Introduction

Second only to nitrogen, phosphorus is a crucial macronutrient for plants. It is directly involved in the synthesis of nucleic acids, cell division, and the development of new tissues. It is also required for a number of cellular functions, including photosynthesis, energy production, carbohydrate metabolism, redox homeostasis, and signaling1,2,3. Phosphorus is also necessary for plant growth and development; levels can reach 0.2% of the dry weight of plants4. However, arable land around the world has more total phosphorus and less available phosphorus, and the concentration of plant-available phosphorus rarely surpasses 10 µM, which has a significant impact on agricultural development and plant growth5. Thirty to sixty-five% of soil phosphorus is organic, and the remaining thirty to seventy% is inorganic. Long-term application of phosphorus fertilizer overwhelmingly produces phosphate that is difficult for plants to directly absorb and cannot fully address phosphorus deficiencies in soils because organic phosphorus typically exists in the soil in an inert form and is fixed to form insoluble phosphorus forms, which are difficult for plants to directly absorb and use, and inorganic phosphorus readily reacts with ions in the soil, such as Fe3+, Al3+, and Ca2+ 6, 7.

In an effort to increase crop productivity, farmers frequently apply excessive amounts of fertilizer. Over application of fertilizer can result from erroneous perceptions about its efficacy. A common misconception is that increased fertilizer equals increased output. This problem is additionally exacerbated by inadequate soil testing and knowledge8, furthermore, some people can continue to use antiquated methods without taking into account the particular nutrient requirements of their crops. Sometimes there is a lack of trustworthy information, which leads to bad decisions. Farmers who are under pressure to satisfy consumer demands may put short-term outcomes ahead of long-term strategies. Excessive use of phosphate and nitrogen fertilizers can cause eutrophication, imbalances in soil nutrients, and adverse effects on the food chain. When fertilizers are utilized excessively, nutrient contamination becomes a serious issue. Runoff from agriculture can cause this kind of contamination by introducing phosphorus and nitrogen into streams. This type of drainage causes toxic algal blooms. Toxins produced by these blooms pose a risk to aquatic ecosystems and public health. Hypoxia, or low oxygen levels, are also caused by the decomposition and death of algae, which uses up oxygen in the water. These circumstances make it difficult for fish and other marine life to survive, which frequently leads to die-offs. Therefore, one of the main causes of nutrient contamination in agriculture is the overuse of nutrients, especially nitrogen and phosphorus. The main cause of this problem is agricultural runoff, which occurs when fertilizers used on crops wash into adjacent streams during irrigation or rainfall. Both freshwater and marine ecosystems are impacted by such nutrient overload, which causes major problems with water quality. Additionally, overuse of phosphate fertilizers may cause the microbiological ecology in the soil to be destroyed. Furthermore, the overabundance of nutrients upsets the environment’s natural equilibrium, which results in a decline in biodiversity. Nutrient imbalances cause the health of the soil to deteriorate, which might damage beneficial microbes that are necessary for successful farming. Concerns about climate change may worsen if greenhouse gas emissions rise.

The world’s phosphate supplies are finite and will likely run out in 50–100 years, or roughly 2059–21099. There are currently an estimated 370 years of globally reserves left at current extraction rates, up from less than 100 years with earlier estimates due to significant improvements in the USGS’s phosphate rock reserve estimations in 2011. These reserves are geographically concentrated, with Morocco alone controlling around three-quarters of them. Output is similarly notably concentrated, with two-thirds of output coming from three countries: China, the US, and Morocco. The current disparity in reserve-to-production (R/P) ratios among nations is probably going to change how reserves and production are distributed in the future. According to the analysis, reserves may last 300–400 years globally, but most nations will have used them up in 100 years, leaving a significant output shortfall. Morocco’s R/P ratio of almost 2000 years significantly distorts the worldwide figure. Since more than 70% of world output is now produced in nations with R/P ratios under 100 years, the depletion of these reserves would cause a significant production deficit of about 125 Mt year in the twenty-first century. Together, China and the US produce more than half of the world’s current supply, and at present extraction rates, both will run out in 60 years. The future production landscape will therefore be significantly altered as merely maintaining present levels of production will require large increases in production within some counties to balance depletion within others10, the projected production shortfall will be exacerbated by the growing demand for phosphate rock. Future demand for phosphate rock is probably going to be significantly higher due to growing populations and the per capita need for phosphorus. The demand for phosphate rock is projected to rise at a 1% annual rate from 2010 to 2050, reaching 262 Mt, and then leveling out at 0% by 2050, causing a production deficit by 2075. Morocco’s share of world production is projected to rise from 15% in 2010 to nearly 40% in 2050 and 80% in 2100, despite slow depletion of reserves and its largest R/P ratio.In conclusion, Morocco’s monopoly on phosphate rock stocks and production is posing geopolitical risks, increasing dependence on one nation for vital resources. Prioritizing a food-production system that improves phosphorus use efficiency and recycles phosphorus-containing waste is crucial for future phosphorus and food security.

However, as new phosphate ores have lately been found in Scandinavian nations like Norway, these statistics may need to be updated. Finding alternate, economically feasible, and environmentally acceptable methods to raise phosphorus levels in low-phosphorus or phosphorus-deficient soils for plant growth is necessary due to the negative effects of applying phosphorus fertilizers and the decrease in global phosphorus stocks1. Phosphate solubilizing bacteria (PSB), which play a crucial role in the soil phosphorus cycle by hydrolyzing inorganic phosphorus minerals through enzyme activity and mineralizing organic phosphorus through acid secretion, provide a biological solution to phosphorus deficiency in soils11,12. This increases the amount of available phosphorus in soils by solubilizing insoluble phosphorus. The ability of PSB to transform the insoluble phosphorus forms found in soils into accessible phosphorus that may be directly absorbed and used by plants through various pathways has been validated by a number of recent research. Examples include the production of dissolved phosphate by the release of enzymes (phytase and phosphatase), acids (organic and inorganic), and chelation (siderophores, extracellular polysaccharides)12,13. PSB can work in concert with phosphate fertilizers to promote phosphorus use in addition to their own capacity to change phosphorus speciation14. Additionally, PSB are progressively being used as raw materials for biofertilizers and inoculants; their application in agricultural fields has a notable impact and can greatly boost the productivity of agronomic crops in agro-ecological niches15,16. There are currently few thorough studies from several angles and a narrow focus on PSB gene regulation and phosphatase enzyme protein engineering processes. The ecological environment in soil is so complex that both the exogenous addition of PSB and fertilization will cause changes in microbial communities, which indirectly alter the phosphorus content in soil. PSB metabolites have varying levels of capacity due to the interference of PSB species and external factors, and many functional genes have different effects at different levels. Based on these results, the current study’s objective was to evaluate the 3D structure of the simulated pho proteins using the genes encoding the sequences of acid phosphatase (ACPase) and alkaline phosphatase (ALPase) from Streptomyces sp. MMA-NRC. The ACPase and ALPase proteins’ ideal binding affinities with rock phosphate were found through docking studies. To increase phosphatase activity and accessible phosphorus, the Streptomyces sp. MMA-NRC ACPase and ALPase genes were molecularly cloned and expressed in E. coli DH5α.

