A gram-positive, nonmotile, irregular, short, rod-shaped new strain of
The
First proposed by Collins et al. [
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 [
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 [
The strain Cab3 was tested for its ability to ferment various carbohydrates using the method of Kandler and Weiss [
The antibiotic tests were performed using commercially available antibiotic octadiscs containing Amoxyclav (Ac), cephalexin (Cp), ciprofloxacin (Cf), clindamycin (Cd), cloxacillin (Cx), erythromycin (E), tetracycline (T), ampicillin (A), carbenicillin (Cb), cefotaxime (Ce), chloramphenicol (C), co-Trimazine (Cm), gentamicin (G), norfloxacin (Nx), oxacillin (Ox), amikacin (Ak), amoxycillin (Am), bacitracin (B), cephalothin (Ch), novobiocin (Nv), oxytetracycline (O), vancomycin (V), penicillin-G (P), tobramycin (Tb), cephaloridine (Cr), kanamycin (K), lincomycin (L), methicillin (M), norfloxacin (Nf), and oleandomycin (Ol) from Hi-media Pvt. Ltd. India.
The bacterial cell pellet was lysed using a solution containing guanidium thiocyanate (a chaotropic agent) and SDS (a detergent), to extract DNA [
The genomic DNA of isolate Cab3 was used for amplification of 16S rRNA gene. The universal 16S rDNA primers, forward primer 8F (5′AGTTGATCCTGGCTCAG3′), and reverse primer 1492R (5′ACCTTGTTACGACTT3′) 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
Fifteen
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 [
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
The glucansucrase was produced in the enzyme production medium as described by Tsuchiya et al. [
The production glucansucrase was compared under shaken-flask 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 cell-free extract was analysed for enzyme activity and protein concentration as described earlier.
The polysaccharide content of the isolated strain (Cab3) was determined by phenol-sulphuric acid method [
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. [
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 sucrose concentration in 20 mM sodium acetate buffer at 30°C for 15 min. The enzyme assay was performed within the temperature range of 20°C to 50°C. The enzyme assay was done in 5% (w/v) sucrose solution in 20 mM sodium acetate buffer (pH 5.4) for 15 min.
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 [
Differential characteristics of
Characteristics | 1 | 2 [ | 3 [ | 4 [ | 5 [ | 6 [ | 7 [ | 8 [ | 9 [ | 10 [ |
---|---|---|---|---|---|---|---|---|---|---|
Acid from: | ||||||||||
Ribose | + | + | + | + | − | − | + | NT | NT | NT |
D-Xylose | + | + | − | + | − | + | − | + | + | + |
Galactose | + | − | + | + | − | + | + | + | + | + |
D-Fructose | + | − | + | + | + | NT | − | + | + | + |
Cellobiose | + | − | − | + | − | + | − | + | − | + |
Melibiose | − | + | − | − | − | + | + | + | + | + |
Sucrose | + | + | − | + | + | + | D | + | + | + |
Trehalose | − | + | − | − | + | − | D | + | + | + |
D-Raffinose | − | + | − | − | − | − | + | + | + | − |
Maltose | + | NT | − | + | + | + | + | − | + | + |
Dextran formation | + | − | + | + | − | + | − | + | + | + |
Cell morphology | Short rods | Short rods, often thickened to one end | Irregular rods | Short rods, thickened at one end | Large spherical or lenticular cells | NT | Spherical | Cocci-shaped and random arrangement in groups or chains | NT | NT |
1,
Scanning electron microscopic image of
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
Full-length 16S rRNA gene (1500 bp) of the isolate cab3 amplified with universal primers. The amplicon was electrophoretically resolved on a 1.2% agarose gel in 1X TBE buffer (a) Lane1: DNA ladder (b) Lane 2: amplified product of full length 16S rRNA gene.
16S rRNA gene sequence investigation of
Evolutionary relationships of 11 taxa.
Temperatures ranging from 22°C to 40°C were studied for the production of enzyme under shaking at 180 rpm (Figure
Effect of temperature on glucansucrase production from
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
Comparison of
Fermentation parameters | ||||
---|---|---|---|---|
Duration (h) | 12 | 12 [ | 14 [ | 16 [ |
Maximum enzyme activity (U/mL) | 6.1 | 4.1 | 4.8 [ | 3.4 [ |
pH range for maximum enzyme production | 5.2–5.6 | 6.0–6.5 [ | 4.4–5.9 [ | 4-5 [ |
Temperature (°C) | 25°C | 23°C [ | 25°C [ | 25°C [ |
Cell density ( | 5.4 | 5.8 | 5.0 | 8.1 |
Glucan concentration (mg/mL) | 34 | 6.9 [ | 7.0 [ | 10.2 [ |
Effect of shaken-and static-flask culture on glucansucrase production. Enzyme activity under shaken-(—●—) and static-(—▲—) flask culture is shown.
Fermentation profile of Weissella confusa. The enzyme activity (—▲—), cell growth (—♦—), pH (—■—), sucrose conc. (- - -
The cell-free supernatant of isolated strain
The enzyme was purified from cell-free supernatant with 1.0 U/mg specific activity. The fractionation by 33% (w/v) PEG-400 gave 10.4 U/mg specific activity with 10-fold purification and 26% overall yield. The purified enzyme showed, approximately, 180 kDa molecular size on the nondenaturing SDS-PAGE gel when stained with Coomassie Brilliant Blue (Figure
Nondenaturing SDS-PAGE analyses using 7% acrylamide gel. Lane (1) protein molecular weight marker (29–205 kDa), (2) 33% (w/v) PEG-400 fraction; identification of glucansucrase of purified glucansucrase by staining of glucan formed using: lane (3) Sucrose, (4) raffinose.
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
Effect of different enzyme assay conditions on specific activity (U/mg). The effect of ionic strength (—▲—), temperature (—●—), and pH (—■—) are shown.
The isolated bacterium (Cab3) was identified at species and genus levels based on 16S rDNA sequencing to be
This research work was supported by a project grant by the Council of Scientific and Industrial Research (CSIR), New Delhi, India, to A. Goyal Fellowship to S. Shukla by Indian Institute of Technology Guwahati is thankfully acknowledged.