accordingly, some studies conduct one-step purification to reduce enzyme costs [28]. In this study, the cellulase was purified in a single step by ion chromatography from the crude enzyme, and its biochemical properties were analyzed. Finally, the potential of the obtained cellulase was assessed in terms of its applications in the clarification of apple juice.

2. Materials and Methods

2.1. Bacteria Strain

P. elgii TKU051 was isolated at Tamkang University (New Taipei, Taiwan) and described in an earlier report [29].

2.2. Cellulase Assay

The activity of cellulase form P. elgii (PeCel) was assessed in terms of the quantity of reducing sugar liberated from the carboxymethyl cellulose (CMC) hydrolysis. The reaction setup comprised 50 µL of enzyme and 150 µL of 1% CMC prepared in phosphate buffer (pH 6, 100 mM). The mixture was incubated at 60 °C for 30 min. Subsequently, 350 µL of 3,5-dinitrosalicylic acid (DNS) was introduced to stop the reaction, followed by incubation in a water bath at 100 °C for 10 min. The color intensity of the obtained mixture was measured at 540 nm. The negative control was identical to the method described, except the cellulase solution was substituted with a heat-denatured cellulase solution. The cellulase activity was quantified in terms of a unit, which is the enzyme quantity required to generate 1 μmol of reducing sugar as glucose per minute under standard assay conditions.

2.3. Screening Fermentation Conditions

The fermentation conditions were screened using the one factor at a time (OFAT) method. The factors included the type of carbon source (sugarcane bagasse powder (SCBP), rice bran powder (RBP), rice husk powder (RHP), corn cob powder (CCP), and carboxymethyl cellulose (CMC)), the amount of CMC (0.25–3%), pH (pH 6–9), temperature (28–40 °C), and incubation time (0–5 days). The basal medium consisted of 0.5% NH4NO3, 0.1% K2HPO4, and 0.05% MgSO4.

2.4. Enzyme Purification

On day 4 of the incubation, 1 L of enzyme solution was harvested from the P. elgii TKU051 culture medium by centrifugation (6000 rpm in 20 min). Cold ethanol (−20 °C) was introduced to the enzyme solution in a 3/1 ratio (v/v) to precipitate the cellulase. The sediment was then solubilized in acetate buffer (50 mM, pH = 5.8) and applied to a High Q column rinsed with the same buffer. Cellulase was eluted by a gradient of sodium chloride (0 to 1 M). The cellulolytic fractions were collected for further experiments. The purity and mass of the obtained enzyme were checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) [30]. The in-gel cellulolytic activity was examined by zymography using native-PAGE containing 0.05% CMC and 0.1% congo red as the staining reagent.

2.5. Enzyme Characterization

The optimum temperature for the activity of PeCel was identified by evaluating its activity across a temperature range of 37 °C to 90 °C. Additionally, the thermal stability of PeCel was determined by incubating it at temperatures from 4 °C to 90 °C for 1 h. To determine the optimal pH, PeCel was evaluated over a pH range of 2.0–10.6 using 50 mM buffers. Simultaneously, the pH stability was studied, and PeCel was incubated at a pH range from 2.0 to 10.6 for 1 h, followed by the estimation of the residual activity of PeCel.
To find out the effect of various chemical additives, PeCel was incubated with each chemical, including NaCl, KCl, ZnCl2, FeCl2, CaCl2, BaCl2, MgCl2, MnCl2, CuCl2, E-64, 2-mercaptoethanol (2-ME), ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline, and phenylmethanesulfonyl fluoride (PMSF), at a final concentration of 5 mM. Regarding surfactants (cetyltrimethylammonium bromide (CTAB), Triton X-100, sodium dodecyl sulfate (SDS), Tween 20, and Tween 40), a final concentration of 1% was used. This incubation took place at 4 °C for 30 min. The enzyme activity measured with no chemical additions served as the control.
The substrate specificity of PeCel was confirmed using CMC, cellulose powder, chitin, chitosan, xylan, alginate, dextran, gum arabic, pectin, β-1,3-glucan, starch, 2-nitrophenyl-β-D-galactopyranoside, 4-nitrophenyl N-acetyl-β-D-glucosaminide, 4-nitrophenyl-α-D-glucopyranoside, and 4-nitrophenyl-β-D-glucopyranoside. The activity was measured using 1% CMC as the control.

2.6. High-Performance Liquid Chromatography and Viscosity Analysis

CMC was used at a 1% concentration to study the hydrolysis mechanism of PeCel. The resulting hydrolysis products were examined at various time intervals, from 0 h to 4 h, using HPLC. The analysis settings included a KS-803 column, water as the mobile phase, and detection by a refractive index detector. The viscosity of the reaction was determined using a rotational viscometer (Brookfield, MA, USA).

2.7. Clarification of Apple Juice

The enzyme was added at different concentrations (0–10 U) to the apple juice, and the mixtures were incubated at 50 °C for 2 h. The clarity of the juice was determined by the transmittance (%T) at 660 nm against distilled water. The concentration of reducing sugar was determined using Miller’s method (1959) [31]. The α, α-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging activity was performed according to a previous report [32].

