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 ei...

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Main Author:	Noorizhab, Mohd Nur Fakhruzzaman
Format:	Thesis
Language:	English
Published:	2012
Subjects:	Proteolytic enzymes Proteases Bacteria
Online Access:	https://ir.uitm.edu.my/id/eprint/13370/2/13370.pdf
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id	my-uitm-ir.13370
record_format	uketd_dc
spelling	my-uitm-ir.133702022-12-22T06:36:17Z Characterization of protease from thermophiles isolated from Local Hot Springs / Mohd Nur Fakhruzzaman Noorizhab 2012 Noorizhab, Mohd Nur Fakhruzzaman Proteolytic enzymes. Proteases Bacteria 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. 2012 Thesis https://ir.uitm.edu.my/id/eprint/13370/ https://ir.uitm.edu.my/id/eprint/13370/2/13370.pdf text en public mphil masters Universiti Teknologi MARA Faculty of Health Sciences
institution	Universiti Teknologi MARA
collection	UiTM Institutional Repository
language	English
topic	Proteolytic enzymes Proteases Bacteria
spellingShingle	Proteolytic enzymes Proteases Bacteria Noorizhab, Mohd Nur Fakhruzzaman Characterization of protease from thermophiles isolated from Local Hot Springs / Mohd Nur Fakhruzzaman Noorizhab
description	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.
format	Thesis
qualification_name	Master of Philosophy (M.Phil.)
qualification_level	Master's degree
author	Noorizhab, Mohd Nur Fakhruzzaman
author_facet	Noorizhab, Mohd Nur Fakhruzzaman
author_sort	Noorizhab, Mohd Nur Fakhruzzaman
title	Characterization of protease from thermophiles isolated from Local Hot Springs / Mohd Nur Fakhruzzaman Noorizhab
title_short	Characterization of protease from thermophiles isolated from Local Hot Springs / Mohd Nur Fakhruzzaman Noorizhab
title_full	Characterization of protease from thermophiles isolated from Local Hot Springs / Mohd Nur Fakhruzzaman Noorizhab
title_fullStr	Characterization of protease from thermophiles isolated from Local Hot Springs / Mohd Nur Fakhruzzaman Noorizhab
title_full_unstemmed	Characterization of protease from thermophiles isolated from Local Hot Springs / Mohd Nur Fakhruzzaman Noorizhab
title_sort	characterization of protease from thermophiles isolated from local hot springs / mohd nur fakhruzzaman noorizhab
granting_institution	Universiti Teknologi MARA
granting_department	Faculty of Health Sciences
publishDate	2012
url	https://ir.uitm.edu.my/id/eprint/13370/2/13370.pdf
_version_	1783733173600911360

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

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