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Characterization and application of dextranase produced by Chaetomium globosum mutant through combined application of atmospheric and room temperature plasma and ethyl methyl sulfone
Process Biochemistry 85 (2019) 116–124
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Characterization and application of dextranase produced by Chaetomium globosum mutant through combined application of atmospheric and room temperature plasma and ethyl methyl sulfone Liu Yanga, Nandi Zhoub, Yaping Tiana, a b
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The Key Laboratsory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Chaetomium globosum Dextranase Atmospheric and room temperature plasma treatment-ethyl methyl sulfone compound mutagenesis DNA structure Dental caries
Dextranase has extensive applications in the food, pharmaceutical, and chemical industries. However, enzyme production by the wild-type strain is generally low and few studies have conducted structural analysis of dextranase. This study describes an efficient and stable mutagenesis method for producing dextranase and the relationship between the enzyme structure and enzymatic properties before and after mutagenesis. Wild-type Chaetomium globosum was mutated by combined atmospheric and room temperature plasma treatment and ethyl methyl sulfone application. Mutant strains were screened for optimal dextranase production ability and genetic stability. The maximum yield of dextranase reached 824.73 U/mL. Dextranase produced by the mutant strain displayed the same optimum pH and temperature values of 5.5 and 60℃, respectively, as dextranase produced by the wild-type strain. However, dextranase from the mutant strain showed greater heat stability at 70℃. Furthermore, the three-dimensional structure of the enzyme changed slightly after mutagenesis, with the amino acid residue at position 578 changing from His to Leu. This change in the three-dimensional structure affected the properties of dextranase. Finally, dextranase significantly inhibited the growth of Streptococcus mutans. The results indicated dextranase from Chaetomium globosum would give potential implications in the medical, prevent dental caries and food industries.
1. Introduction Dextranase (α-1,6-d-glucan-6-glucanohydrolase; EC 3.2.1.11) catalyzes the degradation of dextran (a polymer of glucose) within or at the end of the chain into low-molecular-weight fractions. It has important applications in various fields, such as reducing the viscosity of sugarcane juice in the sugar industry [1], preparing low-molecular-weight medicinal dextran [2], and inhibiting or removing dental plaque [3]. Continuous developments in the food and pharmaceutical industries have led to an increase in the demand for dextran. As dextranase has been isolated from different mold strains, Catenovulum, yeast, Streptococcus, and Arthrobacter [4–6], the enzymatic biocatalysis of dextran should be thoroughly evaluated and optimized. However, the reported enzyme activity of dextranase from wild-type strains is relatively low. Dextranase from Talaromyces pinophilus shows an activity of 4.60 U/mL, with an optimum temperature of 45 °C and optimum pH of 6.0 [7], whereas dextranase from Aspergillus allahabadii X26 has an activity of 110 U/mL, with an optimum pH and temperature of 5.8 and 55 °C,
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respectively [4], Sufiate et al. [8] isolated a novel dextranase from Pochonia chlamydosporia, which showed a specific activity of 358.63 U/ mg and high activity at 50 °C and pH 5.0. Improving productivity is a constant requirement in the fermentation industry, and efficient methods for creating mutants are necessary to improve dextranase yield and enzymatic characterization. Traditional methods for inducing mutagenesis, such as physical or chemical methods, combined with rational screening processes, are cost-effective methods for adapting strains or increasing the yields of valuable metabolites [9]. Atmospheric and room temperature plasma treatment (ARTP) uses a new type of atmospheric pressure: a nonequilibrium discharge plasma source [10]. Under normal atmospheric pressure and at a temperature of 25–40 °C, excited nitrogen, oxygen, and nitrogen atoms and the OH radical are highly active particles. The main mechanism of action of this technique involves altering the structure and permeability of microbial cell walls and membranes as well as DNA damage caused by active ions in the plasma, which significantly alter microbial gene sequences and metabolic networks.
Corresponding author. E-mail addresses: [email protected] (L. Yang), [email protected] (N. Zhou), [email protected] (Y. Tian).
https://doi.org/10.1016/j.procbio.2019.06.026 Received 20 February 2019; Received in revised form 29 June 2019; Accepted 29 June 2019 Available online 01 July 2019 1359-5113/ © 2019 Published by Elsevier Ltd.
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centrifuged for 10 min at 5000 ×g. The final concentration of the spore suspension was adjusted to 106 CFU/mL with phosphate buffer (pH 7.0). Mutagenic treatment was performed as described previously by Christoph et al. [10], with slight modifications. On a clean bench, spore suspensions and 10% glycerol were evenly mixed to a final glycerol concentration of 5%. Approximately 10 μL of the mixture was then spread on a 1-cm plate and later exposed to the ARTP system’s nozzle exit. The working parameters for Pin, Pout, and distance (D) were set to 100 W, 20 W, and 2 mm, respectively. The flow rate of helium gas was 10 slm. High-purity helium plasma mutagenesis treatment was performed for 0 (control group), 30, 60, 90, 120, 150, 180, and 210 s. After treatment, the plates were placed in a new sterile tube containing 300 μL of sterile water and spread on blue dextran screening culture medium. After 3–5 days of cultivation at 28 °C, the ability of the strains to hydrolyze blue dextran was assessed. Colony numbers were calculated on a solid medium plate using the colony-forming unit method. Mutation and fatality rates were calculated based on the different halo sizes.
These changes eventually lead to microbial genetic mutations [11]. ARTP has been successfully employed in 40 different types of microorganisms including bacteria, fungi, yeast, and microalgae [12]. In chemical mutagenesis, ethyl methyl sulfone (EMS) is a widely used alkylating agent shown to be an effective mutagen. At present, EMS mutagenesis is widely used in microbial applications and to select improved crop varieties [13,14]. A single mutagenesis method often induces resistance to the chemical or lethal agent used and the resulting mutant strain may be unstable. Therefore, two or more methods are often combined to induce mutagenesis, a process known as composite inducement. In recent years, as the incidence of dental caries has increased, oral health has received more attention. Dental caries may cause diseases, such as periodontitis and odontitis, and are considered by the World Health Organization to be the third most concerning disease after cardiovascular diseases and cancers. The oral cavity provides an environment suitable for the growth of resident facultative anaerobic bacteria (Streptococcus), which cause dental caries [15]. Oral pathogens may assist in the breakdown of food after the generation of sucrose from α1,6-glucoside and α-1,3-glucoside. Additionally, chewed food residues, saliva, and inorganic salts adhere to teeth and form dental plaque. Several types of oral bacteria secrete acids that destroy dentin and result in caries and periodontal disease [16]. Dextranase can degrade glucan, which may help prevent the formation of dental plaque and thus has important potential applications for preventing and treating dental caries [17,18]. In this study, ARTP coupled with EMS application was first used to produce dextran anhydrase through multiple rounds of mutagenesis fermentation. The correlation between the transparent circle size of blue dextran tablets and enzyme activity was evaluated to obtain a mutant strain producing high levels of dextranase. Studies of the properties of dextranase and its applications in the prevention and treatment of dental caries provides a foundation for future industrial applications.
2.3.2. EMS mutagenesis Various concentrations of EMS were prepared by dilution with phosphoric acid buffer (pH 7.0), and 100 μL of these solutions was mixed with 50 μL of spore suspension in sterilized Eppendorf tubes (sealed with sealing film). The final concentrations of EMS were 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 M. The tubes were shaken for 4 h at 28 °C while lying flat. After treatment, 50 μL of 10% sodium thiosulfate was added to terminate the mutagenesis reaction. Samples were then diluted 10−1, 10−2, and 10−3 with phosphate buffer (pH 7.0) to obtain three parallel conditions at each concentration. A coating (100 μL) was applied to the screening medium plates, after which the plates were incubated for 3–5 days at 28 °C. Mutation and fatality rates were calculated based on the different halo sizes. 2.3.3. Combined ARTP and EMS mutagenesis For ARTP mutagenesis, the mutation time showing the highest positive mutation rate was considered as the optimal condition. Colonies exhibiting large halos were selected and inoculated into 250-mL shaker flasks containing 50 mL of standard medium. Spore suspensions were prepared and treated with optimal concentrations of EMS. Colonies obtained after combined ARTP-EMS mutagenesis were cultivated in screening medium at 28 °C and 220 r min−1 for 8 days. After fermentation, the culture broth was centrifuged at 12,000 ×g for 10 min to remove the bacterial cells. The cell-free culture supernatant was used to calculate the enzyme activities of the mutant strains.
