Production, characterization and techno-economic evaluation of Aspergillus fusant L-asparaginase.

Protoplast fusion is one of the most reliable methods of introducing desirable traits into industrially-promising fungal strains. It harnesses the entire genomic repertoire of fusing microorganisms by routing the natural barrier and genetic incompatibility between them. In the present study, the axenic culture of a thermo-halotolerant strain of Aspergillus candidus (Asp-C) produced an anti-leukemic L-asparaginase (L-ASNase) while a xylan-degrading strain of Aspergillus sydowii (Asp-S) produced the acrylamide-reduction type. Protoplast fusion of the wild strains generated Fusant-06 with improved anti-leukemic and acrylamide reduction potentials. Submerged fed-batch fermentation was preferred to batch and continuous modes on the basis of impressive techno-economics. Fusant-06 L-ASNase was purified by PEG/Na+ citrate aqueous two-phase system (ATPS) to 146.21-fold and global sensitivity analysis report revealed polymer molecular weight and citrate concentration as major determinants of yield and purification factor, respectively. The enzyme was characterized by molecular weight, amino acid profile, activity and stability to chemical agents. Michaelis-Menten kinetics, evaluated under optimum conditions gave Km, Vmax, Kcat, and Kcat/Km as 6.67 × 10-5 M, 1666.67 µmolmin-1 mg-1 protein, 3.88 × 104 min-1 and 5.81 × 108 M-1.min-1 respectively. In-vitro cytotoxicity of HL-60 cell lines by Fusant-06 L-ASNase improved significantly from their respective wild strains. Stability of Fusant-06 L-ASNase over a wide range of pH, temperature and NaCl concentration, coupled with its micromolar Km value, confers commercial and therapeutic value on the product. Free-radical scavenging and acrylamide reduction activities were intermediate and the conferred thermo-halo-stability could be exploited for sustainable clinical 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.

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.

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

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.

Awareness, Knowledge, and Acceptance of Haematopoietic Stem Cell Transplantation for Sickle Cell Anaemia in Nigeria.

Background. Sickle cell anaemia (SCA) is an inherited condition whose clinical manifestations arise from the tendency of haemoglobin to polymerize and deform red blood cells into characteristic sickle shape. Allogeneic bone marrow transplantation offers a cure. The aim of this study was to determine the level of awareness, knowledge, and acceptance of this beneficial procedure in Nigeria. Materials and Methods. This multicentre cross-sectional study was conducted in 7 tertiary hospitals in Nigeria in 2015. Approval was obtained from each institution's research and ethics committee. A pretested structured questionnaire was administered to respondents aged 18 years and above and to the parents or guardians of those below 18 years of age. Results. There were 265 respondents comprising 120 males and 145 females. One hundred and seventy-one (64.5%) respondents were aware of BMT for the treatment of SCA. About 67.8% (116 of 171) of those who were aware believed SCA can be cured with BMT (p = 0.001) and 49.7% (85 of 171) of the respondents accepted BMT (p = 0.001). Conclusion. Awareness of BMT in Nigeria is low when compared with reports from developed countries. The knowledge is poor and acceptance is low. With adequate information, improved education, and psychological support, more Nigerians will embrace BMT.

Utilization of banked blood in pediatric surgical procedures in Calabar, Nigeria.

BACKGROUND: Performing major surgery in a child demands that blood is cross-matched and saved to be transfused as indicated. Because the cost of cross-matching and donation of blood can be enormous and may equal or surpass the cost of surgery in our setting, it is pertinent to evaluate its utilization. The aim of this study was to determine how banked blood meant for pediatric surgical procedures was utilized with the hope of streamlining our blood requisition policy. This may be useful to pediatric and other surgeons involved in the operative care of children in similar settings. MATERIALS AND METHODS: This was a prospective study of all children who had ELECTIVE or EMERGENCY surgical procedures between January 2009 and June 2010. The age, sex, nature of surgery, blood loss, banked units of blood and amount transfused were collected and analyzed. RESULTS: Eighty two patients had 81 units of blood banked for them. Forty - eight and half units (59.9%) of the banked blood were for the emergency group but only 18 units (22.2%) were actually transfused at the end (P = 0.044) leading to inadequate use of the product. CONCLUSION: Banking large quantities of blood but utilizing only little is tantamount to inadequate use and delays surgical intervention. Indirectly, it increases cost of surgery. There is need to rationalize our blood ordering habits without causing harm to patients.