جداسازی و شناسایی قارچ‌های تولیدکننده بتاگالاکتوزیداز و بررسی فعالیت آنزیم حاصل از جدایه منتخب

نوع مقاله : مقاله کامل علمی پژوهشی

نویسندگان

1 دانش‌آموخته کارشناسی‌ارشد، گروه علوم و صنایع غذایی، دانشگاه علوم کشاورزی و منابع طبیعی گرگان

2 دانشیار، گروه علوم و صنایع غذایی، دانشگاه علوم کشاورزی و منابع طبیعی گرگان

3 استاد، گروه علوم و صنایع غذایی، دانشگاه علوم کشاورزی و منابع طبیعی گرگان

چکیده

سابقه و هدف: بتا-گالاکتوزیدازها آنزیم هایی از خانواده گلیکوزید هیدرولاز EC (3.2.1.23) هستند که هیدرولیز برخی دی ساکاریدها را کاتالیز نموده و کاربردهای گسترده‌ای در صنایع غذایی دارند. همچنین برخی بتا-گالاکتوزیدازها واکنش ترانس-گالاکتوزیلاسیون را کاتالیز می کنند. این مطالعه با هدف جداسازی قارچ‌های مولد بتا-گالاکتوزیداز از چند نمونه پنیر سنتی و آب پنیر و تعیین فعالیت آنزیمی تحت شرایط مختلف (دما، زمان، pH و کاتیون‌های مختلف) انجام شد.
مواد و روش ها: ابتدا مخمرها و کپک ها جداسازی و از نظر توانایی تولید بتا-گالاکتوزیداز با استفاده از محیط کشت کروموژنیک حاوی 5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside (X-gal) غربالگری شدند. پس از غربالگری، شناسایی ژنوتیپی قوی ترین کپک ها و مخمرهای جدا شده با توالی یابی قطعه ای از ژن rDNA ITS انجام گرفت. سپس، فعالیت هیدرولیزی محلول خام آنزیم بتا-گالاکتوزیداز به دست آمده از جدایه های منتخب با استفاده از ortho-Nitrophenyl-b-galactoside (ONPG) به عنوان سوبسترا در شرایط مختلف (سطوح مختلف دما، زمان، pH و کاتیون های مختلف (Mn2+، Mg2+، Zn2+، Cu2+) ارزیابی شد. در نهایت، فعالیت ترانس-گالاکتوزیلاسیون محلول خام آنزیمی منتخب مورد بررسی قرار گرفت.
یافته ها و نتیجه گیری: نتیجه توالی یابی محصول PCR منجر به شناسایی ایزوله های MM24، MT12 و MW14 به ترتیب به عنوان Kluyveromyces lactis H1-3 (99.57%)، Kluyveromyces lactis E3 (97.53%) و Penicillium (brevicom) (100%) شد. بررسی فعالیت آنزیم های بتاگالاکتوزیداز تولید شده توسط جدایه های MM24، MT12 و MW14، فعالیت بالاتری را برای آنزیم خام به دست آمده از K. lactis MM24 نشان داد. همچنین بیشترین فعالیت در دمای 37 درجه سانتی گراد، pH 7 و زمان واکنش 30 دقیقه مشاهده شد. کاتیون های مختلف بر فعالیت آنزیمی در غلظت های 0.1 و 1 میلی مولار تاثیری نداشتند، با این حال فعالیت آنزیم به طور کامل در برابر Cu2 + و Zn2 + در غلظت 10 میلی مولار مهار شد. علاوه بر این در غلظت 10 میلی مولار، Mn +2 وCa+2 فعالیت آنزیمی را به ترتیب حدود 80% و 65% مهار کردند.نتایج به دست آمده از کروماتوگرافی مایع با کارایی بالا (HPLC) فعالیت هیدرولیتیک آنزیم خام منتخب را روی لاکتوز تایید نمود. با این حال، فعالیت ترانس گالاکتوزیلاسیون در این مطالعه مشاهده نشد. به طور کلی، K. lactis MM24 جداسازی شده در پژوهش حاضر می تواند به عنوان سویه جدید مولد بتا-گالاتوزیداز در نظر گرفته شود.
به طور کلی، K. lactis MM24 جداسازی شده در پژوهش حاضر می تواند به عنوان سویه جدید مولد بتا-گالاتوزیداز در نظر گرفته شود.
به طور کلی، K. lactis MM24 جداسازی شده در پژوهش حاضر می تواند به عنوان سویه جدید مولد بتا-گالاتوزیداز در نظر گرفته شود.

کلیدواژه‌ها

موضوعات

عنوان مقاله [English]

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

نویسندگان [English]

  • Forough Yaghoubi 1
  • Ali Moayedi 2
  • Morteza Khomeiri 3
  • Alireza Sadeghi 2
1 MSc. Graduate, Department of Food Science and Technology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran.
2 Associate Professor, Department of Food Science and Technology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran
3 Professor, Department of Food Science and Technology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran.
چکیده [English]

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.

کلیدواژه‌ها [English]

  • ß-galactosidase
  • Transgalactosylation
  • Molecular identification
  • Enzyme activity

مراجع

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نشریه فرآوری و نگهداری مواد غذایی
دوره 16، شماره 2
تیر 1403
صفحه 87-101

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