2. Materials and methods 2.1. Substrates and chemicals Dextran T2000 (molecular weight = 2000 kDa) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Dextran T40 and T20 (with average molecular weights of 40 and 20 kDa, respectively) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Blue dextran was obtained from Pharmacia (Uppsala, Sweden). All other reagents were of the highest analytical grade available and used as recommended by their respective manufacturers.
2.4. Calculation of mutation and fatality rates 2.2. Strain and growth conditions The mutation rates of the spores after different mutagenesis treatment times were evaluated using the following equation:
Wild-type C. globosum (CGMCCC No. 15867) was isolated by our laboratory [19]. Chaetomium globosum M101, induced from C. globosum, was identified and cultured in our laboratory. Mutated strains were screened using screening medium, which was the same medium used to culture the wild-type strain. The standard medium for liquid culture and fermentation medium have been described previously for cultivating the wild-type strain [19]. Among the mutated dextranase-producing strains, one fungal strain, C. globosum M101, showed higher dextranase activity than the wild-type strain in repetitive cultures and thus was chosen for further analysis.
Mutation rate (%) = (Np + Nn)/N0 × 100%, where Np is the total colony count of the positive mutant, in which the diameter of the transparent circle was more than 5% of the wild-type; Nn is the total colony count of the negative mutant, in which the diameter of the transparent circle was less than 5% of the wild-type; and N0 is the total colony count of the sample without treatment. The fatality rate was calculated according to the following equation: Fatality rate (%) = (N0 − Nm)/N0 × 100%,
2.3. Mutagenesis of C. globosum using the compound method
where Nm is the total colony count after treatment, including those with positive mutations, negative mutations, and no mutations. Positive and negative mutation rates were calculated according to the following equations: Positive mutation rate (%) = (Np/N0) × 100%,
2.3.1. ARTP mutagenesis Chaetomium globosum was cultured in screening medium for 5 days at 28 °C under aerobic conditions. Spores were washed with 0.9% normal saline, collected into sterilized 1.5-mL Eppendorf tubes, and 117
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incubating the enzyme in reaction buffers ranging from pH 3.0 to 9.5 at 50 °C for 1 h without substrate. Dextran T2000 was added to determine the relative residual enzyme activity under enzyme assay conditions as described above.
Negative mutation rate (%) = (Nn/N0) × 100%. 2.5. Dextranase activity assay Reducing sugar quantities were determined using 3,5-dinitrosalicylic acid as previously described [20]. Enzyme activity was determined as previously described for wild-type C. globosum [19]. One unit of dextranase activity was defined as the amount of enzyme that liberated reducing sugars equivalent to 1 μmoL of glucose per minute at pH 5.5 and 50 °C.
2.9. Structure of dextranase before and after mutagenesis Genomic DNA was obtained from the original and mutant strains. The dextranase gene was amplified by PCR using the sense primer, 5′-CCGGAATTCATGTTTTCTGCTGTTCTTCTGGGCTGGC-3′ and antisense primer, 5′-CCCAAGCTTTTAACGAATGACCCACTGCCCCCAAT AAG-3′. The reaction conditions were as follows: (94 °C, 3 min) × 1 cycle, (98 °C, 10 s; 58 °C, 30 s; 72 °C, 2 min) × 32 cycles, and (72 °C, 5 min) × 1 cycle. PCR products were separated using 1% agarose gels and products approximately 1,800-base pairs were excised, purified, and ligated into the T-tailed vector PMD-19. Recombinant plasmids were transformed into competent DH5α cells, and cells carrying the recombinant plasmid were used for sequencing analysis. Protein sequences were submitted to the NCBI server (https://www.ncbi.nlm.nih. gov) and multiple sequence alignment was performed by Clustalx version 1.81 and DNAMAN. Homology modeling was performed by submitting the deduced protein sequence of laccase to the Swiss Model server (http://swissmodel.expasy.org/interactive). The 3D protein structure-viewing software Swiss-PdbViewer 4.0.1 was used to visualize the hydrogen bonds and calculate the force field energy of the protein.
2.6. Optimization of culture medium and fermentation conditions to enhance dextranase production by the mutant strain To optimize dextranase production, different carbon and nitrogen sources and different concentrations of K2HPO4 and MgSO4 were added to the medium to determine their effects on dextranase production and cell growth. Orthogonal experiments were performed with different culture medium formulations to determine the optimum nutrient concentrations. In optimization experiments, initial pH (4.5, 5, 5.5, 6, 6.5, 7, 7.5, and 8), fermentation volume (20, 30, 40, 50, 60, 70, 80, 90, and 100 mL), rotation speed (140, 160, 180, 200, and 220 rpm), temperature (22 °C, 24 °C, 26 °C, 28 °C, 30 °C, and 32 °C), and inoculation amount (1%, 2%, 3%, 4%, 5%, 6%, and 7%) were all tested. After cultivation, the cell-free culture supernatant was used to calculate enzyme activity.
2.10. Using dextranase to control dental caries 2.7. Production and purification of dextranase 2.10.1. Inhibition and clearance effects of dextranase on biofilm formation The effect of dextranase on the growth of S. mutans was investigated using brain heart infusion (BHI) medium containing 1% sucrose. A 0.22-μm filter was used to filter-sterilize the dextranase sample. The final concentrations of the enzyme were 10, 20, 30, 40, 50, 60, and 70 U/mL. After reaching an optical density of 1.0 at 600 nm (OD600), the S. mutans culture was diluted 1:10 in BHI medium in a test tube and the OD600 in the experimental group was measured after culturing the cells at 37 °C in 5% CO2 for 24 h. Uninoculated medium was used as a control. To examine the inhibitory effect of dextranase on plaque biofilm formation, 180 μL of BHI medium containing 1% sucrose and different concentrations of dextranase were added to a 96-well cell culture plate. Each well was then inoculated with 20 μL of S. mutans. An enzyme-free control sample was also used. After incubation at 37 °C in 5% CO2 for 24 h, the culture medium was removed, and weakly adherent bacteria were removed by washing with distilled water. After air drying, the biofilm in each well was stained with 0.1% crystal violet for 5 min. Excess staining solution was washed off using distilled water and the wells were treated with 200 μL of 95% ethanol. The plate was slowly shaken for 30 min on an Enspire 2300 microplate reader (Perkin Elmer, Waltham, MA, USA) and the OD600 of each well was measured. To examine the scavenging effect of dextranase on biofilms, 180 μL of BHI medium containing 1% sucrose was added to each well of a 96well cell culture plate and the plate was inoculated with 20 μL of an S. mutans suspension. The plate was then incubated at 37 °C for 24 h in an atmosphere of 10% CO2. After incubation, the bacteria were removed and each well was gently washed several times with 200 μL of sterile water, which was followed by the addition of 200 μL of dextranase diluted to different concentrations with 1% sucrose medium. After 24 h of incubation at 37 °C in 10% CO2, the liquid was removed, and each well was washed with distilled water prior to determining the absorbance at 600 nm.
