Purification, characterization, immobilization and applications of an enzybiotic β-1,3–1,4-glucanase produced from halotolerant marine Halomonas meridiana ES021

Abstract Extracellular β-1,3–1,4-glucanase-producing strain Halomonas meridiana ES021 was isolated from Gabal El-Zeit off shore, Red Sea, Egypt. The Extracellular enzyme was partially purified by precipitation with 75% acetone followed by anion exchange chromatography on DEAE-cellulose, where a single protein band was determined with molecular mass of approximately 72 kDa. The Km value was 0.62 mg β-1,3–1,4-glucan/mL and Vmax value was 7936 U/mg protein. The maximum activity for the purified enzyme was observed at 40 °C, pH 5.0, and after 10 min of the reaction. β-1,3–1,4-glucanase showed strong antibacterial effect against Bacillus subtilis, Streptococcus agalactiae and Vibrio damsela. It also showed antifungal effect against Penicillium sp. followed by Aspergillus niger. No toxicity was observed when tested on Artemia salina. Semi-purified β-1,3–1,4-glucanase was noticed to be effective in clarification of different juices at different pH values and different time intervals. The maximum clarification yields were 51.61% and 66.67% on mango juice at 40 °C and pH 5.3 for 2 and 4 h, respectively. To our knowledge, this is the first report of β-1,3–1,4-glucanase enzyme from halotolerant Halomonas species. Graphic Abstract

Using immobilized enzymes in chemical, pharmaceutical and food industries has become a routine process with high substrate specificity, high catalytic activity, and mild optimal reaction conditions (Basso and Serban 2019;Cao et al. 2016). The most significant advantages of enzyme immobilization are reusability and the simplicity with which it can be separated (Cho et al. 2018). Furthermore, the remaining enzyme amount left in the product is reduced, and enzyme immobilization allows enzymes to be reused multiple times, lowering enzyme consumption costs (Sheldon 2007). Furthermore, enzyme immobilization frequently results in increased heat stability or resistance to mechanical inactivation (Tu et al. 2006).
Enzybiotics are a new class of anti-microbials based on enzymes that may present a solution to this global demand. The term enzybiotics is a hybrid from the two words enzyme and antibiotic (Wu et al. 2012). Enzybiotics are a solution for antibiotic resistance problem using natural antimicrobial enzymes or entire bacteriophages to inhibit pathogenic bacteria or fungi growth (Veiga-Crespo and Villa 2009). A study by Jin et al. (2011) showed that β-1,3-1,4-glucanase from endophytic Bacillus subtilis could be a desirable vital agent against microbial pathogens with higher efficiency and lower toxicity, they proved that the bacterial enzyme had a broad antimicrobial spectrum against fungal and bacterial strains. Antifungal effect of β-1,3-1,4-glucanase was also reported by Dewi et al. (2016), Xu et al. (2016), Zalila-Kolsi et al. (2018) and Yuan et al. (2020).
Fruit juices are cloudy in different degrees because they contain polysaccharides (pectin, lignin, cellulose, hemicelluloses and starch), proteins and some metals (Vaillant et al. 2001). So, enzymes play a key role in the production of fruit. Their main goals are increasing juice extraction from raw materials, producing a clear and visually appealing finished product and improving processing efficiency as solid settling, pressing, or removal (Sharma et al. 2014).
In this report, purification, immobilization and characterization of the extracellular β-1,3-1,4-glucanase from the halotolerant marine isolate Halomonas meridiana will be described, seeking for potent features and proposing for multiple applications with highly efficient mode of action. Moreover, we aimed to scan its enzybiotic activity against a broad spectrum of microbial strains.

Purification of extracellular β-1,3-1,4-glucanase produced by Halomonas meridiana ES021
The culture broth was centrifuged at 5000 rpm for 10 min in a cooling centrifuge at 4 °C. 75% acetone concentration was used to precipitate the protein content of supernatant of centrifuged Halomonas meridiana ES021 cultures. The fraction was dialyzed against distilled water (Niu et al. 2016b). The resulting protein fraction was introduced to DEAE-Cellulose A-52 column (28 × 1.8 cm), which was equilibrated with 0.05 M tris-HCl buffer pH 8.0. The gradual elution of the protein was done using 0.05 M tris-HCl buffer pH 8.0 followed by different molarities (0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 M) of NaCl dissolved in the same buffer. The eluent was obtained in 3.0 mL fractions collected at a flow rate of 1.0 mL/min adjusted with a peristaltic pump.

