Mutagenesis of Leuconostoc dextranicum NRRL B-1146 for higher glucan production

摘要

The constitutive mutants of Leuconostoc dextranicum NRRL B-1146 were generated by classical mutagenesis technique using UV radiation for higher production of glucan. The conditions of mutagenesis such as dilution factor and time of UV light exposure were optimized. Three stage screening of mutants was carried out and the mutants were grown and subsequently tested for glucan content produced after the second stage. In the first stage 137 mutants were picked by visual screening based on morphology and colony size and grown. In the second stage of screening, 97 colonies were visually screened from 137 colonies and glucan content was analysed by microtitre format. In the third stage screening, 11 higher glucan producing mutants were selected from 97. Finally, 3 mutants produced significantly high glucan content, out of 11 selected mutants. The wild-type strain gave 1.01 g/l of glucan using statistically optimized medium. The mutant 64 gave maximum glucan content of 5.1 g/l, 5 times higher than that produced by wild-type Leuconostoc dextranicum NRRL B-1146 followed by mutant 88 giving 4.24 g/l and mutant 9 giving 4.1 g/l. Introduction Glucansucrases are extracellular proteins involved in the synthesis of α-glucans. Lactic acid bacteria produce a wide variety of exopolysaccharides [1,2]. Leuconostoc species are commercially exploited for the production of glucans, homopolysaccharides which contain only one type of monosaccharide, glucose. Glucans differ in the type of glucosidic linkages, degree and type of branching, length of glucan chains, molecular mass and conformation of polymers [3]. All these properties strongly contribute to specific polysaccharide properties such as solubility, rheology and other physical characteristics. Depending on the main chain glucosidic linkages in glucan, three different types of α-glucans synthesized by Leuconostoc are known viz. dextran with α-(1-6), mutan with α-(1-3) and alternan with α-(1-6), α-(1-3) linkages [3]. Chemical modifications in polysaccharides have been extensively studied and reviewed [4]. Glucans are potential therapeutic agents and are also used as viscosifying, stabilizing, emulsifying, sweetening, gelling, or water-binding agents, in the food as well as in non-food industries [5,6]. The efficiency of established bioprocesses can be increased either by introducing more productive strains or by optimizing the cultivation medium itself [7]. Development of new functional polysaccharides or glucans is important for food industry [8]. Classical mutagenesis techniques can be used to generate mutants for improvement of enzyme and or glucan production. It can lead to production of new types of glucans. The selection for the desired strain and hunting for mutants however is cumbersome. Efficient mutagenesis and good planning pays off in time and labor. Mutants of Leuconostoc strains NRRL B-512F [9], B-742 [10] and B-1299 [11] and 512FMC [12] are presently used in the industry for their traits. The traditional approach of creating mutants is by exposing the microbes to ultraviolet (UV) light that is fairly efficient in producing substitutions. The glucan produced by Leuconostoc dextranicum NRRL B-1146 is unique with α-(1-6) and α-(1-4) linkages [7] and also contains viscosifying and gelling properties [13]. The present study describes classical mutagenesis of Leuconostoc dextranicum NRRL B-1146 by UV irradiation technique, generating mutant strains with enhanced glucan production. Materials and Methods Mutagenesis of by Ultra Violet (UV) irradiation Leuconostoc dextranicum NRRL B-1146 was grown (at 28?C at 200 rpm) in 10 ml of statistically designed liquid medium as described earlier by Majumder et al. 2009 [7] . A cell concentration of 5.2x10 7 cfu/ml was used and 1 ml of these cells were serially diluted in 9 ml sterile saline (0.85%) contained in test tubes. 100 ?l from each of the tubes with dilution factor of 10 3 , 10 4 , 10 5 and 10 6 was plated on modified MRS agar medium describ