Xylanase from thermotolerant Bacillus haynesii strain, synthesis, characterization, optimization using Box-Behnken Design, and biobleaching activity

Nutrient agar plate, Logule’s iodine solution, dialysis bag, Sephadex G-200 (Sigma Chemicals, Cairo, Egypt), Xylan powder, 3, 5 dinitrosallicylic acid, NaOH, sodium potassium tartrate, and ammonium sulfate (El-Noor Company for Chemicals, in Qasr Al-Aini, Cairo, Egypt).

In the current study, water samples were collected from Ain-Helwan Spring, Helwan, Cairo, Egypt (GPS N 29° 51′, E 31° 19′) in sterilized falcon tubes. The pH (6–7) and temperature (35–40 °C) of the collected water samples were measured upon collection. The collected water samples were transferred directly to laboratory for bacterial isolation. Approximately, 200 µL of each sample was spread over the surface of nutrient agar plates (contained g/L beef extract 3.0, peptone 5.0, NaCl 0.5, agar 20.0, and distilled H₂O 1 L; pH = 7) and incubated at 45 °C for 24 h. The different bacterial colonies that appeared at the end of incubation period were picked up and re-inoculated onto new plate to check the purity. The purified bacterial isolates were inoculated on nutrient agar slants and preserved at −4 °C for further study.

The efficacy of the purified bacterial isolates to secretion of xylanase enzymes was screened on mineral salt media (contained g/L NaNO₃, 5; KH₂PO₄, 1; K₂HPO₄, 2, MgSO₄.7H₂O, 0.5; KCl, 0.1; CaCl₂, 0.01; FeSO₄.7H₂O, 0.02; agar, 17, distilled H₂O, 1 L; pH = 7) supplemented with 2 g, w/v xylan powder, and sterilized at 120 °C (1.5 psi) for 20 min [18], pouring the sterilized media onto Petri-dishes under aseptic conditions, left to solidify, inoculated with bacterial strains, and incubated for 24 h at 45 °C. At the end of incubation period, the inoculated plates were flooded with Logule’s iodine solution, and the results were recorded as a diameter of clear zone around bacterial growth which indicate the successful xylanase production.

The most potent bacterial isolate (high xylanase enzyme producers) was subjected to morphological, physiological, and biochemical identification according to standard keys [19]. The molecular identification by amplification and sequencing of 16S rRNA gene was accomplished to confirm the traditional identification. The 16S rDNA gene was amplified using universal primers of 27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492r (5′-GGTTACCTTGTTACGACTT-3′) [20]. The obtained sequence was compared with those deposited in GeneBank database by ClustalX 1.8 software package (http://​www-igbmc.​u-strasbg.​fr/​BioInfo/​clustalx). The phylogenetic tree was drawn by the neighbor-joining method using MEGA (Version 6.1) software. The confidence level of each branch (1000 repeats) was tested by bootstrap analysis.

The production of xylanase enzyme was achieved by mineral salt broth media supplemented with xylan (2 g/L, w/v). The prepared broth media were inoculated by bacterial culture (1 %, v/v) and incubated for 24 h at 45 °C under 200 rpm shaking condition. Before assay, the inoculated media were centrifuged at 5000 rpm for 10 min at 4 °C to obtain the supernatant (crude enzyme) which used to xylanase enzyme assay [17].

The quantitative activity of xylanase enzyme was achieved by detecting the amount of reducing sugars from xylan substrate using the dinitrosalcylic acid (DNS) method [21]. The DNS assay was carried out as follows: 0.2 mL of culture filtrate was mixed with 1 mL of 1 %, w/v xylan (prepared in phosphate buffer, pH 7) in a test tube, and incubated at 45 °C for 30 min. At the end of incubation period, 1 mL of DNS reagent was added, and the mixture was subjected to boiling at 100 °C for 5 min. After cooling, the color intensity was measured at 540 nm. The amount of reducing sugars released was measured using glucose as xylanase activity standard. One unit of xylanase enzyme activity was defined as the amount of enzyme that released 1µmol of reducing sugar per minute. The total soluble protein was determined by Bradford method [22]. This procedure depends upon using the protein reagent Coomassie Brilliant Blue (CBB) G-250 prepared by dissolving the CBB G-250 in 50mL of 95% ethanol. A total of 100mL of o-phosphoric acid was added later and the whole reagent was diluted to 200 mL to make 5× concentrated dye, where the CBB G-250 bind to protein and the color of reaction changes from light green to blue. The absorbance was read off a spectrophotometer at wavelength of 595 nm.

