16S rRNA-Based Identification of a Glucan-Hyperproducing Weissella confusa

A gram-positive, nonmotile, irregular, short, rod-shaped new strain of Weissella confusa bacterium was isolated from fermented cabbage. The isolate was physiologically and biochemically characterised. The 16S rDNA was amplified by polymerase chain reaction (PCR). The isolate was identified as Weissella confusa (GenBank accession number: GU138518.1) based on nucleotide homology and phylogenetic analysis. The isolate produces glucansucrase when grown in sucrose-supplemented culture medium which catalyses glucan formation. This novel isolate possesses high capacity of industrial use due to its high productivity of glucan (34 mg/mL) as compared to other strains reported. The optimum temperature for glucansucrase production was 25°C. The shaking condition gave an enzyme activity of 6.1 U/mL which was 1.5 times higher than that given by static condition (4.1 U/mL). The temperature 35°C, pH 5.4, and ionic strength 10–20 mM were optimum for enzyme assay. This investigation unraveled the abundance of industrially valuable microflora of the north east India.


Introduction
The Weissella species are gram-positive, non-spore-forming, heterofermentative, nonmotile, and irregular or coccoid rodshaped organisms. Members of the genus Weissella have been isolated from a variety of sources, such as fresh vegetables, fermented silage, meat, or meat products. Several lactic acid bacteria belonging to the genera Weissella have been introduced to wheat sourdough baking for in situ production of exopolysaccharides [1]. Weissella kimchii PL9023 was selected as a prebiotic as it mostly produced hydrogen peroxide which inhibited the growth and adherence of vaginal isolates of Candida albicans, Escherichia coli, Staphylococcus aureus, and Streptococcus agalactiae [2]. Lactic acid bacteria (LAB) that produce dextrans include various Leuconostoc, Streptococcus, and Lactobacillus species [3]. Smitinont et al. [4] had emphasised on dextran-synthesizing ability of the Pediococcus genus. Patel et al. [5] reported the dextranproduction ability of Pediococcus pentosaceus for the first time. Additionally, dextran production has typically served as a phenotypic test in the identification of bacteria classified in the genus Weissella [6]. Maina et al. [7] reported industrially useful dextran-producing Weissella confusa E392 which could be a suitable alternative to the widely used L. mesenteroides B512F in the production of linear dextran.

Isolation and Maintenance of Microorganism.
Fresh chopped cabbage was subjected to fermentation in 2.5% (w/v) NaCl solution. One gram of fermented cabbage was ground to paste and mixed in 10 mL of saline (0.9% w/v) homogeneously in test tubes. The serial dilutions were made, till the dilution factor 10 −5 . One hundred microliter from all the dilutions of cabbage from 10 −0 to 10 −5 was taken and spread plated on petri plates of 1.7% (w/v) MRS agar with glucose as carbon source [13]. Petri plates were incubated at 28 • C for 24 h. Based on the higher-glucansucrase activity and glucan concentration determined (as described later), a microbe Cab3 (named after its colony number) was isolated. The isolated bacterium was propagated as stab in MRS agar supplemented with sucrose as carbon source [14] at 25 • C and stored at 4 • C. For long-term preservation, the isolate was kept in 20% (v/v) glycerol at −80 • C.

Morphological, Physiological, and Biochemical
Characterization. The colony morphology of isolates grown on MRS medium for overnight at 25 • C was observed directly and by light microscopy. Cell mobility and gliding movement were assessed by phase-contrast microscopy (1000x) using cells of MRS broth. The Gram reaction was performed using the KOH method of Gregesen [15]. The cell morphology of the selected isolate was examined by scanning electron microscopy (Leo1430 VP, Leo Electron Microscopy Ltd., Cambridge, UK) operated at 10.0 kV. The catalase activity was determined by transferring fresh colonies from MRS agar to a drop of 5% (v/v) H 2 O 2 on a glass slide. For the temperature tolerance of the microorganism, the growth of selected isolate was carried out using MRS broth at different temperatures, that is, 4 • C, 10 • C, 15 • C, 20 • C, 30 • C, 37 • C, and 42 • C for 2 days. For salt tolerance of the isolate, the growth was observed using MRS broth in the presence of 4.0% and 6.5% (w/v) of NaCl incubated at 25 • C for 2 d. The production of glucan (slimy layer) by isolate Cab3 from sucrose was observed on MRS agar in which glucose was replaced by sucrose [14].
The strain Cab3 was tested for its ability to ferment various carbohydrates using the method of Kandler and Weiss [21]. From the overnight-grown MRS broth containing 2% (w/v) glucose as carbohydrate source, 50 µL was inoculated in 5.0 mL liquid MRS medium lacking glucose but containing phenol red and other test carbohydrates to give a final inoculum to medium ratio of 1% (v/v). The test media were incubated for 2 days at 30 • C without shaking. The acid production was recorded between 24 and 48 h. The acid production was indicated by a change in the colour of the phenol red indicator dye from red to yellow. The isolated strain cab3 was tested for susceptibility to 30 antibiotics using a standardized filter-paper disc-agar diffusion assay, to determine the drug susceptibility of microorganisms [22].

