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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| Format: | Thesis |
| Language: | English English |
| Published: |
2023
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| Subjects: | |
| 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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| Summary: | 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. |
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