Materials and methods

Plasmids, reagents, media and strains

Streptomyces sp. MMA-NRC (accession no. OR770185), as reported by17, which might effectively solubilizing the rock phosphate (RP) was kept in our laboratory according to the specified instructions at -20 °C glycerol stocks. E. coli DH5a was cultivated at a temperature of 37 degrees Celsius for a duration of 12 to 18 h in either Luria-Bertani (LB) broth or low-salt LB medium. The following concentrations (g/L) are found in the National Botanical Research in P medium (NBRIP media), the basic medium used to isolate and ferment that solubilizes the rock phosphate17,18: glucose (10 g), MgSO4.7H2O 0.25 g, (NH4)2SO4 0.1 g, KCl 0.2 g, and agar (15 g) supplemented with 5 g rock phosphate (RP) from Khouribga phosphate mine, pH 7, and agar 15 g.The rock phosphate is dissolved in 1,000 ml of distilled water19. They were cultured for three to seven days in orbital shaker (180 rpm) at 37 °C. For actinomycetes growth was used plate count (P.C.) agar medium (Himedia, West Chester, Pennsylvania, USA). Promega Co. in Madison, WI, USA, provided the plasmid known as pGEM-T Easy-cloning.It has 3015 base pairs and was manufactured by Ampr. Both wild-type Streptomyces sp. has genomic DNA extracted. With the use of a genomic DNA isolation kit made in Taoyuan, Taiwan by GeneDireX, Inc. The plasmid DNA of E. Coli DH5a was extracted using the QIA prep spin miniprep kit. The T4 DNA ligase was supplied by TaKaRa in China and NEB in the US.

Quantitative estimation of available phosphorus solubilizing assessment using rock phosphate

The quantitative solubilization ability of RP by phosphate-solubilizing bacteria was tested in a liquid medium containing NBRIP. To do this, a pre culture in plate count medium was prepared overnight and subsequently transferred to NBRIP medium. For seven days of incubation at 37 °C with shaking at 150 rpm, 20 ml of the bacterial cultures were removed and centrifuged for fifteen minutes at 12,000xg. After being extracted, supernatants were measured for pH and their available P content, samples done in three replicates and take average of three replicates. The control treatments were administered without inoculation in the same way. The available phosphorus was measured spectrophotometrically in accordance with20, and extracted using 0.5 M NaHCO3 at pH 8.521. The ascorbic acid colorimetric method was used to determine the as available P concentration20. After a few minutes of incubation at room temperature, around 1 mL of supernatant is combined with 160 µL of a reaction solution, and the optical density (OD) was measured at 880 nm using Shimadzu UV-visible spectrophotometer. The calculation of available phosphorus content was performed using a standard curve of KH2PO4 betewwn absorption at 880nm and concentration, with calibration curve equation of: mg/kg− 1= (a-b)*(v*df*mcv/w) where: a = P concentration in extracted sample (mg L− 1), b = P concentration in the target (mg L− 1 ), V = extraction volume (mL), W = soil sample weight (g), DF = dilution factor = total solution volume/aliquot (extract) and mcf = humidity correction factor. R2 ≥ 0.995. Phosphate concentration was expressed in term of mg/L− 1 phosphate released in culture.

Acid phosphatase (ACPase) and alkaline phosphatase (ALPase) encoding genes amplification

Initially, the product’s design was determined by the DNA sequence of the primers intended for the acid phosphatase (ACPase) and alkaline phosphatase (ALPase) genes of Streptomyces sp. 4 F.Using a genomic DNA isolation kit (GeneDireX, Inc., Taoyuan, Taiwan), genomic DNA was recovered from the Streptomyces sp. MMA-NRC strain (NCBI Accession No. OR770185), as described by17. The acid phosphatase (ACPase) and alkaline phosphatase (ALPase) genes were amplified using the isolated material. The ACPase and ALPase gene DNA sequences of the Streptomyces sp. MMA-NRC strain were found to be significantly similar, leading to the development of particular primers. These primers designed using Primer3 based on conserved regions of ACPase and ALPase genes as follows: ACPase-F (5′- GAGGCGGACGGCGGG-3′) and the reverse primer ACPase-R (5′- CGAGGCGGCGGACAGTT-3′), and the forward primer ALPase-F (5′- TTCCTGCACGGCGTCGC − 3′) and the reverse primer ALPase-R (5′- GCGGGTGCGGTAGGAGC − 3′).The analysis revealed ACPase and ALPase genes have an open reading frame (ORF) of 1464 and 1680 bp, respectively. Using a GeneAmp PCR System 2400 heat cycler from PerkinElmer in Norwalk, Connecticut, USA, the PCR was performed using 100 ng of genomic DNA in a 100 µl reaction with 5 µM primers and master mix (TIANGEN, Beijing, China)17. The thermal protocol called for five minutes of denaturation at 95 °C, one minute of annealing at 57.5 °C, two minutes of extension at 72 °C per kilobase pair (kbp), and three minutes of extension at 72 °C. There were thirty-five cycles in all. The amplified genes were eluted using a FavorPrep gel purification kit (FAVORGEN, Biotech Corp., Ping Tung, Taiwan) prior to sequencing. The PCR results were then examined following electrophoresis on a 1% agarose gel and 10.000 bp DNA ladder (Invitrogen, California, USA), as shown in Fig. 122.

Bioinformatics analysis

The ACPase and ALPase protein sequences were obtained from the experimentally determined data phosphatase (pho) protein of Streptomyces sp. strain MMA-NRC using the online translation tool ExPASY (http://web.expasy.org/translate). The NCBI protein blast tool (BLAST: Basic Local Alignment Search Tool) was used to analyze the inferred amino acids. Blast performs duplicate-free searches of the protein sequence database, classifying finds containing identical sequences as matches23,24. The ACPase and ALPase protein’s PDBsum home page (ebi.ac.uk) secondary structure prediction was performed using the online PDBsum tool25. Using the BLAST program, a cluster analysis (phylogenetic tree) of the wild-type ACPase and ALPase protein sequences were performed.