16S rDNA-Based Sequence Analyses
2.3.1. Extraction of Genomic DNA. The bacterial cell pellet was lysed using a solution containing guanidium thiocyanate (a chaotropic agent) and SDS (a detergent), to extract DNA [23]. This lysis solution was used to disrupt the cell, remove proteins and polysaccharides, and partial hydrolysis of RNA. The genomic DNA was extracted by GeNeiTM Genomic DNA Extraction kit (Bangalore Genei Pvt. Ltd.). DNA was then precipitated using alcohol and washed with 70% (v/v) alcohol to remove contaminants. DNA pellet was solubilized in sodium acetate buffer at pH 5.5 added to a final concentration of 0.3 M in isopropanol at higher temperature (50-55 • C), to increase the solubility of genomic DNA. The extracted DNA was purified using Ultrapure prep kit (KT83B, Bangalore Genei, India). Genomic DNA was purified in a gravity flow column using elution buffer, followed by treatment with lysozyme, proteinase K, and RNase A supplied with the kit. DNA concentrations were determined in duplicate using a spectrophotometer. The quality of purified DNA was evaluated on 1.2% Agarose gel, a single band of high-molecular-weight DNA was observed.

PCR Amplification.
The genomic DNA of isolate Cab3 was used for amplification of 16S rRNA gene. The universal 16S rDNA primers, forward primer 8F (5 AGTTGA-TCCTGGCTCAG3 ), and reverse primer 1492R (5 ACC-TTGTTACGACTT3 ) were used for the polymerase chain reaction (PCR). The PCR amplification was carried out in a reaction mixture containing ∼10 ng genomic DNA as template, 1 µL dNTP mix (2.5 mM each), 100 ng/µL each of forward and reverse primer, 1X Taq DNA polymerase assay buffer (10X), 3U Taq DNA polymerase enzyme (Bangalore Genei Ltd.,Bangalore, India), and distilled water enough to make up the volume to 50 µL reaction mixture. Amplification conditions were 5 min initial denaturation at 94 • C, 30 s denaturation at 94 • C, 30 s of primer annealing at 54 • C, 1 min elongation at 72 • C for 35 cycles, and a final extension of 10 min at 72 • C. The reactions were carried out in a Thermal Cycler (Applied Biosystems, model ABI 2720).
Enzyme Research 3

Electrophoresis and Sequencing of 16S rRNA Gene.
Fifteen µL of PCR amplified product was electrophoresed on 1.2% (w/v) low-EEO agarose gel in 1X TBE buffer (45 mM Tris-borate, pH 8.3, and 1 mM Na 2 EDTA) at 100 V for 2 h. The gel was stained with ethidium bromide in a final concentration of 0.5 µg/mL, visualized, and photographed under UV light. The amplified 16S rRNA gene from the isolate was eluted and purified from the gel slice using the GeneiPure TM Gel Extraction Kit (QIAGEN). Forward and reverse DNA sequencing reaction of PCR amplicon was carried out with 8F and 1492R primers using BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer. Consensus sequence of 1288 bp of 16S rDNA gene was generated from forward and reverse sequence data using aligner software.

Sequence Alignment of 16S rRNA
Gene. The 16S rDNA gene sequence of the isolate cab3 was used to carry out BLAST with the nr database of NCBI GenBank database. Based on maximum identity score, first ten sequences were selected and aligned using multiple alignment software program ClustalW. Distance matrix was generated using RDP database, and the phylogenetic tree was constructed using MEGA 4 [28].

Production of Glucansucrase and Its Assay
2.4.1. Enzyme Activity Assay. The enzyme assay was carried out in 1 mL reaction mixture containing 5% (w/v) sucrose, 20 mM sodium acetate buffer (pH 5.4), and 20 µL cell-free supernatant. The enzymatic reaction was performed at 30 • C for 15 min. 100 µL aliquot from the reaction mixture was taken for reducing sugar estimation. The enzyme activity was determined by estimating the released reducing sugar by Nelson [29] and Somogyi [30] method. The absorbance of the colour developed was measured by spectrophotometer at 500 nm. Fructose was used to plot the standard graph.