3. Results and Discussion

3.1. Screening the Cellulase Biosynthesis Conditions

P. elgii TKU051 was cultured on media containing different carbon sources, including SCBP, RBP, RHP, CCP, and CMC to explore the cellulase productivity. This strain showed poor cellulase productivity in the SCBP-, RBP-, RHP-, and CCP-containing media (Figure 1a). The maximum cellulase content was observed in the CMC-containing medium on the fourth day of the incubation (Figure 1a). This result agrees with previous reports that CMC is the best carbon source for cellulase synthesis [33,34,35]. CMC dissolves in water, providing easy access and utilization by bacterial cells, whereas the other substrates remain insoluble. Further assays showed that 2% CMC was most suitable for cellulase biosynthesis, and the maximum cellulase activity was observed on day 4 (Figure 1b). Similarly, the highest cellulase productivity by P. barcinonesis was noted when using 2% CMC [20].
The culture parameters of pH and temperature could alter the enzyme productivity [36]. Growth media containing 2% CMC at different pH levels (pH 6, pH 7, pH 8, and pH 9) were prepared. P. elgii TKU051 could produce cellulase in the media within the pH range of 6–8, with pH 8 yielding optimum cellulase production (Figure 1c). Previous studies have reported pH 7 as optimum for cellulase production [36,37,38]. Finally, an assessment of the impact of temperature on cellulase productivity revealed the highest cellulase productivity of P. elgii TKU051 at 31–34 °C (Figure 1d). This finding is almost identical to that reported by Asha et al. (2012) [20], Boondaeng et al. (2024) [38], and Sohail et al. (2016) [39], who observed that the optimal temperature for cellulase synthesis was 35 °C. At lower temperatures, substrate access by cells is restricted, leading to a decline in enzyme synthesis. At the same time, at higher temperatures, the enzyme activity declines due to thermal denaturation [38]. In this study, the most suitable incubation time for harvesting cellulase was on day 4 (3.601 ± 0.153 U/mL) (Figure 1d).

3.2. Enzyme Purification

The crude enzyme was obtained from the culture medium through ethanol precipitation and could be directly subjected to the purification step without the need for salt removal beforehand. The purification process was accomplished in a single step using a High Q column. As shown in Figure 2a, a high amount of protein was washed out before the gradient was applied, and during the elution stage, the protein peak and the cellulase activity peak coincided, suggesting that the purification process may be highly effective. As mentioned in Table 1, the recovery yield of purified cellulase (PeCel) was 35%, with 42.2-fold purification. These recovery yield and fold-purification values were better than the values reported by Dar et al. (2019) [40], Elsababty et al. (2022) [41], and Nisar et al. (2022) [10]. The specific activity of the obtained cellulase was 358.7 U/mg, surpassing that of P. barcinonensis (16.88 U/mg) [20] and comparable to that of B. licheniformis Z9 cellulase (356.5 U/mg) [41].
The activity fraction from the chromatography step revealed a unique protein band of 45 kDa on SDS-polyacrylamide gel (Figure 2b). Zymography analysis reveals a distinct band on the congo red theme, confirming the cellulolytic activity of the purified cellulase (Figure 2c). Therefore, a cellulase of 45 kDa molecular weight was successfully purified in a single step using a High Q column. As illustrated in Table 2, the MW of P. elgii TKU051 cellulase differs from those produced by other Paenibacillus and Bacillus strains. Only a cellulase of Bacillus tequilensis G9 (43 kDa) [40] closely resembles that obtained in this study. This could be a novel cellulase produced by the Paenibacillus genus.

