Mutation of AN39-1 for production and characterization of constitutive, thermostable and pH-resistant dextransucrase

: Objective: Leuconostoc mesenteroides AN39-1 has recently been isolated from Crataegus orientalis var . Orientalis. It produces inducible extracellular dextransucrase (EC 2.4.1.5) forming dextran from sucrose. The aim of this study was (1) to obtain constitutive, pH-resistant and thermostable dextransucrase, (2) to characterization of these dextransucrase. Methods: Mutagenesis was carried out on the parent strain (AN39-1) using UV, ethyl methane sulfonate, and N- meth-yl-N´-nitro-N-nitrosoguanidine. Dextransucrases from wild type (AN39-1) and the mutant strain (A26-2/11) were purified by polyethylene glycol (PEG) precipitation and characterized. Results: Mutants (A26, A26-2, and A26-2/11) hyper producing and constitutive for dextransucrase were isolated. The mutants (A26, A26-2, A26-2/11) produced 7.2, 8.1, and 2.0 times more dextransucrase activity as compared to parent strain on sucrose medium, respectively. In addition, the mutants produced dextransucrase on glucose medium with higher activities (3.0-5.8 times) than what the paren-tal strain produced on sucrose medium. The mutant enzyme (A26-2/11) was much more thermostable than the native enzyme and resistant to pH more than dextransucrase of AN39-1. The dextransucrase from mutant strain was stable up to 35°C and pH of 7.5 for 3 hr. Conclusion: The structures of dextrans produced by wild type and mutant enzymes were similar to commercially produced B-512 F dextran. Thus, the newly dextransucrases produced by mutant strain could find industrial applications at higher temperature and pH.


Introduction
Dextrans are easily soluble, biodegradable and biocompatible biopolymers comprised by glucose units. There has been incredible increase in the demand of biodegradable biotechnological products including enzymes and biopolymers. Exopolysaccharides produced from lactic acid bacteria (LAB) have gain popularity among industrial sectors due to its generally regarded as safe status (GRAS). LAB and their bio-products are used in several food industries.
Dextrans are polymers composed of glucose units connected by α (1→6) linkages with varying amounts and arrangements of α (1→2), α (1→3), or α (1→4) branch linkages [1]. They are produced from sucrose by dextransucrases (EC 2.4.1.5) which are produced by various species of Leuconostoc, Streptococcus, and Lactobacillus. The dextransucrases from Streptococcus are produced constitutively. However, dextransucrases from Leuconostoc mesenteroides (L. mesenteroides) can only be produced by sucrose induction. Strains of L. mesenteroides that must be induced by sucrose in the culture medium have at least three major problems: Only one half of the carbon source, the fructose part of sucrose, can be utilized for growth. The culture fluid becomes highly viscous during growth due to an increase in the amount of dextran. The enzyme produced has dextran attached due to the presence of dextran produced from sucrose which is substrate for dextran production and an inducer for dextransucrase production. Thus, enzyme production from Leuconostoc mesenteroi-des is accompanied with dextran production to which the enzyme has a high affinity. The enzyme is difficult to purify because of the high viscosity and presence of a large amount of dextran [2]. In order to overcome the problems associated with dextran, several mutation techniques have been used to produce constitutive dextransucrases.
Recently, our research group has isolated a strain from Crataegus orientalis var. Orientalis, (AN39-1: Leuconostoc mesenteroides ssp. mesenteroides; GenBank accession no: GQ280007) producing dextransucrase which catalyzes the formation of dextran from sucrose [8]. The enzyme is produced extracellularly and is inducible by sucrose. The structure of the dextran produced by this enzyme is similar to commercial dextran produced by Lm B-512F dextransucrase. It was also found that the newly produced AN39-1 dextransucrase could carry out dextran synthesis at low temperatures (4°C) better than Lm B-512 F dextransucrase.
The purpose of this study was to find mutant strains of AN 39-1 constitutive for dextransucrase to overcome problems associated with the production of inducible dextransucrase. The present work describes mutagenesis of L. mesenteroides AN39-1 using UV, EMS, and NTG to isolate hyperproducing strains constitutive for dextransucrase. Dextransucrase produced by wild type (AN39-1) and the mutant strain (A26-2/11) were purified and characterized.
Dextranase 50 L, a commercial enzyme produced by submerged fermentation of a selected strain of Penicillium lilacinum, was a gift of Novozyme (Bagsvaerd, Denmark). The activity of the enzyme was 63 IU/ml. The activity unit (IU) is defined as the amount of enzyme forming 1 μmol isomaltooligosaccharide (isomaltose and isomaltotriose) from dextran solution [Mw: 5,000, 2%(w/v)] in 1 min at pH 5.4 and 30°C.