Protein electrophoresis
According to the described protocol steps of Laemmli (1970), Purified protein was detected after running on SDS-PAGE (8%) (Huang et al. 2022).

Characterization of purified extracellular β-1,3-1,4-glucanase
To determine the optimal substrate concentration, different substrate concentrations varying from 0.1 to 3.0 mg/ mL were added to 100 µL equivalent to 12 µg of purified enzyme solution. Lineweaver and Burk (1934) method was applied for kinetic parameters determination providing the Michaelis-Menten equation: where the Michaelis-Menten constant (K m ) and maximal velocity (V max ) were calculated upon the equation symbols V 1 , [S], K m and V max that were identified as the reaction velocity, the substrate concentration, the substrate concentration at half-maximal velocity, and the maximal velocity, respectively. To determine the optimal temperature for the β-1,3-1,4-glucanase activity, enzyme reactions were carried out at different temperatures ranging from 30 to 70 °C for 10 min. The pH dependence of β-1,3-1,4-glucanase activity was determined, the range of studied pHs were from 3.0 to 8.0 (Lim et al. 2022).
Thermal stability of the enzyme was evaluated by preheating certain portions of the enzyme preparation without substrate separately at different temperatures (40, 50, 60 and 70 °C), for various time periods (15, 30 and 60 min), respectively. The optimal incubation period for β-1,3-1,4glucanase activity was determined by carrying out the reaction at different time intervals ranging from 5 till 80 min. Without adding the substrate, different NaCl concentrations ranging from 10 to 90 ppt were exposed to the enzyme to evaluate the effect of salinity.
Immobilization of semi-purified β-1,3-1,4-glucanase by entrapment 0.1 g agar or agarose was dissolved in 10 mL distilled water to form 1% agar or agarose gel. 2.0 mL of each gel material were mixed with 1.0 mL of semi-purified β-1,3-1,4-glucanase solution, then poured into a Petri-dish. After gel solidification, equal cubes cut with 0.5 cm diameter using sterile cutter were applied to the reaction after washing several times to get rid of any unbound enzyme. Ca-alginate gel was prepared by stirring 0.1 g Na-alginate in 10 mL distilled water to form 1% Naalginate gel. Kappa-carrageenan gel was prepared by dissolving 0.1 g kappa-carrageenan in 10 mL distilled water to form 1% kappa-carrageenan gel. The entrapment was structured by adding the mixture composed of 2.0 mL of each gel material mixed with 1.0 mL of semi-purified β-1,3-1,4-glucanase enzyme solution, through a syringe into 50 mL of 2% calcium chloride solution (for Ca-alginate) or 2% potassium chloride solution (for kappa-carrageenan) and left for 2 h. The beads formation of 1.0-1.5 mm diameter were collected and used in the reaction after washing several times to remove the unbound enzyme.

Immobilization of semi-purified β-1,3-1,4-glucanase by covalent bonding
The gel was prepared by dissolving 0.5 g chitosan in 15 mL acetic acid (2.5%) then dropped through a syringe into 100 mL sodium hydroxide solution (1.5%) to form beads and left for 1 h. The resulting beads were divided into 3 portions (1.0 g each), added to different concentrations of glutaraldehyde solution (1, 3 and 5%) and left overnight at ambient temperature. After that, the beads were rinsed with distilled water, 0.5 g of each portion was mixed with 1.0 mL semi-purified β-1,3-1,4glucanase enzyme solution and left at ambient temperature overnight. After equilibration, glutaraldehyde was decanted and beads were used in reaction after washing several times.

Assay of immobilized enzyme
The immobilized enzyme was incubated with 1.0 mL of 0.2% barley β-glucan dissolved in 0.1 M phosphate buffer pH 6.0 for 10 min. After the desired time of incubation, the reaction solution was separated from the immobilized enzyme; the enzyme activities of unbound and immobilized enzyme were assayed using DNS method described by Miller (1959).