Different environmental conditions were investigated to detect the best condition for xylanase enzyme by the most potent bacterial strain. To achieve this goal, the mineral salt broth media supplemented with xylene were prepared, autoclaved, and inoculated by 2 % v/v of overnight bacterial culture. The analyzed environmental conditions were incubation period (6, 12, 18, 24, and 30 h), different temperatures (30, 35, 40, 45, and 50 °C), different substrate concentration (1, 2, 3, and 4 g/L, w/v), different pH values (6, 7, 8, 9, and 10), different inoculums size (1, 2, 3, and 4 %, v/v), and different nitrogen sources (ammonium nitrate, ammonium chloride, ammonium hydrogen orthophosphate, beef extract, urea, and peptone). Each experiment was achieved in triplicate. At the end of incubation period, 2 mL of inoculated media was withdrawn and centrifuged at 5000 rpm for 10 min at 4 °C to collect a clear supernatant which was used after that to detect enzyme activity using DNS method [23].

The Box-Behnken design (BBD) was used for major factors determining which influence on xylanase production. The factors and their levels are found in Table 1 used in BBD for xylanase production. Where these factors of basal medium were six numerical factors, the factors include temperature, pH, inoculum size, substrate concentration (xylan), incubation period, and different nitrogen source (peptone). Fifty-four experimental trials comprised as the experimental design, where all runs involve three levels (−1, 0, +1) as shown in Table 1. In the BBD, three levels were used for maximum xylanase production determining what was obtained at −1 or +1 of the variables by comparing them with the experimental results obtained from 0 point values. According to this rule R=n+1, a 54 run experiment was generated, where R is the run numbers and n is the number of variables. The Box-Behnken experimental design was achieved based on the following first-order model:

where Y represents the response (xylanase activity U/mL), β0 is the model intercept, Bi is the linear coefficient, Xi is the level of independent variable, and k is the number of involved variables.

The crude enzyme (supernatant collected from inoculated media after centrifugation) was mixed with ammonium sulfate (60 %) which added drop-wisely under stirring conditions till saturation [24]. The previous mixture was stored in cold conditions for 3 h followed by centrifugation at 10,000 rpm for 30 min and the residue was collected. The collected final product was dissolved in phosphate buffer (pH 7.0) to form turbid suspension which subjected to centrifugation for 10 min at 10,000 rpm to form clear supernatant which was undergone to more purification by dialysis bag and column chromatography.

The crude extract from ammonium sulfate step was put into dialysis bag against distilled water for 3 h, followed by transfer to phosphate buffer at pH 7. The obtained xylanase was concentrated by putting dialysis bag against sucrose crystals and kept in the refrigerator at 4 °C for further purification.

By using phosphate buffer, a sephadex G-100 column (1.5× 50 cm) was equilibrated. After purification with dialysis bag, the concentrated xylanase fraction was loaded onto the column and chromatographed by using phosphate buffer subsequently. At a flow rate, 5 mL/h fractions of 5 mL were collected and the active fractions pooled [24].

In this study, the molecular weight of the purified enzyme was detected by an Advion compact mass spectrometer (CMS) provided with TLC reader. First, make adjustment parameters with mode of fragmentation: typical. Its mass ranges from 100 to 1200, with mass type (ESI). NB standard sulphadiazine (M. Wt = 250) was injected to assure the quality of analysis.

Standard preparation: where the stock solution contains 18 amino acids (aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histadine, lysine, ammonia, arginine), all amino acid concentration are 2.5 µMol/mL, except cystine 1.25 µMol/mL, then dilute 60 µL in a 1.5-mL vial with sample dilution buffer then filtered using a 0.22-µm syringe filter then 100 µL was injected. Sample preparation: 1gm of sample was mixed with 5mL hexane. The mixture was allowed to macerate for 24 h. Then, the mixture was filtered on Whatman No. 1 filter paper and the residue was transferred into a test tube where it was incubated in an oven with 10 mL 6N HCl for 24h at 110°C. After the incubation, the sample was filtered on Whatman No. 1 filter paper, evaporated on rotary evaporator, and dissolved completely in 20-mL dilution buffer, filtered using 0.22 µm syringe filter, and 100 µL was injected. Xylanase amino acids were identified by Sykam Amino Acid Analyzer (Sykam GmbH, Germany) equipped with Solvent Delivery System S 2100 (quaternary pump with flow range 0.01 to 10.00 mL/min and maximum pressure up to 400 bar), Autosampler S 5200, Amino Acid Reaction Module S4300 (with built-in dual filter photometer between 440 and 570 nm with constant signal output and signal summary option), and Refrigerated Reagent Organizer S 4130.