Mutagenesis using UV
UV mutagenesis on AN39 -1 was carried out on cultures grown overnight at 30°C in MRS broth (100 ml) as described by Smith et al. [4]. The cells were harvested aseptically by centrifugation at 4.000 x g for 20 min and then washed three times using cold (4°C), sterile NaCl solution [150 ml, 0.85% (w/v)]. The final pellets were resuspended in sterile NaCl solution (100 ml) and diluted to a concentration of approximately 10 8 CFU/ml. The suspensions (10 ml) were placed into sterile glass petri dishes (150 mm in diameter) and irradiated by 254-nm UV light using Mini UV/Vis ® ultraviolet light lamp (Desaga GmbH, Heidelberg, Germany) for different time intervals at varied distances ranging from 20 to 360 sec and 7 to 40 cm. The radiation dose was chosen to give rise to a 0.1% surviving rate (distance: 40 cm; time: 160 sec) as estimated from a UV killing curve. Irradiation and subsequent steps were carried out in darkness to prevent photoreactivation. After irradiation, the cell suspensions (1 ml) were transfered to a MRS broth (4 ml) and then incubated in darkness at 30°C for 48 h. Cell suspensions were then diluted serially (10-fold dilutions) into sterile MRS broth and the dilutions were plated onto MRS agar containing sucrose [10% (w/v)] and maltose [10% (w/v)]. The plates were incubated in darkness at 30°C for 48 h. Approximately 200 colonies were screened and 105 colonies were isolated as mutants based on differences such as size and appearances. Unirradiated control cultures were also studied at the same time as the irradiated cultures to serve as a standard for comparing colony morphologies.

Mutagenesis using ethyl methane sulfonate
Mutagenesis with ethyl methane sulfonate (EMS) on the mutant strain, A26, was carried out as described by Kim & Robyt, [1]. The mutant A26, a UV variant of AN39-1, was grown on glucose medium and then collected from 1-day-old liquid culture by centrifugation at 10,000 rpm for 10 min. The cell suspension (1.5 ml), washed twice with sterile sodium phosphate buffer solution (1.5 ml, 100 mM, pH 7.0), was treated with EMS solution (40 µl, 1.17 gr EMS /ml) at 28°C for 1 h. The treated cell suspension (1 ml) was then transferred to a sterile sodium thiosulfate solution [3 ml, 10% (w/v)] to neutralize the remaining EMS. The cells, then were centrifuged (10.400 g), washed twice with sodium phosphate buffer solution (100 mM, pH 7.0) and plated onto glucose and sucrose agar. The plates were incubated at 30°C for 48 h. Colonies on the plates were examined on the basis of morphology. Slimy shiny colonies on sucrose agar were then picked and single colony was restreaked on each agar plates for isolation of dextran producing Leuconostoc strains. 46 colonies having dextransucrase activities were isolated.

Selection of mutants constitutive for dextransucrase production
Constitutive mutants were selected using the agar overlay method of Mizutani et al. [10]. Serial dilutions 10-

Determination of dextransucrase activity
The assay mixture (0.5 ml) contained enzyme solution (50 μl) and sucrose (150 mM) in calcium acetate buffer (25 mM, pH 5.4). The reaction mixture was incubated at 30°C for 20 min. Aliquots (0.1 ml) from the reaction mixture were analyzed for reducing sugar by DNS method [11]. The activity unit (IU) is defined as the amount of enzyme catalyzing the formation of 1 μmol of D-fructose from sucrose (150 mM) in acetate buffer (25 mM, pH 5.4) at 30°C per min.

Protein concentration determination
The protein concentration was assayed by the method of Bradford using bovine serum albumin (Sigma) as the standard [12]. Absorbance at 595 was measured after 5 min of color development.

Zymographic analysis
Non-denaturing sodium dodecyl sulfate-polycrylamide gel electrophoresis (SDS-PAGE) was performed on a resolving gel [1.5 mm thick, 7.5%(w/v) acrylamide] at room temperature (25°C) using a vertical slab mini gel unit (BioRad), according to the method of Laemmli [13]. Proteins were stained using Coomassie brilliant blue G 250. In situ detection of dextransucrase activity in the gel was carried out using the protocol described by Purama & Goyal [9]: The gel was washed with acetate buffer containing CaCl 2 (0.