Homology modelling and validation of acid phosphatase (ACPase) and alkaline phosphatase (ALPase)

In order to forecast the tertiary configuration of ACPase and ALPase protein, ROBETTA (http://robetta.bakerlab.org) was used26,27. After five models were generated, the best model was determined by looking at the model with the highest confidence score (C-score, a statistic showing the correctness of the expected model)28,29. The C-score typically ranges from zero to one. An obvious relationship exists between the C-score and structural quality. Among the five generated models, an optimized ACPase and ALPase homology model was constructed using the “DOPE profile” options. The optimal validation framework for ACPase and ALPase proteins were verified using the SAVES v6.0 (structure analysis and verification server version 6) web tool (http://SAVESv6.0http://SAVESv6.0.ucla.edu - Structure Validation Server (ucla.edu) as well as the ProSA server. A total of five programs make up SAVES v6.0, and their purpose is to evaluate the protein structure’s overall integrity. Subsequently, the model that was produced underwent evaluation using general stereo chemical parameters using the PROCHECK website. In addition, the Ramachandran plot was developed for the energy-minimized model of ACPase and ALPase structures. The x-axis of the plot is divided into four quadrants, which correspond to the low-energy area, permitted region, usually allowed region, and prohibited region30, when amino acids located in allowed rejoin this indicated for high quality of protein model, but when amino acids located in disallowed rejoin this indicated for low or bad quality of protein model. Out of the five tools we employed, two stood out: VERIFY-3D, which analyzed the three-dimensional sequence profiles of protein models, and PROCHECK, which evaluated the structure using the Ramachandran plot30,31. Using the “PyMol” software developed by DeLano Scientific LLC, a stereo picture of the ACPase and ALPase models were created to display the surface groove structure32.

Molecular docking studies

To process the 3D structure of the ACPase and ALPase proteins, the MOE program was used (Molecular Operating Environment (MOE) | MOEsaic | PSILO (chemcomp.com), Decanting the concentration of ions, water molecules, and present ligands. The receptor molecule was functionalized with hydrogen atoms by means of PyMOL (PyMOL | pymol.org). The rock phosphate ligand was created in pdb format using MOE and PyMOL; its PubChem SID is 459143506. The HDock online program was used to perform protein-ligand docking investigations (http://HDOCK.hust.edu.cn Server (hust.edu.cn)30,33, in order to learn how rock phosphate interacts with ACPase and ALPase. Docking was the reason for saving the macromolecule file in PDB format28,32,34, .

Preparation of competent cells methods

Competent cells were prepared from E. coli DH5α by using traditional CaCl2 solution method of35. All preparations were performed in triplicates; all steps should be carried out on ice and eluted in 100 l of sterile Calcium Chloride Solutions. The quality of competent cells was assessed by calculating transformation efficiency36,37.

Cloning and expression of acid phosphatase (ACPase) and alkaline phosphatase (ALPase) encoding genes

Using a kit from Promega Co., based in Madison, WI, USA, ligation cloning was performed using the amplified product ACPase and ALPase of Streptomyces sp. MMA-NRC, the pGEM®-T Easy cloning vector. The insert was inserted into the vector at the proper spots. The T4 ligase enzyme is used in a ligation process to create the recombinant plasmid. Twenty micrograms of ligated DNA and 0.1 millilitres of newly produced competent cells were mixed together; the transformation efficiency of the cells was 2.98*105 colony-forming units per microgram of DNA38. After introducing the recombinant plasmid into E. coli DH5a cells using the heat shock protocol, the cells were incubated for one hour at 37 °C. After the incubation period, the samples were transferred onto LB agar plates that were enhanced with 40 µg X-gal, 50 µg/mL ampicillin, and 0.5 mM IPTG. Next, the plates were incubated at 37 °C for the entire night. The blue-white screening method was used to determine whether colonies were positive. ACPase and ALPase colonies were streaked once more on an appropriate plate that contained antibiotics, and they were thereafter incubated for 16 to 20 h at 37 degrees Celsius. In positive colonies, the extracellular expression of ACPase and ALPase activity was assessed using rock phosphate substrate at temperature 37 °C for seven days incubation23,32,39,40. After transforming E. Coli DH5ɑ cells with plasmids pGEM-T- ACPase and pGEM-T-ALPase, overnight-grown single colonies were inoculated into Luria-Bertani (LB) broth medium supplemented with ampicillin (50 µg/mL) to create a 50 mL culture at OD600 harvest of the bacteria. After that, the culture was incubated for 16 h at 37 °C while being shaken. Centrifugation was applied to the mature culture for 10 min at 4 degrees Celsius, with a speed of 4000 revolutions per minute. Following the manufacturer’s instructions, the liquid portion above the sediment cells was then removed, and the solid cell material was utilized to extract the plasmid DNA molecule23. Colony PCR screening was used to analyze the colonies that were transformed using the plasmid carrying the ACPase and ALPase genes (pGEM-T-), using specific primers for the ACPase and ALPase genes and the same PCR conditions as for amplifying these genes were part of this screening procedure.

Results and discussion

Acid phosphatase (ACPase) and alkaline phosphatase (ALPase) encoding genes amplification and alignment in Genbank (Blast)

In a previous study conducted by17 actinomycetes Streptomyces sp. MMA-NRC strain was isolated and identified. The designed primers were for the ACPase and ALPase genes. The genes were successfully enhanced and sequenced. After that, we used BLAST to compare the obtained sequence to known sequences in the NCBI database. The analysis revealed ACPase and ALPase genes have an open reading frame (ORF) of 1464 and 1680 bp, respectively. In the anticipated duration of the ACPase and ALPase genes in Streptomyces sp. MMA-NRC, at the first time. These genes sequence encodes a protein comprising 488 and 560 amino acids, respectively. The comparison also showed that the ACPase and ALPase genes and translated proteins from Streptomyces sp. MMA-NRC shared 99% similarity with ACPase and ALPase genes of Streptomyces sp. 4 F, as descriped in Fig. 1, and showed 0.0 E-values, 2710 and 3103 alignment score for ACPase and ALPase genes of Streptomyces sp. MMA-NRC; respectively. Consequently, the obtained sequence for the ACPase and ALPase genes was submitted to the NCBI database with the accession numbers PV646716 and PV646717 for Streptomyces sp. MMA-NRC, respectively. Subsequently, Protein ACPase and ALPase was represented by their respective amino acid sequences, which were generated from the nucleotide sequences of the genes. We used the UniProt protein database to find three more ACPase and ALPase sequences that corresponded with the amino acid sequence. Additionally, the InterProScan server (EMBL) identified amino acid 1-488 and 1-560 residues as belonging to the ACPase and ALPase families.

