Screening of Alginate Lyase-Producing Bacteria and Optimization of Media Compositions for Extracellular Alginate Lyase Production

Abstract

Background: Alginate is a linear polysaccharide consisting of guluronate (polyG) and mannuronate (polyM) subunits. Methods: In the initial screening of alginate-degrading bacteria from soil, 10 isolates were able to grow on minimal medium containing alginate. The optimization of cell growth and alginate lyase (algL) production was carried out by the addition of 0.8% alginate and 0.2-0.3 M NaCl to the culture medium. Of 10 isolates, one was selected based on its fast growth rate on minimal 9 medium containing 0.4% sodium alginate. The selected bacterium, identified based on morphological and biochemical characteristics, as well as 16S rDNA sequence data, was confirmed to be an isolate belonging to the genus Bacillus and designated as Bacillus sp. TAG8. Results: The results showed the ability of Bacillus sp. TAG8 in utilizing alginate as a sole carbon source. Bacillus sp. TAG8 growth and algL production were augmented with an increase in sodium alginate concentration and also by the addition of 0.2-0.3 M NaCl. Molecular analysis of TAG8 algL gene showed 99% sequence identity with algL of Pseudomonas aeruginosa PAO1. The algL produced by Bacillus sp. TAG8 cleaved both polyM and polyG blocks in alginate molecule, as well as acetylated alginate residues, confirming the bifunctionality of the isolated lyase. Conclusion: The identification of novel algL genes from microbial communities constitutes a new approach for exploring lyases with specific activity against bacterial alginates and may thus contribute to the eradication of persistent biofilms from clinical samples.

Biosynthesised AgCl NPs using Bacillus sp. 1/11 and evaluation of their cytotoxic activity and antibacterial and antibiofilm effects on multi‐drug resistant bacteria

“…To do molecular identification [8], Bacillus isolate was cultured in Lysogeny Broth (LB) medium (37°C, 150 rpm) and the biomass was harvested by centrifugation (4500 g , 10 min) after 18 h and used for DNA extraction. The extraction of genomic DNA was accomplished by polymerase chain reaction (PCR) template purification kit (Qiagen, Iran).…”

“…Bacillus subtilis ATCC 6633, Halobacillus karajensis and ten Bacillus spp. isolated from soil located in Malayer, Hamedan city, Iran, 34°17′ latitude and 48°17′ longitude have chosen for AgNP synthesis screening [8]. Each bacterial extract adjusted to pH12 were subjected to 5 mM AgNO 3 final concentration in dark at room temperature.…”

“…Aliquots of premier bacterial extract were prepared for different pH (6)(7)(8)(9)(10)(11)(12), and then dispersed in test tubes containing different final concentrations of AgNO 3 (1-5 mM) and incubated in dark at room temperature. The results were investigated by UV-vis spectroscopy.…”

Production, Purification and Inhibition of Alginate Ly-Ase From Local Isolate of Pseudomonas Aeruginosa Na11

“…Bacterial isolates Forty local isolates of P.aeruginosa were taken from Biotechnology Department/College of Science /Baghdad University, Al-Yarmouk Hospital Baghdad, AL-Kadhimi Hospital-Baghdad, includes seventeen were isolated from burns and twenty-three isolated from wounds. P.aeruginosa cells were activated at 37 Cº in brain heart infusion broth.= Primary screening of alginate lyaseproduction The competency of forty P.aeruginosa local isolates for alginate lyase production were screened according to the method portray by Tavafi et al (23). A 75 µl of cell suspensions (1.6 × 10 6 cells/ml) of all isolates were transferred into holes (3mm) of the alginate agar medium containing of 0.5 % sodium alginate, 1 % peptone, 0.5 % yeast extract, 0.5 % NaCl, and 1.5 % agar with pH 7.0.…”

“…Production media (type of substrate) Five different types of media were used for alginate lyase production from local isolate of P.aeruginosa NA11, including:-A: 0.5 % sodium alginate, 0.1 % peptone, 0.5 % yeast extract, 0.5 % Nacl, 0.1 % MgSO 4 , 0.1 % KH 2 PO 4 ; B: 0.5 % Nacl, 1 % ammonium sulphate , 0.5 % KH 2 PO 4 , 1.5 % K 2 HPO 4 , 0.2 % MgSO 4 , 1 % sodium alginate, 0.5 % yeast extract; C: 0.1 % peptone, 0.5 % sodium alginate, 0.5 % yeast extract ; D: 0.5 % sodium alginate, 0.5 % peptone, 0.1 % yeast extract, 0.5 % Nacl, 0.1 % MgSO 4 , 0.1 % KH 2 PO 4 ; E:1 % peptone, 0.1 % yeast extract, 3 % Nacl, 0.5 % KH 2 PO 4 , 1 % ammonium sulphate, 0.5 % sodium alginate. Each media were inoculated with 1.6 × 10 6 cells/ml of local isolate of P.aeruginosa NA11 and incubated at 37 0 C for 48 h.. Alginate lyase activity and protein concentration were estimated (23).…”