The Optimization of Growth Condition of the Bacteria Producing Cold-Active Proteolytic Enzyme from the Antarctic Region

Muhammad Asyraf Abd Latip; Noor Faizul Hadry Nordin; Siti Aisyah Alias; Jerzy Smykla; Faridah Yusof; Mohd Azrul Naim Mohamad

doi:10.31436/iiumej.v24i1.2447

Authors

Muhammad Asyraf Abd Latip Mariculture Research Division
Noor Faizul Hadry Nordin International Institute for Halal Research and Training (INHART) https://orcid.org/0000-0002-2067-5692
Siti Aisyah Alias Institute of Ocean and Earth and Sciences https://orcid.org/0000-0002-6759-5745
Jerzy Smykla Institute of Nature Conservation, Polish Academy of Sciences https://orcid.org/0000-0001-8228-6695
Faridah Yusof International Islamic University Malaysia https://orcid.org/0000-0001-6946-360X
Mohd Azrul Naim Mohamad International Islamic University Malaysia https://orcid.org/0000-0003-0361-6284

DOI:

https://doi.org/10.31436/iiumej.v24i1.2447

Keywords:

cold-active protease, Antarctica, one factor at time, response surface method

Abstract

The growth conditions of bacteria producing cold-active protease isolated from an Antarctic sample were screened using one-factor-at-time (OFAT). Then, crude protease of the strain was extracted during the late logarithmic phase for enzymatic assay. A strain that showed the highest enzyme activity was selected for optimization via response surface method (RSM). The parameters studied were incubation temperature (4 – 36 °C), pH media (4 – 10) and NaCl concentration (0 – 8%). Based on the OFAT results, all eight strains showed the highest growth rate at 20 °C, pH 7 and 4% (w/v) NaCl. The assay showed that the crude enzyme extracted from strain SC8 exhibited significantly higher activity (0.20 U and 0.37 U) than the positive control (0.11 U and 0.31 U) at -20 °C and 20 °C. RSM suggested that the optimized setting for growth of SC8 were at 20.5 °C, pH 6.83 and 2.05% (w/v) of NaCl with the results of the bacterial growth rate value was 3.70 ± 0.06 x 10⁶ cells/hr. Optimal growth conditions of SC8 from this study are useful for the large-scale production of cold-active protease in future.

ABSTRAK: Keadaan pertumbuhan bakteria yang menghasilkan enzim protease aktif sejuk daripada sampel Antartika disaring menggunakan satu faktor pada masa (OFAT). Kemudian, enzim protease ini diekstrak pada lewat fasa logaritma untuk ujian enzimatik. Strain yang menunjukkan aktiviti enzim tertinggi telah dipilih untuk tujuan pengoptimuman melalui kaedah permukaan tindak balas (RSM). Parameter yang dikaji ialah suhu pengeraman (4 – 36 °C), pH media (4 – 10) dan kepekatan NaCl (0 – 8%). Berdasarkan OFAT, kesemua lapan bakteria menunjukkan kadar pertumbuhan tertinggi pada 20 °C, pH 7 dan 4% NaCl. Hasil ujian enzimatik menunjukkan bahawa enzim protease yang diekstrak daripada SC8 mempamerkan aktiviti yang jauh lebih tinggi (0.20 U dan 0.37 U) daripada kawalan positif (0.11 U dan 0.31 U) pada -20 °C dan 20 °C. RSM mencadangkan tetapan optimum untuk pertumbuhan SC8 adalah pada 20.5 °C, pH 6.83 dan 2.05% NaCl dengan keputusan kadar pertumbuhan bakteria ialah 3.70 ± 0.06 x 10⁶ sel/jam. Keadaan pertumbuhan optimum SC8 daripada kajian ini bermanfaat untuk menghasilkan produk protease aktif sejuk secara besar-besaran pada masa hadapan.