Carbohydrate analysis
Thin layer Chromatography (TLC) was used for analysis of dextran hydrolysis products [14]. TLC plates were developed using 3 ascents of nitromethane-water-1-propanol [2:3:5 (v/v)] up to two thirds of the plate, and then 3 ascents of acetonitrile/water [85:15 (v/v)] to the top of the plate. Carbohydrates on TLC plates were visualized by dipping the plates into sulphuric acid [5% (v/v)] in ethanol containing α-naphthol [0.5% (w/v)], followed by heating on a hot plate at 110°C for 10 min. TLC-imaging densitometer, Bio-Rad GS-670, (Bio-Rad, Hercules, CA, USA) was used for quantitative determination of carbohydrates.

Purification of dextransucrases
Dextransucrases (AN39-1 and A26-2/11) produced in sucrose medium were purified by PEG fractionation. The cell-free extracts (200 ml) were added ice cold polyethylene glycol solutions (PEG-400 and PEG-6000) so as to obtain cell free extract having PEG [5 and 25% (w/v)] and incubated for 16 h at 4°C to allow the dextransucrase fraction to precipitate. The mixture was centrifuged at 13.200 g at 4°C for 30 min to separate the dextransucrase fraction. The pellets were dissolved in calcium acetate buffer (25 mM, pH 5.4). These fractions were analyzed for dextransucrase activity and protein content and subjected to dialysis using 12 kDa cutoff membrane. The dextransucrase fractions were analyzed for activity and protein content.

Effects of temperature and pH on dextransucrase activities
Effects of temperature and pH on dextransucrase activities were determined by changing individually the conditions of the dextransucrase activity assay (pH from 3.0 to 8.0; temperature from 20 to 50°C). The stabilities of the enzymes were established by measuring the residual activities at optimum pH (pH 5.5 for AN 39-1 and 4.0 for A26-2/11) and temperature (35°C) after incubating the enzymes at different pH (3.0-8.0) for 3 h and temperatures (20-50°C) for 40 min.

Results and Discussion
AN39-1 (Leuconostoc mesenteroides ssp. mesenteroides), recently isolated by our group from Crataegus orientalis var. Orientalis, produces dextransucrase catalyzing the formation of dextran from sucrose [8]. The enzyme is produced extracellularly and is inducible by sucrose [8]. The structure of the dextran produced by this enzyme is similar to commercial dextran produced by B-512F Leuconostoc mesenteroides dextransucrase.
In this research, mutagenesis using various mutagens on AN39-1 was carried out to obtain mutants constitutive for dextransucrase. Cell suspensions of AN39-1 were exposed to UV light from 20 sec to 6 min and found that irritation time of 160 sec gave 0.1% survivors from which 105 colonies were selected at the basis of colony morphology (size and opacity). The enzyme activities were determined in the cell free culture broth. It was found that 2 out of 105 colonies were constitutive for dextransucrase as indicated by agar overlay method. The constitutive mutant strains, called A6 and A26, showed 32% and 620% increase in dextransucrase production, respectively. In mutagenesis of A26 using EMS, 46 colonies were isolated and A26-2 having 711% increased dextransucrase activity was selected for further mutagenesis. Treatment of A26-2 with NTG resulted in new mutant strains. 24 colonies were chosen and A26-2/11 was further studied Table  1. shows dextransucrase activities produced by mutant bacteria grown in sucrose and glucose mediums. Grown on sucrose, all the mutants produced more dextransucrase than the parent strain, AN39-1. The activities of mutant enzymes (A26 and A26-2) were higher (7.2 and 8.1 times) when grown on sucrose medium than that of parent strain. A26 and A26-2 grown on sucrose had twice more enzyme activities than those grown on glucose. Mutagenesis studies on dextransucrases were also carried out by other researchers. Kamal et al. [15] mutated Leuconostoc mesenteroides PTCC 1059 with UV light and found that dextransucrase activities of mutant strains showed 20% to 40% increase [15]. Iliev et al. [3] reported that treatment of L. mesenteroides strain Lm 28 with EMS resulted in mutant strains producing 40% more dextransucrase than the parent strain [3]. Mizutani et al. [10] obtained constitutive mutants for dextransucrase from Leuconostoc mesenteroides NRRL-B-512F using NTG. Dextransucrase activity of one mutant strain was 3-fold higher than that of the wild strain [10]. Kim & Robyt [1] studied on the production and selection of mutants of Leuconostoc mesenteroides constitutive for glucansucrases. They isolated mutants constitutive for glucansucrases from Leuconostoc mesenteroides NRRL B-512FM, B-1142, and B-1355. The mutants produced higher glucansucrase activities (3 to 22 times) when grown on glucose than the parent strains grown on sucrose [1].

