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Optimization of Cultural Conditions of Gellan Gum Production from Recombinant Sphingomonas Paucimobilis ATCC 31461 and Its Characterization

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Optimization of Cultural Conditions of Gellan Gum Production from Recombinant Sphingomonas Paucimobilis ATCC 31461 and Its Characterization Journal of Applied Biology & Biotechnology Vol. 9(1), pp. 58-67, Jan-Feb, 2021 Available online at http://www.jabonline.in DOI: 10.7324/JABB.2021.9108 Optimization of cultural conditions of gellan gum production from recombinant Sphingomonas paucimobilis ATCC 31461 and its characterization Soumiya Sukumar, Santhiagu Arockiasamy*, Manjusha Chemmattu Moothona School of Biotechnology, National Institute of Technology Calicut, Calicut, Kerala, India. ARTICLE INFO ABSTRACT Article history: Microorganisms that produce exopolysaccharides have currently gained broad attention and are a subject of great Received on: September 02, 2020 concern for modern-day biotechnologists and microbiologists. Sphingomonas paucimobilis is a Gram-negative Accepted on: November 01, 2020 rod-shaped bacterium isolated from the Elodea plant tissue. The effect of the culture conditions on gellan gum Available online: January 17, 2021 production by recombinant S. paucimobilis ATCC 31461 in a stirred-type bioreactor was investigated. The products of fermentation were characterized by Fourier transform infrared (FTIR), scanning electron microscopy (SEM), Key words: thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC). The FTIR spectrum revealed Exopolysaccharide, Recombinant, characteristic peaks of different functional groups in the gellan. SEM was carried out to observe the morphology Rheology, of the gellan gum, while the thermal stability of the biopolymer was determined by TGA and DSC analysis. The Compression, compositional analysis of the purified gellan showed that it is a heteropolymer containing glucose, rhamnose, and Thermogravimetric. glucuronic acid. The compression test of the gellan revealed its textural property. Maximum gellan gum was attained after 48 h of incubation at 30°C with a pH of 6.5, 500 rpm, and 100% dissolved oxygen. The rheology study indicated the non-newtonian nature of gellan gum. The solubility of gellan gum in different polar and non-polar solvents was also investigated. The findings in this study showed that gellan gum produced by recombinant S. paucimobilis ATCC 31461 has a high potential to be utilized in the pharmaceutical and food industry. 1. INTRODUCTION pharmaceutical industry. Gellan gum in its native form comprises of tetrasaccharide repeating unit of β-D-glucose, β-D-glucuronic acid, Microbial exopolysaccharides are water-soluble, high molecular α-L-rhamnose, and acyl groups, namely, acetate and glycerate [2]. weight, natural, or semi-synthetic long-chain polymers. They are composed of sugar residues and are economically valuable The simple media containing a carbon source, nitrogen source, and due to their functional properties and unique structure. Microbial inorganic salts are used for the production of gellan gum [3]. The carbon exopolysaccharides are secreted by microorganisms into the external sources such as glucose, fructose, maltose, sucrose, and mannitol play environment through sugar fermentation. Gellan gum from the strain an essential role in the production yield, composition, structure, and of Sphingomonas paucimobilis, bacterial alginates from Azotobacter properties of gellan gum. The choice of nitrogen sources and their vinelandii, xanthan from Xanthomonas campestris, and wellan from concentration influences gellan broth characteristics and the flow of Alcaligenes are some of the widely used biopolymers which have been carbon to either biomass or product formation. The pH of the media the subject of extensive research. Physiochemical factors such as pH, has a strong effect on product formation and cell proliferation. The temperature, incubation time, agitation speed, medium, carbon, and temperature has a profound influence on the activity of the enzymes nitrogen sources play a key role in the exopolysaccharides yield [1]. involved in the production of gellan gum and microbial growth by Gellan gum has specific characteristics such as biocompatibility, Sphingomonas paucimobilis ATCC 31461 [4]. biodegradability, temperature resistance, stability at acidic conditions, Agitation at the lower levels provides inadequate homogenous rigidity, and clarity makes the polymer widely used in the food and conditions, whereas at higher levels, shear forces inside the fermentation broth increase, which can lead to mechanical damage of *Corresponding Author the cells and thus affecting polysaccharide synthesis [5]. Aeration is Santhiagu Arockiasamy, School of Biotechnology, a prerequisite for aerobic species such as S. paucimobilis for cellular National Institute of Technology Calicut, growth