The mutant strain was cultured under the pre-determined optimum fermentation conditions. A large quantity of crude enzyme was produced after 8 days at 28 °C and thalli were separated by centrifugation (8000 ×g, 20 min, 4 °C). The supernatant was filtered through a membrane filter with 0.22-μm pores (Millipore, Billerica, MA, USA) and used for enzyme purification. Dextranase from the culture supernatant was initially concentrated by ammonium sulfate saturation and Sephadex G-75 chromatography. Fractions with high specific activity were collected and their molecular weights were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native electrophoresis. Following electrophoresis, gels were stained with Coomassie Brilliant Blue G-250. 2.8. Enzymatic properties 2.8.1. Effect of temperature on enzyme activity and stability The effect of temperature on dextranase activity was evaluated by incubating the reaction mixtures at 20–90 °C and measuring enzyme activity using standard methods [19]. To determine the thermostability of dextranase, the enzyme was incubated without substrate in 20 mM acetate buffer (pH 5.5) for 1 h at temperatures of 20–90 °C. Dextran T2000 was added to determine the relative residual enzyme activity under the enzyme assay conditions described above. The sample showing the maximum enzyme activity was used as the control sample (100% activity). Relative activity was expressed as the percentage of the maximum activity. To determine temperature stability, the enzyme was incubated at 4 °C and 25 °C for different times until dextranase activity was reduced to half of its initial activity. Activity was measured under the enzyme assay conditions described above. 2.8.2. Effect of pH on enzyme activity and stability Dextranase activity was determined at pH values of 3.0–9.5 (acetate buffer, pH 3.0–5.5; phosphate buffer, pH 6.0–8.0; Tris-HCl, pH 7.5–9.5) under the enzyme assay conditions described above. The sample showing the maximum enzyme activity was used as the control sample (100% activity). The pH stability of the enzyme was evaluated by
2.10.2. Electron microscopic observation of the effects of dextranase on S. mutans Slides were placed in a 96-deep-well cell culture plate and 180 μL of 118
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Fig. 1. Lethal effects of atmospheric and room temperature plasma treatment (ARTP) and ethyl methyl sulfone (EMS) on Chaetomium globosum. (a) Lethal effects of ARTP on Chaetomium globosum. (b) Lethal effects of EMS on Chaetomium globosum.
BHI medium containing 1% or 5% sucrose and 20 or 50 U/mL dextranase, respectively, was added. Approximately 20 μL of the above suspension was inoculated with S. mutans. Control bacteria were incubated without added enzyme. After incubation for 24 h at 37 °C in 10% CO2, the wells were washed with physiological saline to remove non-adherent bacteria. Samples were then examined by scanning electron microscopy (SEM; FEI Quanta-200, Thermo Fisher Scientific, Waltham, MA, USA; 10 kV, 5,000×) as described by Bernardi et al. [21]. 3. Results and discussion 3.1. ARTP and EMS mutagenesis of C. globosum The results of ARTP and EMS mutagenesis are shown in Fig. 1. After 120 s of ARTP mutagenesis, the lethality rate reached 83% and the forward mutation rate reached 18% (Fig. 1a). Based on previous studies [22,23], a lethality rate of approximately 90% was expected. These observations were consistent with those of previous reports of mutagenesis in Arthrobacter KQ11 [24]. Using blue dextran screening culture medium, which can be degraded by dextranase-producing strains to produce transparent rings, 47 colonies with significantly large transparent zones were selected. We chose 120 s as the optimal treatment time for mutagenesis. EMS mutagenesis had a fatality rate of 75% and forward mutation rate of 15% when using a final EMS concentration of 0.5 M (Fig. 1b). Using early screening medium containing blue dextran, 36 colonies showing distinctly large transparent zones were selected. Therefore, 0.5 M was chosen as the optimal treatment concentration for EMS mutagenesis. The results of ARTP-EMS mutagenesis are shown in Fig. 2. The mutation rate for ARTP mutagenesis after 120 s of treatment was 45.2%, while the positive and negative mutation rates were 25% and 20.2%, respectively. The mutation rate for 0.5 M EMS treatment was 32.5%, with positive and negative mutation rates of 15% and 17.5%, respectively. The mutation rate when using combined ARTP-EMS treatment reached 56.7%, with positive and negative mutation rates of 32% and 24.7%, respectively. The original strain showed high mutation rates, possibly because of DNA and protein damage caused by the active ions injected into the target cells. This damage may lead to cell death, as demonstrated by the decreased survival rate. Maintenance of a low survival rate is necessary for mutation induction and mutant strain selection. These results indicate that combined mutagenesis treatment results in higher mutation and positive mutation rates than single-treatment mutagenesis. This
Fig. 2. Effects of mutagenesis on dextranase production in Chaetomium globosum. ARTP, atmospheric and room temperature plasma treatment; EMS, ethyl methyl sulfone treatment; ARTP-EMS, combined ARTP and EMS treatment.
phenomenon is similar to that observed by Christoph et al. [10]. 3.2. Screening and genetic stability of mutant strains After screening for ARTP-induced mutants by transparent circle comparisons and enzyme activity determination, a positive mutant strain was identified with an enzyme activity 15.3% higher than that of the wild-type strain. A second EMS treatment step was performed on this mutant strain and 21 mutant colonies were then selected. Of these 21 colonies, approximately 7 mutant strains showed positive mutations and 5 showed relatively lower dextranase production capacity. The sizes of the transparent zones of positive mutant strains were compared to those of the original strain and one positive mutant strain, named as C. globosum M101, was selected. The yield of dextranase in this mutant strain reached 49.53 U/mL, which was 30.31% higher than the dextranase yield of the wild-type strain (38.01 U/mL). Energy and active ions introduced by plasma treatment may cause DNA alterations, such as transversions, transitions, frameshifts, insertions, and deletions. During these processes, damage to the dextranase gene may lead to increased dextranase production [25]. This phenomenon was more common in the present study than in previous studies using the ARTP method with Arthrobacter KQ11 [24]. The selected mutant strain was then isolated to assess its genetic stability. After five generations of 119
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Fig. 3. Optimization of the conditions for dextranase production. Effect of initial pH (a), culture volume (b), shaking speed (c), culture temperature (d), and inoculation amount (e) on Chaetomium globosum M101 growth and dextranase production.
after loading equal amounts of protein. This suggests that the activity of dextranase produced by the mutant strain was higher than that of the enzyme from the wild-type strain. This is consistent with results reported for Escherichia coli K-12 [27] and Arthrobacter KQ11 [24].
culture, the strain retained genetic stability and maintained high enzyme activity. 3.3. Optimization of culture medium and fermentation conditions for dextranase production
3.5. Effects of temperature and pH on dextranase activity and enzyme stability before and after mutagenesis
The culture medium conditions of the enzyme-producing strain were then optimized. The best carbon and nitrogen sources for enzyme production were dextran T20 and yeast extract at optimum concentrations of 20 and 10 g/L, respectively. The optimum concentrations of K2HPO4 and MgSO4 were 2.0 and 0.5 g/L, respectively (data not shown). The enzyme activity of dextranase under these optimum culture conditions reached 427.31 U/mL. Under different fermentation conditions, the mutant strain showed different dextranase production capacities. In the presence of optimum growth medium, the optimum conditions of fermentation were as follows (Fig. 3): initial pH, 7.0 (Fig. 3a); volume, 70/250 mL (Fig. 3b); shaking speed, 200 rpm (Fig. 3c); culture temperature, 28 °C (Fig. 3d); and inoculation amount, 6% (Fig. 3e). Three parallel experiments were performed under optimum conditions. Under these conditions, the enzyme activity reached 824.73 U/mL (relative standard deviation was 3%), which was 16.65-fold higher than the enzyme activity before optimization (49.53 U/mL). This production capacity was higher than that of Chaetomium gracile [26].
Fig. 5 shows the effects of temperature and pH on dextranase activity before and after mutagenesis. As shown in Fig. 5a, the optimum temperature for dextranase activity before and after mutagenesis was 60 °C. The enzyme was more stable at 70 °C after mutagenesis than before mutagenesis, while the overall enzyme activity was higher before mutagenesis. Dextranases from fungi generally show maximum activity between 55 °C and 60 °C. Before and after mutagenesis, dextranase incubated at 20–50 °C for 1 h retained over 80% of its initial enzyme activity. Before mutagenesis, the relative enzyme activity following incubation at 60 °C for 1 h was less than 20%; however, after mutagenesis, the enzyme retained more than 60% of its activity under similar conditions. Dextranase is typically thermotolerant at temperatures under 50 °C, but the mutant enzyme was more heat stable than the wild-type enzyme. The effect of pH on dextranase activity before and after mutagenesis is shown in Fig. 5b. The optimum pH of dextranase from both wild-type and mutagenic strains was 5.5, which is consistent with the values reported for Arthrobacter KQ11 [24] and C. gracile dextranases [28]. After mutagenesis, dextranase showed higher activity at pH 4.5–7.0 than before mutagenesis. The mutant enzyme retained more than 80% of its activity between pH 4.5 and 7.0, but the range of pH stability was slightly reduced compared to the wild-type enzyme. In general, the effect of pH on dextranase was consistent before and after mutagenesis. Dental caries are typically formed in an environment with a pH below 5.5 [16], and the mutant enzyme was more suited to such an environment.