3.3. Enzyme Characterization

The PeCel obtained in this study exhibited optimal activity at 60 °C and remained stable up to 40 °C (Figure 3a). After heating the purified enzyme at 50 °C for 1 h, approximately 80% of its activity was retained. The optimal temperature of 60 °C is in close agreement with that for cellulases produced by P. polymyxa GS01 [41], P. barcinonensis [20], P. campinasensis BL11 [46], and B. licheniformis ATCC 14580 [48]. Figure 3b shows that PeCel exhibits optimal activity at pH 6. The optimal pH range of cellulase from Paenibacillus seems to be in the range of 5.0–7.0 (Table 2). The activity of PeCel stabilized over the pH range of 4–10.
The substrate specificity of PeCel was explored by utilizing different kinds of polysaccharides as substrate, including CMC, cellulose powder, chitin, chitosan, xylan, alginate, dextran, gum arabic, pectin, β-1,3-glucan, and starch. The enzyme activity was the highest for CMC (100.00 ± 4.65%), followed by cellulose powder (29.47 ± 0.58%). This result is in agreement with those reported by Kim et al. (2023) [8] and Ko et al. (2010) [46]. PeCel could also use other polysaccharide substrates (chitin, chitosan, xylan, alginate, dextran, gum arabic, pectin, β-1,3-glucan, and starch), albeit to a lesser extent (Table 3). In addition, four types of chromogenic substrates (2-nitrophenyl-β-D-galactopyranoside, 4-nitrophenyl N-acetyl-β-D-glucosaminide, 4-nitrophenyl-α-D-glucopyranoside, and 4-nitrophenyl-β-D-glucopyranoside) were also used to assess the substrate specificity of PeCel. PeCel did not catalyze the cleavage activity on all four chromogenic substrates (Table 3), suggesting that it may lack β-galactosidase, β-glucosaminidase, α-glucosidase, and β-glucosidase activities. In fact, Lee et al. (2008) also indicated that the endo-type glucanase from B. amyloliquefaciens DL-3 could not hydrolyze 4-nitrophenyl-β-D-glucopyranoside [9].
Table 4 presents the effect of various chemicals on the activity of PeCel. Considering the effect of metal ions, the enzyme activity was significantly enhanced and reduced in the presence of Mn2+ (140.00 ± 0.27%) and Cu2+ (35.88 ± 2.64%), respectively. The impact of metal ions on cellulase activity may appear to differ among various bacterial strains. Indeed, in stark contrast, Cu2+ was reported to act as an activator while Mn2+ served as an inhibitor of cellulases from P. peoriae MK1 [4], P. xylanilyticus KJ-03 [38], and B. licheniformis Z9 [41]. Enzyme inhibitors (E-64, EDTA, PMSF, and 1,10-phenanthroline) and a reducing agent (2-ME) mildly affected the activity of PeCel with the residual activity in the range of 82.16–88.89%. According to recent studies, EDTA could inhibit more than 50% of cellulase activity from B. tequilensis SB125 [36] and B. subtilis [49]. Among the examined surfactants, the anionic one (SDS) exerted a significant inhibition effect on the activity of PeCel, resulting in a residual activity of 15.39%. In contrast, the effect of cationic surfactant (cetyltrimethylammonium bromide or CTAB) was relatively mild (86.13 ± 3.14%), while the non-ionic surfactants (Triton X-100 and Tween 20) slightly enhanced the activity (117.98 ± 5.19% and 104.34 ± 3.33, respectively). However, Tween 40 (a non-ionic surfactant) decreased the activity of PeCel slightly (90.38 ± 5.20%). Likewise, the stimulatory effect of Triton X-100 and the inhibition effect of SDS on the activity of cellulases have been reported by Malik and Javed (2024) [36].
The mode of action of PeCel was investigated by incubating this enzyme with the substrate CMC for periods ranging from 0 to 4 h. The viscosity profile of the hydrolysate is presented in Figure 4a. After 0.5 h of hydrolysis, PeCel could decrease the CMC viscosity by over 75%, suggesting its endo-acting function [50]. The HPLC profile of the hydrolysate is shown in Figure 4b. Initially, at 0 h, no product peaks were observed; however, from 0.5 h onward, two peaks emerged (8.05 min and 7.45 min), representing those of cellobiose and cellotriose (respectively). These results indicate that PeCel hydrolyzes CMC to primarily produce two products: cellotriose (C3) and predominantly cellobiose (C2). In contrast to exoglucanases, cellulose hydrolysis by endoglucanases results in a mixture of products [51]. Wu et al. (2018) reported that a GH 5 family endoglucanase from B. subtilis BS-5 could hydrolyze cellulose to produce cellobiose and cellotriose [50]. Likewise, another GH 5 family endoglucanase from Cytophaga hutchinsonii could also hydrolyze cellulose to produce cellobiose and cellotriose as the main products [52]. This result suggests that PeCel may be an endoglucanase.

3.4. Clarification of Apple Juice

The viscosity and turbidity of a juice are related to the presence of cellulose [53], which can be clarified by cellulase [14,54]. After cellulase treatment, the clarification values for apple juice (T%) improved and were directly related to the enzyme concentration. In other words, the clarity of apple juice increased by 34.54 ± 0.62%, 62.52 ± 1.54%, 68.19 ± 1.85%, and 71.33 ± 1.92% upon treatment with 0 U, 1 U, 5 U, and 10 U of PeCel, respectively. The amount of reducing sugar in apple juices treated with different enzyme concentrations (0 U, 1 U, 5 U, and 10 U) increased by 0.82 ± 0.08 mM, 1.02 ± 0.09 mM, 1.31 ± 0.07 mM, and 1.51 ± 0.10 mM, respectively.
Apples are abundant in antioxidants [55], and their fruit possesses significant free radical scavenging ability [56]. Thus, this study also investigated the effect of PeCel-mediated enzymatic clarification on the DPPH scavenging ability of apple juice. Table 5 shows that the difference between the DPPH radical scavenging activity values of apple juice treated with different enzyme concentrations was non-significant. Studies have reported that enzymatic treatment may have a detrimental influence on antioxidant activity [54,57]. The antioxidant ability of a fruit is linked to the ascorbic acid, phenolic, and flavonoid contents, and the clarifying process may cause the deposition of scavengers such as phenolics. In contrast, Chen et al. (2023) found that treatment by pectinase enhances the antioxidant ability of guava juices [58]. Pectinases degrade the cell wall, thereby releasing polyphenols and flavonoids, improving the antioxidant content and activity in the juice.

4. Conclusions

P. elgii TKU051 could synthesize the cellulase PeCel with the highest productivity of 3.601 U/mL in a medium containing 2% CMC as the sole carbon source. This cellulase, isolated and purified from the culture medium in a single step, exhibited a molecular weight of 45 kDa and was biochemically characterized. PeCel exhibited optimal activity at pH 6.0 and 60 °C and was considered an endoglucanase, as it efficiently catalyzed the conversion of CMC into cellobiose and cellotriose as the main products. Moreover, PeCel also significantly improved the clarification of apple juice, indicating its high potential for usage in the fruit juice industry.