Graphing and statistical analysis
Excel software was used for graphing and expressing results as mean ± SD.

Applications of extracellular β-1,3-1,4-glucanase enzyme produced by Halomonas meridiana ES021 cultures
Enzybiotic activity of purified β-1,3-1,4-glucanase Antimicrobial susceptibility testing was performed with agar well diffusion method. Nutrient agar plates for bacterial pathogens, and potato dextrose agar plates for fungal pathogens were inoculated by the microbial pathogens using pour plate technique. 25 µg (200 µL) of enzyme protein solution was introduced into the well and allowed to diffuse in agar media before incubation (Jin et al. 2011). The bioassay plates were incubated overnight at 37 °C for bacteria and for 24 and 48 h at 30 °C for fungi to measure the diameter of the inhibition zone (mm) to evaluate antimicrobial effect.

Juice clarification by semi-purified β-1,3-1,4-glucanase
Juice clarification was carried out by adding 25 mg enzyme protein to 5.0 mL of each fruit juice in different sets and control was done (1.0 mL of distilled water was added to each of the fruit juice). Reaction mixtures were incubated for 2 and 4 h at 40 °C. Transmittance of sample is determined at 650 nm.
Juice clarification in percent (%) was calculated by formula: (%) Clarification = (T t -T c /T c ) × 100, where, T t is transmittance of test and T c is transmittance of control (Kothari et al. 2013).

Toxicity of purified β-1,3-1,4-glucanase
The test was performed using Artemia salina larvae (Obtained from NIOF, Alexandria, Egypt). An artificial saline solution was prepared using sea water and distilled water in ratio 1:2, 10 of Artemia salina larvae were added to enzyme protein solutions at different concentrations (20-120 µg/mL) prepared by diluting the extract in artificial saline solution. The larvae were incubated with the saline solution in negative control (without added enzyme) (Meyer et al. 1982;Cavalcante et al. 2019). The assay was maintained under artificial lighting with aeration at 27 ± 2 °C and mortality rates were determined. After 24 and 48 h the number of dead and live larvae in each vial was counted and the probability of mortality was calculated according to the formula: Mortality probability (%) = (r/n) × 100, where, r is number of dead larvae and n is total number of Artemia salina in each vial.

Anion exchange chromatography on DEAE-cellulose
The crude enzyme was first partially purified by fractional precipitation with 75% acetone fraction, which yielded the highest specific activity (1425.60 U/mg protein) hitting 2.39fold higher than that obtained from the crude enzyme (data not shown).
A certain weight 8.12 mg protein of the partially purified enzyme was dissolved in 0.05 M tris-HCl buffer pH 8.0 and was loaded on DEAE-cellulose A-52 column that was equilibrated with the same buffer. The eluted fractions were 70 fractions as shown in Fig. 1, where about 58.6% of the applied enzyme protein was recovered by the eluting solutions and was separated to two protein components. The first protein component was the major one; it was covered by fractions from 1 to 10 representing about 77.52% of the total recovered protein. The second protein peak was a minor one and was covered by fractions from 22 to 26 representing about 22.48% of the total recovered protein. The total recovered β-1,3-1,4-glucanase activity from the column was fractionated in the column into 1 peak synchronized with the first protein peak, where it represented about 65.28% of the original activity. 3.4 mg of the purified enzyme was obtained with specific activity reached about 2409 ± 15.54 U/mg and a purification of 3.87-fold of the crude enzyme and a recovery yield about 5.96 ± 0.12%. A summary of the purification steps of β-1,3-1,4-glucanase enzyme is shown in Table 1.  Fig. 1 Purification of the semi-purified β-1,3-1,4-glucanase enzyme using on exchange chromatography on DEAE Cellulose A-52; showed the first protein peak which accompanied with the only activity peak recovered from the column, the second protein peak and no enzyme activity noticed with this peak

Gel electrophoresis
The purity, integrity and molecular weight of the β-1,3-1,4glucanase enzyme was examined by gel electrophoresis. β-1,3-1,4-glucanase enzyme obtained from the ion exchange column gave a single band on SDS-PAGE gel, indicating the purity and integrity of the isolated β-1,3-1,4-glucanase, and the molecular weight of the purified enzyme was estimated to be 72 kDa (Fig. 2).