Fig. 1
figure 1

Agarose gel electrophoresis of; lan 1, amplified purified PCR ACPase (1464 bp); lan 2, amplified purified PCR ALPase (1680 bp); M, 10.000 bp DNA ladder (Invitrogen, California, USA).

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In a related study of41, performed a phylogenetic analysis using bacterial PP2C phosphatase sequences from the InterPro database, classifying the family into five groupings. investigated the evolutionary links, species distribution, sequence and structural changes, and domain distribution features of bacterial PP2C phosphatases based on this classification. Evidence of a shared evolutionary origin for bacterial PP2C phosphatases was found by our analysis. By providing important information for upcoming functional investigations of bacterial serine/threonine phosphatases and supporting the development of targeted treatments for harmful bacteria, these discoveries enhance our knowledge of PP2C phosphatases42, reported the gene encoding alkaline phosphatase designated phoK was amplified and sequenced from of Sphingomonas sp. strain BSAR-143, using genomic DNA as a template, the gene encoding putative thermostable Alkaline Phosphatase APase was sequenced from B. licheniformis MTCC1483. According to sequence analysis, the gene was 1662 bp long (GenBank accession number KF577606) and was expected to code for a 553 amino acid polypeptide44, reported the olpA gene of Chryseobacterium meningosepticum, encoding a molecular class C phosphatase, was amplified and sequenced45, reported M2-32 is a non-specific acid phosphatase was amplified and sequenced, with a rare ability to function across a broad pH range (3.5–8.5).Analysis using SWISS-PROT Prf Profiles classifies it as a class A acid phosphatase (Z-score: 78.97), sharing 50%-60% sequence similarity with enzymes such as PhoC and PhoN46, reported twelve clinical isolates and two reference strains of S. aureus had their SapS gene extracted and sequenced; in silico PCR was used to test 49 S. aureus strains and 11 coagulase-negative staphylococci. Semi-purified protein extracts from the clinical strains’ culture medium were tested for phosphatase activity using a variety of phosphatase inhibitors in combination with p-nitro-phenylphosphate, O-phospho-L-tyrosine, O-phospho-L-serine, and OphosphoL-threonine47, reported genomic sequence analysis of Acinetobacter baumannii revealed the presence of a putative Acid Phosphatase (AcpA; EC 3.1.3.2)48, initially, 21 amino acid sequences of the alkaline phosphatase enzyme from several P. aeruginosa strains were obtained. In BLAST, the amino acid sequences displayed a minimum similarity of 99% and a maximum similarity of 100%. P. aeruginosa PAO1, P. aeruginosa UCBPP-PA14, P. aeruginosa VRFPA05, P. aeruginosa PAO579, P. aeruginosa HB13, P. aeruginosa DHS01, P. aeruginosa CI27, P. aeruginosa MPAO1/P2, P. aeruginosa PADK2_CF510, P. aeruginosa CIG1, P. aeruginosa E2, P. aeruginosa PA45, P. aeruginosa VRFPA02, P. aeruginosa VRFPA03, P. aeruginosa VRFPA07, and P. aeruginosa VRFPA08.

Cluster analysis (phylogenetic tree) of Streptomyces sp. strain MMA-NRC ACPase and ALPase proteins

After sequence analysis, a phylogenetic investigation was performed to ascertain the position of the ACPase and ALPase proteins within the larger framework of known phosphatase family members. In this investigation, a carefully selected dataset of 10 ACPase and ALPase proteins from various species was chosen from the UniProt protein database. One of the proteins in question is ACPase and ALPase proteins derived from Streptomyces sp. strain MMA-NRC. A comparison revealed that the ACPase and ALPase protein of Streptomyces sp. MMA-NRC has a sequence similarity of 99% with the ACPase and ALPase proteins of Streptomyces sp. strain 4 F, which are also part of the streptomyces genus. The BLAST program was used to generate the phylogenetic tree, utilizing the ACPase and ALPase proteins sequences from the 10 streptomyces strains. The results revealed a Streptomyces sp. strain MMA-NRC and Streptomyces sp. strain 4 F were classified and closely associated with putative ACPase and ALPase proteins.Additional information on the phylogenetic connections among these strains is provided in Fig. 2.

Fig. 2
figure 2

Phylogenetic tree of ten Streptomyces strains and Streptomyces MMA-NRC (a) ALPase and (b) ACPase protein sequences and other phosphatase proteins, constructed using the neighbor-joining method (BLAST) software.

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In a related study of41, reported 57,520 amino acid sequences were obtained from the PP2C family entry PF13672 at InterPro using phylogenetic analysis. Bacteria from nearly every taxonomic group provide the majority of these sequences. Bacterial PP2C phosphatases were shown to have a common ancestor in the resultant phylogenetic tree, indicating that these genes may have shared an ancestry and have maintained important biological activities across time. The phylogenetic tree also revealed that the PP2C family may be categorised into five groups, which are designated Group I, II, III, IV, and V. There are 801, 764, 612, 647, and 749 sequences in each of these groupings, respectively, suggesting a fairly even distribution throughout the categories49, using a variety of bioinformatics methods, thirty-three sequences six from each of the three classes of bacterial acid phosphatase and seven from archaeal phosphoesterases were examined. Three classes of bacterial acid phosphatases and archaeal phosphoesterases were compared and contrasted using phylogenetic analysis, dot plot comparisons, and motif analysis. Using a bioinformatics method, we have tried to determine the evolutionary relationships between three groups of bacterial acid phosphatase and archaeal phosphoesterases. The following are the names of bacteria and archaea, accompanied by the accession numbers in parenthesis: Class A bacterial acid phosphatase is represented by the digits 1 through 10. Class B acid phosphatase is represented by numbers 11–17, Class C acid phosphatase by numbers 18–26, and Archaeal phosphoesterase by numbers 27–3313, reported class C genes are more widely distributed; their association with the outer-membrane of cells gives them a clear role in the cycling of organic phosphorus, especially in soils. Bacterial Non-Specific Acid Phosphatase (NSAP) enzymes were used to determine NSAP diversity and subcellular localization in microbial genomes; corresponding NSAP gene sequences were used to census metagenomes from diverse ecosystems; and researchers examined the impact of long-term land management on NSAP diversity and abundance41, study the structural similarities and differences among PP2C phosphatases in the five identified groups, five PP2C phosphatases Q8VQA1 (PDB ID: 2PK0), P9WHW5 (PDB ID: 1TXO), A0A1L7GEB2, A0A7V6HC85, and P76395 corresponding to five groups of Group I, II, III, IV, and V, separately, were selected as representative proteins for detailed structural characterization48. A branching diagram called a phylogenetic tree aids in understanding the evolutionary relationships between biological species. Here, the phylogenetic tree was constructed using the MEGA6 software. Two phylogenetic trees were created in total. One phylogenetic tree comprising the alkaline phosphatase amino acid sequences from various P. aeruginosa strains. Another one made up the cDNA of the protein found in various P. aeruginosa strains. In the phylogenetic tree of the amino acid sequences of alkaline phosphatase of selected strains of P. aeruginosa showed mainly three major groups present.

