Strain improvement and statistical optimization as a combined strategy for improving fructosyltransferase production by Aureobasidium pullulans NAC8

Abstract

Strain improvement of a low fructosyltransferase-producing Aureobasidium pullulans NAC8 (Accession No. KX023301) was carried out using chemical mutagens such as ethidium bromide and ethyl methane sulfonate. The wild-type and mutant strain were distinguished using Random amplified polymorphic DNA PCR and DNA fingerprinting analysis. Plackett-Burman and Box Behnken design were statistical tools used to determine important media parameters and optimization, respectively. Phenotypically and genetically, the new improved strain was different from the wild-type. The most important media parameters from PDB influencing fructosyltransferase production were ammonium chloride, sucrose and yeast extract at p < 0.05. Some significant parameters obtained with the BBD exhibited quadratic effects on FTase. The F values (35.37 and 32.11), correlation coefficient (0.98 and 0.97) and the percent coefficient of variation (2.53% and 2.40%) were obtained for extracellular and intracellular FTase respectively. The validation of the model in the improved strain resulted in an overall 6.0 and 2.0-fold increase in extracellular and intracellular FTase respectively compared to the wild-type.A relatively low FTase-producing strain of Aureobasidium pullulans NAC8 was enhanced for optimum production using a two-pronged approach involving mutagenesis and statistical optimization. The improved mutant strain also had remarkable biotechnological properties that make it a suitable alternative than the wild-type.

Preparation, characterization and optimization of cross-linked fructosyltransferase aggregates for the production of prebiotic fructooligosaccharides

“…The fungus, Aureobasidium pullulans, used in the study was genetically modified using chemical mutagenesis of the wild-type Aureobasidium pullulans NAC8 strain (Accession no. KX023301) (Ademakinwa and Agboola, 2016;Ademakinwa et al, 2017). The fungus was maintained on the malt extract agar slant at 4EC and was routinely subcultured after every 7 days.…”

“…The so-obtained homogenate was centrifuged at 4000 × g for 20 min, and the resulting supernatant was used as the source of intracellular enzyme. All operations were performed on the ice at 4EC (Ademakinwa et al, 2017).…”

“…The released glucose was quantified using a glucose oxidase-peroxidase (GOD-POD) kit (Sigma), using a spectrophotometer at 505 nm. One unit of fructosyltransferase was defined as the amount of enzyme activity required to produce 1 μmol of glucose per minute under the described conditions (Ganaie et al, 2014, Ademakinwa et al, 2017.…”

“…where X is the predicted response; $ 0 is the model constant; Y 1 , Y 2 , and Y 3 are independent variables; $ 1 , $ 2 , and $ 3 are linear coefficients; $ 12 , $ 13 , and $ 23 are cross product coefficients; and $ 11 , $ 22 , and $ 33 are the quadratic coefficients. The model equation for the analysis is given as representing the constant process effect in total, the linear (Y i ), quadratic effect (Y j ), and the effect of interaction between Y i and Y j for the aggregation yield (Haaland, 1989;Ademakinwa et al, 2017).…”

“…Therefore, in this study, we synthesized cross-linked fructosyltransferase aggregates from an improved mutant strain of Aureobasidium pullulans NAC8 (Ademakinwa and Agboola, 2016;Ademakinwa et al, 2017), and we developed a mathematical model for the industrial production by optimizing the scale-up conditions using statistical techniques. The characterization of CLEAs was performed using photomicroscope and scanning electron microscopy energy-dispersive X-ray spectroscopy (SEM-EDX).…”