Author Contributions

Conceptualization, methodology, C.T.D., T.N.T. and S.-L.W.; software, validation, formal analysis, investigation, resources, data curation, C.T.D., T.N.T., T.P.P., T.T.T.T., B.P.T., T.T.N., T.M.N., T.Q.H.B., A.D.N. and S.-L.W.; writing—original draft preparation; writing—review and editing; visualization C.T.D., S.-L.W. and T.N.T.; supervision, project administration, funding acquisition, S.-L.W. and C.T.D. 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) and by a grant from Tay Nguyen University, Vietnam (T2023-42CBTÐ).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Jakob, M.; Mahendran, A.R.; Gindl-Altmutter, W.; Bliem, P.; Konnerth, J.; Müller, U.; Veigel, S. The strength and stiffness of oriented wood and cellulose-fibre materials: A review. Prog. Mater. Sci. 2022, 125, 100916. [Google Scholar] [CrossRef]
  2. Li, H.; Zhang, M.; Zhang, Y.; Xu, X.; Zhao, Y.; Jiang, X.; Zhang, R.; Gui, Z. Characterization of cellulose-degrading bacteria isolated from silkworm excrement and optimization of its cellulase production. Polymers 2023, 15, 4142. [Google Scholar] [CrossRef] [PubMed]
  3. Nishiyama, Y.; Langan, P.; Chanzy, H. Crystal structure and hydrogen-bonding system in cellulose iβ from synchrotron x-ray and neutron fiber diffraction. J. Am. Chem. Soc. 2002, 124, 9074–9082. [Google Scholar] [CrossRef] [PubMed]
  4. Nishiyama, Y.; Sugiyama, J.; Chanzy, H.; Langan, P. Crystal Structure and Hydrogen Bonding System in Cellulose Iα from Synchrotron X-ray and Neutron Fiber Diffraction. J. Am. Chem. Soc. 2003, 125, 14300–14306. [Google Scholar] [CrossRef] [PubMed]
  5. Deligey, F.; Frank, M.A.; Cho, S.H.; Kirui, A.; Mentink-Vigier, F.; Swulius, M.T.; Nixon, B.T.; Wang, T. Structure of in vitro-synthesized cellulose fibrils viewed by cryo-electron tomography and 13C natural-abundance dynamic nuclear polarization solid-state NMR. Biomacromolecules 2022, 23, 2290–2301. [Google Scholar] [CrossRef]
  6. Berruyer, P.; Gericke, M.; Moutzouri, P.; Jakobi, D.; Bardet, M.; Karlson, L.; Schantz, S.; Heinze, T.; Emsley, L. Advanced characterization of regioselectively substituted methylcellulose model compounds by DNP enhanced solid-state NMR spectroscopy. Carbohydr. Polym. 2021, 262, 117944. [Google Scholar] [CrossRef] [PubMed]
  7. Blasi, A.; Verardi, A.; Lopresto, C.G.; Siciliano, S.; Sangiorgio, P. Lignocellulosic agricultural waste valorization to obtain valuable products: An overview. Recycling 2023, 8, 61. [Google Scholar] [CrossRef]
  8. Kim, S.J.; Shin, K.C.; Kim, D.W.; Kim, Y.S.; Park, C.S. Cloning and characterization of cellulase from Paenibacillus peoriae MK1 isolated from soil. Fermentation 2023, 9, 873. [Google Scholar] [CrossRef]
  9. Lee, Y.J.; Kim, B.K.; Lee, B.H.; Jo, K.I.; Lee, N.K.; Chung, C.H.; Lee, Y.C.; Lee, J.W. Purification and characterization of cellulase produced by Bacillus amyoliquefaciens DL-3 utilizing rice hull. Bioresour. Technol. 2008, 99, 378–386. [Google Scholar] [CrossRef]
  10. Nisar, K.; Abdullah, R.; Kaleem, A.; Lqtedar, M.; Aftab, M.; Saleem, F. Purification, characterization and thermodynamic analysis of cellulases produced from Thermomyces dupontii and its industrial applications. Saudi J. Biol. Sci. 2022, 29, 103483. [Google Scholar] [CrossRef]
  11. Ilić, N.; Milić, M.; Beluhan, S.; Dimitrijević-Branković, S. Cellulases: From lignocellulosic biomass to improved production. Energies 2023, 16, 3598. [Google Scholar] [CrossRef]
  12. Sutaoney, P.; Rai, S.N.; Sinha, S.; Choudhary, R.; Gupta, A.K.; Singh, S.K.; Banerjee, P. Current perspective in research and industrial applications of microbial cellulases. Int. J. Biol. Macromol. 2024, 264, 130639. [Google Scholar] [CrossRef] [PubMed]
  13. Ding, S.Y.; Liu, Y.S.; Zeng, Y.N.; Himmel, M.E.; Baker, J.O.; Bayer, E.A. How does plant cell wall nanoscale architecture correlate with enzymatic digestibility? Science 2012, 338, 1055–1060. [Google Scholar] [CrossRef] [PubMed]
  14. Campos, A.O.; Asevedo, E.A.; Souza Filho, P.F.; Santos, E.S.d. Extraction of cellulases produced through solid-state fermentation by Trichoderma reesei CCT-2768 using green coconut fibers pretreated by steam explosion combined with alkali. Biomass 2024, 4, 92–106. [Google Scholar] [CrossRef]
