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

Abstract

In this study, the preparation, characterization, and evaluation of the cross-linked enzyme aggregates (CLEAs) of fructosyltransferase isolated from the mutant-type Aureobasidium pullulans NAC8 for the production of fructooligosaccharides (FOS) with prebiotic properties were investigated. The reaction conditions were optimized statistically by using response surface methodology. CLEAs were characterized by using both photo-microscopy and scanning electron microscopy energy-dispersive X-ray spectroscopy (SEM-EDX). The secondary structure of the protein was predicted by using Fourier transform infrared spectroscopy (FTIR). FOSs produced as a result of the biotransformation of sucrose was quantified by using a chemometric analysis of FTIR spectral data. The prepared FOSs were investigated for the prebiotic effects under anaerobic conditions using a standard strain of Lactobacillus sp. Following were the statistically determined, optimum conditions for the production of FOSs: 5% (v/v) glutaraldehyde, pH 5.5, and a temperature of 32EC. The values of R 2 , F value, adjusted precision, and coefficient of variation were found to be 0.94, 11.50, 9.34, and 35.34%, respectively. Validation of the model resulted in the reusability of the CLEAs in six reaction cycles as it retained 70% of the residual activity. The SEM-EDX and photomicrographic analysis showed that the sizes of CLEAs varied, which ranged between 10 and 40 μm. Aggregation leads to the formation of a higher number of beta sheets in the CLEAs than that in the free enzyme. The total FOSs produced by the CLEAs was around 57% (w/v). A. pullulans FOS supplemented to the medium had a stimulatory effect on the growth of bi dobacteria compared with the control medium. The growth curves of Lactobacillus sp. reached the plateau phase after 24 h of cultivation with FOS. The CLEA of fructosyltransferase catalyzed the biocatalysis of sucrose to FOSs, a process with a biotechnological importance as prebiotics.

Biochemical characterization and kinetic studies on a purified yellow laccase from newly isolated Aureobasidium pullulans NAC8 obtained from soil containing decayed plant matter

“…The reaction medium was incubated at room temperature (25 °C) for 15 min and absorbance of the reaction medium was taken at 450 nm. The extinction coefficient of guaiacol is 12,100 M −1 cm −1 [16]. One unit of laccase activity is defined as the amount of laccase that catalyzed the oxidation of one micromole of guaiacol per minute [17].…”

“…Since yellow laccases are often less studied with more focus on the blue laccases, this study investigated the yellow laccase elaborated by A. pullulans NAC8, which was subsequently purified, biochemically characterized and the catalytic properties determined. Preliminary investigations on the utilization of this enzyme in decolorization of textile dyes and textile waste water effluents have been carried out in our laboratory [16]. The catalytic properties and laccase type of this enzyme from A. pullulans NAC8, has not been reported in any literature.…”

Laccase immobilization via cross-linked aggregate preparation: Characterization, thermodynamic/kinetic properties and application in removal of bisphenol A from solutions

“…It was concluded that the laccase CLEA was more thermostable than the free enzyme. The thermostability of CLEA or other immobilized enzymes over their free enzyme has been reported in recent literature [17,21,[23][24][25] Heat inactivation studies Table 2 shows the summary of the kinetic and thermodynamic parameters for both the free and immobilized laccase. Both enzyme form's enzymatic activity decayed in a typical first-order heat inactivation pattern.…”

“…For the SEM studies, the dried CLEA was coated with gold, viewed under 2500 X magnification under 10 kV using scanning electron microscope model TESCAN. For the FTIR studies, the dried CLEA was ground in a mortar containing KBr (in 1:1) and screwed to pellets [17]. Scans were recorded from 4000-400 cm in transmittance mode using a Shimadzu FTIR-8400S spectrophotometer.…”

Recent advances in β-galactosidase and fructosyltransferase immobilization technology

“…The lower enzyme activity in other supports was attributed to enzyme inactivation during the process and diffusional problems. Ademakinwa et al (2018) prepared and evaluated cross-linked enzyme aggregates (CLEAs) of fructosyltransferase to produce FOS using GA as cross-linking agent. Authors indicated that the best precipitant for CLEAs production was ammonium sulfate being able to maintain 100% of residual activity over four rounds of catalysis.…”