Secondary structure prediction of ACPase and ALPase protein

Use the template Streptomyces sp.4F and wild type Streptomyces sp MMA-NRC to align the ACPase 488 residues and ALPase 500 residues (from 560 residues) proteins and predict their secondary structures. The experiments were conducted using the PDBsum service.

According to the ACPase model characterization in the PDBsum, the predicted ACPase enzyme for template Streptomyces sp.4F has the following secondary structure: has 23 helix and 35beta turn, as shown in Fig. 3a. In contrast, wild type Streptomyces sp MMA-NRC has 24 α-helices and 23 β-turn, as shown in Fig. 3b.

Fig. 3
figure 3figure 3

Predicted ACPase and ALPase proteins secondary structure of (A) template Streptomyces sp.4 F ACPase; (B) wild type Streptomyces sp MMA-NRC ACPase; (C) template Streptomyces sp.4 F ALPase; (D) wild type Streptomyces sp MMA-NRC ALPase.

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According to the ALPase model characterization in the PDBsum, the ALPase enzyme in template Streptomyces sp.4F is expected to have the following secondary structure: has 15 helix and 51 beta turn, as shown in Fig. 3c. In contrast, wild type Streptomyces sp MMA-NRC has 13 α-helices and 47 β-turn, as shown in Fig. 3d.

Reference41, study the structural similarities and differences among PP2C phosphatases in the five identified groups. As depicted in result of structure superposition, a core β sandwich made up of anti-parallel β slices and two anti-parallel helices on either side of the β slices in an alpha-β-β-α configuration were among the secondary structural units that the bacterial PP2C phosphatases had in common. However, the five groups’ secondary structural components also exhibit variations. In the core β-sandwich, there were varying numbers of β-strands: Groups I and II had ten β-strands, Groups III and V had nine, and Group IV had eleven. These variations could be the cause of these phosphatases’ functional peculiarities. The flap subdomain in PP2C phosphatases is a characteristic of proteins belonging to Groups I, II, and III that includes frontal extension α-helices in addition to the pair of antiparallel helices flanking the β-sheet45. , determined the x-ray crystal structure of non-specific acid phosphatase M2-32 by molecular replacement using an AlphaFold2-guided truncated model, achieving a resolution of 2.2 Å. The protein crystallized as a dimer-of-dimers. Each monomer (residues 38–274) adopts an all-alpha-helical fold composed of 14 helices and two disulfide bonds48, alkaline phosphatase were predicted by PSI-blast based secondary structure PREDiction (PSIPRED) and Chou and Fasman secondary structure prediction (CFSSP) server (http://cho-fas.sourceforge.net/index.php). The findings of the secondary structure prediction showed that the percentage of alpha helices was significantly higher than the percentage of other protein conformations, such as sheet and turn. Most of The strains of P. aeruginosa displayed the 63.7% helix. However, EME95396 and ERF08779.1 were the exceptions.Alpha helices were present in 64.7% of P. aeruginosa HB13 and P. aeruginosa PA21_ST175 samples. The percentage of betasheet was 54%, the same for all selected strains of P. aeruginosa.Additionally, the proportion of turn was nearly the same across all P. aeruginosa strains. The only one with a 14.5% turn was ETD45171.1 (P. aeruginosaVRFPA07). A detailed determination of the secondary structure of the amino acid sequence of P. aeruginosa PAO1’s alkaline phosphatase was made. There were no abnormal protein binding sites found.

Homology modeling and validation of ACPase and ALPase proteins

Using Robetta server, five models were generated for the acid phosphatase (ACPase) 488 residues and alkaline phosphatase (ALPase) 500 residues from 560 residues (Robetta server do not accept more than 500 amino acids for protein modeling), for both the template Streptomyces sp.4F and wild type Streptomyces sp MMA-NRC; respectively.

Determined by the density of their clusters. The confidence ratings (C-scores) for the five various models of the ACPase residues. Model 5, with the highest C-score and low estimation error, the sample was chosen for additional examination because to its high C-score, which indicates a strong level of confidence in the model was 0.73 for template strain Streptomyces sp.4F, as illustrated in Fig. 4a. Model 1, with the highest C-score and low estimation error, the data was chosen for additional examination because to its high C-score, which indicates a strong level of confidence in the model was 0.72 for wild type strain Streptomyces sp MMA-NRC, as illustrated in Fig. 4b.

Fig. 4
figure 4

Modelled 3D structure of ACPase and ALPase proteins: (A) template Streptomyces sp.4 F ACPase; (B) wild type Streptomyces sp MMA-NRC ACPase; (C) template Streptomyces sp.4 F ALPase; (D) wild type Streptomyces sp MMA-NRC ALPase.

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Determined by the density of their clusters. The confidence ratings (C-scores) for the five various models of the ALPase residues. Model 1, with the highest C-score and low estimation error, the sample was chosen for additional examination because to its high C-score, which indicates a strong level of confidence in the model was 0.86 for template strain Streptomyces sp.4F as illustrated in Fig. 4c. Model 1, with the highest C-score and low estimation error, the sample was chosen for additional examination because to its high C-score, which indicates a strong level of confidence in the model was 0.85 for wild type strain Streptomyces sp MMA-NRC as illustrated in Fig. 4d.

Subsequently, the model that was produced underwent evaluation using general stereo chemical parameters using the PROCHECK website. In addition, the Ramachandran plot was developed for the energy-minimized model of ACPase and ALPase structures for Streptomyces sp.4F and Streptomyces sp.MMA-NRC. The x-axis of the plot is divided into four quadrants, which correspond to the low-energy area, permitted region, usually allowed region, and prohibited region, as shown in Table 1.

Table 1 Ramachandran plot energy-minimized model of ACPase and alpase of Streptomyces sp.4f and Streptomyces sp.-MMA-NRC.
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The 3D modeled for template strain Streptomyces sp.4F ACPase protein underwent analysis using PROCHECK, which indicated that 488 residues, accounting for 100% overall quality factor, representing 91.04% of the total quality factor, were 92.2% located in the most favorable areas of the Ramachandran plot. In addition, 7.5% of residues were found in the further permitted areas, 0.2% in the generously allowed regions, and lowest 0.0% in the disallowed regions, which indicate model stability, as illustrated in Fig. 5a.