3.4. Protein molecular weight The final Sephadex G-75 chromatography-purified dextranase preparation from the mutant strain showed higher specific activity (8350.8 U/mg) compared to dextranase from the wild-type strain (7535.8 U/ mg). The molecular weight of purified dextranase from both the wildtype and mutant strains was approximately 53 kDa, as estimated from the SDS-PAGE results, suggesting that C. globosum dextranase is a monomer. Dextranase from the mutant strain showed a molecular weight similar to that of dextranase from C. globosum [19]. Moreover, as shown in Fig. 4, the white protein band obtained from the mutant strain was much brighter than that obtained from the wild-type strain 120
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Fig. 4. SDS-PAGE and native PAGE of dextranases from Chaetomium globosum and C. globosum M101. (a) Dextranase collected from gel filtration chromatography visualized by 12.0% SDS-PAGE. M, Marker protein (kDa); Lane 1, crude dextranase extract; Lane 2, ammonium sulfate precipitation; Lane 3, Sephadex G-75 chromatography. (b) SDS-PAGE of dextranases from wild-type C. globosum and C. globosum M101. M, Marker protein (kDa); Lane 1, Sephadex G-75 chromatography of dextranase from C. globosum; Lane 2, Sephadex G-75 chromatography of dextranase from C. globosum M101. (c) Native-PAGE of dextranase from wildtype C. globosum and C. globosum M101; Lane 1, native dextranase from C. globosum. Lane 2, native dextranase from C. globosum M101.
Asp 431, which interacted to form hydrogen bonds. The modeling results indicated that Asp 408, Asp 409, and Asp 431 are catalytic residues. It has been reported that Asp395, Asp396, and Asp375 form the active centers of Penicillium minioluteum dextranase [29], whereas Asp385 is the key nucleophile of S. mutans dextranase [30]. A recent site-directed mutagenesis study showed that Asp243 is necessary for the dextranase activity of Thermotoga lettingae [31]. Asp residues of enzymes are essential for the hydrolysis of dextran [32]. After mutagenesis, the nucleotide base at position 1,733 in the dextranase gene changed from A to T, resulting in a change in the amino acid residue at position 578 from His to Leu. By simulating the secondary structure of dextranase before and after mutagenesis, this residue was found to be located in the protein helix, close to the catalytic groove, which typically stabilizes the protein conformation and affects the catalytic function of the enzyme. This phenomenon may be responsible for the slight change in the enzymatic properties of the mutant dextranase. These results are similar to those obtained by Suzuki et al. [33].
3.6. Protein structure analysis Dextranase genes from the wild-type and mutant strains were sequenced. Amino acid alignment showed that C. globosum dextranase (GenBank accession no. MH122516) belongs to glycosyl hydrolase family 49 (GH 49). The structure of C. globosum dextranase was predicted using SWISS-MODEL and analyzed with Autodock software. The C. gracile dextranase was found to have 87.06% sequence identity and 72% homology with C. globosum dextranase. The three-dimensional structures of dextranases from C. globosum and C. globosum M101 are shown in Fig. 6. The structure of dextranase before and after mutagenesis was mainly comprised of β-pleated sheets and random coils, including 5 α-helices and 43 β-helix folds. The active site of the protein was between the helix and folded lamella and a small molecular ligand may act on the active site to antagonize or activate the target. The ligand may enter the active site of the target protein mainly through hydrophobic and Van der Waals forces. The structure of C. globosum dextranase (Fig. 6a) showed that the ligand interacts with the amino acid residues Trp 438, Lys 412, Tyr 414, Asp 408, Asp 409, and Asp 431 at the active site of the protein to form hydrogen bonds, which are advantageous for catalytic protein hydrolysis. However, the structure of C. globosum M101 dextranase was slightly different (Fig. 6b), as the active sites involved Asn 361, Lys 412, Tyr 414, Asp 408, Asp 409, and
3.7. Use of dextranase to control dental caries 3.7.1. Inhibition and clearance effects of dextranase on biofilm formation As shown in Fig. 7, increased concentrations of dextranase resulted in decreased growth rates of S. mutans. Dextranase was shown to inhibit
Fig. 5. Effects of temperature and pH on dextranase before and after mutagenesis. (a) Effect of temperature on the activity (round) and stability (square) of dextranase from Chaetomium globosum. (b) Effect of pH on the activity (round) and stability (square) of dextranase from C. globosum M101. 121
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Fig. 6. Three-dimensional modeling and molecular docking with isomaltose of dextranase from wild-type Chaetomium globosum (a) and C. globosum M101 (b).
biofilm inhibition by dextranase, and inhibition and clearance tend to be stable when the concentration is increased beyond this range. The results reported here are similar to those reported when using dextranase to treat Arthrobacter oxydans [18] and Arthrobacter sp. biofilms [34]. 3.7.2. Electron microscopic observation of the effects of dextranase on biofilm clearance The clearing effect of dextranase on biofilm is shown in Fig. 8. The biofilm that formed when 5% sucrose was used as the substrate was denser than the biofilm formed when using 1% sucrose. The efficiency of S. mutans biofilm degradation increased with increasing concentrations of dextranase. This result was similar to those reported by Jiao et al. [34] and Walker et al. [35]. At a dextranase concentration of 20 U/mL, the biofilms were significantly degraded, and the inhibition rate reached more than 90% when the dextranase concentration was increased. The dextranase tested in the present study more effectively inhibited biofilm formation than a previously characterized marinebacteria-derived dextranase [34]. These results indicate that dextranase produced by C. globosum M101 effectively inhibited biofilm formation.
Fig. 7. Inhibition and removal of Streptococcus mutans biofilm by dextranase from Chaetomium globosum. Effect of dextranase on the growth of Streptococcus mutans (bar) and the inhibition and reduction of Streptococcus mutans biofilm (line).
4. Conclusions and remove biofilms of S. mutans. These results are consistent with those reported by Jiao et al. [34] and Walker et al. [35] in studies of the prevention and treatment of dental caries using dextranase from the marine bacteria and Lipomyces starkeyi. These studies showed that biofilm formation was inhibited by dextranase in a dose-dependent manner. In the present study, at 50 U/mL, the rate of inhibition of S. mutans biofilm formation by dextranase was 71.58%, while the clearance rate was 49.07%. The inhibition and clearance rate slightly decreased with increasing dextranase concentrations, but the difference was not significant. There may be an optimal concentration range for
This study provides a promising strategy for obtaining C. globosum strains with a high yield of dextranase by combining ARTP and EMS mutagenesis methods. The fatality rate of C. globosum M101, generated using the combined ARTP-EMS mutagenesis system, was evaluated to determine the optimum mutagenesis treatment time and concentration. Under the optimum conditions of combined ARTP-EMS mutagenesis, a strain with a high yield of dextranase was obtained. This mutagenesis method served was an efficient strategy for generating high-dextranaseproducing C. globosum strains. The temperature and pH stability of the enzyme slightly differed before and after mutagenesis. The mutant 122
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Fig. 8. Electron microscopic observation of the effect of dextranase on biofilm clearance. The effect of different concentrations of dextranase on the biofilm formed by 1% and 5% sucrose were used as the substrate, respectively.
enzyme was found to be more heat stable than the wild-type enzyme, which may be because of a change in the secondary structure of the enzyme caused by amino acid changes. However, dextranase was stable at various temperatures and pH conditions before and after mutagenesis. The ability of dextranase to inhibit S. mutans indicates that it may be an effective treatment for inhibiting and removing plaque biofilms. The toxicity of dextranase towards microorganisms was also demonstrated in a safety test. Most importantly, these results provide an important basis for further scientific research of the industrial application of dextranase.
