Diversity analysis of phytase-producing bacteria from coastal seawater and sediment and characterization of their phytases

Highlights

• •Diversity of phytate-utilizing bacteria in coastal marine environments was analyzed.
• •Twenty-seven phytase-producing bacterial strains were isolated.
• •Five phytases were expressed and characterized.
• •Phytase 3919 has strong potential as a feed additive.

Abstract

Phytic acid, also known as inositol hexaphosphate (IP6), is one of the most abundant organophosphorus compounds in nature. Its degradation by phytase plays a key role in the natural phosphorus cycle. In addition, phytases are widely used in livestock and poultry feed to enhance phosphorus utilization. While most reported and commercial phytases are derived from terrestrial organisms, relatively few originate from marine microorganisms, and information on the diversity of phytase-producing marine bacteria remains limited. In this study, following enrichment with sodium phytate, we analyzed the bacterial diversity in seawater and sediment samples collected from the coast of Aoshan Bay in Qingdao, China, using 16S rRNA gene amplicon sequencing. A total of 138 OTUs representing 10 phyla, 15 classes, 37 orders, 55 families, and 70 genera were identified. Furthermore, 27 phytase-producing bacterial strains were isolated from the enrichment cultures, primarily belonging to the phyla Firmicutes (14/27) and Proteobacteria (12/27). Five extracellular phytase genes were identified through genome sequencing of three representative strains. These phytases were subsequently expressed and characterized. All were classified as histidine acid phosphatase-type phytases, exhibiting optimal activity at temperatures of 50–60 °C and pH values of 4.0–5.0. Notably, phytase 3919 showed a specific activity as high as 2485.25 U/mg, indicating strong potential for practical applications. This study provides insight into the diversity of coastal bacteria involved in phytic acid degradation, contributing to our understanding of bacterial-driven phosphorus cycling in coastal ecosystems and facilitating the discovery of phytases with industrial potential.

Graphical abstract

1. Introduction

Phytic acid, also known as inositol hexaphosphate (IP6), is one of the most abundant organophosphorus compounds in nature [1]. It serves as the primary phosphorus storage form in plants, accounting for up to 90% of the total phosphorus in seeds [2,3]. Phytic acid is released into the environment through feces and decaying plant matter [4], and phytates constitute 20–80% of the organic phosphorus in soil and manure. A considerable amount of phytic acid also enters aquatic systems via surface runoff and subsurface flow from land, eventually reaching the ocean [5]. Phytate is thus the main source of biophosphorus input from terrestrial runoff into aquatic environment [6]. Despite their abundance, phytates cannot be directly absorbed by plants and animals due to their complex structure [3]. In natural ecosystems, phytic acid is hydrolyzed by phytases, enzymes produced by plants and microorganisms, into lower inositol phosphate derivatives and inorganic phosphate, which can then be utilized by plants and animals. Consequently, phytases play a vital role in recycling organophosphorus in both terrestrial and aquatic environments [7]. In addition, phytases are widely used in livestock and poultry feed to enhance phosphorus bioavailability.

Based on their structure and catalytic mechanisms, phytases are classified into four subclasses: histidine acid phytases (HAPhys), β-propeller phytases (BPPhys), protein tyrosine phosphatase-like phytases (PTPhys), and purple acid phytases (PAPhys) [3]. Among these, HAPhys, PTPhys, and PAPhys are acid phytases primarily derived from terrestrial organisms, while BPPhys are alkaline phytases and represent the only phytase group commonly found in aquatic environments. HAPhys, PTPhys, and PAPhys can each hydrolyze five phosphate groups from a phytic acid molecule, producing inositol 1-phosphate (Ins(2)P) as the final product. In contrast, BPPhys typically remove only three specific phosphate groups, resulting in the formation of inositol-2,4,6-trisphosphate (Ins(2,4,6)P₃). HAPhys represent the most diverse and extensively studied phytase group. These enzymes, with an optimal pH range of 2.5–5.5, are primarily produced by fungi, yeasts, and bacteria. HAPhys belong to clade 2 of the histidine phosphatase superfamily (HP2) and are further categorized into two subclades: histidine acid phosphatases with phytase activity (HP2Ps) and multiple inositol polyphosphate phosphatases (MINPPs) [8,9]. BPPhys are mainly identified in bacteria, particularly Bacillus species, and exhibit high activity in neutral to alkaline pH ranges (6.5–8.5) along with high thermostability [10,11]. PTPhys were first discovered in the anaerobic ruminant bacterium Selenomonas ruminantium and later in soil metagenomes. They belong to the cysteine phosphatase superfamily and exhibit peak activity at pH 4.0–5.5 [3,12,13]. PAPhys are widely distributed in plants and have also been detected in fungi and bacteria. However, only plant-derived PAPhys have been shown to exhibit phytase activity to date [1,12,14]. They belong to the metallophosphoesterase superfamily and are most active at pH 5.0–6.0.