Optimum conditions
The effect of substrate concentration on the activity of the purified β-1,3-1,4-glucanase enzyme illustrated the correlation between the rate of the reaction and the substrate concentration. The optimum substrate concentration for the pure enzyme was 0.6 mg/reaction mixture giving β-1,3-1,4-glucanase specific activity about 4954 ± 162 U/ mg which was about 2.07-fold of that obtained by initial substrate concentration (Fig. 3). The K m and V max values of the purified enzyme were found to be 0.62 mg β-1,3-1,4-glucan/mL and 1111 U/mL equivalent to specific activity 7936 U/mg protein, respectively (Fig. 4).
The influence of temperature, pH and incubation time of the reaction using a substrate concentration of 0.62 mg/ mL reaction mixture was studied. The optimum temperature was 40 °C with maximum β-1,3-1,4-glucanase activity which was 5131 ± 147 U/mg which was 1.04-fold of that obtained by initial incubation temperature (Fig. 5). At 70 °C, the enzyme activity was 77.28% of that obtained at 40 °C. The optimum pH value was pH 5.0 with β-1,3-1,4glucanase activity equals to 6759 ± 109 U/mg which was about 1.3-fold increase than that obtained by initial pH value (Fig. 6). β-1,3-1,4-glucanase enzyme showed to be a relatively stable at pH range from 4.0 to 6.0. The lowest enzyme activity was observed at pH 8.0 showing about 31.59% decrease of value obtained at pH 5.0. The optimum incubation time of reaction mixture was 10 min with β-1,3-1,4-glucanase activity about 6765 ± 130 U/ mg (Fig. 7). Further increase in incubation period causes gradual slight decrease in enzyme activity.

Thermal stability
Thermal stability of β-1,3-1,4-glucanase enzyme was affected by different temperatures and different periods of exposure. β-1,3-1,4-glucanase enzyme when exposed to 40 °C for up to 15 min lost 7.67% of its activity, while it lost 9.32% and 12.79% of its activity after 30 min and 60 min of exposure, respectively. Also, by increasing the temperature to 50 °C, the enzyme lost 8.04%, 9.32% and 18.44% of its activity after 15, 30 and 60 min of exposure, respectively. A further increase in temperature to 60 °C, it lost 11.24%, 17.16% and 19.35% of its activity after 15, 30 and 60 min of exposure, respectively. At treatment temperature of 70 °C, it lost 12.68%, 22.27% and 25.18% of its activity after 15, 30 and 60 min of exposure, respectively (Fig. 8).

Immobilization by physical adsorption and ionic bonding
The immobilized enzyme prepared by adsorption on chitosan showed the highest immobilization activity reached about 429.5 ± 21.0 U/g carrier and the highest immobilization yield reached about 48.71 ± 1.80% among supports used for physical adsorption followed by the immobilized enzyme prepared by adsorption on chitin (230.4 ± 6.0 U/g carrier) with immobilization yield about 26.73 ± 0.09%. The immobilized enzyme prepared by ionic bonding with DEAE-cellulose showed the highest immobilization activity reached about 455.5 ± 21.0 U/g carrier and the highest immobilization yield reached about 51.79 ± 1.77%, even higher than that obtained by physical adsorption on chitosan. However, the immobilized enzyme prepared by ionic bonding with carboxy methyl cellulose (CMC) showed a lower immobilization activity (275.9 ± 10.8 U/g carrier) with immobilization yield about 31.34 ± 0.50% (Table 2).

Immobilization by entrapment in gel material
The immobilized enzyme prepared by entrapment in agarose gel showed the highest immobilization activity reached about 667.08 ± 16.30 U/g carrier and the highest immobilization yield reached about 50.19 ± 0.09%, followed by the immobilized enzyme prepared by entrapment in agar gel (540.42 ± 10.00 U/g carrier) with immobilization yield about 40.66 ± 0.16%. The immobilized enzyme prepared by entrapment in Ca-alginate beads showed the lowest immobilization activity (19.17 ± 0.83 U/g carrier) and the lowest immobilization yield (1.44 ± 0.03%) ( Table 3).