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Declaration of Competing Interest The authors declare no conflicts of interest. Acknowledgements This work was supported by the National Natural Science Foundation of China [grant number 31601558] and Fundamental Research Funds for the Central Universities [grant number JUSRP51402A]. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.procbio.2019.06.026. References [1] L. Liu, Y. Ding, S. Liu, S. Wang, Y. Fang, M. Lyu, Dextrans removal from sugarcane juice using dextranase from marine bacterium Arthrobacter oxydans KQ11, Qual. Assur. Safe. Crops. Foods 11 (2019) 53–59. [2] M. Bashari, Z.Y. Jin, J.P. Wang, X.B. Zhan, A novel technique to improve the biodegradation efficiency of dextranase enzyme using the synergistic effects of ultrasound combined with microwave shock, Int. F. Sci. Emerg. Technol. 35 (2016) 125–132. [3] A. Yuko, S.J. Zheng, N. Yasuo, S. Kenichiro, J. GotaroIiizumi, D.B. Liu, F.B. Li, An, decomposition and removal of dental model plaque by using toothpaste containing dextranase, Oral. Care Industry 28 (2018) 21–24. [4] N. Siwames, N. Suwanna, A. Tomohiro, B.K. Mallika, M. Nipa, Purification and characterization including dextran hydrolysis of dextranase from Aspergillus
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Characterization and application of dextranase produced by Chaetomium globosum mutant through combined application of atmospheric and room temperature plasma and ethyl methyl sulfone Liu Yanga, Nandi Zhoub, Yaping Tiana, a b
T
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The Key Laboratsory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Chaetomium globosum Dextranase Atmospheric and room temperature plasma treatment-ethyl methyl sulfone compound mutagenesis DNA structure Dental caries
Dextranase has extensive applications in the food, pharmaceutical, and chemical industries. However, enzyme production by the wild-type strain is generally low and few studies have conducted structural analysis of dextranase. This study describes an efficient and stable mutagenesis method for producing dextranase and the relationship between the enzyme structure and enzymatic properties before and after mutagenesis. Wild-type Chaetomium globosum was mutated by combined atmospheric and room temperature plasma treatment and ethyl methyl sulfone application. Mutant strains were screened for optimal dextranase production ability and genetic stability. The maximum yield of dextranase reached 824.73 U/mL. Dextranase produced by the mutant strain displayed the same optimum pH and temperature values of 5.5 and 60℃, respectively, as dextranase produced by the wild-type strain. However, dextranase from the mutant strain showed greater heat stability at 70℃. Furthermore, the three-dimensional structure of the enzyme changed slightly after mutagenesis, with the amino acid residue at position 578 changing from His to Leu. This change in the three-dimensional structure affected the properties of dextranase. Finally, dextranase significantly inhibited the growth of Streptococcus mutans. The results indicated dextranase from Chaetomium globosum would give potential implications in the medical, prevent dental caries and food industries.
1. Introduction Dextranase (α-1,6-d-glucan-6-glucanohydrolase; EC 3.2.1.11) catalyzes the degradation of dextran (a polymer of glucose) within or at the end of the chain into low-molecular-weight fractions. It has important applications in various fields, such as reducing the viscosity of sugarcane juice in the sugar industry [1], preparing low-molecular-weight medicinal dextran [2], and inhibiting or removing dental plaque [3]. Continuous developments in the food and pharmaceutical industries have led to an increase in the demand for dextran. As dextranase has been isolated from different mold strains, Catenovulum, yeast, Streptococcus, and Arthrobacter [4–6], the enzymatic biocatalysis of dextran should be thoroughly evaluated and optimized. However, the reported enzyme activity of dextranase from wild-type strains is relatively low. Dextranase from Talaromyces pinophilus shows an activity of 4.60 U/mL, with an optimum temperature of 45 °C and optimum pH of 6.0 [7], whereas dextranase from Aspergillus allahabadii X26 has an activity of 110 U/mL, with an optimum pH and temperature of 5.8 and 55 °C,
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respectively [4], Sufiate et al. [8] isolated a novel dextranase from Pochonia chlamydosporia, which showed a specific activity of 358.63 U/ mg and high activity at 50 °C and pH 5.0. Improving productivity is a constant requirement in the fermentation industry, and efficient methods for creating mutants are necessary to improve dextranase yield and enzymatic characterization. Traditional methods for inducing mutagenesis, such as physical or chemical methods, combined with rational screening processes, are cost-effective methods for adapting strains or increasing the yields of valuable metabolites [9]. Atmospheric and room temperature plasma treatment (ARTP) uses a new type of atmospheric pressure: a nonequilibrium discharge plasma source [10]. Under normal atmospheric pressure and at a temperature of 25–40 °C, excited nitrogen, oxygen, and nitrogen atoms and the OH radical are highly active particles. The main mechanism of action of this technique involves altering the structure and permeability of microbial cell walls and membranes as well as DNA damage caused by active ions in the plasma, which significantly alter microbial gene sequences and metabolic networks.
Corresponding author. E-mail addresses: [email protected] (L. Yang), [email protected] (N. Zhou), [email protected] (Y. Tian).
https://doi.org/10.1016/j.procbio.2019.06.026 Received 20 February 2019; Received in revised form 29 June 2019; Accepted 29 June 2019 Available online 01 July 2019 1359-5113/ © 2019 Published by Elsevier Ltd.
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centrifuged for 10 min at 5000 ×g. The final concentration of the spore suspension was adjusted to 106 CFU/mL with phosphate buffer (pH 7.0). Mutagenic treatment was performed as described previously by Christoph et al. [10], with slight modifications. On a clean bench, spore suspensions and 10% glycerol were evenly mixed to a final glycerol concentration of 5%. Approximately 10 μL of the mixture was then spread on a 1-cm plate and later exposed to the ARTP system’s nozzle exit. The working parameters for Pin, Pout, and distance (D) were set to 100 W, 20 W, and 2 mm, respectively. The flow rate of helium gas was 10 slm. High-purity helium plasma mutagenesis treatment was performed for 0 (control group), 30, 60, 90, 120, 150, 180, and 210 s. After treatment, the plates were placed in a new sterile tube containing 300 μL of sterile water and spread on blue dextran screening culture medium. After 3–5 days of cultivation at 28 °C, the ability of the strains to hydrolyze blue dextran was assessed. Colony numbers were calculated on a solid medium plate using the colony-forming unit method. Mutation and fatality rates were calculated based on the different halo sizes.
These changes eventually lead to microbial genetic mutations [11]. ARTP has been successfully employed in 40 different types of microorganisms including bacteria, fungi, yeast, and microalgae [12]. In chemical mutagenesis, ethyl methyl sulfone (EMS) is a widely used alkylating agent shown to be an effective mutagen. At present, EMS mutagenesis is widely used in microbial applications and to select improved crop varieties [13,14]. A single mutagenesis method often induces resistance to the chemical or lethal agent used and the resulting mutant strain may be unstable. Therefore, two or more methods are often combined to induce mutagenesis, a process known as composite inducement. In recent years, as the incidence of dental caries has increased, oral health has received more attention. Dental caries may cause diseases, such as periodontitis and odontitis, and are considered by the World Health Organization to be the third most concerning disease after cardiovascular diseases and cancers. The oral cavity provides an environment suitable for the growth of resident facultative anaerobic bacteria (Streptococcus), which cause dental caries [15]. Oral pathogens may assist in the breakdown of food after the generation of sucrose from α1,6-glucoside and α-1,3-glucoside. Additionally, chewed food residues, saliva, and inorganic salts adhere to teeth and form dental plaque. Several types of oral bacteria secrete acids that destroy dentin and result in caries and periodontal disease [16]. Dextranase can degrade glucan, which may help prevent the formation of dental plaque and thus has important potential applications for preventing and treating dental caries [17,18]. In this study, ARTP coupled with EMS application was first used to produce dextran anhydrase through multiple rounds of mutagenesis fermentation. The correlation between the transparent circle size of blue dextran tablets and enzyme activity was evaluated to obtain a mutant strain producing high levels of dextranase. Studies of the properties of dextranase and its applications in the prevention and treatment of dental caries provides a foundation for future industrial applications.