While most reported phytases are derived from terrestrial organisms, only a limited number have been isolated from microorganisms in marine environments. Hirimuthugoda et al. identified ten phytase-producing yeast strains from diverse marine sources, including the guts of marine fish and sea cucumbers, as well as waters from the Pacific, Indian, and South China Seas, and salt farms, suggesting that phytase-producing yeasts are widely distributed across different marine environments [15]. Kailasam et al. isolated a phytase-producing Curtobacterium luteum strain from seagrass meadow sediment [16]. Farha et al. recovered a phytase-producing fungal strain, Penicillium arCSPf, from marine sediment on the continental slope of the Eastern Arabian Sea [17]. Lin et al. found that seagrass-associated BPPhy communities displayed higher diversity and evenness at sampling sites impacted by greater human activity [18]. Nam et al. characterized a BPPhy enzyme from marine Pseudomonas sp. BS10-3 [19]. Yu et al. isolated a phytase-producing yeast, Rhodotorula mucilaginosa strain JMUY14, from Antarctic deep-sea sediment and characterized the secreted phytase from this strain [20]. Despite these studies, our understanding of the diversity of phytase-producing bacteria in marine environments remains limited.

This study aimed to investigate the diversity of phytase-producing bacteria in coastal seawater and sediment, along with characterization of their phytases. Seawater and sediment samples were collected from the coast of Aoshan Bay in Qingdao, China. Following enrichment with sodium phytate, the bacterial diversity in the enrichment cultures was analyzed using 16S rRNA gene amplicon sequencing. Phytase-producing bacterial strains were then isolated from these cultures. Three representative strains were selected for whole-genome sequencing to identify putative phytase genes. The identified genes were heterologously expressed in Escherichia coli, and the resulting recombinant phytases were subsequently characterized.

2. Materials and methods

2.1. Sample collection, enrichment cultivation, and diversity analysis of phytate-utilizing bacteria

Seawater and sediment samples were collected from the intertidal zone of Aoshan Bay, Qingdao, China (36°22′12"N, 120°42′19"E), on September 29, 2022. The samples were placed in sterile plastic bags and transported to the laboratory under low-temperature conditions (4–6 °C). In the laboratory, seawater samples were filtered through a 0.22 μm membrane to collect microbial cells. The membrane was then placed into a flask containing 10 mL sterile artificial seawater (3% Sigma sea salts, w/v) and 30 sterile glass beads. Simultaneously, approximately 1 g of sediment sample was added to another flask containing 10 mL sterile artificial seawater and 30 glass beads. Both flasks were shaken (180 rpm) at 25 °C for 20 min. From each flask, 1 mL of suspension was transferred into 50 mL liquid phytase screening medium (PSM), which consisted of 2% glucose, 0.5% NH₄NO₃, 0.4% sodium phytate, 0.2% CaCl₂∙2H₂O, 0.05% MgSO₄∙7H₂O, 0.05% KCl, 0.001% MnSO₄∙7H₂O, and 0.001% FeSO₄∙7H₂O (pH 6.5) [20]. Sodium phytate (P8810) was purchased from Sigma (St. Louis, MO, USA), and all other reagents were of analytical grade. The cultures were incubated at 25 °C with continuous shaking (180 rpm) for 10 days. Subsequently, 0.2 mL of culture was transferred into 50 mL of the same fresh PSM medium and incubated under the same conditions for further enrichment. After repeated enrichment steps, bacterial diversity was assessed by 16S rRNA gene amplicon sequencing, conducted by Wuhan Onemore-Tech Co., Ltd. (Wuhan, China), following a previously described protocol [21].