Effect of Temperature on Enzyme
Production. The glucansucrase was produced in the enzyme production medium as described by Tsuchiya et al. [31]. Fermentations were carried out in triplicate sets of 60 mL enzyme production medium in a 250 mL Erlenmeyer flask incubated at 25 • C under shaking condition at 180 rpm. The samples (1 mL) were withdrawn at indicated time intervals and centrifuged at 8000 g for 10 min at 4 • C to separate the cells. The cellfree supernatant was analyzed for enzyme activity. Weissella confusa was grown at various temperatures: 22 • C, 25 • C, 28 • C, 30 • C, 32 • C, 37 • C, and 40 • C in the flasks as described previously in an orbital shaking incubator at 180 rpm to determine the optimum incubation temperature for the maximum enzyme production. The culture (1.0 mL) was withdrawn at the indicated time intervals and centrifuged at 8000 g for 10 min at 4 • C to pellet out the cells. The cell-free supernatant was analyzed for enzyme activity.

Effect of Shaken-Flask Culture on Enzyme Production.
The production glucansucrase was compared under shakenflask condition with the static flask culture at 25 • C in triplicate sets of 60 mL enzyme production medium in 250 mL Erlenmeyer flasks. The shaking was carried out in an orbital shaking incubator at 180 rpm. The samples (1.0 mL) were withdrawn at indicated time intervals and centrifuged at 8000 g for 10 min at 4 • C to pellet out the cells. The cellfree extract was analysed for enzyme activity and protein concentration as described earlier.

Analysis of Glucan Content of the Isolated Strain.
The polysaccharide content of the isolated strain (Cab3) was determined by phenol-sulphuric acid method [32] in a microtitre plate [33]. The isolated strain was grown in 60 mL liquid medium described by Tsuchiya et al. [31] at 25 • C and 180 rpm up to 32 h. The samples (1 mL) were withdrawn at regular intervals. To 200 µL of the culture supernatant, three volumes of the prechilled ethanol were added and centrifuged at 12,000 g. The supernatant was discarded, and the precipitate was resuspended in 200 µL distilled water. The process was repeated two more times. The final precipitate was air dried and dissolved in 200 µL distilled water. Dextran T-40 was used to plot the standard.

Purification and Identification of Glucansucrase by Activity Staining.
The crude 50 mL cell-free supernatant with enzyme activity 6 U/mL and specific activity 1 U/mg was subjected to purification by fractionation using PEG-400 (33%, w/v). The enzyme activity was determined as described earlier. The protein concentration was determined by the method of Lowry et al. [34]. To confirm the existence of glucansucrase, the PAS staining of the sucrose-and raffinoseincubated gel was carried out on 7% nondenaturing SDS-PAGE [35]. The electrophoresis was performed on a mini gel unit (BioRad) using 1.5 mm thick gels, following the method of [36]. After run, the gel was treated thrice with a solution containing 20 mM sodium acetate buffer, pH 5.4, 0.1% (w/v) Triton X-100, and 0.005% (w/v) calcium chloride for 20 min to remove SDS. Then, the gel was incubated with 5% (w/v) sucrose/raffinose solution in 20 mM sodium acetate buffer, pH 5.4 for 10-12 h. Following incubation, the gel was washed twice with 75% (v/v) ethanol for 20 min and incubated in solution with 0.7% (w/v) periodic acid in 5% (v/v) acetic acid for 20 min at room temperature. The gel was then washed thrice with 0.2% (w/v) sodium bisulfate in 5% (v/v) acetic acid solution and finally stained with Schiff 's reagent (0.5% w/v basic fuchsin, 1% (w/v) sodium bisulfate, and 0.1 N HCl) until the discrete magenta bands appeared within the gel.

