Characterization of newly isolated protease producing marine bacteria and expression of a neutral protease from Bacillussp PPB15 isolated from mangroves in Sabah

Characterization of newly isolated protease producing marine bacteria and expression of a neutral protease from Bacillussp PPB15 isolated from mangroves in Sabah

A total of 112 species of marine bacteria were isolated from the mangrove habitats along the east coast of Sabah, East Malaysia. Eighteen of these isolates were protease producing bacteria (PPB). Molecular identification of these protease producing bacteria based on 16S rDNA was carried out in orde...

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Main Author: Shuhadah Mustapha
Format: Thesis
Language:English
Published: 2012
Subjects:
QH301-705.5 Biology (General)
Online Access:https://eprints.ums.edu.my/id/eprint/11308/1/ph000000066.pdf
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id my-ums-ep.11308
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institution Universiti Malaysia Sabah
collection UMS Institutional Repository
language English
topic QH301-705.5 Biology (General)
spellingShingle QH301-705.5 Biology (General)
Shuhadah Mustapha
Characterization of newly isolated protease producing marine bacteria and expression of a neutral protease from Bacillussp PPB15 isolated from mangroves in Sabah
description A total of 112 species of marine bacteria were isolated from the mangrove habitats along the east coast of Sabah, East Malaysia. Eighteen of these isolates were protease producing bacteria (PPB). Molecular identification of these protease producing bacteria based on 16S rDNA was carried out in order to facilitate the identification of the bacterial strains. PPB1, PPB6, PPB11 and PPB13 were identified as Bacillus cereus with 99% similarity, whereas PPB3 with 99% similarity with Proteus mirabilis H4320. Strain PPB2, PPB4 and PPB10 have shown similarity with Bacillus GIDM. Strain PPBS, PPB7 and PPB18 were identified as Bacillus megaterium whereas PPB9 and PPB14 were classified as Staphylococcus saprophyticus subsp. saprophyticus ATCC 15305. Strain PPB16 and PPB17 have shown that these species were 99% similar to Bacillus sp CNJ845PL04. Strain PPBS, PPB12 and PPB15 have shown 99% similarity with Bacillus sp 41 KBZ. Assays for total protein and proteases activity of these isolated PPBs were conducted. Results on the protease activity study showed that Proteus mirabilis PPB3, Bacillus sp PPB8, Bacillus sp PPB15 and Bacillus megaterium PPB18 exhibited the highest protease activity with reading of 0.63, 0.61, 0.64 and 0.62 U/ml respectively. These strains grew up to 50°C with a broad pH range between 5 to 7.5. The optimal temperature and pH for growth were 35°C and 5.0 respectively. A study to determine the effect of various protease inhibitors, namely phenylmethylsulfonylflouride (PMSF), Pepstatin A, E-64 (trans-epoxysuccinyl Leucylamido (4-guanidino) butane and EOTA on the activity of these proteases clearly indicated the compound E-64 inhibited the protease activity of isolates from Bacillus sp strain PPB15 to a significant degree. EDTA and Pepstatin inhibited the protease isolated from Bacillus megaterium strain PPB 18. PMSF had no significant effect on the proteases derived from all PPBs. These results implied that the proteases derived from bacterial Bacillus sp PPB12, Bacillus sp PPB15 and Bacillus megaterium PPB18 can be categorized as belonging to the family of acidic mesophilic proteases and metalloprotease mesophilic proteases. Further characterization of the proteases was carried out by utilising seven different types of p-nitroanilide synthetic substrates. Results have shown that the amino acids in the position PI have a strong influence on the catalytic activity of proteases. The neutral protease derived from Bacillus sp PPB15 indicated preference for Leucine, phenylalanine and arginine at position PI and exhibited high activity for Sar-Pro-Arg-pNA dihydrochloride (1.05 Units/ml enzyme) L-Leucine-pNA (0.74 Units/ml enzyme), N-Suc-Ala-Ala-Pro-Leu-pNA (0.83 Units/ml enzyme), and N-Suc-Gly-Gly-phe-pNA (0.44 Units/ml enzyme). Lower activity was observed when Ala or Gly was the amino acids residues at position PI, notably the N-Suc-gly-gly-gly-pNA or N-Suc-Ala-Ala-Ala-pNA. The Km value for L Leucine-pNA and N-Suc-gly-gly-gly-pNA as subsrate were 3.31µm and I8.50µm, respectively. The corresponding Vrnax value were 78.31µM/min and 3.58µM/min, respectively. Two pairs of gene specific primers were designed to target the neutral protease genes of Bacillus sp PPB15 and Bacillus sp PPB12. PCR generated an amplicon of around 1638bp, which confirmed the identity of a neutral protease B in the genome of Bacillus sp PPBIS. The protease gene was cloned in to Pexp5-NT vector which was expressed in Ecoli BL21(DE3) under control of T7 promoter. SDS-PAGE analysis showed a strong neutral protease gene expression after induction by 1 mM IPTG for 5 hrs at 3PC with molecular mass approximately of 62 kDa. Further investigation on the activity of purified protease from recombinant protein (pEXP5NT-NprB) indicated that the protease activity was at 1.3U with the concentration of 0.625 ug.
format Thesis
author Shuhadah Mustapha
author_facet Shuhadah Mustapha
author_sort Shuhadah Mustapha
title Characterization of newly isolated protease producing marine bacteria and expression of a neutral protease from Bacillussp PPB15 isolated from mangroves in Sabah
