Enhancement of Clavulanic Acid Production by Streptomyces Sp MU-NRC77 Via Mutation and Medium Optimization

Said et al Tropical Journal of Pharmaceutical Research January 2017; 16 (1): 31-42 ISSN: 1596-5996 (print); 1596-9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. All rights reserved. Available online at http://www.tjpr.org http://dx.doi.org/10.4314/tjpr.v16i1.5 Original Research Article Enhancement of clavulanic acid production by Streptomyces sp MU-NRC77 via mutation and medium optimization Ismail G Said1, Nayera AM Abdelwahed1, Hassan M Awad1,2*, Madgy A Shallan3, Kamal YI El-Shahed1 and Emam A Abdel-Rahim3 1Chemistry of Natural and Microbial Products Dept., Pharmaceutical Industries Div., National Research Centre, 33 EL Bohouth St. (Former EL Tahrir St.), Dokki, PO Box 12622, Giza, Egypt, 2Deanship of Preparatory Year, Al jouf University, Al Jouf, Sakaka, PO Box 2014, Kingdom of Saudi Arabia, 3Biochemistry Department, Faculty of Agriculture, Cairo University, Giza, Egypt *For correspondence: Email: [email protected] Received: 6 September 2016 Revised accepted: 19 December 2016 Abstract Purpose: To enhance clavulanic acid production using UV-mutagenesis on Streptomyces sp. NRC77. Methods: UV-mutagenesis was used to study the effect of Streptomyces sp. NRC77 on CA production. Phenotypic and genotypic identification methods of the promising mutant strain were characterized. Optimization of the fermentation medium and culture conditions were investigated Results: Out of the screened mutants, 120A3 mutant isolate was selected as promising. The phenotypic properties of 120A3 mutant showed culture characteristics similar to those of Streptomyces species. Phylogenetic analysis of 16S rRNA gene sequence indicate that this strain has similarity (99 %) to Streptomyces sp.T2-7; therefore it was suggested as Streptomyces sp. MU-NRC77 and has Gen Bank accession no. KT953342. Conclusion: Improvement of CA yield by 48 % was obtained from fermentation medium and culture condition optimization. Further optimization by addition of H2O2 and activated charcoal to the production medium increased CA yield to 646.12 and 682.94 mg/L respectively, i.e., 83 % more than that obtained prior to addition. Keywords: Clavulanic acid, Medium optimization, Phenotypic and Genotypic identification, Streptomyces sp. MU-NRC77, UV-Mutagenesis Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopus, International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals (DOAJ), African Journal Online, Bioline International, Open-J-Gate and Pharmacy Abstracts INTRODUCTION successfully employed in clinical practice. It is produced by the actinomycete Streptomyces β-lactam ring-containing molecules such as clavuligerus, and is used in combination with those of penicillins and cephalosporins were beta-lactam antibiotics that are sensitive to attack hydrolyzed by the effect of the catalytic ability of by beta-lactamase enzymes. It acts by enzymes produced by many pathogenic bacteria, specifically inhibiting the active centers of these resulting in the blocking of their antibacterial enzymes, hence avoiding loss of the beta-lactam property, in turn causing resistance to antibiotics antibiotic and restoring its antimicrobial action. and creating a problem in the treatment of The combined action as ß-lactamase inhibitor infectious diseases [1]. Clavulanic acid (CA) is a and antibacterial agent makes CA very important potent ß-Lactamase inhibitor that has been both clinically and economically [2]. Trop J Pharm Res, January 2017; 16(1): 31 Said et al Media composition, growth conditions different exposure times (30, 60, 90, 120, 150, optimization and strain improvement increased 180, 210 and 240 seconds) at a distance of 20 the production of CA (up to approx. 1.4 g/l) [3]. cm from the UV lamp with wavelength of 254 nm Strain improvement was achieved by and power of 30 W. All these exposures were mutagenesis, the most generally utilized tool for performed in a dark room to avoid any better yield of secondary metabolites in the photoreaction in the production of mutants. fermentation process [4]. A relatively safe Spore suspension, exposed or not exposed to mutagen like ultraviolet (UV) irradiation UV irradiation, was also spread over the surface represents a very convenient system to enhance of an ISP2 medium for 10 days at 28 oC. strains through random mutation. But only a Colonies which survived at each dose of the UV small number of researchers have utilized UV irradiation were transferred to ISP2 slants irradiation to overproduce clavulanic acid [3]. The composed of the following (g/L): D (+) glucose, 4; taxonomic properties of S. olivaceus, such as the malt extract, 10; yeast extract, 4 and agar, 20, for color of the aerial mycelium, liquefaction of 10 days. The direct demonstration of CA gelatin, growth of cellulose, production of production was achieved by sub-culturing streaks ammonia and nitrate reduction were changed of each mutant, on