Whatman filter paper was treated with 3 mL of xylanase enzyme and incubated under optimal conditions at pH 7, temperature 45 °C, for 1.5 h. A similar type of Whitman filter paper treated with distilled water instead of enzyme was running as a control. After incubation, the treated samples and control investigated by testing machines instruments (TMI) device for softness measurement, and Techno bright device for measuring brightness and darkness [25].

Totally, 40 bacterial isolates were obtained from collected water samples and showed high efficacy to grown at 45 °C. Among these isolates, 21 (52 %) bacterial isolates have the efficacy to produce xylanase enzyme detected by rapid qualitative agar plate after flooding with Logule’s iodine solution (Table 2). The bacterial isolate designated as K6 was selected based on high producing of xylanase enzyme and kept for further study. Compatible with our study, out of 257 isolates obtained from soil samples, 112 isolates represented by 44 % of total bacterial isolates showed xylanase enzyme activity detected by agar plate method. Among 112 isolates, 19 isolates were selected based on highest clear zone formed around their growth [26]. Also, out of 6 isolates, one isolate designated as Bact-1 was xylanase producer detected by agar plate methods [27]. In the current study, the isolate K6 was subjected to morphological, physiological, and biochemical identification. Data showed that the bacterial strain was Gram-positive, bacilli, spore former, facultative anaerobic, the growth was inhibited at 20 °C, and weakly growth was observed at 55–60 °C. The bacterial strain has the efficacy to fermenting different sugars including glucose, galactose, fructose, xylose, arabinose, maltose, mannose, and starch forming acid and gas, whereas lack the potentiality to ferment mannitol and lactose. The physiological tests including methyl red, Vogues-Proskauer, citrate utilization, and nitrate reduction were positive. Based on the above results, the bacterial strain was identified as Bacillus haynesii [19]. The traditional identification was confirmed by amplification and sequencing of 16S rRNA gene. Data showed that the strain K6 has a similarity percentage of 99.8 % with bacterial strain Bacillus haynesii (accession number NR157609). The obtained sequence showed that the bacterial strain K6 was clustered in the group of B. haynesii. Therefore, the most potent bacterial strain in the current study was identified as B. haynesii strain K6 (Fig. 1). The obtained sequence in the current study was registered in GenBank under accession number of OM469329. Xylanases are synthesized by different microbial strains such as bacteria, fungi, actinomycetes, yeasts, and algae. The production of xylanase enzymes using different microbial strains showed varying biochemical characteristics which enables it to integrate into various biotechnological and industrial applications [28]. Although the fungal xylanases have taken more attention because of their high activity, the restrictions that faced the large scale limited their productions and this paved the way for bacterial xylanase production [29]. Various bacterial species such as Bacillus, Micrococcus, Paenibacillus, Rhodothermus, Microbacterium, Arthrobacter, Staphylococcus, Anoxybacillus, and Pseudoxanthomonas have been reported as xylanase producers [24, 29, 30]. Bacillus are considered the most bacterial strains for xylanase production. Various Bacillus spp. such as Bacillus stearothermophilus, Bacillus circulans, Bacillus halodurans, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus safensis, Bacillus altitudinis, and Bacillus pumilus have been showed xylanolytic activity [23, 29, 31, 32]. To the best of our knowledge, this is the first report for production of xylanase using thermotolerant B. haynesii.