2.2. Isolation of phytase-secreted bacteria and phylogenetic analysis

Phytase-secreting bacterial strains were isolated from the enrichment cultures by plating serial dilutions (10⁻² to 10⁻⁸) onto solid PSM medium and incubating at 25 °C for 4–10 days. Colonies exhibiting clear hydrolysis zones were selected and purified. For each strain, the diameters of the hydrolysis zone and the corresponding colony were measured, and the hydrolysis-to-colony (H/C) ratio was calculated. The phylogenetic tree of the isolated strains was constructed using the Neighbor-Joining method in MEGA 7, following the procedure described previously [21].

2.3. Genomic sequencing and analysis of putative extracellular phytases

The genomes of bacterial strains PZQ1, P4A1, and P1A3 were sequenced and assembled by Wuhan Onemore-Tech Co., Ltd. (Wuhan, China). Putative phytase genes were identified through functional annotation using BLASTp searches against the Nr (http://ftp.ncbi.nih.gov/blast/db/) and Swiss-Prot (http://www.uniprot.org/) databases [22,23]. Signal peptides in the predicted phytase proteins were identified using SignalP 5.0 [24].

2.4. Overexpression and characterization of extracellular phytases

Phytase genes 4182, 148, 634, 3898, and 3919, excluding their native signal peptide sequences, were amplified from their respective bacterial genomes using PCR. The genes were cloned into the pET-22b(+) expression vector and transformed into Escherichia coli BL21(DE3) for heterologous expression. Recombinant strains were cultured at 18 °C and 110 rpm for 16 h with 300 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) used as the inducer. The expressed phytase proteins were purified using Ni-NTA His-tag affinity chromatography. Protein purity was assessed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and protein concentrations were measured using the bicinchoninic acid (BCA) protein assay kit (Thermo, Waltham, MA, USA), with bovine serum albumin as the standard.

Phytase activity was measured spectrophotometrically according to a previously described method [25]. Briefly, 100 μL of enzyme solution was mixed with 200 μL of sodium phytate solution (7.5 mM, pH 5.5) and incubated at 37 °C for 30 min. The reaction was terminated by adding 200 μL of a chromogenic reagent consisting of nitric acid (33.3%, v/v), ammonium molybdate (10%, w/v), and ammonium vanadate (0.24%, w/v) in a 2:1:1 volume ratio. A control was prepared by adding the chromogenic reagent to the reaction mixture prior to incubation. Absorbance was measured at 415 nm. One unit (U) of phytase activity was defined as the amount of enzyme that liberates 1 μmol of phosphate per minute from sodium phytate. Optimal temperatures for phytase activity were determined by assaying enzyme activity across a temperature range of 30–70 °C using 5 mM sodium phytate dissolved in 100 mM acetate buffer (pH 5.5). Optimal pH values were determined by measuring enzyme activity at the respective optimal temperatures across a pH range of 2.0–7.0 using the following buffers: 100 mM glycine-HCl (pH 2.0–4.0), 100 mM sodium acetate (pH 4.0–6.0), and 100 mM Tris–acetate (pH 6.0–7.0). The substrate (5 mM sodium phytate) was freshly dissolved in the corresponding buffer before use.

3. Results

3.1. Diversity analysis of phytate-utilizing bacteria in coastal seawater and sediment samples from the Aoshan Bay

Using sodium phytate as the sole phosphorus source, enrichment cultivation was performed to recover microorganisms from seawater and sediment samples collected from Aoshan Bay. To investigate the diversity of phytate-utilizing bacteria present in these samples, the bacterial community composition in the enrichment cultures was analyzed via 16S rRNA gene amplicon sequencing. In total, 138 OTUs were identified, representing 10 phyla, 15 classes, 37 orders, 55 families, and 70 genera. At the phylum level, the most abundant groups were Firmicutes (88.42% in seawater, 66.33% in sediment), Proteobacteria (11.37% in seawater, 21.07% in sediment), and Actinobacteriota (11.74% in sediment). Other phyla each accounted for less than 1% of the community. At the family level, 50 families were identified in seawater samples, 53 in sediment samples, and 47 were common to both. Several families were sample-specific: Acholeplasmataceae (0.002%) and Leuconostocaceae (0.003%) were detected only in the seawater sample, whereas Cellulomonadaceae (0.07%), Deferribacteraceae (0.003%), Desulfovibrionaceae (0.002%), Vagococcaceae (0.005%), and uncultured members of the Bacillales order (0.051%) were exclusive to the sediment sample. Bacillaceae was notably abundant in both sample types, being the most dominant family in the sediment (65.63%) and the second most abundant in the seawater sample (33.22%). In seawater, the three most prevalent families were Paenibacillaceae (54.89%), Bacillaceae (33.22%), and Pseudomonadaceae (11.08%). In the sediment sample, the dominant families were Bacillaceae (65.63%), Enterobacteriaceae (20.89%), and Microbacteriaceae (11.24%) (Fig. 1). These results reveal a high diversity of phytate-utilizing bacteria in coastal seawater and sediment, suggesting their potential ecological role in phytate degradation and phosphorus cycling within coastal ecosystems.