Analysis of zymogram
Zymogram was used for in situ detection of dextransucrase activity in SDS-PAGE gel. The gel was treated first with sucrose and then periodic acid-Schiff's base reagents, resulting in staining in purple in color. The formation of purple bands on the gel shows the presence of dextran synthesis by active dextransucrase from sucrose within PAGE (Fig. 1a). The molecular sizes of dextransucrases were determined using activity staining gel and non-denaturing SDS-PAGE containing protein standards, stained with Coomassie brilliant blue (Fig. 1b). It was found that AN39-1 had 1 major (184 kDa) and 1 minor active band (180 kDa). Its mutants (A26, A26-2, and A26-2/11) had also 1 major (184 kDa) and 1 minor (150 kDa) active bands. In situ detection of enzyme activity for the characterization of dextran-producing Leuconostoc strains has been used in some studies [16,17]. Activity staining gel (Fig. 1a) and nondenaturing SDS-PAGE (Fig. 1b) show that isolated mutants produce much more dextransucrase than AN39-1.

Enzyme purification and characterization
The purification of crude enzymes from AN39-1 and A26-2/11S by polyethylene glycol fractionation resulted in significant increase in dextransucrase activities. The results of purification process are presented in Table 2. The fractionations of AN39-1 and A26-2/11S with 25% (V/V) PEG 400 gave maximum specific activities of 29.77 IU/mg and 27.00 IU/mg resulting in 50 and 29-fold purifications, respectively. The fractionations of AN39-1 and A26-2/11S with 5% (V/V) PEG 6000 gave the maximum specific activities of 5.85 IU/mg and 14.54 IU/mg, resulting in 10 and 16-fold purifications, respectively. The characterization studies were carried out using enzymes purified using 25% (V/V) PEG-400 (Fig. 2). The optimum temperature for both of the enzymes was 35°C (Fig. 2a). The enzyme activities gradually decreased after 35°C. This result is supported by the findings that optimum temperature for dextransucrase activity from Leuconostoc mesenteroides B-512F is in the range of 30-35°C [5]. The mutant enzyme (A26-2/11) was much more thermostable than the native enzyme, AN39-1 (Fig.  2b). At 50°C, AN39-1 lost all of its initial activity. However, A26-2/11S lost about 90% of its initial activity at the same temperature.

Analysis of dextranase hydrolyzates of dextrans prepared by new isolates
The structures of dextrans formed by AN39-1 and its mutants were studied by dextranase hydrolysis. Hydrolysis products of dextrans were analyzed by TLC (Fig. 3).
It is known that highly branched dextrans (B-1142, B-742, and B-1299) are resistant to endo-dextranase hydrolysis [19]. The dextrans produced by new enzymes were purified and hydrolyzed using Penicillium dextranase covalently immobilized onto Eupergit C. Soluble dextranase was not used in the analysis since it had invertase and glucose isomerase activities. However, the immobilized enzyme had only dextranase activity. TLC showed that the hydrolysis products (oligosaccharides) for all the dextrans produced by AN39-1 and its mutants were the same as the hydrolysis products of B-512F dextran, indicating that dextrans produced by AN39-1 and its mutants have mainly α-(1→6) linkages between glucose units (Fig. 3). Commercial dextran, produced by B-512F dextransucrase, has 95% α-(1→6) linkages and 5% α-(1→3) branch linkages. B-512 F dextran is completely hydrolyzed by dextranase, yielding oligosaccharides. Dextranase hydrolysis products of dextrans synthesized by AN39-1 and its mutants were similar to those of commercial B-512 F dextran.

Conclusion
In the present study, mutant strains of AN 39-1 constitutive for dextransucrase were obtained to overcome problems of producing inducible enzyme. The mutant (A26-2/11), grown on glucose, produced 5,8 times more enzyme activities than the parent strain (AN39-1), grown on sucrose. Furthermore, enzyme produced by this mutant was more pH and temperature stable than the native enzyme. Dextran synthesized by the mutant enzyme was similar to commercial B-512 F dextran, possibly leading to be used in industrial applications.