2.3.2. EMS mutagenesis Various concentrations of EMS were prepared by dilution with phosphoric acid buffer (pH 7.0), and 100 μL of these solutions was mixed with 50 μL of spore suspension in sterilized Eppendorf tubes (sealed with sealing film). The final concentrations of EMS were 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 M. The tubes were shaken for 4 h at 28 °C while lying flat. After treatment, 50 μL of 10% sodium thiosulfate was added to terminate the mutagenesis reaction. Samples were then diluted 10−1, 10−2, and 10−3 with phosphate buffer (pH 7.0) to obtain three parallel conditions at each concentration. A coating (100 μL) was applied to the screening medium plates, after which the plates were incubated for 3–5 days at 28 °C. Mutation and fatality rates were calculated based on the different halo sizes. 2.3.3. Combined ARTP and EMS mutagenesis For ARTP mutagenesis, the mutation time showing the highest positive mutation rate was considered as the optimal condition. Colonies exhibiting large halos were selected and inoculated into 250-mL shaker flasks containing 50 mL of standard medium. Spore suspensions were prepared and treated with optimal concentrations of EMS. Colonies obtained after combined ARTP-EMS mutagenesis were cultivated in screening medium at 28 °C and 220 r min−1 for 8 days. After fermentation, the culture broth was centrifuged at 12,000 ×g for 10 min to remove the bacterial cells. The cell-free culture supernatant was used to calculate the enzyme activities of the mutant strains.
2. Materials and methods 2.1. Substrates and chemicals Dextran T2000 (molecular weight = 2000 kDa) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Dextran T40 and T20 (with average molecular weights of 40 and 20 kDa, respectively) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Blue dextran was obtained from Pharmacia (Uppsala, Sweden). All other reagents were of the highest analytical grade available and used as recommended by their respective manufacturers.
2.4. Calculation of mutation and fatality rates 2.2. Strain and growth conditions The mutation rates of the spores after different mutagenesis treatment times were evaluated using the following equation:
Wild-type C. globosum (CGMCCC No. 15867) was isolated by our laboratory [19]. Chaetomium globosum M101, induced from C. globosum, was identified and cultured in our laboratory. Mutated strains were screened using screening medium, which was the same medium used to culture the wild-type strain. The standard medium for liquid culture and fermentation medium have been described previously for cultivating the wild-type strain [19]. Among the mutated dextranase-producing strains, one fungal strain, C. globosum M101, showed higher dextranase activity than the wild-type strain in repetitive cultures and thus was chosen for further analysis.
Mutation rate (%) = (Np + Nn)/N0 × 100%, where Np is the total colony count of the positive mutant, in which the diameter of the transparent circle was more than 5% of the wild-type; Nn is the total colony count of the negative mutant, in which the diameter of the transparent circle was less than 5% of the wild-type; and N0 is the total colony count of the sample without treatment. The fatality rate was calculated according to the following equation: Fatality rate (%) = (N0 − Nm)/N0 × 100%,
2.3. Mutagenesis of C. globosum using the compound method
where Nm is the total colony count after treatment, including those with positive mutations, negative mutations, and no mutations. Positive and negative mutation rates were calculated according to the following equations: Positive mutation rate (%) = (Np/N0) × 100%,
2.3.1. ARTP mutagenesis Chaetomium globosum was cultured in screening medium for 5 days at 28 °C under aerobic conditions. Spores were washed with 0.9% normal saline, collected into sterilized 1.5-mL Eppendorf tubes, and 117
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incubating the enzyme in reaction buffers ranging from pH 3.0 to 9.5 at 50 °C for 1 h without substrate. Dextran T2000 was added to determine the relative residual enzyme activity under enzyme assay conditions as described above.
Negative mutation rate (%) = (Nn/N0) × 100%. 2.5. Dextranase activity assay Reducing sugar quantities were determined using 3,5-dinitrosalicylic acid as previously described [20]. Enzyme activity was determined as previously described for wild-type C. globosum [19]. One unit of dextranase activity was defined as the amount of enzyme that liberated reducing sugars equivalent to 1 μmoL of glucose per minute at pH 5.5 and 50 °C.
2.9. Structure of dextranase before and after mutagenesis Genomic DNA was obtained from the original and mutant strains. The dextranase gene was amplified by PCR using the sense primer, 5′-CCGGAATTCATGTTTTCTGCTGTTCTTCTGGGCTGGC-3′ and antisense primer, 5′-CCCAAGCTTTTAACGAATGACCCACTGCCCCCAAT AAG-3′. The reaction conditions were as follows: (94 °C, 3 min) × 1 cycle, (98 °C, 10 s; 58 °C, 30 s; 72 °C, 2 min) × 32 cycles, and (72 °C, 5 min) × 1 cycle. PCR products were separated using 1% agarose gels and products approximately 1,800-base pairs were excised, purified, and ligated into the T-tailed vector PMD-19. Recombinant plasmids were transformed into competent DH5α cells, and cells carrying the recombinant plasmid were used for sequencing analysis. Protein sequences were submitted to the NCBI server (https://www.ncbi.nlm.nih. gov) and multiple sequence alignment was performed by Clustalx version 1.81 and DNAMAN. Homology modeling was performed by submitting the deduced protein sequence of laccase to the Swiss Model server (http://swissmodel.expasy.org/interactive). The 3D protein structure-viewing software Swiss-PdbViewer 4.0.1 was used to visualize the hydrogen bonds and calculate the force field energy of the protein.
2.6. Optimization of culture medium and fermentation conditions to enhance dextranase production by the mutant strain To optimize dextranase production, different carbon and nitrogen sources and different concentrations of K2HPO4 and MgSO4 were added to the medium to determine their effects on dextranase production and cell growth. Orthogonal experiments were performed with different culture medium formulations to determine the optimum nutrient concentrations. In optimization experiments, initial pH (4.5, 5, 5.5, 6, 6.5, 7, 7.5, and 8), fermentation volume (20, 30, 40, 50, 60, 70, 80, 90, and 100 mL), rotation speed (140, 160, 180, 200, and 220 rpm), temperature (22 °C, 24 °C, 26 °C, 28 °C, 30 °C, and 32 °C), and inoculation amount (1%, 2%, 3%, 4%, 5%, 6%, and 7%) were all tested. After cultivation, the cell-free culture supernatant was used to calculate enzyme activity.
2.10. Using dextranase to control dental caries 2.7. Production and purification of dextranase 2.10.1. Inhibition and clearance effects of dextranase on biofilm formation The effect of dextranase on the growth of S. mutans was investigated using brain heart infusion (BHI) medium containing 1% sucrose. A 0.22-μm filter was used to filter-sterilize the dextranase sample. The final concentrations of the enzyme were 10, 20, 30, 40, 50, 60, and 70 U/mL. After reaching an optical density of 1.0 at 600 nm (OD600), the S. mutans culture was diluted 1:10 in BHI medium in a test tube and the OD600 in the experimental group was measured after culturing the cells at 37 °C in 5% CO2 for 24 h. Uninoculated medium was used as a control. To examine the inhibitory effect of dextranase on plaque biofilm formation, 180 μL of BHI medium containing 1% sucrose and different concentrations of dextranase were added to a 96-well cell culture plate. Each well was then inoculated with 20 μL of S. mutans. An enzyme-free control sample was also used. After incubation at 37 °C in 5% CO2 for 24 h, the culture medium was removed, and weakly adherent bacteria were removed by washing with distilled water. After air drying, the biofilm in each well was stained with 0.1% crystal violet for 5 min. Excess staining solution was washed off using distilled water and the wells were treated with 200 μL of 95% ethanol. The plate was slowly shaken for 30 min on an Enspire 2300 microplate reader (Perkin Elmer, Waltham, MA, USA) and the OD600 of each well was measured. To examine the scavenging effect of dextranase on biofilms, 180 μL of BHI medium containing 1% sucrose was added to each well of a 96well cell culture plate and the plate was inoculated with 20 μL of an S. mutans suspension. The plate was then incubated at 37 °C for 24 h in an atmosphere of 10% CO2. After incubation, the bacteria were removed and each well was gently washed several times with 200 μL of sterile water, which was followed by the addition of 200 μL of dextranase diluted to different concentrations with 1% sucrose medium. After 24 h of incubation at 37 °C in 10% CO2, the liquid was removed, and each well was washed with distilled water prior to determining the absorbance at 600 nm.