Fig. 1.

The bacterial communities recovered from the coastal seawater and sediment samples from the Aoshan Bay with sodium phytate as the sole phosphorus source. Bacterial communities were analyzed based on 16S rRNA gene amplicon sequencing. Relative abundance of recovered bacteria was shown at the family level. Families with a relative abundance greater than 0.05% are shown.

3.2. Isolation of phytase-secreting bacteria

Following enrichment cultivation, phytase-secreting bacteria were isolated by plating the cultures onto media containing sodium phytate as the sole phosphorus source. After incubation at 25 °C for 4–10 days, more than 1000 bacterial colonies were observed. Morphologically distinct colonies were selected, purified, and identified by 16S rRNA gene sequencing. In total, 27 bacterial strains were obtained. Aside from one strain classified as Actinobacteria, the majority were assigned to Firmicutes (14 out of 27) and Proteobacteria (12/27), representing 12 different genera (Table 1).

Table 1.

Information on the isolated phytase-secreting bacterial strains.

Strain	Top-hit species	Similarity (%)	Completeness (%)
P6B1	Bacillus siamensis KCTC 13613	99.65	96.50
P6B2	Bacillus siamensis KCTC 13613	99.86	96.60
P2D1	Christensenella intestinihominis AF73-05CM02	96.48	98.60
P2A2	Curtobacterium oceanosedimentum ATCC 31317	99.93	95.00
ZQ-1	Enterobacter hormaechei subsp. hormaechei ATCC 49162	99.10	98.60
ZQ-3	Enterobacter hormaechei subsp. oharae DSM 16687	99.43	96.10
P5C2	Escherichia fergusonii ATCC 35469	99.79	96.10
P4A1	Escherichia fergusonii ATCC 35469	99.57	95.90
P4A2	Escherichia fergusonii ATCC 35469	99.79	96.10
P4A4	Escherichia fergusonii ATCC 35469	99.57	96.20
P4A3	Shigella flexneri ATCC 29903	99.86	96.10
P1B1	Niallia circulans ATCC 4513	99.50	95.50
P1B2	Niallia circulans ATCC 4513	99.64	95.30
P5A1	Paenibacillus lupini RLAHU15	99.15	96.40
P2A3	Paenibacillus glycanilyticus subsp. glycanilyticus DS-1	98.51	96.30
P1A1	Flavobacterium acidificum LMG 8364	99.28	99.20
P1A2	Flavobacterium acidificum LMG 8364	99.35	98.70
P1A3	Flavobacterium acidificum LMG 8364	99.35	98.40
P1A4	Flavobacterium acidificum LMG 8364	99.32	98.40
P2A1	Priestia aryabhattai B8W22	99.93	96.50
P2C1	Priestia aryabhattai B8W22	99.93	93.60
P2C3	Priestia aryabhattai B8W22	99.86	96.50
P2C4	Priestia aryabhattai B8W22	100.00	95.40
P2C5	Priestia aryabhattai B8W22	100.00	96.50
P2C6	Priestia aryabhattai B8W22	99.72	96.50
P2B5	Pseudomonas lalkuanensis PE08(T)	99.50	100.00
P5B1	Staphylococcus caprae ATCC 35538	99.86	96.40

To assess their phytase-secreting capacity, the isolated strains were re-inoculated onto sodium phytate-containing plates. Of the 27 strains, 18-belonging to 10 genera-produced visible hydrolytic zones around the colonies (Fig. 2, Table 2), indicating extracellular phytase activity. In general, strains from the genera Enterobacter and Flavobacterium exhibited relatively high H/C ratios, exceeding 3.14, suggesting strong phytase-secreting capability. Strains from Christensenella, Escherichia, Pseudomonas, and Shigella showed moderate H/C ratios ranging from 2.0 to 3.11. In contrast, strains from Bacillus, Paenibacillus, Priestia, and Staphylococcus had lower H/C ratios (less than 1.86), suggesting weaker phytase-secreting ability.