  15. Singh, A.; Bajar, S.; Devi, A.; Pant, D. An overview on the recent developments in fungal cellulase production and their industrial applications. Bioresour. Technol. Rep. 2021, 14, 100652. [Google Scholar] [CrossRef]
  16. Benatti, A.L.T.; Polizeli, M.d.L.T.d.M. Lignocellulolytic biocatalysts: The main players involved in multiple biotechnological processes for biomass valorization. Microorganisms 2023, 11, 162. [Google Scholar] [CrossRef]
  17. Zhang, L.; Cao, G.; Liu, H.; Wu, Z.; Gong, D.; Ru, X.; Gong, X.; Pi, Q.; Yang, Q. Application of Aspergillus niger in practical biotechnology of industrial recovery of potato starch by-products and its flocculation characteristics. Microorganisms 2022, 10, 1847. [Google Scholar] [CrossRef]
  18. Santos, G.B.; de Sousa Francisco Filho, Á.; Rêgo da Silva Rodrigues, J.; Rodrigues de Souza, R. Cellulase production by Aspergillus niger using urban lignocellulosic waste as substrate: Evaluation of different cultivation strategies. J. Environ. Manag. 2022, 305, 114431. [Google Scholar] [CrossRef]
  19. Lee, D.S.; Song, Y.; Lee, Y.G.; Bae, H.J. Comparative evaluation of adsorption of major enzymes in a cellulase cocktail obtained from Trichoderma reesei onto different types of lignin. Polymers 2022, 14, 167. [Google Scholar] [CrossRef]
  20. Asha, B.M.; Revathi, M.; Yadav, A.; Sakthivel, N. Purification and characterization of a thermophilic cellulase from a novel cellulolytic strain, Paenibacillus barcinonensis. J. Microbiol. Biotechnol. 2012, 22, 1501–1509. [Google Scholar] [CrossRef]
  21. Doan, C.T.; Tran, T.N.; Wang, C.L.; Wang, S.L. Microbial conversion of shrimp heads to proteases and chitin as an effective dye adsorbent. Polymers 2020, 12, 2228. [Google Scholar] [CrossRef]
  22. Doan, C.T.; Tran, T.N.; Wang, S.L. Production of thermophilic chitinase by Paenibacillus sp. TKU052 by bioprocessing of chitinous fishery wastes and its application in N-acetyl-D-glucosamine production. Polymers 2021, 13, 3048. [Google Scholar] [CrossRef]
  23. 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] [PubMed]
  24. Ugwuoji, E.T.; Nwagu, T.N.; Ezeogu, L.I. Detergent-stable amylase production by Paenibacillus lactis strain OPSA3 isolated from soil; optimization by response surface methodology. Biotechnol. Rep. 2023, 39, e00808. [Google Scholar] [CrossRef]
  25. Lee, T.H.; Lee, Y.E. Cloning, sequencing and expression of the gene encoding a thermostable β -Xylosidase from Paenibacillus sp. DG-22. J. Life Sci. 2007, 17, 1197–1203. [Google Scholar] [CrossRef]
  26. Mostafa, Y.S.; Alamri, S.A.; Hashem, M.; Nafady, N.A.; Abo-Elyousr, K.A.M.; Mohamed, Z.A. Thermostable cellulase biosynthesis from Paenibacillus alvei and its utilization in lactic acid production by simultaneous saccharification and fermentation. Open Life Sci. 2020, 15, 185–197. [Google Scholar] [CrossRef] [PubMed]
  27. Tran, M.D.; Pham, T.P.; Vu, B.T.; Le, N.T.N. Data on annotation and analysis of genome sequence of Paenibacillus elgii YSY-1.2, a promising chitinase-producing, plant-growth-promoting, and biocontrol agent. Data Brief 2024, 54, 110285. [Google Scholar] [CrossRef] [PubMed]
  28. Sanghi, A.; Garg, N.; Gupta, V.K.; Mittal, A.; Kuhad, R.C. One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash. Braz. J. Microbiol. 2010, 41, 467–476. [Google Scholar] [CrossRef] [PubMed]
  29. Lee, D.H.; Doan, C.T.; Tran, T.N.; Nguyen, V.B.; Nguyen, A.D.; Wang, C.L.; Wang, S.L. Proteases production and chitin preparation from the liquid fermentation of chitinous fishery by-products by Paenibacillus elgii. Mar. Drugs 2021, 19, 477. [Google Scholar] [CrossRef]
  30. 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]
  31. Miller, G.L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 1959, 31, 426–428. [Google Scholar] [CrossRef]
  32. Tran, T.N.; Doan, C.T.; Nguyen, V.B.; Nguyen, A.D.; Wang, S.L. Antioxidant and anti-diabetes potential of water-soluble chitosan–glucose derivatives produced by Maillard reaction. Polymers 2019, 11, 1714. [Google Scholar] [CrossRef] [PubMed]