The mutant strain was cultured under the pre-determined optimum fermentation conditions. A large quantity of crude enzyme was produced after 8 days at 28 °C and thalli were separated by centrifugation (8000 ×g, 20 min, 4 °C). The supernatant was filtered through a membrane filter with 0.22-μm pores (Millipore, Billerica, MA, USA) and used for enzyme purification. Dextranase from the culture supernatant was initially concentrated by ammonium sulfate saturation and Sephadex G-75 chromatography. Fractions with high specific activity were collected and their molecular weights were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native electrophoresis. Following electrophoresis, gels were stained with Coomassie Brilliant Blue G-250. 2.8. Enzymatic properties 2.8.1. Effect of temperature on enzyme activity and stability The effect of temperature on dextranase activity was evaluated by incubating the reaction mixtures at 20–90 °C and measuring enzyme activity using standard methods [19]. To determine the thermostability of dextranase, the enzyme was incubated without substrate in 20 mM acetate buffer (pH 5.5) for 1 h at temperatures of 20–90 °C. Dextran T2000 was added to determine the relative residual enzyme activity under the enzyme assay conditions described above. The sample showing the maximum enzyme activity was used as the control sample (100% activity). Relative activity was expressed as the percentage of the maximum activity. To determine temperature stability, the enzyme was incubated at 4 °C and 25 °C for different times until dextranase activity was reduced to half of its initial activity. Activity was measured under the enzyme assay conditions described above. 2.8.2. Effect of pH on enzyme activity and stability Dextranase activity was determined at pH values of 3.0–9.5 (acetate buffer, pH 3.0–5.5; phosphate buffer, pH 6.0–8.0; Tris-HCl, pH 7.5–9.5) under the enzyme assay conditions described above. The sample showing the maximum enzyme activity was used as the control sample (100% activity). The pH stability of the enzyme was evaluated by
2.10.2. Electron microscopic observation of the effects of dextranase on S. mutans Slides were placed in a 96-deep-well cell culture plate and 180 μL of 118
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Fig. 1. Lethal effects of atmospheric and room temperature plasma treatment (ARTP) and ethyl methyl sulfone (EMS) on Chaetomium globosum. (a) Lethal effects of ARTP on Chaetomium globosum. (b) Lethal effects of EMS on Chaetomium globosum.
BHI medium containing 1% or 5% sucrose and 20 or 50 U/mL dextranase, respectively, was added. Approximately 20 μL of the above suspension was inoculated with S. mutans. Control bacteria were incubated without added enzyme. After incubation for 24 h at 37 °C in 10% CO2, the wells were washed with physiological saline to remove non-adherent bacteria. Samples were then examined by scanning electron microscopy (SEM; FEI Quanta-200, Thermo Fisher Scientific, Waltham, MA, USA; 10 kV, 5,000×) as described by Bernardi et al. [21]. 3. Results and discussion 3.1. ARTP and EMS mutagenesis of C. globosum The results of ARTP and EMS mutagenesis are shown in Fig. 1. After 120 s of ARTP mutagenesis, the lethality rate reached 83% and the forward mutation rate reached 18% (Fig. 1a). Based on previous studies [22,23], a lethality rate of approximately 90% was expected. These observations were consistent with those of previous reports of mutagenesis in Arthrobacter KQ11 [24]. Using blue dextran screening culture medium, which can be degraded by dextranase-producing strains to produce transparent rings, 47 colonies with significantly large transparent zones were selected. We chose 120 s as the optimal treatment time for mutagenesis. EMS mutagenesis had a fatality rate of 75% and forward mutation rate of 15% when using a final EMS concentration of 0.5 M (Fig. 1b). Using early screening medium containing blue dextran, 36 colonies showing distinctly large transparent zones were selected. Therefore, 0.5 M was chosen as the optimal treatment concentration for EMS mutagenesis. The results of ARTP-EMS mutagenesis are shown in Fig. 2. The mutation rate for ARTP mutagenesis after 120 s of treatment was 45.2%, while the positive and negative mutation rates were 25% and 20.2%, respectively. The mutation rate for 0.5 M EMS treatment was 32.5%, with positive and negative mutation rates of 15% and 17.5%, respectively. The mutation rate when using combined ARTP-EMS treatment reached 56.7%, with positive and negative mutation rates of 32% and 24.7%, respectively. The original strain showed high mutation rates, possibly because of DNA and protein damage caused by the active ions injected into the target cells. This damage may lead to cell death, as demonstrated by the decreased survival rate. Maintenance of a low survival rate is necessary for mutation induction and mutant strain selection. These results indicate that combined mutagenesis treatment results in higher mutation and positive mutation rates than single-treatment mutagenesis. This
Fig. 2. Effects of mutagenesis on dextranase production in Chaetomium globosum. ARTP, atmospheric and room temperature plasma treatment; EMS, ethyl methyl sulfone treatment; ARTP-EMS, combined ARTP and EMS treatment.
phenomenon is similar to that observed by Christoph et al. [10]. 3.2. Screening and genetic stability of mutant strains After screening for ARTP-induced mutants by transparent circle comparisons and enzyme activity determination, a positive mutant strain was identified with an enzyme activity 15.3% higher than that of the wild-type strain. A second EMS treatment step was performed on this mutant strain and 21 mutant colonies were then selected. Of these 21 colonies, approximately 7 mutant strains showed positive mutations and 5 showed relatively lower dextranase production capacity. The sizes of the transparent zones of positive mutant strains were compared to those of the original strain and one positive mutant strain, named as C. globosum M101, was selected. The yield of dextranase in this mutant strain reached 49.53 U/mL, which was 30.31% higher than the dextranase yield of the wild-type strain (38.01 U/mL). Energy and active ions introduced by plasma treatment may cause DNA alterations, such as transversions, transitions, frameshifts, insertions, and deletions. During these processes, damage to the dextranase gene may lead to increased dextranase production [25]. This phenomenon was more common in the present study than in previous studies using the ARTP method with Arthrobacter KQ11 [24]. The selected mutant strain was then isolated to assess its genetic stability. After five generations of 119
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Fig. 3. Optimization of the conditions for dextranase production. Effect of initial pH (a), culture volume (b), shaking speed (c), culture temperature (d), and inoculation amount (e) on Chaetomium globosum M101 growth and dextranase production.
after loading equal amounts of protein. This suggests that the activity of dextranase produced by the mutant strain was higher than that of the enzyme from the wild-type strain. This is consistent with results reported for Escherichia coli K-12 [27] and Arthrobacter KQ11 [24].
culture, the strain retained genetic stability and maintained high enzyme activity. 3.3. Optimization of culture medium and fermentation conditions for dextranase production
3.5. Effects of temperature and pH on dextranase activity and enzyme stability before and after mutagenesis
The culture medium conditions of the enzyme-producing strain were then optimized. The best carbon and nitrogen sources for enzyme production were dextran T20 and yeast extract at optimum concentrations of 20 and 10 g/L, respectively. The optimum concentrations of K2HPO4 and MgSO4 were 2.0 and 0.5 g/L, respectively (data not shown). The enzyme activity of dextranase under these optimum culture conditions reached 427.31 U/mL. Under different fermentation conditions, the mutant strain showed different dextranase production capacities. In the presence of optimum growth medium, the optimum conditions of fermentation were as follows (Fig. 3): initial pH, 7.0 (Fig. 3a); volume, 70/250 mL (Fig. 3b); shaking speed, 200 rpm (Fig. 3c); culture temperature, 28 °C (Fig. 3d); and inoculation amount, 6% (Fig. 3e). Three parallel experiments were performed under optimum conditions. Under these conditions, the enzyme activity reached 824.73 U/mL (relative standard deviation was 3%), which was 16.65-fold higher than the enzyme activity before optimization (49.53 U/mL). This production capacity was higher than that of Chaetomium gracile [26].
Fig. 5 shows the effects of temperature and pH on dextranase activity before and after mutagenesis. As shown in Fig. 5a, the optimum temperature for dextranase activity before and after mutagenesis was 60 °C. The enzyme was more stable at 70 °C after mutagenesis than before mutagenesis, while the overall enzyme activity was higher before mutagenesis. Dextranases from fungi generally show maximum activity between 55 °C and 60 °C. Before and after mutagenesis, dextranase incubated at 20–50 °C for 1 h retained over 80% of its initial enzyme activity. Before mutagenesis, the relative enzyme activity following incubation at 60 °C for 1 h was less than 20%; however, after mutagenesis, the enzyme retained more than 60% of its activity under similar conditions. Dextranase is typically thermotolerant at temperatures under 50 °C, but the mutant enzyme was more heat stable than the wild-type enzyme. The effect of pH on dextranase activity before and after mutagenesis is shown in Fig. 5b. The optimum pH of dextranase from both wild-type and mutagenic strains was 5.5, which is consistent with the values reported for Arthrobacter KQ11 [24] and C. gracile dextranases [28]. After mutagenesis, dextranase showed higher activity at pH 4.5–7.0 than before mutagenesis. The mutant enzyme retained more than 80% of its activity between pH 4.5 and 7.0, but the range of pH stability was slightly reduced compared to the wild-type enzyme. In general, the effect of pH on dextranase was consistent before and after mutagenesis. Dental caries are typically formed in an environment with a pH below 5.5 [16], and the mutant enzyme was more suited to such an environment.