and gellan production. Rho and Loung [6] observed a complete Calicut, Kerala - 673 601,India. decrease in gellan gum production when the dissolved oxygen in the E-mail: [email protected] bioreactor was set at zero. © 2021 Sukumar, et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial-ShareAlike Unported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). Sukumar, et al.: Gellan gum production from recombinant Sphingomonas paucimobilis ATCC 31461 2021;9(1):58-67 59 During gellan gum production, the viscosity of the polymer reduces the transformed cells was done using the alkali lysis method [12]. The with an increase in shear rate, that is, they exhibit pseudoplastic clones were confirmed by doubles digestion with restriction enzymes, behavior, and the rheology of the fermentation broth was found to colony PCR, and sequencing. The transformation of the recombinant be dependent on media components, dissolved oxygen levels, and plasmid (pBBR122-gelD) into S. paucimobilis electrocompetent cells agitation rate [7]. The water-holding characteristics of the gellan was carried out by electroporation [13]. The recombinant protein gum maintain the shape, consistency, and freshness of the food for was purified by affinity chromatography using Ni-NTA resin [14], a longer duration of time. The ability of gellan gum to solubilize and the purified protein was analyzed by SDS-polyacrylamide gel in water has created various applications in the pharmaceutical and electrophoresis [15]. food industries [8]. 2.3. Maintenance of Culture Gellan gum promptly forms a gel with calcium ions, and the produced gels exhibit accurate edges and sublime shapes [9]. The textural The recombinant S. paucimobilis ATCC 31461 culture was maintained properties and gelation of the gel samples were evaluated using in YPG agar slants. The YPG medium contained the following compression tests. The compression behavior of the gellan gum is composition (g/l): Glucose 30; peptone 5; yeast extract 3; NaCl 5; and based on the speed of the applied compressive force [10]. agar 20. The pH of the media was adjusted to 6.5. The YPG agar slants were incubated at 30°C for 48 h. The agar slants were stored at 4°C for This study was undertaken with the objectives to investigate the routine use and maintained as glycerol stocks at −80°C for long-term optimum medium components and culture conditions during the storage. fermentation process for maximum gellan gum production by recombinant S. paucimobilis ATCC 31461. The characterization of 2.4. Inoculum Preparation gellan gum in terms of rheological, textural, and structural properties The inoculum of recombinant S. paucimobilis ATCC 31461 was was also investigated. prepared by transferring a 4–5 loopful of YPG slant culture into a 1 L Erlenmeyer flask containing 500 ml of YPG broth. The inoculated 2. MATERIALS AND METHODS flasks were incubated at a temperature of 30 ± 1°C on a rotary shaker 2.1. Bacterial Strains, Vectors, and Reagents at 300 rpm for 24 h. The strain S. paucimobilis ATCC 31461 was procured from LGC, The impact of process parameters on gellan gum production by Promochem Pvt. Ltd., Bengaluru. The primers for polymerase chain recombinant S. paucimobilis ATCC 31461 was studied. The various reaction (PCR) were purchased from Eurofins. DNA isolation, PCR process parameters evaluated include the effect of media, incubation purification, gel extraction, and plasmid isolation kits were procured time, size and age of inoculum, temperature, pH of the medium, from Thermo Fisher Scientific (USA). The vector pBBR122 was carbon source, nitrogen source, agitation speed, dissolved oxygen, and obtained from MoBiTec. The mediums Yeast peptone glucose (YPG), C/N ratio. The effect of one parameter was studied at a time by keeping Luria-Bertani (LB), and nutrient broth (NB) were obtained from other parameters constant. HIMEDIA India. All the chemicals and reagents used in the present study were of analytical reagent grade quality and obtained from 2.5. Optimization of Parameters Merck, India. To determine the optimum biomass and gellan gum production, recombinant S. paucimobilis ATCC 31461 was grown in a different 2.2. Cloning and Expression of the Gellan Gum Biosynthetic medium (YPG, LB, and NB) at various incubation time (12–96 h) with Gene Gel D different incubation temperature (25–50°C), medium pH (4.0–8.0), The genomic DNA from S. paucimobilis ATCC 31461 was isolated dissolved oxygen (20–100%), and agitation speed (100–600 rpm). using the Wizard® DNA isolation kit. The primers for gene gelD Various size (2–12%) and age (6–27 h) of inoculum were investigated encoding the protein for polymerization and export of gellan were to study their effect on gellan gum production. To determine the designed using the Primer Premier Software. The PCR for gene requirement of additional nutrients for gellan production, different gel D was
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