No abstract

The Antarctic region is a new frontier as natural sources for bio-prospecting purposes. Its extreme cold temperature may provide unique enzyme characteristics that have valuable potential for industrial and biotechnological applications. This study was designed to discover proteases that are activate and can work at very low temperatures. Soil samples from the Antarctic region were screened for protease activity on skim milk agar at 4°C. Bacteria that showed clear halo zone around the colonies were selected and identified through 16S rDNA sequencing. Out of 35 bacteria, 10 bacteria that showed rapid halo zone formation were selected and further analyzed by enzymatic assay. By using azocasein as a substrate, the reaction was measured using spectrophotometer at OD340 nm. Based on the 16S rDNA sequence, phylogenetic analysis showed that 88% of the bacteria producing protease were from Pseudomonas sp., 9% from Arthrobacter sp. and 3% from Paenibacillus sp. For enzymatic assay analysis, sample SC8 showed the highest protease activity compared to other 10 samples. This preliminary study successfully demonstrated cold active protease producers that can be further investigated for bioprospecting. In future, purification and characterization of this enzyme is required in order to optimize the enzyme activity.

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.