Effect of Temperature, pH, and Ionic Strength on Enzyme
Assay. The enzyme assay conditions were optimized to find maximum enzyme activity. The purified enzyme having 10.4 U/mg specific activity (1.0 mg/mL) was used for further optimization of assay conditions. The ionic strength of the buffer was varied from 10 mM to 400 mM. The assay was carried out in 1 mL reaction mixture containing 5% (w/v) final sucrose concentration, in sodium acetate buffer (pH 5.4) at 30 • C for 15 min. To study the effect of varied pH on enzyme activity, the pH of buffer was varied from 4 to 7 in 1 mL reaction mixture containing 5% (w/v) final 4 Enzyme Research Acid from:

Results and Discussion
3.1. Isolation, Morphological, Phenotypic, and Chemotaxonomic Characterization of Isolate Cab3. Based on higher glucansucrase activity and glucan concentration determined, a microbe Cab3 was isolated from the fermented cabbage. The isolated culture was identified according to their morphological, cultural, physiological, and biochemical characteristics [37,38]. The tests used were gram reaction, production of catalase and growth at different temperatures and salt concentrations, and acid production from carbohydrates (2% w/v). W. confusa can be identified and differentiated from other species such as Enterococcus spp., Streptococcus spp., Lactococcus spp., and Leuconostoc spp. by its biochemical and physiological properties like arginine deamination, esculin hydrolysis, growth at 42 • C, and acidification of certain carbohydrates [39]. The results of both physiological and biochemical tests are shown in Table 1. The microorganism was identified by comparing the results with those from the literature ( Table 1). The results of the comparison showed that the isolated strain Cab3 has the same characteristics as W. confusa (Table 1, Column 5, which is, 4 [21]). From scanning electron microscopy, the isolate Cab3 was found to be phenotypically homogeneous with irregular short rod cells in pairs or chains with a width and length of 0.5-0.6 µm and 1.2-1.4 µm, respectively ( Figure 1). The colonies on MRS agar medium were opaque, circular, and slightly convex. The isolate showed Gram-positive, catalase-negative, and nonmotile characteristics. The isolate showed growth between 15 and 42 • C. The colony produced slimy layer when grown on MRS agar plate was supplemented with sucrose. The strain could grow in presence of 4.0%, 5.0%, 6.0%, and 6.5% (w/v) NaCl, but not beyond 8.0% NaCl. The strain very efficiently utilised cellobiose, galactose, maltose, ribose, sucrose, and xylose but poorly fermented mellibiose, raffinose, and trehalose which was in accordance with fermentation pattern of Weissella confusa reported earlier [40]. The Cab3 did not utilize rhamnose which is also characteristic of Weissella sp. [39]. The isolate Cab3 was sensitive to chloramphenicol, erythromycin, and tetracycline and moderately resistant to gentamicin, kanamycin, norfloxacin, and vancomycin which is characteristic of Weissella confusa [40].

16S rDNA Sequence Analyses.
16S rRNA gene sequence analyses after PCR amplification was performed for identifying the isolate Cab3. The profiles obtained by PCR amplification allowed the identification of the isolate at both genus and species levels. The amplified product of 16S rRNA showed a single band of 1,500 bp ( Figure 2). Forward and reverse DNA sequencing reaction of PCR amplicon was carried out with 8F and 1492R primers using BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer. The consensus sequence of 1288 bp of 16S rDNA gene was generated from forward and reverse sequence data using aligner software (Figure 3). The 16S rRNA sequence was compared with reference bacteria from the National Centre for Biotechnological Information (NCBI) Genbank (http:// www.ncbi.nlm.nih.gov/) and Ribosomal Database Project (RDP). Similarity searches were carried out using the BLAST algorithms available at (http://www.ncbi.nlm.nih.gov/blast). Multiple sequence alignment was performed using ClustalW (http://www.ebi.ac.uk/clustalw) [41]. The alignment was checked visually and corrected manually using the sequence editor. The homology in sequences identified by the above methods was used to find out common ancestry. The evolutionary history was inferred using the neighbor-joining method [42]. The bootstrap consensus tree inferred from 500 replicates was taken to represent the evolutionary history of the taxa analyzed [43]. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches [43]. The evolutionary distances were computed using the Kimura 2parameter method [44] and are in the units of the number of base substitutions per site. Codon positions included were 1st+2nd+3rd+ noncoding. All positions containing gaps and missing data were eliminated from the dataset (complete-deletion option). There were a total of 1288 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4 [28]. The isolate cab3 and the strain Weissella confusa IMAU:10190 (Genbank accession number GU 138518.1) clustered together (Figure 4) Figure 3).

Effect of Temperature.
Temperatures ranging from 22 • C to 40 • C were studied for the production of enzyme under shaking at 180 rpm ( Figure 5). At 25 • C, the activity achieved was maximum (6.2 U/mL), at a temperature lower than 25 • C, the cell growth was slow which might be the cause for lower enzyme activity as shown in Figure 5. At higher temperature, the enzyme activity decreased which might be due to the deactivation of the enzyme at higher temperatures.

