Isolation and identification of ß-galactosidase producing fungi and investigating the activity of enzyme from the selected isolate

Background and Objective: ß-galactosidases are enzymes of the glycoside hydrolase family EC (3.2.1.23) that catalyze the hydrolysis of some disaccharides with wide applications in the food industry. Also, some ß-galactosidases have transgalactosylation activity. This study aimed to isolate ß-galactosidase-producing fungi from several traditional cheese and whey samples and to determine the enzymatic activity under the various conditions (temperature, time, pH, and different cations).
Material and Methods: Yeasts and molds were isolated and screened for the ability to produce beta-galactosidase by a chromogenic test containing 5-bromo-4-chloro-3-indolyl-Beta-D-galacto-pyranoside (X-gal) in the media. After screening, the most promising isolated molds and yeasts were identified genotypically by ITS rDNA sequencing. Then, the hydrolytic activity of crude beta-galactosidase was evaluated using ortho-Nitrophenyl-β-galactoside (ONPG) as the substrate under different conditions (different levels of temperature, time, pH, and various cations Mn2+, Mg2+, Zn2+, Cu2+). Finally, the possibility of transgalactosylation activity of the selected enzyme was investigated.
Results: The results of PCR product sequencing resulted in the identification of isolates MM24, MT12, and MW14 as Kluyveromyces lactis H1-3 (99.57%), Kluyveromyces lactis E3 (97.53%), and Penicillium brevicompactum (100%), respectively. Investigating the activity of ß-galactosidase produced by MM24, MT12, and MW14 isolates showed a higher activity for the crude enzyme from K. lactis MM24. Moreover, the highest activity was found at 37°C, pH 7, and 30 min reaction time. Different cations did not significantly influence the enzymatic activity at 0.1 and 1mM, however it was completely inhibited against Cu+2 and Zn+2 at 10 Mm. Furthermore, 10 mM Mn+2 and Ca+2 inhibited enzymatic activity by about 80% and 65%, respectively. High-performance liquid chromatography (HPLC) analysis confirmed ß-galactosidase activity on lactose, whereas no transgalactosylation activity was observed in this study.
Conclusion: Overall, K. lactis MM24 isolated in the current study can be considered as a new isolate for ß-galactosidase production.
Background and Objective: ß-galactosidases are enzymes of the glycoside hydrolase family EC (3.2.1.23) that catalyze the hydrolysis of some disaccharides with wide applications in the food industry. Also, some ß-galactosidases have transgalactosylation activity. This study aimed to isolate ß-galactosidase-producing fungi from several traditional cheese and whey samples and to determine the enzymatic activity under the various conditions (temperature, time, pH, and different cations).
Material and Methods: Yeasts and molds were isolated and screened for the ability to produce beta-galactosidase by a chromogenic test containing 5-bromo-4-chloro-3-indolyl-Beta-D-galacto-pyranoside (X-gal) in the media. After screening, the most promising isolated molds and yeasts were identified genotypically by ITS rDNA sequencing. Then, the hydrolytic activity of crude beta-galactosidase was evaluated using ortho-Nitrophenyl-β-galactoside (ONPG) as the substrate under different conditions (different levels of temperature, time, pH, and various cations Mn2+, Mg2+, Zn2+, Cu2+). Finally, the possibility of transgalactosylation activity of the selected enzyme was investigated.
Results: The results of PCR product sequencing resulted in the identification of isolates MM24, MT12, and MW14 as Kluyveromyces lactis H1-3 (99.57%), Kluyveromyces lactis E3 (97.53%), and Penicillium brevicompactum (100%), respectively. Investigating the activity of ß-galactosidase produced by MM24, MT12, and MW14 isolates showed a higher activity for the crude enzyme from K. lactis MM24. Moreover, the highest activity was found at 37°C, pH 7, and 30 min reaction time. Different cations did not significantly influence the enzymatic activity at 0.1 and 1mM, however it was completely inhibited against Cu+2 and Zn+2 at 10 Mm. Furthermore, 10 mM Mn+2 and Ca+2 inhibited enzymatic activity by about 80% and 65%, respectively. High-performance liquid chromatography (HPLC) analysis confirmed ß-galactosidase activity on lactose, whereas no transgalactosylation activity was observed in this study.
Conclusion: Overall, K. lactis MM24 isolated in the current study can be considered as a new isolate for ß-galactosidase production.