This study was conducted to enhance the production of tacrolimus in Streptomyces tsukubaensis by strain mutagenesis and optimization of the fermentation medium. A high tacrolimus producing strain S. tsukubaensis FIM-16-06 was obtained by ultraviolet mutagenesis coupled with atmospheric and room temperature plasma mutagenesis.Then, nine variables were screened using Plackett–Burman experimental design, in which soluble starch, peptone and Tween 80 showed significantly affected tacrolimus production. Further studies were carried out employing central composite design to elucidate the mutual interaction between the variables and to work out optimal fermentation medium composition for tacrolimus production. The optimum fermentation medium was found to contain 61.61 g/L of soluble starch, 20.61 g/L of peptone and 30.79 g/L of Tween 80. In the optimized medium, the production of tacrolimus reached 1293 mg/L in shake-flask culture, and reached 1522 mg/L while the scaled-up fermentation was conducted in a 1000 L fermenter, which was about 3.7 times higher than that in the original medium. Combining compound mutation with rational medium optimization is an effective approach for improving tacrolimus production, and the optimized fermentation medium could be efficiently used for industrial production.

Improvement of tacrolimus production in Streptomyces tsukubaensis by mutagenesis and optimization of fermentation medium using Plackett–Burman design combined with response surface methodology

Abstract Downstream processing is a significant part of a production process and accounts for 50–90% of the production cost of biotechnological products. Post-fermentation localization of a microbial metabolite contributes significantly to the recovery cost of the product. Enterobacter cloacae produced naturally, acidic lipase with a 0.023:1 extracellular localization ratio. This research aimed to re-direct the localization of lipase to the extracellular milieu to reduce recovery costs using multi-objective response surface optimization (MO-RSM). The approach resulted in a 1:0.32 extracellular: intracellular lipase ratio, with product formation kinetics of Luedeking-Piret function showing a significant switch from a completely growth-associated intracellular production to a predominantly non-growth-associated extracellular localization. The enzyme was purified by an aqueous two-phase system which extracted 95.22% lipase with 72.36 purity. Characterization of the enzyme showed a molecular weight of 55.7 kDa, kcat of 68.59 s−1, and a Km of 0.63 mmol. Lipase activity occurred optimally at pH 2.5–3.5 and 50 °C, and was stable in most organic solvents tested. The acidic lipase demonstrated pH-dependent enantioselective esterification in resolving (R, S)-ibuprofen (E = 14, pH 4.5) and (R, S)-Naproxen (E = 13, pH 2.5), with an enantioselective preference for (S)-enantiomer in both drugs thus underpinning its potential for pharmaceutical applications.

Intracellular-to-extracellular localization switch of acidic lipase in Enterobacter cloacae: evaluation of production kinetics and enantioselective esterification potential for pharmaceutical applications

Intracellular-to-extracellular localization switch of acidic lipase in Enterobacter cloacae through multi-objective medium optimization: aqueous two-phase purification and activity kinetics

Menaquinone-7 (MK-7) has an important role in preventing diseases such as cardiovascular disease and osteoporosis. In this study, a combination strategy of strain improvement and medium optimization is investigated to increase MK-7 production in Bacillus amyloliquefaciens. Conventional breeding method was first used to modify the biosynthetic pathway to construct a MK-7 high-producing strain by atmospheric and room temperature plasma mutagenesis and protoplast fusion. The resulted strain Ba-4 with resistance to sulfaguanidine, 1-hydroxy-2-naphthoic acid, menadione, 2-deoxy-d-glucose and rifampicin as well as sensitive to β-fluoropyruvate produced 73.57 ± 1.61 mg/L of MK-7, which was 1.36 times more than that of the parent strain H.β.D.R.-5 (i.e., 31.12 ± 1.40 mg/L). Subsequently, single-factor optimization and response surface methodology (RSM) were used to optimize the medium components for increasing MK-7 production by strain Ba-4. Strain Ba-4 produced 90.43 ± 1.32 mg/L of MK-7 under the single-factor optimized medium. Moreover, the results of response surface methodology indicated that glycerol, soy peptone and Tween-80 had significant effects on MK-7 production, and the highest MK-7 production (i.e., 95.03 ± 1.01 mg/L) was obtained under the optimized medium, which was 0.29 times higher than that of the initial medium. These results confirmed that the conventional breeding methods and fermenter control system are effective strategies in improving MK-7 production by B. amyloliquefaciens. 