Fig. 5
figure 5

Ramachandran plot of ACPase and ALPase proteins: (A) template Streptomyces sp.4 F ACPase; (B) wild type Streptomyces sp MMA-NRC ACPase; (C) template Streptomyces sp.4 F ALPase; (D) wild type Streptomyces sp MMA-NRC ALPase.

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The 3D modeled strain for wild type strain Streptomyces sp MMA-NRC ACPase protein was analyzed using PROCHECK, which revealed that 488 residues, accounting for 94.37% overall quality factor (94.5% The majority of residues were located inside the most favorable sections of the Ramachandran plot. In addition, 4.8% of the residues were found in the extra permitted areas, 0.8% in the generously allowed regions, and lowest 0.0% in the disallowed regions, which indicate model stability, as illustrated in Fig. 5b.

The 3D modeled for template strain Streptomyces sp.4F ALPase protein was analyzed using PROCHECK, which revealed that 500 residues, accounting for 92.16% overall quality factor (86.8% The majority of residues were located inside the most preferred areas of the Ramachandran plot. In addition, 11.8% of the residues were located in the extra permitted zones, 1.2% in the generously allowed regions, and lowest 0.2% in the disallowed regions, which indicate model stability), as illustrated in Fig. 5c.

The 3D modeled wild type strain Streptomyces sp MMA-NRC ALPase protein was analyzed using PROCHECK, which revealed that 500 residues, accounting for 93.08% overall quality factor (87.8% The majority of residues were located inside the most preferred areas of the Ramachandran plot. In addition, 10.4% of the residues were located in the extra permitted zones, 0.9% in the generously allowed regions, and 0.9% in the disallowed regions, which indicate model stability), as illustrated in Fig. 5d.

In related studies of41, the UniProt database was used to retrieve representative protein structures from five phylogenetic groups: P9WHW5 (PDB ID: 1TXO) from M. tuberculosis, A0A1L7GEB2 (AlphaFold ID: AF-A0A1L7GEB2-F1) from Streptomyces sp. TN58, A0A7V6HC85 (AlphaFold ID: AF-A0A7V6HC85-F1) from the Clostridiaceae bacterium, Q8VQA1 (PDB ID: 2PK0) from S. agalactiae, and P76395 (AlphaFold ID: AF-P76395-F1) from Escherichia coli. The other three proteins were predicted structures derived from AlphaFold, with the exception of the PP2C from S. agalactiae (PDB ID: 2PK0) and M. tuberculosis (PDB ID: 1TXO), which represented the known structure in bacteria. The amino acid residues from the active centre were then labelled based on the sequence alignment results after the five typical protein structures were superimposed using ChimeraX software. Lastly, the primary to quaternary protein structure data of five sample proteins were examined using ESPript50. , the native enzyme structures were superimposed with their corresponding AlphaFold2-predicted QTY variants, demonstrating a high degree of structural similarity. These findings support the effectiveness of the QTY code for modeling β-barrel membrane enzymes and highlight its potential utility in designing water-soluble membrane proteins for a broad range of biological applications51. , using a combination of forward genetics, biochemical reconstitution, and AlphaFold2 structure prediction, identified a conserved, tripartite substrate docking interface comprised of three variable loops on the surface of the PPM phosphatase domains of SpoIIE and RsbU that recognize the 3D three-dimensional structure of the substrate protein43. , comparison modeling of the recombinant of BlAPase amino acid sequence with that of a few other bacterial alkaline phosphatases. Their active site areas involved in phosphate binding, phosphorylation, and metal binding are substantially conserved, according to the multiple alignment study. The recombinant BlAPase showed the highest level of similarity with B. licheniformis MC14 APase. Compared to Bacillus subtilis, C. marina, E. coli, antarctic strain Table 5, T. thermophilus HB27, and Meiothermus ruber, the enzyme’s amino acid sequence showed 37, 32, 31, 31, 28, and 27% similarity, respectively. In recombinant BlAPase, the amino acid residues Asp49, Ser89, Thr150, Arg168, Glu283, Asp288, His292, and Asp307 are highly conserved48. , the comparative homology protein model of P. aeruginosa PAO1 alkaline phosphatase was predicted using the SWISS-Model Workspace. In this case, 1alh.1. A, the crystal structure of a mutant E. coli alkaline phosphatase, was chosen as the template sequence. 73.93% of the template sequence and the query sequence were identical. The structure’s resolution was 2.50 ̺. The projected protein model’s oligo state was homo dimer. The alkaline phosphatase of P. aeruginosa PAO1 predicted protein model was assessed and confirmed using the QMEAN and SAVES servers. From SAVES, ERRAT, Ramachandran plot, and 3D verification were assessed. The Ramachandran plot analysis showed that 97.0% of the residues were in the favored (red) region, 4.7% were in the allowed (brown) regions, and the remaining 2.6% were in the generously allowed (yellow) or outlier region. A high-quality model has more than 90% of its residues in the favored region.

Docking and molecular interaction studies

Using the HDock online tool, docking studies were carried out to investigate how the substrate rock phosphate binds to the 3D model of the ACPase and ALPase protein. For the docking procedure, the receptor and ligand files were saved in pdb format. The nonpolar hydrogen atoms were mixed with the carbon atoms, and the polar H atoms were given Gasteiger charges. The required adjustments were made to the internal degrees of freedom and torsions. Low distance and strong inertaction between the ligand and protein receptor were suggested by active site amino acids with the lowest degree of interface residues.

The docking results of rock phosphate with the 3D model of ACPase showed the patterns of interaction. With an affinity score of -116.76 kcal/mol, a confidence score of 0.3397, a ligand RSMD (Å) of 34.22, and active site amino acids of ARG291 and PHE456 with low interface residues of 2.345 and 2.103, respectively, the template strain Streptomyces sp. 4f ACPase.

The docking results of rock phosphate with the 3D model of ACPase showed the patterns of interaction. The wild type strain Streptomyces sp. MMA-NRC ACPase had an affinity score of -110.86 kcal/mol, a confidence score of 0.3137, a ligand RSMD (Å) of 69.31, and active site amino acids ARG91 and THR298 with low interface residues of 2.105 and 2.262, respectively, as shown in Table 2. Demonstrated aromatic interaction, H-Bond interaction, and hydrophobic interaction, as shown in Fig. 6.