Immobilization by covalent bonding
The immobilized enzyme prepared by covalent bonding with 1% glutaraldehyde showed the highest immobilization yield reached about 62.09 ± 0.26% followed by the immobilized enzyme prepared by covalent bonding  Fig. 4 Lineweaver-Burk plot for evaluation of kinetic constants (K m and V max ) β-1,3-1,4-glucanase from Halomonas meridiana ES021. Enzyme activity was determined at different β-1,3-1,4-glucan concentrations of 0.1-3.0 mg/ml. V max value of the enzyme was estimated to be 1111 U/mL (7936 U/mg protein) when the K m is 0.62 mg β-1,3-1,4-glucan/mL Effect of incubation temperature on the activity of purified β-1,3-1,4-glucanase from Halomonas meridiana ES021, the enzymatic reaction was carried out at temperature range from 30 to 70 ℃, the enzyme retained more than 80% of its activity in a temperature within this range, and the maximal enzyme activity was reached at 40 ℃ Effect of incubation time on the activity of purified β-1,3-1,4glucanase from Halomonas meridiana ES021, the enzymatic reaction was carried out at 40 ℃ and pH 5.0 for time intervals ranging from 5 to 80 min, and the maximal enzyme activity was achieved after 10 min of incubation, further increase in incubation time causes gradual slight decrease in enzyme activity with 3% glutaraldehyde with immobilization yield about 52.67 ± 0.63%. On the other hand, the immobilized enzyme prepared by covalent bonding with 1% glutaraldehyde showed the lowest immobilization activity (420.4 ± 8.0 U/g carrier) followed by the immobilized enzyme prepared by covalent bonding with 5% glutaraldehyde with immobilization activity about 441.2 ± 11.0 U/g carrier (Table 4).

Enzybiotic activity of purified β-1,3-1,4-glucanase enzyme
The antibacterial and antifungal effects of purified β-1,3-1,4-glucanase were evaluated. The purified enzyme has antimicrobial effect and it could be used as enzybiotic alternative for treating some bacterial and fungal infections. It has a strong antibacterial effect against Bacillus subtilis, Streptococcus agalactiae and Vibrio damsela strains and weak antibacterial effect on Escherichia coli, Enterococcus faecalis and Klebsiella pneumonia. On the other hand, it has no effect on Pseudomonas fluorescence, Aeromonas hydrophilia, Staphylococcus aureus and Pseudomonas aeruginosa. The highest inhibition zone was detected against Vibrio damsela followed by Streptococcus agalactiae and Bacillus subtilis. It also showed high antifungal effect against Penicillium sp. followed by Aspergillus niger. On the other hand, it has no effect on Aspergillus oryzae. Data shown in Table 5 and Fig. 11, 12.