The growth conditions of bacteria producing cold-active protease isolated from an Antarctic sample were screened using one-factor-at-time (OFAT). Then, crude protease of the strain was extracted during the late logarithmic phase for enzymatic assay. A strain that showed the highest enzyme activity was selected for optimization via response surface method (RSM). The parameters studied were incubation temperature (4 – 36 °C), pH media (4 – 10) and NaCl concentration (0 – 8%). Based on the OFAT results, all eight strains showed the highest growth rate at 20 °C, pH 7 and 4% (w/v) NaCl. The assay showed that the crude enzyme extracted from strain SC8 exhibited significantly higher activity (0.20 U and 0.37 U) than the positive control (0.11 U and 0.31 U) at -20 °C and 20 °C. RSM suggested that the optimized setting for growth of SC8 were at 20.5 °C, pH 6.83 and 2.05% (w/v) of NaCl with the results of the bacterial growth rate value was 3.70 ± 0.06 x 10⁶ cells/hr. Optimal growth conditions of SC8 from this study are useful for the large-scale production of cold-active protease in future.

ABSTRAK: Keadaan pertumbuhan bakteria yang menghasilkan enzim protease aktif sejuk daripada sampel Antartika disaring menggunakan satu faktor pada masa (OFAT). Kemudian, enzim protease ini diekstrak pada lewat fasa logaritma untuk ujian enzimatik. Strain yang menunjukkan aktiviti enzim tertinggi telah dipilih untuk tujuan pengoptimuman melalui kaedah permukaan tindak balas (RSM). Parameter yang dikaji ialah suhu pengeraman (4 – 36 °C), pH media (4 – 10) dan kepekatan NaCl (0 – 8%). Berdasarkan OFAT, kesemua lapan bakteria menunjukkan kadar pertumbuhan tertinggi pada 20 °C, pH 7 dan 4% NaCl. Hasil ujian enzimatik menunjukkan bahawa enzim protease yang diekstrak daripada SC8 mempamerkan aktiviti yang jauh lebih tinggi (0.20 U dan 0.37 U) daripada kawalan positif (0.11 U dan 0.31 U) pada -20 °C dan 20 °C. RSM mencadangkan tetapan optimum untuk pertumbuhan SC8 adalah pada 20.5 °C, pH 6.83 dan 2.05% NaCl dengan keputusan kadar pertumbuhan bakteria ialah 3.70 ± 0.06 x 10⁶ sel/jam. Keadaan pertumbuhan optimum SC8 daripada kajian ini bermanfaat untuk menghasilkan produk protease aktif sejuk secara besar-besaran pada masa hadapan.

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Author Biographies

Muhammad Asyraf Abd Latip, Mariculture Research Division

Mariculture Research Division, Fisheries Research Institute Langkawi, Kompleks Perikanan Bukit Malut, 07000 Langkawi, Kedah, Malaysia

Biotechnology Engineering Department, Kulliyyah of Engineering, International Islamic University Malaysia, Jalan Gombak, 53100 Kuala Lumpur

Noor Faizul Hadry Nordin, International Institute for Halal Research and Training (INHART)

International Institute for Halal Research and Training (INHART), Block A, Level 3, KICT Building, International Islamic University Malaysia, Jalan Gombak, 53100 Kuala Lumpur

Siti Aisyah Alias, Institute of Ocean and Earth and Sciences

Institute of Ocean and Earth and Sciences, C308, Level 3, Block C, Institute for Advanced Studies Building, 50603 Kuala Lumpur, Malaysia

National Antarctic Research Centre, B303, Level 3, Block C, Institute for Advanced Studies Building, 50603 Kuala Lumpur, Malaysia

Jerzy Smykla, Institute of Nature Conservation, Polish Academy of Sciences

Institute of Nature Conservation, Polish Academy of Sciences, al. A. Mickiewicza 33 PL-31-120 Krakow, Poland

Faridah Yusof, International Islamic University Malaysia

Biotechnology Engineering Department, Kulliyyah of Engineering, International Islamic University Malaysia, Jalan Gombak, 53100 Kuala Lumpur

Mohd Azrul Naim Mohamad, International Islamic University Malaysia

Department of Biotechnology, Kulliyyah of Science, International Islamic University Malaysia, Jalan Sultan Ahmad Shah, Bandar Indera Mahkota, 25200 Kuantan, Pahang, Malaysia

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