  33. Islam, F.; Roy, N. Screening, purification and characterization of cellulase from cellulase producing bacteria in molasses. BMC Res. Notes 2018, 11, 445. [Google Scholar] [CrossRef]
  34. Datsomor, O.; Yan, Q.; Opoku-Mensah, L.; Zhao, G.; Miao, L. Effect of different inducer sources on cellulase enzyme production by white-rot basidiomycetes Pleurotus ostreatus and Phanerochaete chrysosporium under submerged fermentation. Fermentation 2022, 8, 561. [Google Scholar] [CrossRef]
  35. Lugani, Y.; Singla, R.; Sooch, B.S. Optimization of cellulase production from newly isolated Bacillus sp. Y3. J. Bioproc. Biotech. 2015, 5, 1000264. [Google Scholar] [CrossRef]
  36. Malik, W.A.; Javed, S. Enhancement of cellulase production by cellulolytic bacteria SB125 in submerged fermentation medium and biochemical characterization of the enzyme. Int. J. Biol. Macromol. 2024, 263, 130415. [Google Scholar] [CrossRef] [PubMed]
  37. Maravi, P.; Kumar, A. Optimization and statistical modeling of microbial cellulase production using submerged culture. J. Appl. Biol. Biotechnol. 2021, 9, 142–152. [Google Scholar]
  38. Boondaeng, A.; Keabpimai, J.; Trakunjae, C.; Vaithanomsat, P.; Srichola, P.; Niyomvong, N. Cellulase production under solid-state fermentation by Aspergillus sp. IN5: Parameter optimization and application. Heliyon 2024, 10, e26601. [Google Scholar] [CrossRef] [PubMed]
  39. Sohail, M.; Ahmad, A.; Khan, S.A. Production of cellulase from Aspergillus terreus MS105 on crude and commercially purified substrates. 3 Biotech 2016, 6, 103. [Google Scholar] [CrossRef]
  40. Dar, M.A.; Pawar, K.D.; Rajput, B.P.; Rahi, P.; Pandit, R.S. Purification of a cellulase from cellulolytic gut bacterium, Bacillus tequilensis G9 and its evaluation for valorization of agro-wastes into added value byproducts. Biocatal. Agric. Biotechnol. 2019, 20, 101219. [Google Scholar] [CrossRef]
  41. Elsababty, Z.E.; Abdel-Aziz, S.H.; Ibrahim, A.M.; Guirgis, A.A.; Dawwam, G.E. Purification, biochemical characterization, and molecular cloning of cellulase from Bacillus licheniformis strain Z9 isolated from soil. J. Genet. Eng. Biotechnol. 2022, 20, 34. [Google Scholar] [CrossRef] [PubMed]
  42. Liang, Y.L.; Zhang, Z.; Wu, M.; Wu, Y.; Feng, J.X. Isolation, screening, and identification of cellulolytic bacteria from natural reserves in the subtropical region of China and optimization of cellulase production by Paenibacillus terrae ME27-1. BioMed Res. Int. 2014, 2014, 512497. [Google Scholar] [CrossRef] [PubMed]
  43. Cho, K.M.; Hong, S.J.; Math, R.K.; Islam, S.M.; Kim, J.O.; Lee, Y.H.; Kim, H.; Yun, H.D. Cloning of two cellulase genes from endophytic Paenibacillus polymyxa GS01 and comparison with cel 44C-man 26A. J. Basic Microbiol. 2008, 48, 464–472. [Google Scholar] [CrossRef] [PubMed]
  44. Park, I.H.; Chang, J.; Lee, Y.S.; Fang, S.J.; Choi, Y.L. Gene cloning of endoglucanase Cel5A from cellulose-degrading Paenibacillus xylanilyticus KJ-03 and purification and characterization of the recombinant enzyme. Protein J. 2012, 31, 238–245. [Google Scholar] [CrossRef]
  45. Dhar, H.; Kasana, R.C.; Dutt, S.; Gulati, A. Cloning and expression of low temperature active endoglucanase EG5C from Paenibacillus sp. IHB B 3084. Int. J. Biol. Macromol. 2015, 81, 259–566. [Google Scholar] [CrossRef] [PubMed]
  46. Ko, C.H.; Tsai, C.H.; Lin, P.H.; Chang, K.C.; Tu, J.; Wang, Y.N.; Yang, C.Y. Characterization and pulp refining activity of a Paenibacillus campinasensis cellulase expressed in Escherichia coli. Bioresour. Technol. 2010, 101, 7882–7888. [Google Scholar] [CrossRef] [PubMed]
  47. Pham, V.H.T.; Kim, J.; Shim, J.; Chang, S.; Chung, W. Coconut mesocarp-based lignocellulosic waste as a substrate for cellulase production from high promising multienzyme-producing Bacillus amyloliquefaciens FW2 without pretreatments. Microorganisms 2022, 10, 327. [Google Scholar] [CrossRef]
  48. Kim, D.; Ku, S. Bacillus cellulase molecular cloning, expression, and surface display on the outer membrane of Escherichia coli. Molecules 2018, 23, 503. [Google Scholar] [CrossRef]