Accelerating the menaquinone-7 production in Bacillus amyloliquefaciens by optimization of the biosynthetic pathway and medium components

The axenic culture of Aspergillus candidus (Asp-C) produced an anti-leukemic L-asparaginase while Aspergillus sydowii (Asp-S) produced the acrylamide-reduction type. Upon mutagenesis by atmospheric and room-temperature plasma (ARTP), their individual L-asparaginase activities improved 2.3-folds in each of Ile-Thr-Asp-C-180-K and Val-Asp-S-180-E stable mutants. Protoplast fusion of selected stable mutants generated fusant-09 with improved anti-leukemic activity, acrylamide reduction, higher temperature optimum and superior kinetic parameters. Submerged (SmF) and solid-state fermentation (SSF) types were compared; likewise batch, fed-batch and continuous fermentation modes; and fed-batch submerged fermentation was selected on the basis of impressive techno-economics. Fusant L-asparaginase was purified by PEG/Na+ citrate aqueous two-phase system and molecular exclusion chromatography to 69.96 and 146.21-fold, respectively, and characterized by molecular weight, specificity, activity and stability to chemical and physical agents. Michaelis-Menten kinetics, evaluated under optimum conditions gave Km, Vmax, Kcat, and Kcat/Km as 1.667 × 10-3 M, 1666.67 µmol min-1 mg-1 protein, 645.99 s-1 and 3.88 × 105 M-1 s-1 respectively. In-vitro cytotoxicity of HL-60 cell lines by fusant-09 L-asparaginase improved 3.00 and 18.71-folds from mutants Ile-Thr-Asp-C-180-K and Val-Asp-S-180-E, and from 5.73 and 32.55 from respective original strains. Free-radical scavenging and acrylamide reduction improvements were intermediate. Fusant-09 L-asparaginase is strongly recommended for sustainable economic anti-leukemic and food industry applications.

Interspecific protoplast fusion of atmospheric and room-temperature plasma mutants of Aspergillus generates an L-asparaginase hyper-producing hybrid with techno-economic benefits.

Sequential optimization of bioprocess nutritional conditions for production of glutaminase-near-free L-asparaginase by Aspergillus candidus UCCM 00117 was conducted under shake flask laboratory conditions. Catalytic and anti-cancer activities of the poly-peptide were evaluated using standard in vitro biochemical methods. Medium nutrients were selected by one-factor-at-a-time (OFAT) approach while Plackett–Burman design (PBD) screened potential factors for optimization. Path of steepest ascent (PSA) and response surface methodology (RSM) of a Min-Run-Res V fractional factorial of a central composite rotatable design (CCRD) were employed to optimize factor levels towards improved enzyme activity. A multi-objective approach using desirability function generated through predictor importance and weighted coefficient methodology was adopted for optimization. The approach set optimum bioprocess conditions as 49.55 g/L molasses, 64.98% corn steep liquor, 44.23 g/L asparagine, 1.73 g/L potassium, 0.055 g/L manganese and 0.043 g/L chromium (III) ions, at a composite desirability of 0.943 and an L-asparaginase activity of 5216.95U. The Sephadex-200 partially-purified polypeptide had a specific activity of 476.84 U/mg; 0.087U glutaminase activity, 36.46% yield and 20-fold protein purification. Anti-cancer activity potentials of the catalytic poly-peptide were dose-dependent with IC50 (µg/mL): 4.063 (HL-60), 13.75 (HCT-116), 15.83 (HeLa), 11.68 (MCF-7), 7.61 (HepG-2). The therapeutic enzyme exhibited 15-fold more cytotoxicity to myeloid leukemia cell line than to normal (HEK 238 T) cell. Optimum temperature and pH for activity were within physiological range. However, significant interactions between exposure time and levels of each of temperature and pH made interpretations of residual enzyme activities difficult. The manganese-dependent L-asparaginase from Aspergillu s candidus UCCM 00117 is recommended for further anticancer drug investigations.

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Bioprocess Optimization of Nutritional Parameters for Enhanced Anti-leukemic L-Asparaginase Production by Aspergillus candidus UCCM 00117: A Sequential Statistical Approach

This research was carried out on the antibacterial and phyto-chemical properties of aqueous and methanol extracts of the rhizome of Zingiber officinale. The methanol and aqueous extracts of the spice demonstrated high antibacterial potency against Staphylococcus aureous, Bacillus subtilis and Escherichia coli. The effect of the extracts on the test organisms varied with the dilution level used, and methanol extract was generally more inhibitive than the aqueous extract. Phyto-chemical analysis of the extracts confirmed the presence of saponins, flavonoid, cardiac glycosides, polyphenols and anthraquinones. These are anti-oxidative compounds which possibly might have contributed to the high antibacterial potency of the spice.

Ubong Ekong

Papers

Bioprocess Optimization of Nutritional Parameters for Enhanced Anti-leukemic L-Asparaginase Production by Aspergillus candidus UCCM 00117: A Sequential Statistical Approach

Antibacterial and Phytochemical Properties of Crude Extract of Zingiber Officinale