3.4. Protein molecular weight The final Sephadex G-75 chromatography-purified dextranase preparation from the mutant strain showed higher specific activity (8350.8 U/mg) compared to dextranase from the wild-type strain (7535.8 U/ mg). The molecular weight of purified dextranase from both the wildtype and mutant strains was approximately 53 kDa, as estimated from the SDS-PAGE results, suggesting that C. globosum dextranase is a monomer. Dextranase from the mutant strain showed a molecular weight similar to that of dextranase from C. globosum [19]. Moreover, as shown in Fig. 4, the white protein band obtained from the mutant strain was much brighter than that obtained from the wild-type strain 120
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Fig. 4. SDS-PAGE and native PAGE of dextranases from Chaetomium globosum and C. globosum M101. (a) Dextranase collected from gel filtration chromatography visualized by 12.0% SDS-PAGE. M, Marker protein (kDa); Lane 1, crude dextranase extract; Lane 2, ammonium sulfate precipitation; Lane 3, Sephadex G-75 chromatography. (b) SDS-PAGE of dextranases from wild-type C. globosum and C. globosum M101. M, Marker protein (kDa); Lane 1, Sephadex G-75 chromatography of dextranase from C. globosum; Lane 2, Sephadex G-75 chromatography of dextranase from C. globosum M101. (c) Native-PAGE of dextranase from wildtype C. globosum and C. globosum M101; Lane 1, native dextranase from C. globosum. Lane 2, native dextranase from C. globosum M101.
Asp 431, which interacted to form hydrogen bonds. The modeling results indicated that Asp 408, Asp 409, and Asp 431 are catalytic residues. It has been reported that Asp395, Asp396, and Asp375 form the active centers of Penicillium minioluteum dextranase [29], whereas Asp385 is the key nucleophile of S. mutans dextranase [30]. A recent site-directed mutagenesis study showed that Asp243 is necessary for the dextranase activity of Thermotoga lettingae [31]. Asp residues of enzymes are essential for the hydrolysis of dextran [32]. After mutagenesis, the nucleotide base at position 1,733 in the dextranase gene changed from A to T, resulting in a change in the amino acid residue at position 578 from His to Leu. By simulating the secondary structure of dextranase before and after mutagenesis, this residue was found to be located in the protein helix, close to the catalytic groove, which typically stabilizes the protein conformation and affects the catalytic function of the enzyme. This phenomenon may be responsible for the slight change in the enzymatic properties of the mutant dextranase. These results are similar to those obtained by Suzuki et al. [33].
3.6. Protein structure analysis Dextranase genes from the wild-type and mutant strains were sequenced. Amino acid alignment showed that C. globosum dextranase (GenBank accession no. MH122516) belongs to glycosyl hydrolase family 49 (GH 49). The structure of C. globosum dextranase was predicted using SWISS-MODEL and analyzed with Autodock software. The C. gracile dextranase was found to have 87.06% sequence identity and 72% homology with C. globosum dextranase. The three-dimensional structures of dextranases from C. globosum and C. globosum M101 are shown in Fig. 6. The structure of dextranase before and after mutagenesis was mainly comprised of β-pleated sheets and random coils, including 5 α-helices and 43 β-helix folds. The active site of the protein was between the helix and folded lamella and a small molecular ligand may act on the active site to antagonize or activate the target. The ligand may enter the active site of the target protein mainly through hydrophobic and Van der Waals forces. The structure of C. globosum dextranase (Fig. 6a) showed that the ligand interacts with the amino acid residues Trp 438, Lys 412, Tyr 414, Asp 408, Asp 409, and Asp 431 at the active site of the protein to form hydrogen bonds, which are advantageous for catalytic protein hydrolysis. However, the structure of C. globosum M101 dextranase was slightly different (Fig. 6b), as the active sites involved Asn 361, Lys 412, Tyr 414, Asp 408, Asp 409, and
3.7. Use of dextranase to control dental caries 3.7.1. Inhibition and clearance effects of dextranase on biofilm formation As shown in Fig. 7, increased concentrations of dextranase resulted in decreased growth rates of S. mutans. Dextranase was shown to inhibit
Fig. 5. Effects of temperature and pH on dextranase before and after mutagenesis. (a) Effect of temperature on the activity (round) and stability (square) of dextranase from Chaetomium globosum. (b) Effect of pH on the activity (round) and stability (square) of dextranase from C. globosum M101. 121
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Fig. 6. Three-dimensional modeling and molecular docking with isomaltose of dextranase from wild-type Chaetomium globosum (a) and C. globosum M101 (b).
biofilm inhibition by dextranase, and inhibition and clearance tend to be stable when the concentration is increased beyond this range. The results reported here are similar to those reported when using dextranase to treat Arthrobacter oxydans [18] and Arthrobacter sp. biofilms [34]. 3.7.2. Electron microscopic observation of the effects of dextranase on biofilm clearance The clearing effect of dextranase on biofilm is shown in Fig. 8. The biofilm that formed when 5% sucrose was used as the substrate was denser than the biofilm formed when using 1% sucrose. The efficiency of S. mutans biofilm degradation increased with increasing concentrations of dextranase. This result was similar to those reported by Jiao et al. [34] and Walker et al. [35]. At a dextranase concentration of 20 U/mL, the biofilms were significantly degraded, and the inhibition rate reached more than 90% when the dextranase concentration was increased. The dextranase tested in the present study more effectively inhibited biofilm formation than a previously characterized marinebacteria-derived dextranase [34]. These results indicate that dextranase produced by C. globosum M101 effectively inhibited biofilm formation.
Fig. 7. Inhibition and removal of Streptococcus mutans biofilm by dextranase from Chaetomium globosum. Effect of dextranase on the growth of Streptococcus mutans (bar) and the inhibition and reduction of Streptococcus mutans biofilm (line).
4. Conclusions and remove biofilms of S. mutans. These results are consistent with those reported by Jiao et al. [34] and Walker et al. [35] in studies of the prevention and treatment of dental caries using dextranase from the marine bacteria and Lipomyces starkeyi. These studies showed that biofilm formation was inhibited by dextranase in a dose-dependent manner. In the present study, at 50 U/mL, the rate of inhibition of S. mutans biofilm formation by dextranase was 71.58%, while the clearance rate was 49.07%. The inhibition and clearance rate slightly decreased with increasing dextranase concentrations, but the difference was not significant. There may be an optimal concentration range for
This study provides a promising strategy for obtaining C. globosum strains with a high yield of dextranase by combining ARTP and EMS mutagenesis methods. The fatality rate of C. globosum M101, generated using the combined ARTP-EMS mutagenesis system, was evaluated to determine the optimum mutagenesis treatment time and concentration. Under the optimum conditions of combined ARTP-EMS mutagenesis, a strain with a high yield of dextranase was obtained. This mutagenesis method served was an efficient strategy for generating high-dextranaseproducing C. globosum strains. The temperature and pH stability of the enzyme slightly differed before and after mutagenesis. The mutant 122
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Fig. 8. Electron microscopic observation of the effect of dextranase on biofilm clearance. The effect of different concentrations of dextranase on the biofilm formed by 1% and 5% sucrose were used as the substrate, respectively.
enzyme was found to be more heat stable than the wild-type enzyme, which may be because of a change in the secondary structure of the enzyme caused by amino acid changes. However, dextranase was stable at various temperatures and pH conditions before and after mutagenesis. The ability of dextranase to inhibit S. mutans indicates that it may be an effective treatment for inhibiting and removing plaque biofilms. The toxicity of dextranase towards microorganisms was also demonstrated in a safety test. Most importantly, these results provide an important basis for further scientific research of the industrial application of dextranase.
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