  49. Malik, W.A.; Javed, S. Biochemical characterization of cellulase from Bacillus subtilis strain and its effect on digestibility and structural modifications of lignocellulose rich biomass. Front. Bioeng. Biotechnol. 2021, 9, 800265. [Google Scholar] [CrossRef]
  50. Wu, B.; Guddat, L.W.; Chang, S.; He, B.; Schenk, G. Processivity and enzymatic mechanism of a multifunctional family 5 endoglucanase from Bacillus subtilis BS-5 with potential applications in the saccharification of cellulosic substrates. Biotechnol. Biofuels 2018, 11, 20. [Google Scholar] [CrossRef]
  51. Wu, S.; Wu, S. Processivity and the mechanisms of processive endoglucanases. Appl. Biochem. Biotechnol. 2020, 190, 448–463. [Google Scholar] [CrossRef] [PubMed]
  52. Zhang, C.; Wang, Y.; Li, Z.; Zhou, X.; Zhang, W.; Zhao, Y.; Lu, X. Characterization of a multi-function processive endoglucanase CHU_2103 from Cytophaga hutchinsonii. Appl. Microbiol. Biotechnol. 2014, 98, 6679–6687. [Google Scholar] [CrossRef] [PubMed]
  53. Li, C.; Kumar, A.; Luo, X.; Shi, H.; Liu, Z.; Wu, G. Highly alkali-stable and cellulase-free xylanases from Fusarium sp. 21 and their application in clarification of orange juice. Int. J. Biol. Macromol. 2020, 155, 572–580. [Google Scholar] [CrossRef] [PubMed]
  54. Ozyilmaz, G.; Gunay, E. Clarification of apple, grape and pear juices by co-immobilized amylase, pectinase and cellulase. Food Chem. 2023, 398, 133900. [Google Scholar] [CrossRef] [PubMed]
  55. Aleksandra, D.C.; Tomasz, T.; Tadeusz, T. Antioxidant activity of apples-an impact of maturity stage and fruit part. Acta Sci. Pol. Technol. Aliment. 2011, 10, 443–454. [Google Scholar]
  56. Asale, Y.; Dessalegn, E.; Assefa, D.; Abdisa, M. Phytochemicals and antioxidant activity of different apple cultivars grown in South Ethiopia: Case of the wolayta zone. Int. J. Food Prop. 2020, 24, 354–363. [Google Scholar] [CrossRef]
  57. Hosseini, S.S.; Khodaiyan, F.; Mousavi, S.M.; Azimi, S.Z. Clarification of the pomegranate juice in a bioreactor packed by pectinase enzymes immobilized on the glass bead activated with polyaldehyde polysaccharides. LWT-Food Sci. Technol. 2021, 137, 110500. [Google Scholar] [CrossRef]
  58. Chen, X.; Xu, Y.; Wu, J.; Yu, Y.; Zou, B.; Li, L. Effects of pectinase pre-treatment on the physicochemical properties, bioactive compounds, and volatile components of juices from different cultivars of guava. Foods 2023, 12, 330. [Google Scholar] [CrossRef]
Polymers 16 02037 g001
Figure 1. Effect of the carbon source (a), CMC ratio (b), pH (c), and temperature (d) on cellulase productivity by P. elgii TKU051. Data are the mean of three replications, and the error bars represent the SD (standard deviation).
Figure 1. Effect of the carbon source (a), CMC ratio (b), pH (c), and temperature (d) on cellulase productivity by P. elgii TKU051. Data are the mean of three replications, and the error bars represent the SD (standard deviation).
Polymers 16 02037 g001
Polymers 16 02037 g002aPolymers 16 02037 g002b
Figure 2. High Q column chromatography profile of the crude cellulase from Paenibacillus elgii TKU051. (a) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (b) and zymography (c) profiles of PeCel.
Figure 2. High Q column chromatography profile of the crude cellulase from Paenibacillus elgii TKU051. (a) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (b) and zymography (c) profiles of PeCel.
Polymers 16 02037 g002aPolymers 16 02037 g002b
Polymers 16 02037 g003
Figure 3. The impact of temperature (a) and pH (b) on the activity of Paenibacillus elgii TKU051 cellulase (PeCel).
Figure 3. The impact of temperature (a) and pH (b) on the activity of Paenibacillus elgii TKU051 cellulase (PeCel).
Polymers 16 02037 g003
Polymers 16 02037 g004
Figure 4. Viscosity profile (a) and high-performance liquid chromatography profile (b) of carboxymethyl cellulose (CMC) hydrolysates catalyzed by Paenibacillus elgii TKU051 cellulase (PeCel). C2, cellobiose; C3, cellotriose.
Figure 4. Viscosity profile (a) and high-performance liquid chromatography profile (b) of carboxymethyl cellulose (CMC) hydrolysates catalyzed by Paenibacillus elgii TKU051 cellulase (PeCel). C2, cellobiose; C3, cellotriose.
Polymers 16 02037 g004
Table 1. A summary of the purification of cellulase produced by Paenibacillus elgii TKU051.