Table 2 Active site amino acids and receptor interface residue(s) of Docking interaction of ACPase and ALPase of wild type strain Streptomyces sp. MMA-NRC with rock phosphate substrate.
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Fig. 6
figure 6figure 6figure 6

protein–ligand docking by HDock of Streptomyces sp.MMA-NRC ACPase. (A) 3D surface. (B) 3D cartoon complex interaction showing binding ACPase with ligand rock phosphate displaying (red color) the most effective binding mode in the protein cavity (active site displayed by cyan color). C; Aromatic interaction, (D) H-Bond interaction and (E) Hydrophobic interaction.

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The docking results of rock phosphate with the 3D model of ALPase showed the patterns of interaction. For the template strain Streptomyces sp. 4f ALPase strain, the affinity score of -129.64 kcal/mol, confidence score of 0.3996, ligand RSMD (Å) of 29.86, and active site amino acids of HIS385 and ASP417 with low interface residues of 2.135 and 2.033, respectively,

The docking results of rock phosphate with the 3D model of ALPase showed the patterns of interaction. For the wild type Streptomyces sp. strain MMA-NRC ALPase, the affinity score of -108.55 kcal/mol, confidence score of 0.3039, ligand RSMD (Å) of 33.68, and active site amino acids of GLN354 and HIS385 with low interface residues of 2.146 and 1.465, respectively, as shown in Table 2. Demonstrated aromatic interaction, H-Bond interaction, and hydrophobic interaction, as shown in Fig. 7.

Fig. 7
figure 7figure 7figure 7

protein–ligand docking by HDock of Streptomyces sp.MMA-NRC ALPase. A; 3D surface. B; 3D cartoon complex interaction showing binding ALPase with ligand rock phosphate displaying (red color) the most effective binding mode in the protein cavity (active site displayed by cyan color). C; Aromatic interaction, D; H-Bond interaction and E; Hydrophobic interaction.

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In another investigation conducted by41, found that conserved Asp and Gly residues were primarily grouped at the active core locations within motifs 1, 2, 5, 8, and 11 when we looked at the superposition diagram with labelled conserved amino acids. In line with the active center of eukaryotic PP2C phosphatase, conserved Asp residues found in motifs 2, 8, and 11 joined forces with two manganese ions to form a binuclear metal centre. Notably, examination of the PDB structures of the representative proteins from Groups I and II revealed that the active center of bacterial PP2C phosphatase contained a third metal ion. When the active centers of five protein groups were superimposed, it was revealed that conserved Ser residues in Group IV and conserved Asp residues in Motif 5 helped Groups I, II, III, and V bind the third metal ion. Furthermore, a comparison of the flap subdomains of the five typical proteins showed that Groups I, II, and III shared a lot in common, while Groups IV and V proteins showed a lot of variances. According to recent research, the flap subdomain was essential for the third metal ion’s binding. We therefore speculate that the third metal ion would be common in the PP2C phosphatases of Groups I, II, and III based on these data. Additionally45, , determined of non-specific acid phosphatase M2-32 docking studies with adenosine monophosphates, combined with site-directed mutagenesis, identified His174, Arg207, His213, Asp217 as critical catalytic residues, and Tyr136 and Ser172 probably involved in substrate recognition. Mutations at these positions resulted in over 90% loss of enzymatic activity, highlighting their functional significance51. , reported the PPM phosphatase domains of SpoIIE and RsbU, nonconserved amino acids in these loops facilitate the accommodation of the cognate substrate and prevent dephosphorylation of the noncognate substrate. Together, single amino acid substitutions in these three elements cause an over 500-fold change in specificity. Data additionally suggest that substrate-docking interactions regulate phosphatase specificity through a conserved allosteric switch element that controls the catalytic efficiency of the phosphatase by positioning the metal cofactor and substrate. A generalizable mechanistic model for PPM family phosphatase substrate specificity.Importantly, the substrate docking interface with the phosphatase is only partially over lapping with the much more extensive interface with the upstream kinase, suggesting the possibility that kinase and phosphatase specificity evolved independently.

Cloning of ACPase and ALPase encoding genes in E. coli DH5α

The ACPase and ALPase encoding gene that scientists are studying is 1464 and 1680 bp long and comes from the Streptomyces sp. MMA-NRC. It is improved by using primers that are specific to the gene of interest. An analysis of the PCR results was performed using gel electrophoresis, which revealed the presence of two products measuring 1464 and 1680 bases in length, respectively, derived from genomic DNA. From what we can tell, the optimal circumstances for amplifying the target DNA amplicon were achieved at an annealing temperature of 57.5 °C. The Qiagen gel purification kit was used to remove the DNA band of the Streptomyces sp. MMA-NRC from the agarose gel. After that, a ligation cloning kit was used to clone it into the pGEM®-T Easy Vector. The cloning was done under T7 and SP6 strong promoters to enable the expression of ACPase and ALPase. The recombinant plasmid obtained was designated as pGEM-T-ACPase and pGEM-T-ALPase. The recombinant plasmid was introduced into the host organism for protein synthesis, E. coli DH5α, using the heat-shock method. The next step was to find transformants that could withstand ampicillin by using the white/blue screening approach, which included IPTG and X-gal. Different transformants derived from the E. coli strain were successfully produced by efficient transformation. The plasmids were extracted from E. coli transformants that were chosen at random and then subjected to analysis using agarose gel electrophoresis. PCR amplification was used to screen E. coli transformants containing genes for ACPase and ALPase. Specific primers utilized for the isolation of ACPase and ALPase, were employed for this purpose.

ACPase and ALPase expression

The ACPase and ALPase activity of Streptomyces sp. MMA-NRC was conducted by using a fermentation medium that was enhanced with 0.5% rock phosphate. The experiment included examining the E. coli recipient strains, the donor Streptomyces sp. MMA-NRC, and four E. coli strains harboring the ACPase and ALPase plasmids (E.coli pGEM-T-ACPase and E.coli pGEM-T-ALPase). The cultures were placed in an incubator at a temperature of 37 °C and subjected to shaking at a speed of 120 revolutions per minute for a maximum duration of 7 days. Each culture was sampled and the obtained supernatant was obtained using centrifugation. This supernatant was used as the crude enzyme for the phosphatase activity test, following the previously described method. The findings revealed that the donor Streptomyces sp. MMA-NRC had ACPase and ALPase activity, but the recipient strains of E. coli showed no activity. Nevertheless, the E. coli cultures that obtained the ACPase and ALPase plasmids (E. coli pGEM-T-ACPase and E. coli pGEM-T-ALPase) exhibited phosphatase functionality. The results validated the biological functionality of the cloned ACPase and ALPase genes. Both E. coli recombinant strains exhibited phosphatase activity increase with the increase of incubation period with rock phosphate. The available phosphor was observed on seven day of incubation, with approximately pf 52.64 and 57.22 mg/L− 1 for E.coli DH5α pGEM-T-ACPase and E.coli DH5α pGEM-T-ALPase, respectively, as shown in Fig. 8. In contrast, the wild-type Streptomyces sp. MMA-NRC displayed available phosphor of 35.44 mg/L− 1 after 7 days of incubation, as reported by17.