Fig. 2.

Hydrolytic zones of the isolated phytase-secreting bacteria cultured on plates containing 0.4% sodium phytate. The strains were cultured in PSM liquid medium for 48 h. Then, 3 μL of the culture solution was added to a PSM solid plate, which was incubated at 25 °C for 5 days. The strain names are shown in the figure, and their top-hit species are shown in Table 2.

Table 2.

The H/C values of the phytase-secreting bacteria shown in Figure 2^a.

Strain	Top-hit species	H/C
PZQ1b	Enterobacter hormaechei subsp. oharae	5.00
P1A3b	Flavobacterium acidificum	4.87
PZQ3	Enterobacter hormaechei subsp. hormaechei	4.60
P1A4	Flavobacterium acidificum	3.56
P1A2	Flavobacterium acidificum	3.28
P1A1	Flavobacterium acidificum	3.14
P4A1b	Escherichia fergusonii	3.11
P4A2	Escherichia fergusonii	2.89
P4A3	Shigella flexneri	2.89
P4A4	Escherichia fergusonii	2.60
P2B5	Pseudomonas lalkuanensis	2.40
P2E1	Christensenella intestinihominis	2.38
P2E2	Christensenella intestinihominis	2.00
P5B1	Staphylococcus caprae	1.86
P5A1	Paenibacillus lupini	1.83
P2C3	Priestia aryabhattai	1.80
P2A1	Priestia aryabhattai	1.57
P5C2	Bacillus velezensis	1.43

^aH/C, the diameter of the hydrolytic zone to that of the colony of a phytase-secreting bacterial strain.

^bThe genome-sequenced strains are bold.

3.3. Genome sequencing and putative extracellular phytase analysis

To investigate the extracellular phytases of the isolated strains, three representative strains from different genera with varying H/C ratios were selected for genome sequencing: Enterobacter strain PZQ1 (H/C ratio: 5.0), Flavobacterium strain P1A3 (H/C ratio: 4.87), and Escherichia strain P4A1 (H/C ratio: 3.11). Genes encoding phytases with predicted signal peptides were identified in the genomes of these strains. These were considered putative extracellular phytases in this study and are listed in Table 3. In strain PZQ1, a single putative extracellular phytase, designated 4182, was identified. This enzyme belongs to the HP2 superfamily and was annotated as a bifunctional glucose-1-phosphatase/inositol phosphatase. It shares 79.18% sequence identity with glucose-1-phosphatase from E. coli strain K12 (P19926) and 61.07% with AgpP from Pantoea sp. 3.5.1 (AIE90144.1) [26,27]. In strain P1A3, two putative extracellular phytases (148 and 634) were identified, both belonging to the HP2 superfamily. Phytase 148 was annotated as a histidine phosphatase and showed 67.32% sequence identity to a 3-phytase from Klebsiella sp. strain ASR1 (AAM23271.1) [28]. Phytase 634 was annotated as a bifunctional glucose-1-phosphatase/inositol phosphatase and showed 87.08% identity to AgpP from Pantoea sp. 3.5.1 (AIE90144.1) [27]. Similarly, two putative extracellular phytases (3898 and 3919) were identified in strain P4A1, both also belonging to the HP2 superfamily. Phytase 3898 was annotated as a bifunctional glucose-1-phosphatase/inositol phosphatase, with 63.99% identity to AgpP from Pantoea sp. 3.5.1 (AIE90144.1) [27]. Phytase 3919 was annotated as a histidine phosphatase, showing 98.61% identity to the acid phosphatase/phytase AppA2 from E. coli (AAR87658.1) [29]. In total, five putative extracellular phytases were identified across the three strains. All belong to the HP2 superfamily, indicating that they are all HAPhys. Sequence alignment with known HAPhys revealed that all five phytases contain the conserved catalytic HD motif and the RHG/NxRxP motif involved in substrate binding (Fig. 3), supporting their classification as HAPhys. Phylogenetic analysis further confirmed that all five enzymes belong to the HP2Ps subclade (Fig. 4).

Table 3.

Information on putative extracellular phytases of the genome-sequenced strains.