Juice clarification by semi-purified β-1,3-1,4-glucanase enzyme
Clarifying of some fruit juices using purified β-1,3-1,4glucanase was studied. Semi-purified β-1,3-1,4-glucanase was effective in clarification of different juices at different pH values and different time intervals (2 and 4 h). The maximum clarification yield (51.61% and 66.67%) was on mango juice at 40 °C and pH 5.3 for 2 and 4 h, Effect of salinity on the activity of purified β-1,3-1,4glucanase from Halomonas meridiana ES021, the purified enzyme was incubated with different NaCl concentrations ranging from 0 to 90 ppt in absence of its substrate, then the enzyme assay was performed at 40 ℃ and pH 5.0 for 10 min, using a substrate concentration of 0.6 mg/mL reaction mixture. β-1,3-1,4-glucanase is a salt activated enzyme, enzyme activity increased gradually by increasing NaCl concentration and reached the maximum activity at 60 ppt respectively. On the other hand, the lowest clarification yield (20.51%) was on orange juice at 40 °C and pH 4.3 for 2 and 4 h ( Table 6).
Halomonas meridiana ES021 β-1,3-1,4-glucanase is a halotolerant enzyme, it retained more than 90% of its activity after exposure to 90 ppt NaCl. Also, gradual increase in enzyme activity by increasing NaCl concentration indicated that it is a salt activated enzyme similarly to the recombinant enzyme cloned from Paenibacillus sp. S09 (Cheng et al. 2014). At 60 ppt, the enzyme activity increased about 122% and this result was higher than that of fungal β-1,3-1,4-glucanase purified from Aspergillus fumigatus which increased only 113.2% when treated with 5 mM NaCl.
Results obtained by enzyme immobilization on porous silica gel and DEAE-cellulose by adsorption indicate that weak adsorption occurred on both carriers, so the enzyme was eluted by washing process and no immobilization yield detected, while immobilization by adsorption on chitosan and ionic binding on DEAE-Cellulose showed relatively high immobilization yields. Good immobilization yields were also achieved by entrapment in agar and agarose gel materials. This research is the first report for β-1,3-1,4glucanases immobilization using these techniques. Covalent bonding on chitosan using 1% glutaraldehyde as crosslinking reagent was the best immobilization technique with the highest immobilization yield, similar technique was used by Cho et al. (2018) to covalently immobilize Bacillus sp. β-1,3-1,4-glucanase enzyme on porous silica using glutaraldehyde as crosslinking reagent. A recent research covalently immobilized β-1,3-1,4-glucanase from Penicillium occitanis successfully on chitosan-clay composite beads using glutaraldehyde as cross-linking reagent (Chaari et al. 2015).
Purified Halomonas meridiana ES021 β-1,3-1,4glucanase enzyme exhibited an enzybiotic activity against some newly studied pathogens, this activity was varying between bacterial and fungal pathogens. β-1,3-1,4glucanase showed an antibacterial effect against Bacillus subtilis, Escherichia coli and Enterococcus faecalis similarly to Jin et al. (2011), while its activity against Streptococcus agalactiae, Vibrio damsela and Klebsiella pneumonia was first approved in this study. On fungi level, antifungal effect was reported against Penicillium sp. (human pathogenic), Yuan et al. (2020) also reported the antifungal effect of β-1,3-1,4-glucanase against Canidia albicans (human pathogenic yeast). The enzyme also showed a weak antifungal effect against the phytopathogenic Aspergillus niger, the antifungal activity of the enzyme against phytopathogenic fungi was previously reported against Curvularia affinis and Colletotrichum gloeosporioides (Dewi et al. 2016), Cryphonectria parasitica, Cylindrocladium quinqueseptatumI, Helicobasidium purpureum , and Alternaria alternata (Zalila-Kolsi et al. 2018). Juice clarification by semi-purified β-1,3-1,4-glucanase enzyme showed variation in clarification yields of different juices at their different pH values. The obtained results observed that the purified β-1,3-1,4-glucanase enzyme could be used to reduce the cost of juice processes. β-1,3-1,4-glucanase enzyme was first used as a juice clarifying agent in this study, while juice clarification was processed by other type of β-glucanases as cellulase (β-1,4-glucanase) (Pradhan et al. 2021), and by combination of cellulase with pectinase (Al-Hooti et al. 2002;Abbès et al. 2011). In order to ensure the enzyme safety, toxicity of purified β-1,3-1,4-glucanase enzyme was tested on Artemia salina showing no toxicity at different tested concentrations. This result indicated that β-1,3-1,4glucanase could be a safe supplement to be used in food and drug industry and other biotechnological applications, as well as it could be applied as a safe prebiotic substance to enhance the immune system of aquacultures of different species also, prebiotics are known as a substrate for probiotic commensal bacteria and can induce immune response (Pujari and Banerjee 2021).

Conclusion
A novel halotolerant β-1,3-1,4-glucanase was successfully purified from the cultures of marine Halomonas meridiana. This study demonstrated the enzybiotic activity of β-1,3-1,4glucanase against newly studied pathogenic bacterial and fungal strains, and its potential to be used as a natural and safe clarifying agent in juice industry. The thermal stability and the high immobilization efficiency make this enzyme of potential interest in a number of industrial applications. Also, β-1,3-1,4-glucanase has no toxicity on Artemia salina, so it could be safely applied in various industries especially in aquaculture.