plates containing a starch after irradiation of the parent strain with UV, nitrate medium. The plates were then cut off causing the creation of mutants different in their using sterile cork borer and the discs were spectrum of antimicrobial activity. Treatment of transferred to Petri dishes containing Muller S. fradiae NRRL 2702 with n-nitroso-guanidine Hinton agar as a bioassay medium, seeded with (NTG), or exposure to UV formed from tylosin a penicillin-resistant test strain of E. coli hyper-producing mutants. Mutagenesis by NTG suspension (OD = 1.0 at 600 nm), then and UV increase the productivity of S. transferred to a refrigerator for 30 min at 4 oC hygroscopicus FC 904 (rapamycin producer) and incubated at 37 oC for 24 h. The diameter of from 60 - 124 % [5]. the resulting inhibition zones were then measured and considered to estimate the The objective of the present study is to improve mutant's CA potentialities [7]. Colonies with a CA production by the Streptomyces sp. MU- diameter of inhibition zone greater than that of NRC77 mutant strain followed by optimization of the parent wild type strain Streptomyces sp. the fermentation medium and culture conditions. NRC-77 were picked up for further secondary screening in shake flasks. EXPERIMENTAL Production medium and cultivation Microorganisms and culture conditions conditions The strain Streptomyces NRC-77 was previously The most potent selected strain obtained after isolated from an Egyptian soil sample by a UV mutagenesis was cultivated in Erlenmeyer screening program [6]. This Streptomyces strain flasks (250 mL) containing 50 ml production produces CA and was deposited in the medium in an Innova 4080 rotary shaker (New Actinomycetes Culture Collection, National Brunswick, NJ, USA) at 200 rpm and 28 °C for Research Centre, Dokki, Giza, Egypt. It was six days. The inoculation was conducted using a 8 maintained in an International Streptomyces spore suspension (1.6 × 10 spores/mL) of the Project medium 4 (ISP4) composed of (g/L): most potent strain slanted in an ISP2 medium. Soluble starch, 10.0; K2HPO4, 1.0; MgSO4.7H2O, The production medium was composed of (in 1.0; NaCl, 1.0; (NH4)2SO4, 2.0; CaCO3, 2.0; agar, g/l): Soluble starch 25; Glycerol 10 mL; Soybean 20.0. A strain of Escherichia coli, maintained in a meal 30; KH2PO4, 0.1, FeSO4 .7H2O, 0.1; and nutrient agar medium capable of resisting 25 the pH was adjusted to 7.0 before sterilization. μg/mL of penicillin-G (Glaxo Wellcome UK Ltd., Culture broth was separated from the mycelium Middlesex, UK), was used as a test strain for by centrifugation at 5000 rpm by refrigerated evaluation of CA concentration [6]. Colonies of centrifuge (Sigma 3-16 KL laboratory centrifuge, these strains were stored at -80 °C in 20 % Germany) for 10 min. The supernatant was glycerol for further studies. filtered and used for the evaluation of the inhibitory activity, which was conducted Strain improvement through UV mutagenesis biologically using the agar diffusion method and spectrophotometrically through the reaction of A sample (10 mL) of spore suspension from a CA with imidazole at 312 nm [7,8]. solid culture of Streptomyces sp. NRC-77 was transferred to a sterilized Petri dish plate under Fermentation assays were carried out in aseptic conditions. The plate, with cover duplicates. The most potent strain, which showed removed, was exposed to UV irradiation for Trop J Pharm Res, January 2017; 16(1): 32 Said et al the greatest CA production, was selected for Master Mix (Thermo K1051). The 16S rDNA was further investigation. amplified by a polymerase chain reaction (PCR) using primers designed to amplify 1500 bp Determination of CA by high performance fragment of the 16S rDNA region. The domain liquid chromatography (HPLC) bacteria-specific primer 27F (forward primer) was 5’AGAGTTTGATCMTGGCTCAG3’ and the Clavulanic acid was determined after reacting universal bacterial primer 1492R (reverse primer) with an imidazole reagent [8] using SYIKIM was 5’TACGGYTACCTTGTTACGACTT3’ [14]. HPLC (Germany), a reversed phase column (Polaris C-18, 5 μm, 250 × 4.6 nm), at 30 ºC. The The PCR reaction was performed with 5μl of peaks were detected using a variable wave genomic DNA as the template, 1μl of 16S rRNA length photos array detector model (Jasko - UV - Forward primer, 1 μL of 16S rRNA reverse 2070 Plus, Intelligent UV/Visible detector, primer, 18 μL nuclease-free water, and 25 μL Japan). For chromatographic separation, a C-18 Maxima® Hot Start PCR Master Mix (2X) in a column (Thermo Hypersil Keystone, 5 μm, 250 × 50μL reaction mixture as follows: activation of 2 4.6 mm) was used. Potassium dihydrogen Taq polymerase at 95 °C for 2 min; 35 cycles of orthophosphate buffer (pH 3.2) and 6 % 95 °C for 1 min, 65 °C, and 72 °C for 1 min each methanol was used as a mobile phase and the were performed, finishing with a 10-minute step flow rate was adjusted to 1 mL/min. The sample at 72 °C.

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