Strain	Enzyme	Amino acid number	Annotated protein name	NCBI /UniProt number of similar sequence	Identity (%)	Coverage (%)
PZQ1	4182	413	Bifunctional glucose-1-phosphatase/inositol phosphatase	P19926	79.18	100
P1A3	148	421	histidine phosphatase family protein	AAM23271.1	67.32	97
P1A3	634	545	Bifunctional glucose-1-phosphatase/inositol phosphatase	AIE90144.1	87.08	76
P4A1	3898	413	Bifunctional glucose-1-phosphatase/inositol phosphatase	AIE90144.1	63.99	99
P4A1	3919	442	AppA family phytase/histidine-type acid phosphatase	AAR87658.1	98.61	100

Fig. 3.

Sequence alignment of putative extracellular phytases 4182, 148, 634, 3898, and 3919 with their most similar phytases using the ClustalX 2.1 program. Similar amino acid residues are boxed and shown in red, and conserved amino acid residues are shown with a red background. The substrate-binding associated conserved motif RHG/NxRxP is marked with a black dotted box. The conserved motif HD associated with catalysis is labeled with a solid black box. AAM23271.1 is from Klebsiella sp. strain ASR1, AAR87658.1 from E. coli, AIE90144.1 from Pantoea sp. 3.5.1, and P19926 from E. coli strain K12.

Fig. 4.

Phylogenetic analysis of putative extracellular phytases 4182, 148, 634, 3898, and 3919 with other HAPhys. The phylogenetic tree was constructed by the Neighbor-Joining method with MEGA 7. Bootstrap analysis of 1000 replicates was conducted.

3.4. Biochemical characterization of the extracellular phytases

To characterize the five putative extracellular phytases, their corresponding genes were heterologously expressed in E. coli BL21(DE3). The recombinant proteins, each containing a His-tag, were purified using Ni-NTA affinity chromatography (Fig. 5). As shown in Table 4, all recombinant proteins exhibited detectable activity toward sodium phytate, confirming their identity as functional phytases. Notably, phytase 3919 exhibited exceptionally high specific activity, reaching 2485.25 U/mg. Phytases 4182, 3898, and 148 displayed moderate specific activities ranging from 20.91 to 44.33 U/mg, while phytase 634 showed a very low specific activity of 0.18 U/mg. The optimal temperature for phytase activity was 55 °C for 634, 4182, and 3898; 50 °C for 148; and 60 °C for 3919 (Fig. 6). Among them, phytase 4182 maintained more than 90% of its peak activity at 60 °C, and 55% at 70°C, suggesting it may be the most thermostable of the group. Additionally, all phytases demonstrated clear activity toward sodium phytate at 25 °C (Table 4), indicating they remain functional at environmentally relevant (in situ) temperatures. The optimal pH values for all five enzymes ranged between 4.0 and 5.0, classifying them as acidic phytases (Fig. 7). Among these, phytase 634 exhibited the broadest pH tolerance, retaining more than 80% of its maximum activity across a pH range of 3.0–5.5, suggesting it has the widest pH adaptability.

Fig. 5.

SDS-PAGE analysis of purified recombinant phytases expressed in E. coli BL21 DE3. Lane M, protein molecular mass marker; Lane 1, phytase 4182; Lane 2, phytase 148; Lane 3, phytase 3898; Lane 4, phytase 3919; Lane 5, phytase 634.

Table 4.

Biochemical characteristics of the phytases.

Enzyme	Optimal temperature (°C)	Optimal pH	Specific activity at optimal temperature and pH (U/mg)	Specific activity at 25°C and optimal pH (U/mg)
4182	55	5	20.91 ± 0.27	8.57 ± 0.29
148	50	4.5	44.33 ± 0.99	6.78 ± 0.01
634	55	4	0.18 ± 0.01	0.01± 0.01
3898	55	4.5	23.12 ± 0.07	2.53 ± 0.10
3919	60	5	2485.25 ± 40.17	341.90 ± 5.97

Fig. 6.

Effect of temperature on the activity of the recombinant phytases 4182 (a), 148 (b), 634 (c), 3898 (d), and 3919 (e). The enzyme assay was performed in 0.1 M acetate buffer of pH 5.5. The data are from triplicate experiments (mean ± S.D.).

Fig. 7.

Effect of pH on the activity of the recombinant phytases 4182 (a), 148 (b), 634 (c), 3898 (d), and 3919 (e). The enzyme assay was performed at the optimal temperature of each enzyme. The data are from triplicate experiments (mean ± S.D.).