Effect of Shaken-Flask Culture.
Shaking condition significantly favoured the enzyme production giving 6.1 U/mL enzyme activity which was 1.5 times higher than the enzyme activity observed under static condition (4.1 U/mL) as shown in Figure 6. The maximum enzyme activity was achieved 4 h later in case of static flask culture. Figure 7 shows a complete fermentation profile of the new strain of Weissella confusa (Cab3). The enzyme production and glucan production were cell growth associated. The fermentation profile of the new strain of Weissella confusa (Cab3) was compared with other lactic acid bacterial strains using the same medium composition and the parameters compared are listed in Table 2. It was reported that 23 • C temperature under static flask culture condition was optimum for the production of dextransucrase from L. mesenteroides NRRL B-512F [16], and 25 • C temperature under shaking condition was found to be optimum for L. mesenteroides NRRL B-640, whereas, for Weissella confuse, the temperature 25 • C and shaking conditions were optimum. From the results, it was clear that the enzyme activity (6.1 U/mL) from Weissella confusa was 50%, 27%, and 65% higher than the enzyme activity from L. mesenteroides NRRL B-512F (4.1 U/mL), L. mesenteroides NRRL B-640 (4.8 U/mL), and P. pentosaceus, respectively, as shown in Table 2. The cell growth obtained in all strains after 12 h was similar. There was a sharp decrease in the pH of the broth with Weissella confusa after 12 h (Figure 7), when the maximum activity was achieved, whereas the pH drop was much lower from the point where the maximum enzyme activity was observed for L. mesenteroides NRRL B-512F. The pH range for maximum enzyme production for Weissella confusa was narrow 5.2-5.6, whereas for L. mesenteroides NRRL B-512F, L. mesenteroides NRRL B-640, and P. pentosaceus, it was 6.0-6.5, 4.4-5.9, and 4.0-5.0, respectively.

Analysis and Comparison of Glucan Content of Selected
Strain. The cell-free supernatant of isolated strain Weissella confusa (Cab3) gave glucan concentration of 34 mg/mL (Figure 7). This was very high as compared to other lactic acid bacteria such as from L. mesenteroides NRRL B-512F (6.9 mg/mL) [19], L. mesenteroides NRRL B-640 (7.0 mg/mL) [20], and Pediococcus pentosaceus (10.2 mg/mL) [5]. The glucan concentration of the strain can be further increased by optimizing the medium composition and culture conditions.

Effect of Temperature, pH, and Ionic Strength on Enzyme
Activity. The partially purified enzyme was maximally active at the temperature 35 • C, at pH 5.4 in 0.2 mM sodium acetate buffer pH 5.4 (Figure 9). The results showed that the glucansucrase activity was stable within the range of 10-20 mM (Figure 9). The enzyme lost 41% of its activity at 50 mM. Beyond 50 mM, the decrease in the enzyme activity was not significant with the increase of ionic strength. The pH 5.4 was optimum for enzyme activity as shown in

Conclusion
The isolated bacterium (Cab3) was identified at species and genus levels based on 16S rDNA sequencing to be Weissella confusa IMAU:10190 (Genbank Accession Number GU 138518.1). The isolated strain could be very promising candidate for glucan production at industrial level as it has high glucan concentration (34 mg/mL). The physiological and biochemical characterisation was carried out to distinguish the isolate from other closely related lactic acid bacteria. Based on biochemical and physiological studies, the strain showed its relation with other Weissella sp. The strain was resistant to gentamicin, kanamycin, norfloxacin, and vancomycin. The isolate Weissella confusa (Cab3) could not ferment mellibiose, raffinose, and trehalose, a common trait of Weissella confusa. The optimum culture conditions for maximum enzyme production were optimized. The 25 • C temperature and shaking condition were optimum for enzyme production. The partially purified enzyme was confirmed as glucansucrase by PAS staining of nondenaturing SDS-PAGE gel incubated with sucrose. The presence of fructosyltransferase was ruled out based on the absence of the band in raffinose-incubated nondenaturing SDS-PAGE gel. The partially purified enzyme showed highest activity at 35 • C, when assayed in 20 mM sodium acetate buffer (pH 5.4). The wide applications of glucan in food, pharmaceutical, and cosmetics industries emphasize the importance of exploration of the new strains and characterization of their traits. This study reveals a novel high glucan producing bacterial isolate which could be a good alternative for glucanproduction in the future.