Although choosing the right strain is important for a successful industrial enzyme factory, it will not ensure maximal enzyme output until the production process is tuned properly. The kind of fermentation technique employed, the temperature, the pH of the production medium, the nutrition supply provided, and the length of fermentation are all aspects that determine the adequacy of a fermentation process. For reaching the optimum amount of enzyme production, proper exploration and adjustment of these factors are critical [33]. In the current study, the maximum xylanase production using Bacillus haynesii was achieved at pH 7.0 (Fig. 2A). The ambient pH has a significant impact on ionization and the transport of nutritional components across the cell membrane; hence, enzyme synthesis is largely reliant on it. As a result, their accessibility to the bacteria cell and enzymatic activities is reduced, which has an impact on cell proliferation and product creation. As shown, the enzyme activity at different pH values was statistically not significant. These findings contrast with those of Limkar et al. [34], who demonstrated that Bacillus spp. produces the most xylanase at pH 8.5. Bacterial xylanases have a pH that is neutral or alkaline, making them suitable for a wide range of industrial applications. The optimal temperature for xylanase synthesis varies per organism [35]. At different temperatures, such as 30, 35, 40, 45, and 50 °C, the effect of temperature on production of xylanase was investigated. The greatest production of xylanase by B. haynesii strain K6 was found at 40 °C with productivity of 11.17 U/mL (Fig. 2B). Temperatures above or below 40 °C signify a decrease in xylanase production; the most powerful strain’s xylanase enzyme is a thermotolerant enzyme. Similarly, the maximum xylanase productivity was attained at temperature of 35 °C by Bacillus subtilis and 40 °C by Bacillus megaterium [36]. For enhancing xylanase production using B. haynesii K6, different inoculum sizes (1–4 %) were tested as shown in Fig. 2C. The maximum xylanase output occurred when the inoculum size was 1 %, and the enzyme production decreased as the inoculum size increased. Bacillus subtilis and Bacillus megaterium exhibited highest xylanase activity at inoculum size of 2 % and 1.5 %, respectively [36]. The findings demonstrated that when the inoculum size was increased by more than 1%, the rate of xylanase enzyme production decreased. Also, the highest xylanase synthesis was accomplished at 2 % inoculum size of Bacillus mojavensis in submerged fermentation [37]. As shown in Figure (2D), the maximum xylanase productivity using B. haynesii strain was 18.63 U/mL and was achieved by adding 3 g/L xylan in fermentative media. Substrate concentrations below and above this value showed decreasing gradually as compared to the optimal one. Incompatible with the current study, the highest xylanase activity (7.3 to 8.4 U/mL) by Bacillus pumilus was attained in broth media supplemented with 0.5 to 1 % xylan, and the amount of xylanase activity decreased as the substrate concentration increased or decreased [38]. The effect of incubation period was studied on the xylanase production as displayed in Figure (2E). Data analysis showed that the maximum xylanase activity (26.27 U/mL) from Bacillus haynesii strain K6 was achieved after 24 h of incubation. The xylanase activity decreased when the incubation time became longer or less than 24 h. The obtained data completely agreed with those reported by Rosli, et al., who reported that the maximum xylanase activity (10.86 U/mL) by Bacillus tequilensis was achieved after 24 h of incubation period [39]. In the current study, the efficacy of extra-inorganic and organic nitrogen source on xylanase activity was investigated. Analysis of variance showed that the maximum xylanase activity (29.57 U/mL) was achieved in presence of peptone, whereas the enzyme activity was decreased in presence of other nitrogen sources (Fig. 2F). In a similar study, the xylanolytic activity of Bacillus sp. was the highest value (7.896 U/mL) in the presence of beef extract in fermentative media [38].

In this study, the BBD of response surface methodology for studying the effect of six factors with six levels affecting on xylanase production was investigated. The six factors were temperature, pH, peptone, inoculum size, xylan, and incubation period. As shown in Table 3 and Fig. 3, the xylanase activity ranged from 17.63 to 35.02 U/mL in all runs. Run No. 17 provided the maximum xylanase activity with values of 35.02 U/mL, where these factors were 40 °C, pH 7, 5 g/L peptone as a best concentration of the best nitrogen source, 1 % inoculum size, 3 g/L xylan concentration, and 24 h of incubation period. Naz et al. [40] reported that the maximum xylanase activity was 175.6945 U/mL in presence of 3 % corncob concentration, 0.05 % peptone, and 0.5 % of KH₂PO₄. The constructed experimental model is highly significant and accurately portrays the real connection between the impacts of factors and enzyme response linked to xylanase activity, according to the findings of ANOVA obtained from the current investigation. The expected vs. actual plots and normal residual plots of xylanase activity of the xylanolytic bacterial strain B. haynesii showed more stability in the residual plot (Fig. 4). The distribution of response variable from several experimental circumstances revealed that most of the components contributed equally. The likelihood graphs also showed that the expected and real xylanase activities were quite comparable.