Fig. 8
figure 8

rock phosphate solubilization of (a) control, (b) Ecoli DH5α, (c) recombinant Ecoli DH5α ACPase and (d) recombinant Ecoli DH5α ALPase on NBRIP rock phosphate broth media without glucose addition after 7 days incubation.

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In a related study of43, the recombinant plas mid pBL-ALP2 and empty vector pET19b were transformed to E. coli BL21 (DE3) for APase expression through induction with 0.5 mM IPTG at 37 °C for 3–4 h after the thermostatable Alkaline Phosphatase APase gene from Bacillus licheniformis MTCC 1483 was cloned into a His-tagged expression vector pET19b.A Ni-NTA affinity column was used to purify the recombinant enzyme, and the resultant enzyme had a specific activity of 24,890 U/mg protein.The enzyme demonstrated great thermostability and peak activity at pH 10.0 and 50 °C. In order to dephosphorylate linear DNA segments, the recombinant alkaline phosphatase from B. licheniformis MTCC 1483 demonstrated a dephosphorylation efficiency of 92.9%44, reported Escherichia coli was cloned and shown to produce the olpA gene of Chryseobacterium meningosepticum, which codes for a molecular class C phosphatase. A 29-kDa polypeptide with an amino-terminal signal peptide characteristic of bacterial membrane lipoproteins is encoded by the gene. A functional product that primarily partitions in the outer membrane is the outcome of expression in E. coli. Two stages of ion exchange chromatography were used to purify a secreted soluble OlpA derivative (sOlpA) that was made in E. coli but lacked the N-terminal cysteine residue for lipid anchoring45, after being cloned into the pET28(b) vector and overexpressed in Escherichia coli BL21 (DE3), the gene encoding M2-32, a non-specific acid phosphatase, was isolated. Size exclusion chromatography, dynamic light scattering, and sedimentation investigations showed a dimeric form of M2-32 in solution despite the monomeric form’s molecular weight of about 28 kDa. Strong activity was shown by enzymatic tests employing p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 3’- and 5’-adenosine monophosphate over a pH range of 4.0–8.0 at both 30 and 50 °C. The enzyme refolded following heat denaturation at 80 °C, despite differential scanning fluorimetry showing an unfolding temperature at 47 °C. In study52, the gene encoding alkaline phosphatase from the psychrotrophic bacterium Shewanella sp. SIB1 was cloned, sequenced, and overexpressed in Escherichia coli.The recombinant protein was purified and its enzymatic properties were compared with those of E. coli alkaline phosphatase (APase), which shows an amino acid sequence identity of 37%. Additionally53, demonstrated overexpressed the phoK gene from Sphingomonas sp., which codes for a new alkaline phosphatase called PhoK, in the radioresistant bacterium Deinococcus radiodurans. Using zymographic and enzyme activity tests, the resulting recombinant strain (Deino-PhoK) demonstrated exceptionally high alkaline phosphatase activity. Uranium may be precipitated effectively by Deino-PhoK cells across a broad range of input U values. The strain precipitated almost 90% of the uranium in 2 h at low uranyl concentrations (1 mM), and at 10 mM U concentration, it demonstrated a high loading capacity of approximately 10.7 g U/g of dry weight of cells42, reported Escherichia coli strain BL21 (DE3) overexpressed the cloned phoK gene, which encodes the alkaline phosphatase from Sphingomonas sp. strain BSAR-1. Compared to BSAR-1, the resulting E. coli strain EK4 produced extracellular PhoK activity 13 times higher and overexpressed cellular activity 55 times higher47, reported the existence of a putative Acid Phosphatase (AcpA; EC 3.1.3.2) was discovered through genomic sequence analysis of Acinetobacter baumannii. After creating a plasmid construct, E. coli showed high expression of the recombinant protein (rAcpA)43, reported represent Asp73, Ser124, Thr177, Arg188, Glu344, Asp349, His353, Asp391, His392, and His434 in E. coli recombinant APase. With the exception of Ser124 and Arg188, which make up catalytic and phosphate-binding sites, respectively, the highly conserved residues form three metal-binding sites. Asp175 is a component of the Mg2+-binding site in E. coli APase, and its substitution with His, as in mammalian APase, increases the activity of the enzyme. Since Asp175 of E. coli APase is equivalent to His155 in recombinant BlAPase, this alteration may help raise the specific activity of recombinant BlAPase by improving its affinity for binding Mg2+. Different sources have different amino acid residues that are closed to metal sites, which could lead to conformational modifications that alter the binding affinities of certain metal ions. The specific activity of recombinant BlAPase in the presence of metal ions may increase or decrease as a result of these conformational changes54, reported Streptomyces isolate KT 6-4-1, Streptomyces isolate L3, Bacillus isolate MC 9 and Streptomyces isolate ST 3 rock Phosphate solubilization ability in PVK Broth of 37.59, 15.37, 5.96 and 30.98; respectively.

Conclusion

Due to the wide range of applications, phosphatase had attracted a lot of attention. Streptomyces sp. MMA-NRC, an actinomycetes strain, was isolated and identified as a potential strain for the production of the phosphatase enzyme. The unique Streptomyces sp. 4 F and Streptomyces sp. MMA-NRC, which encode ACPase and ALPase genes, were isolated, sequenced, and submitted to the NCBI GenBank accession number based on the conserved rejoin. The Ramachandran plot, which demonstrated that the residues were accurately modelled, was used to validate the 3D model. Streptomyces sp. MMA-NRC ACPase and ALPase ultimately exhibited a contact with a high affinity score, according to molecular docking experiments. Streptomyces sp. MMA-NRC ACPase and ALPase expressing genes that were effectively cloned and expressed in E. coli DH5α, yielding greatly elevated levels of phosphatase activity and available phosphorus. This study’s findings highlight the promise of microbial phosphatase, especially ACPase and ALPase, for rock phosphate solubilization. Other agriculture that rock phosphate solubilization, such as plant growth promotion, may be good candidates for further investigation into the potential applications of phosphatase. In order to create sustainable cropping systems, Streptomyces sp. will therefore be promising at the same time that crop productivity and economic-environmental sustainability need to be chosen, characterised, and identified.