New and simple plate test for screening relative transfructosylation activity of fungi

Abstract

Several microorganisms are reported to have transfructosylation activity due to fructosyltransferase and/or fructofuranosidase activities. However, the search for other fungi with higher transfructosylation activity remains a challenge. So, a presumptive and indirect colorimetric plate assay for the evaluation of transfructosylation activity in fungi was developed which involved the simultaneous determination in the same plate of glucose and fructose released from sucrose. The method entailed the (a) glucose oxidase-peroxidase coupled reaction using phenol and 4-aminoantipyrine for determination of glucose; and (b) fructose dehydrogenase oxidation in the presence of a tetrazolium salt for determination of fructose. The presence of enzymes with transfructosylation activity was identified by the formation of pink (presence of glucose) and blue (presence of fructose) halos around the fungal colony. In conclusion, the results showed that the method is suitable for screening a large number of fungi due to its simplicity, reproducibility and rapidity and also gives a relative quantitative idea of the transfructosylation activity of different fungi species.

A molasses habitat-derived fungus Aspergillus tubingensis XG21 with high β-fructofuranosidase activity and its potential use for fructooligosaccharides production

“…It is known that glucose and fructose are the reaction products accompanying FOS synthesis and the excess of glucose over fructose is characteristic of the transfructosylation activity of FFase (Chen and Liu 1996). Thus, the detection of glucose by using the glucose oxidase (GOD)-peroxidase (POD) system could provide a rapid and indirect assay for screening for potential candidate FOS-synthesizing fungi (Dominguez et al 2006). Here, the isolated fungal strains were tested with the biochemical GOD-POD reaction system.…”

“…To rapidly determine the ability of these fungi to produce FFase, an indirect colorimetric plate assay for evaluation of FFase was used, which involved in the detection of glucose released from sucrose. This method entailed a glucose oxidase–peroxidase (Dingguo Corp., Beijing, China) coupled reaction using phenol and 4-aminoantipyrine for determination of glucose with the formation of pink halos around the fungal colony (Dominguez et al 2006). In detail, 1 μL of spores (10 8 /mL) was spotted on the CD agar plates and incubated at 30 °C for 8 h. Then 10 mL of the biochemical reaction system, composed of glucose oxidase (GOD, 10 U/mL), horseradish peroxidase (POD, 1 U/mL), 4-aminoantipyrine (0.16 mg/mL) and phenol (1 mg/mL) in the citric phosphate buffer (pH 5), was placed on the plates.…”

New improved method for fructooligosaccharides production by Aureobasidium pullulans

“…and Fusarium spp. (Dominguez, Santos, Teixeira, & Lima, 2006;Mussatto, Aguilar, Rodrigues, & Teixeira, 2009a;Yun, 1996;Prata, Mussatto, Rodrigues, & Teixeira, 2010;Sangeetha, Ramesh, & Prapulla, 2004). As an example, Yoshikawa, Amachi, Shinoyama, & Fujii, 2006 reported that at least five ␤-fructofuranosidase are produced by Aureobasidium pullulans DSM2404 and three of them show a high transfructosylation activity.…”

Microbial production of fructosyltransferases for synthesis of pre-biotics

“…Recently, Dominguez et al (2006) reported a new and simple method on agar plates for the evaluation of transfructosylation activity in fungi entailed in (a) glucose oxidaseperoxidase coupled reaction using phenol and 4-aminoantipyrine for determination of glucose; and (b) fructose dehydrogenase oxidation in the presence of a tetrazolium salt for determination of fructose. The presence of enzymes with transfructosylation activity can be identified by the formation of pink (presence of glucose) and blue (presence of fructose) halos around the fungal colony.…”

“…Production of enzymes can be improved with strain development and genetic manipulation as an alternative to reduce production costs (Shin et al 2004). Classic mutagenesis with physical and chemical agents has been an important strategy used by several authors to improve productivity of microbial enzymes including beta-fructofuranosidase, fructosyltransferase and inulinase from fungi and survey for suitable strains for industrial purpose (Suntornsuk and Hang 1994;Nakamura et al 1995;Dominguez et al 2006). Rajoka and Yasmeen (2005) showed that a derepressed mutant of A. niger obtained by irradiation with gamma rays resulted in the improvement of bfructofuranosidase activities.…”