Under optimum broth-state fermentation conditions, xylanase enzyme was produced using B. haynesii strain. At the end of experiment, different concentration of (NH₄)₂SO₄ (10–80 %) was used to precipitate the proteins. The obtained data showed that the precipitation of protein was increased by increasing the (NH₄)₂SO₄ concentration, whereas the enzyme activity increased up to 60 % (NH₄)₂SO₄. The crude enzyme precipitated with saturation of 60 % (NH₄)₂SO₄ and showed highest enzyme activity (58.62 U/mL) as compared with those precipitated with different (NH₄)₂SO₄ concentrations (Fig. 5). The obtained data are compatible with those reported by Kapilan [41], who investigate the efficacy of different concentration of (NH₄)₂SO₄ (10–70 %) in protein precipitation and xylanase enzyme activity. Who reported that the protein precipitation was increased by increasing the concentration of (NH₄)₂SO₄, whereas the highest xylanase enzyme activity (33.16 U/mg protein) was increased up to 50 % (NH₄)₂SO₄. In the current study, the xylanase enzyme was subjected to dialysis against sucrose and the result showed that the specific activity after dialysis was 27.9 U/mg as shown in (Table 4). The concentrated enzyme from dialysis process was loaded on sephadex G-100 column and receives 10 fractions, each one containing 5 mL of purified xylanase enzyme and estimated the xylanase activity, protein content, and specific enzyme activity in each fraction (Table 5). Data showed that the highest enzyme activity was attained in fraction number 6 with enzyme activity, protein content, and the specific enzyme activity values of 93.23 U/mL, 0.48 mg/mL, and 204.65 U/mg, respectively (Table 5). According to the obtained data, the xylanase enzyme activity was varied due to purification steps. The obtained finding was in agreement with those reported that the specific activities of xylanase enzyme synthesized by Paenibacillus macquariensis were 3.2 U/mg after precipitation with 30 to 60 % (NH₄)₂SO₄, 14.8 U/mg after DEAE-cellulose chromatography purification, and 25.2 U/mg after passage through sephadex G-100 chromatography [42].

In this study, the molecular weight of xylanase enzyme produced by B. haynesii strain K6 was 439 KDa (Fig. 6A). The molecular weight of xylanase enzyme synthesized by Thermoanaerobacterium sp. was 350 KDa [43], whereas those synthesized by Paenibacillus macerans have a molecular weight of 205 KDa [44]. Other study reported that the molecular weight of xylanase enzyme produced by B. subtilis was 340 KDa [45]. The amino acid sequencing of xylanase enzyme produced by B. haynesii strain K6 is represented in Fig. 6B. Data analysis showed the xylanase enzyme under study containing 15 amino acids which were aspartic acid followed by threonine, serine, glutamic, proline, glycine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenyalanine, histidine, and lysine. The results showed that the highest values were 1940 and 1520 mg/L for proline and cysteine, respectively, while the lowest value for valine, tyrosine, and histidine was 40 mg/L. Similarly, Dutta et al. [46] found that the amino acid composition of xylanase enzyme synthesized by different bacterial species were theronine (9.5 %), followed by glycine (8.8 %), alanine (8.2 %), serine (7.9 %), and aspartic acid (6.54 %).

The xylanase enzymes used in bio-bleaching process should be characterized by active alkaline pH, high temperature, and do not contain a cellulolytic enzyme to avoid cellulose fiber degradation [47]. The researchers were interested in using xylanase enzyme in the bleaching process since it helps to reduce the consumption of chlorine compounds [48, 49]. Xylanase produced by Bacillus pumilus ASH5 was used in bio-bleaching of kraft pulp to improve its whiteness and brightness and hence reduce the usage of chlorine and chlorine dioxide with percentages of 20 % and 10 %, respectively [50]. In the current study, the thermotolerant xylanase enzyme produced by B. haynesii K6 was used to improve the properties of wastepaper. Data showed that the whiteness of wastepaper was enhanced after treatment with xylanase enzyme as compared with untreated sample (control). The whiteness of untreated samples was 48 % and improved after treatment with xylanase up to 64.5%. Interestingly, the darkness of papers after treatment with xylanase tend to highly decrease (96.4%) as compared to control (86.9%) (Table 6). It is clear from the obtained results of bio-bleaching of wastepaper with xylanase enzyme that the physical properties of the treated paper are improved compared with untreated (Table 6). Similarly, the xylanase enzyme demonstrated a remarkable brightness of up to 13 % for the paper pulp using the bio-bleaching approach in a prior study [31]. Xylanase enzymes are helping in breakdown of xylan that linked with lignin and cellulose of the pulp and hence improve the separation of these components that enhance fiber wall swelling and improve extraction of lignin from the treated pulp [51]. The treatment of cellulosic fibers with xylanase enzyme is a useful tool to increase paper strength through breakdown of xylan and lignin removal [52]. Therefore, the pretreatment of paper pulp with xylanase enzyme has been more effective, high selective, eco-friendly, reduces the use of hazard chemicals, and non-toxic method for bio-bleaching [28].