title_short Characterization of newly isolated protease producing marine bacteria and expression of a neutral protease from Bacillussp PPB15 isolated from mangroves in Sabah
title_full Characterization of newly isolated protease producing marine bacteria and expression of a neutral protease from Bacillussp PPB15 isolated from mangroves in Sabah
title_fullStr Characterization of newly isolated protease producing marine bacteria and expression of a neutral protease from Bacillussp PPB15 isolated from mangroves in Sabah
title_full_unstemmed Characterization of newly isolated protease producing marine bacteria and expression of a neutral protease from Bacillussp PPB15 isolated from mangroves in Sabah
title_sort characterization of newly isolated protease producing marine bacteria and expression of a neutral protease from bacillussp ppb15 isolated from mangroves in sabah
granting_institution Universiti Malaysia Sabah
granting_department Biotechnology Research Institute (BRI)
publishDate 2012
url https://eprints.ums.edu.my/id/eprint/11308/1/ph000000066.pdf
_version_ 1747836418902720512
spelling my-ums-ep.113082017-11-07T06:53:38Z Characterization of newly isolated protease producing marine bacteria and expression of a neutral protease from Bacillussp PPB15 isolated from mangroves in Sabah 2012 Shuhadah Mustapha QH301-705.5 Biology (General) A total of 112 species of marine bacteria were isolated from the mangrove habitats along the east coast of Sabah, East Malaysia. Eighteen of these isolates were protease producing bacteria (PPB). Molecular identification of these protease producing bacteria based on 16S rDNA was carried out in order to facilitate the identification of the bacterial strains. PPB1, PPB6, PPB11 and PPB13 were identified as Bacillus cereus with 99% similarity, whereas PPB3 with 99% similarity with Proteus mirabilis H4320. Strain PPB2, PPB4 and PPB10 have shown similarity with Bacillus GIDM. Strain PPBS, PPB7 and PPB18 were identified as Bacillus megaterium whereas PPB9 and PPB14 were classified as Staphylococcus saprophyticus subsp. saprophyticus ATCC 15305. Strain PPB16 and PPB17 have shown that these species were 99% similar to Bacillus sp CNJ845PL04. Strain PPBS, PPB12 and PPB15 have shown 99% similarity with Bacillus sp 41 KBZ. Assays for total protein and proteases activity of these isolated PPBs were conducted. Results on the protease activity study showed that Proteus mirabilis PPB3, Bacillus sp PPB8, Bacillus sp PPB15 and Bacillus megaterium PPB18 exhibited the highest protease activity with reading of 0.63, 0.61, 0.64 and 0.62 U/ml respectively. These strains grew up to 50°C with a broad pH range between 5 to 7.5. The optimal temperature and pH for growth were 35°C and 5.0 respectively. A study to determine the effect of various protease inhibitors, namely phenylmethylsulfonylflouride (PMSF), Pepstatin A, E-64 (trans-epoxysuccinyl Leucylamido (4-guanidino) butane and EOTA on the activity of these proteases clearly indicated the compound E-64 inhibited the protease activity of isolates from Bacillus sp strain PPB15 to a significant degree. EDTA and Pepstatin inhibited the protease isolated from Bacillus megaterium strain PPB 18. PMSF had no significant effect on the proteases derived from all PPBs. These results implied that the proteases derived from bacterial Bacillus sp PPB12, Bacillus sp PPB15 and Bacillus megaterium PPB18 can be categorized as belonging to the family of acidic mesophilic proteases and metalloprotease mesophilic proteases. Further characterization of the proteases was carried out by utilising seven different types of p-nitroanilide synthetic substrates. Results have shown that the amino acids in the position PI have a strong influence on the catalytic activity of proteases. The neutral protease derived from Bacillus sp PPB15 indicated preference for Leucine, phenylalanine and arginine at position PI and exhibited high activity for Sar-Pro-Arg-pNA dihydrochloride (1.05 Units/ml enzyme) L-Leucine-pNA (0.74 Units/ml enzyme), N-Suc-Ala-Ala-Pro-Leu-pNA (0.83 Units/ml enzyme), and N-Suc-Gly-Gly-phe-pNA (0.44 Units/ml enzyme). Lower activity was observed when Ala or Gly was the amino acids residues at position PI, notably the N-Suc-gly-gly-gly-pNA or N-Suc-Ala-Ala-Ala-pNA. The Km value for L Leucine-pNA and N-Suc-gly-gly-gly-pNA as subsrate were 3.31µm and I8.50µm, respectively. The corresponding Vrnax value were 78.31µM/min and 3.58µM/min, respectively. Two pairs of gene specific primers were designed to target the neutral protease genes of Bacillus sp PPB15 and Bacillus sp PPB12. PCR generated an amplicon of around 1638bp, which confirmed the identity of a neutral protease B in the genome of Bacillus sp PPBIS. The protease gene was cloned in to Pexp5-NT vector which was expressed in Ecoli BL21(DE3) under control of T7 promoter. SDS-PAGE analysis showed a strong neutral protease gene expression after induction by 1 mM IPTG for 5 hrs at 3PC with molecular mass approximately of 62 kDa. Further investigation on the activity of purified protease from recombinant protein (pEXP5NT-NprB) indicated that the protease activity was at 1.3U with the concentration of 0.625 ug. 2012 Thesis https://eprints.ums.edu.my/id/eprint/11308/ https://eprints.ums.edu.my/id/eprint/11308/1/ph000000066.pdf text en public Universiti Malaysia Sabah Biotechnology Research Institute (BRI)

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