Enhanced Production of an Extracellular β-d-Fructofuranosidase Fructohydrolase from a 2-Deoxy-d-glucose Stabilized Mutant of Candida utilis

Abstract

The enzyme beta-D-fructofuranosidase fructohydrolase (FFH) cleaves the alpha-1,4 glycosidic linkage between alpha-D-glucose and beta-D: -fructose molecules of sucrose, releasing monosaccharides by hydrolysis. In the present study, FFH production in Candida utilis GC-46, a lipolytic wild yeast strain was improved by exposure to N-methyl N-nitro N-nitroso guanidine (NG) and 2-deoxy-D-glucose (2dg) at various levels. The mutant strain NG-5 was obtained after exposure to 0.06 mg/ml of NG for 20 min. NG-5 offers improved extracellular FFH production (34 +/- 2.6 U/ml/min) when compared to the wild strain (1.15 +/- 0.01 U/ml/min). A 40-fold increase of FFH (45.65 +/- 2.0 U/ml/min) was achieved when the process parameters, including incubation period (48 h), sucrose concentration (5.0 g/l), initial pH (6.0), inoculum size (2.0% v/v, 16 h old), and urea concentration (0.2%, w/v) were identified using Plackett-Burman design. The kinetic parameters viz. Q (p) (0.723 U/g/h), Y (p/s) (2.036 U/g), and q (p) (0.091 U/g yeast cells/h) indicate that NG-5 is a hyperproducer of extracellular FFH with a concomitant increase in growth rate. The volumetric productivity of NG-5 was over sixfold improved over the parental strain. The enzyme production improvement is highly significant (HS, LSD 0.042, p < or = 0.05), indicating commercial utility.