Table 1. A summary of the purification of cellulase produced by Paenibacillus elgii TKU051.
StepTotal Protein
(mg)		Total Activity
(U)		Specific Activity
(U/mg)		Recovery
(%)		Purification
(fold)
Culture supernatant519.54419.38.51001.0
EtOH precipitation262.63652.113.9831.6
High Q column4.31554.5358.73542.2
Table 2. Properties of cellulases from different Paenibacillus and Bacillus strains.
Table 2. Properties of cellulases from different Paenibacillus and Bacillus strains.
Cellulase-Producing StrainMW
(kDa)		Opt. pHOpt. Temp.
(°C)		Metal IonRef.
InhibitorActivator
Paenibacillus elgii TKU051456.060Cu2+Mn2+This study
Paenibacillus peoriae MK1655.040Mn2+Cu2+, Ba2+, Mg2+, and Fe2+[8]
Paenibacillus terrae ME27-1 5.550 [42]
Paenibacillus polymyxa GS01337.050 [43]
606.060
Paenibacillus xylanilyticus KJ-03645.040Fe3+, Mn2+, Zn2+, and Hg2+Ca2+ and Cu2+[44]
Paenibacillus sp. IHB B 308463.5 40 [45]
Paenibacillus barcinonensis58.66.065Hg2+Mg2+, Fe2+, Mn2+, and Zn2+[20]
Paenibacillus campinasensis BL11387.060Hg2+, Cu2+, and Zn2+Mn2+ and Co2+[46]
Paenibacillus sp.677.040 [33]
Bacillus licheniformis Z954.47.430Mg2+ and Na+Fe3+, Cu2+, and Ca2+[41]
Bacillus amyloliquefaciens FW255 Fe2+ and Zn2+Mg2+ and Ca2+[47]
B. licheniformis ATCC 1458061.77.060 [48]
B. tequilensis G9435.040Pb2+Zn2+, Ca2+, and Co2+[40]
Table 3. Substrate specificity of the obtained cellulase (PeCel).
Table 3. Substrate specificity of the obtained cellulase (PeCel).
SubstrateRelative Activity
CMC *100.00 ± 4.65
Cellulose powder29.47 ± 0.58
Chitin12.44 ± 0.50
Chitosan23.96 ± 3.06
Xylan20.26 ± 0.75
Alginate19.44 ± 1.19
Dextran19.82 ± 1.05
Gum arabic17.04 ± 0.48
Pectin17.83 ± 1.60
β-1,3-Glucan11.51 ± 1.02
Starch5.69 ± 0.56
2-Nitrophenyl β-D-galactopyranosideN.D.
4-Nitrophenyl N-acetyl-β-D-glucosaminideN.D.
4-Nitrophenyl-β-D-glucopyranosideN.D.
4-Nitrophenyl-α-D-glucopyranosideN.D.
*, control; N.D., not detected.
Table 4. Effect of various chemicals on the obtained cellulase (PeCel).
Table 4. Effect of various chemicals on the obtained cellulase (PeCel).
ChemicalRelative Activity
Control100 ± 3.06
Na+97.57 ± 8.22
K+96.81 ± 2.45
Zn2+91.82 ± 6.87
Fe2+90.23 ± 2.05
Ca2+97.44 ± 5.18
Ba2+101.46 ± 6.25
Mg2+101.18 ± 3.39
Mn2+140.00 ± 0.27
Cu2+35.88 ± 2.64
E6482.16 ± 5.30
2-ME88.89 ± 7.04
EDTA86.36 ± 2.31
1,10-Phenanthroline82.68 ± 5.22
PMSF88.56 ± 8.24
SDS15.39 ±5.50
CTAB86.13 ± 3.14
Triton X-100117.98 ± 5.19
Tween 20104.34 ± 3.33
Tween 4090.38 ± 5.20
Table 5. Effect of PeCel on apple juice properties.
Table 5. Effect of PeCel on apple juice properties.
Enzyme Concentration
0 U1 U5 U10 U
Clarification (T%)34.54 ± 0.6262.52 ± 1.5468.19 ± 1.8571.33 ± 1.92
Reducing sugar (mM)0.82 ± 0.081.02 ± 0.091.31 ± 0.071.51 ± 0.10
DPPH radical scavenging activity (%)72.78 ± 1.8371.57 ± 1.4570.92 ± 1.3070.02 ± 1.86
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Doan, C.T.; Tran, T.N.; Pham, T.P.; Tran, T.T.T.; Truong, B.P.; Nguyen, T.T.; Nguyen, T.M.; Bui, T.Q.H.; Nguyen, A.D.; Wang, S.-L. Production, Purification, and Characterization of a Cellulase from Paenibacillus elgii. Polymers 2024, 16, 2037. https://doi.org/10.3390/polym16142037

AMA Style

Doan CT, Tran TN, Pham TP, Tran TTT, Truong BP, Nguyen TT, Nguyen TM, Bui TQH, Nguyen AD, Wang S-L. Production, Purification, and Characterization of a Cellulase from Paenibacillus elgii. Polymers. 2024; 16(14):2037. https://doi.org/10.3390/polym16142037

Chicago/Turabian Style

Doan, Chien Thang, Thi Ngoc Tran, Thi Phuong Pham, Thi Thanh Thao Tran, Ba Phong Truong, Thi Tinh Nguyen, The Manh Nguyen, Thi Quynh Hoa Bui, Anh Dzung Nguyen, and San-Lang Wang. 2024. "Production, Purification, and Characterization of a Cellulase from Paenibacillus elgii" Polymers 16, no. 14: 2037. https://doi.org/10.3390/polym16142037

APA Style

Doan, C. T., Tran, T. N., Pham, T. P., Tran, T. T. T., Truong, B. P., Nguyen, T. T., Nguyen, T. M., Bui, T. Q. H., Nguyen, A. D., & Wang, S. -L. (2024). Production, Purification, and Characterization of a Cellulase from Paenibacillus elgii. Polymers, 16(14), 2037. https://doi.org/10.3390/polym16142037

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