Characterization of protease from thermophiles isolated from Local Hot Springs / Mohd Nur Fakhruzzaman Noorizhab

To obtain enzymes with improved thermostability, many have resort to isolate enzymes
from naturally occuring thermophilic organisms. However, the disadvantage is that it is
often difficult to get good yield. Hence this study was undertaken to investigate the
thermophilic protease production in eight isolated thermophiles (A I, A3, A4, AS, A6,
A8, A13 and A14) by both biochemical PCR. All the eight thennophiles were subjected
to skim milk agar assay to detect protease enzyme and further amplified by PCR for
protease gene. All the eight thermophiles were positive for protease gene but in skim
milk assay, only A 1, A3, A4, A6 and A8 demonstrated hydrolysis. Comparative analysis
amongst these eight indicated that AS had the highest proteolytic activity and therefore
was further examined its potentials. A8 was found to be motile, Gram positive rod with
endospore, catalase positive, oxidase negative and is a glucose, sucrose, fructose
fermenter except maltose. The protease enzyme was stable at 55°C to 75°C and was
most active at pH 7. Furthermore, AS protease activity was inhibited by 5 mM
ethyldiaminetetraacetyl acid and 5 mM phenysulphonylmethylfluoride thus indication
that it produced two types of proteases; metalloproteases and serine proteases. Further
indentification by 16S rRNA gene sequencing revealed that it is closely related to
Geobacillus thermocatenulatus strain BGSC 93A 1 with 70% similarity to peptidase of
Geobacillus sp. strain C56-T3 (Accession No. CP002050.1 ), Geobacillus sp. strain
Y 412MC61 (Accession No. CPOO 1794.1) and Geobacillus kaustophilus strain HT A426
(Accession No. BA000043.1 ). The protease fragment sequence of Geobacillus sp. strain
AS was submitted to GenBank (Accession No. JF960945) where it codes for SS peptidase
group (a group for serine-type protease). Molecular weight determination by SDSPAGE
for AS was found to be 20-27 kDa. The total protease activity by fermentation
study was 185 U/ml. In conclusion this study had successfully isolated a thermostable
protease producer identified as Geobacillus sp. A8 based on I 6S rRNA and biochemical
analysis.