Isolation and characterisation of the alkaline phosphatase from phosphate-solubilising bacteria isolated from the soil of Danum Valley Rainforest, Sabah

Despite the essential roles of soil phosphate for plant growth, only 0.1% of soil phosphates are available for direct uptake by plants. This leads to the high use of phosphate chemical fertiliser to promote plant growth. The development of bio-fertiliser for replacing phosphate chemical fertiliser r...

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Main Author:	Herman Umbau anak Lindang
Format:	Thesis
Language:	English English
Published:	2023
Subjects:	QR75-99.5 Bacteria
Online Access:	https://eprints.ums.edu.my/id/eprint/40531/1/24%20PAGES.pdf https://eprints.ums.edu.my/id/eprint/40531/2/FULLTEXT.pdf
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id	my-ums-ep.40531
record_format	uketd_dc
institution	Universiti Malaysia Sabah
collection	UMS Institutional Repository
language	English English
topic	QR75-99.5 Bacteria
spellingShingle	QR75-99.5 Bacteria Herman Umbau anak Lindang Isolation and characterisation of the alkaline phosphatase from phosphate-solubilising bacteria isolated from the soil of Danum Valley Rainforest, Sabah
description	Despite the essential roles of soil phosphate for plant growth, only 0.1% of soil phosphates are available for direct uptake by plants. This leads to the high use of phosphate chemical fertiliser to promote plant growth. The development of bio-fertiliser for replacing phosphate chemical fertiliser requires highly active phosphate-solubilising bacteria (PSB). It is widely reported that soil PSB profiles are closely related to the geological character of the soil. Therefore, the unique PSB is possibly available from the soil with unique characteristics. While the Danum Valley (DV) of Sabah exhibited low base saturation and strongly leached Acrisols soils, it is hypothesised that DV soil may harbour novel microorganisms producing phosphatase that can thrive in the complex soil condition and exhibit unique functional and structural enzyme characteristics. This study is aimed to screen, isolate phosphate-solubilising bacteria (PSB) from DV soil and characterise their alkaline phosphatases. To address, PSB screening was performed using a selective medium, NBRIP agar, which resulted in five PSB isolates displaying remarkable phosphate solubilising activity. Further molecular identification revealed that the five isolates were Bacillus sp. PSB01, Pseudomonas oryzyhabitans PSB02, Staphylococcus pasteuri PSB03, Paenibacillus sp. PSB04, and Staphylococcus pasteuri PSB05. The combination (consortium) of these PSB was then proven to promote plant growth in Oryza sativa and Brassica rapa var. parachinensis under a sterile soil growth medium. One contributor to the activity of these five PSB to promote plant growth is their phosphatase activity. While the structure and function of phosphatases of Bacillus, Pseudomonas and Staphylococcus were extensively studied, to date, no report on the phosphatase of the Paenibacillae family. Accordingly, PSB04 was further studied genomically to identify the genetic regulation behind its phosphatase activity. The whole genome sequence of PSB04 revealed the existence alkaline phosphatase gene (AP-PSB04) but no acid phosphatase, which is predicted to be the main contributor to the phosphatase activity of this strain. Further in silico analysis revealed that PSB04 is about 44 kDa in size and secreted through the Sec pathway. The catalytic mechanism of AP-PSB04 is unique due to the absence of canonical Lys residue at the substrate binding cavity. Further, the protein was in dimeric structure both in the structural model and size exclusion chromatography analysis. Recombinant AP-PSB04 was produced using the E. coli system and used for further analysis, which revealed that this protein is highly active with a specific activity of 395070.35 U/mg against the pNPP substrate. The optimum temperature and pH for the activity of this enzyme were found to be 70 ºC and pH 8.0, respectively. The presence of Zn2+ metal ion has remarkably enhanced the activity of AP-PSB04 by 100% and completely abolished by Mn, EDTA or EGTA. The isothermal titration calorimetry (ITC) experiment further revealed that two binding events of metal ions might occur in AP-PSB04, involving six Zn ions per dimeric molecule per event. Further, a unique structural segment (Asp307-Thr405) of AP-PSB04, namely a crown-like domain, was found to play a major role in the metal ion binding preferences. This crown-like domain demonstrated essential roles in the stability of dimerisation and thermal stability with no serious effect on the catalytic activity. Altogether, this study provides the first-ever PSB isolated from the Sabah rainforest soil, promising to be further applied as a plant growth promoter. Besides, the alkaline phosphatase produced from the PSB04 strain is also promising for further studies and industrial applications.
format	Thesis
author	Herman Umbau anak Lindang
author_facet	Herman Umbau anak Lindang
author_sort	Herman Umbau anak Lindang
title	Isolation and characterisation of the alkaline phosphatase from phosphate-solubilising bacteria isolated from the soil of Danum Valley Rainforest, Sabah
title_short	Isolation and characterisation of the alkaline phosphatase from phosphate-solubilising bacteria isolated from the soil of Danum Valley Rainforest, Sabah
title_full	Isolation and characterisation of the alkaline phosphatase from phosphate-solubilising bacteria isolated from the soil of Danum Valley Rainforest, Sabah
title_fullStr	Isolation and characterisation of the alkaline phosphatase from phosphate-solubilising bacteria isolated from the soil of Danum Valley Rainforest, Sabah
title_full_unstemmed	Isolation and characterisation of the alkaline phosphatase from phosphate-solubilising bacteria isolated from the soil of Danum Valley Rainforest, Sabah
title_sort	isolation and characterisation of the alkaline phosphatase from phosphate-solubilising bacteria isolated from the soil of danum valley rainforest, sabah
granting_institution	Universiti Malaysia Sabah
granting_department	Biotechnology Research Institute
publishDate	2023
url	https://eprints.ums.edu.my/id/eprint/40531/1/24%20PAGES.pdf https://eprints.ums.edu.my/id/eprint/40531/2/FULLTEXT.pdf
_version_	1811770525133832192
spelling	my-ums-ep.405312024-09-24T04:31:42Z Isolation and characterisation of the alkaline phosphatase from phosphate-solubilising bacteria isolated from the soil of Danum Valley Rainforest, Sabah 2023 Herman Umbau anak Lindang QR75-99.5 Bacteria Despite the essential roles of soil phosphate for plant growth, only 0.1% of soil phosphates are available for direct uptake by plants. This leads to the high use of phosphate chemical fertiliser to promote plant growth. The development of bio-fertiliser for replacing phosphate chemical fertiliser requires highly active phosphate-solubilising bacteria (PSB). It is widely reported that soil PSB profiles are closely related to the geological character of the soil. Therefore, the unique PSB is possibly available from the soil with unique characteristics. While the Danum Valley (DV) of Sabah exhibited low base saturation and strongly leached Acrisols soils, it is hypothesised that DV soil may harbour novel microorganisms producing phosphatase that can thrive in the complex soil condition and exhibit unique functional and structural enzyme characteristics. This study is aimed to screen, isolate phosphate-solubilising bacteria (PSB) from DV soil and characterise their alkaline phosphatases. To address, PSB screening was performed using a selective medium, NBRIP agar, which resulted in five PSB isolates displaying remarkable phosphate solubilising activity. Further molecular identification revealed that the five isolates were Bacillus sp. PSB01, Pseudomonas oryzyhabitans PSB02, Staphylococcus pasteuri PSB03, Paenibacillus sp. PSB04, and Staphylococcus pasteuri PSB05. The combination (consortium) of these PSB was then proven to promote plant growth in Oryza sativa and Brassica rapa var. parachinensis under a sterile soil growth medium. One contributor to the activity of these five PSB to promote plant growth is their phosphatase activity. While the structure and function of phosphatases of Bacillus, Pseudomonas and Staphylococcus were extensively studied, to date, no report on the phosphatase of the Paenibacillae family. Accordingly, PSB04 was further studied genomically to identify the genetic regulation behind its phosphatase activity. The whole genome sequence of PSB04 revealed the existence alkaline phosphatase gene (AP-PSB04) but no acid phosphatase, which is predicted to be the main contributor to the phosphatase activity of this strain. Further in silico analysis revealed that PSB04 is about 44 kDa in size and secreted through the Sec pathway. The catalytic mechanism of AP-PSB04 is unique due to the absence of canonical Lys residue at the substrate binding cavity. Further, the protein was in dimeric structure both in the structural model and size exclusion chromatography analysis. Recombinant AP-PSB04 was produced using the E. coli system and used for further analysis, which revealed that this protein is highly active with a specific activity of 395070.35 U/mg against the pNPP substrate. The optimum temperature and pH for the activity of this enzyme were found to be 70 ºC and pH 8.0, respectively. The presence of Zn2+ metal ion has remarkably enhanced the activity of AP-PSB04 by 100% and completely abolished by Mn, EDTA or EGTA. The isothermal titration calorimetry (ITC) experiment further revealed that two binding events of metal ions might occur in AP-PSB04, involving six Zn ions per dimeric molecule per event. Further, a unique structural segment (Asp307-Thr405) of AP-PSB04, namely a crown-like domain, was found to play a major role in the metal ion binding preferences. This crown-like domain demonstrated essential roles in the stability of dimerisation and thermal stability with no serious effect on the catalytic activity. Altogether, this study provides the first-ever PSB isolated from the Sabah rainforest soil, promising to be further applied as a plant growth promoter. Besides, the alkaline phosphatase produced from the PSB04 strain is also promising for further studies and industrial applications. 2023 Thesis https://eprints.ums.edu.my/id/eprint/40531/ https://eprints.ums.edu.my/id/eprint/40531/1/24%20PAGES.pdf text en public https://eprints.ums.edu.my/id/eprint/40531/2/FULLTEXT.pdf text en validuser Universiti Malaysia Sabah Biotechnology Research Institute

Isolation and characterisation of the alkaline phosphatase from phosphate-solubilising bacteria isolated from the soil of Danum Valley Rainforest, Sabah

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