4. Discussion

Phytases are commonly found in plants, fungi, yeasts, and bacteria, with microbial phytases being the most extensively studied and developed due to their high enzymatic activity, ease of production, and various other advantages [30]. Although most investigated phytase-producing microorganisms and their enzymes have been isolated from terrestrial environments, studies on the diversity of phytase-producing microorganisms in marine habitats remain scarce. Lin et al. analyzed the distribution of BPP genes in seagrass rhizosphere sediments and found these genes were associated with Sphingomonas, Pseudoxanthomonas suwonensis, Armatimonadetes, Deltaproteobacteria, Pseudomonas pachastrellae, Pseudomonas lundensis, Janthinobacterium, Acinetobacter, and others [31]. In our study, the analysis of phytate-utilizing bacteria from coastal seawater and sediment samples collected from Aoshan Bay, Qingdao, China, revealed that Paenibacillaceae, Bacillaceae, and Pseudomonadaceae were predominant in the seawater sample, while Bacillaceae, Enterobacteriaceae, and Microbacteriaceae were most abundant in the sediment sample. Among the isolated strains, those belonging to the genera Enterobacter and Flavobacterium exhibited the highest phytase-secreting potential. These bacteria likely contribute to the hydrolysis of phytic acid derived from coastal algae, seagrasses, and possibly terrestrial plant runoff, releasing inorganic phosphate that can be utilized by marine microorganisms, animals, algae, and seagrasses, thereby supporting marine phosphorus cycling. Thus, our findings highlight key bacterial groups involved in phytic acid degradation and their ecological role in coastal phosphorus cycling.

Although some marine microorganisms have been reported to produce BPPs, a few studies have also identified acidic phytases from marine-derived organisms. For example, Rhodotorula mucilaginosa strain JMUY14, isolated from Antarctic deep-sea sediment, produces a phytase with an optimal pH of 5.0 [20]. Li et al. heterologously expressed a phytase from Kodamaea ohmeri BG3 isolated from the gut of the marine fish Hexagrammos otakii-in E. coli BL21(DE3), which also exhibited an optimal pH of 5.0 [32]. These findings demonstrate that marine microorganisms are capable of producing not only BPPs but also acidic phytases. However, given the limited number of reported acidic phytases from marine microorganisms, further exploration in this area is warranted. In our study, we heterologously expressed and characterized five phytases derived from three bacterial strains isolated from coastal seawater and sediment. All five enzymes were identified as acidic phytases belonging to the HAPhy subclass. Of these, only phytase 3919 shared high sequence identity (98.61%) with a previously reported phytase, whereas the other four exhibited less than 80% sequence identity to known enzymes, suggesting they represent novel phytases. Despite originating from strains isolated from the same or adjacent marine environments, the five phytases displayed varying optimal temperatures from 50 to 60 °C, and pH values (4.0–5.0), which may be attributed to structural differences among the enzymes.

Phytases are widely used in livestock and poultry feed to enhance phosphorus utilization. Currently, most commercial feed phytases are acidic enzymes derived from terrestrial microorganisms, such as PhyA from Aspergillus niger [33,34] and AppA from E. coli [35,36]. Given the diversity of phytases present in marine environments, exploring marine-derived phytases with promising functional properties is of considerable interest. Among the five phytases characterized in this study, phytase 3919 exhibited an exceptionally high specific activity of 2485.25 U/mg, surpassing that of many previously reported phytases, including several industrial or commercial enzymes. For comparison, reported specific activities include PhyK from Klebsiella sp. ASR1 (224 U/mg) [29], APPA from Dickeya paradisiaca (769 U/mg) [37], a phytase from Candida krusei WZ-001(1210 U/mg) [38], the Citrobacter gillenii phytase PhyCg (1577 U/mg) [39], the phytase r-phyC from the Citrobacter freundii (2072 ± 18 U/mg) [40], the first industrial phytase PhyA from Aspergillus niger (100 U/mg) produced by BASF [41,42,44], the Peniophora Lycii phytase PhyA (1080 U/mg) produced by Novozyme and DSM [42,43], and the E. coli phytase AppA (1700 U/mg) produced by Dupont Industrial Biosciences and AB Vista [42]. While a few phytases with higher specific activities have been reported, such as those from Yersinia intermedia (3960 ± 248 U/mg) [44], Citrobacter amalonaticus CGMCC 1696 (AppA, 3548 U/mg) [45], and Citrobacter braakii (3457 U/mg) [46], the high activity of marine-derived phytase 3919 suggests strong potential for its use as a feed additive. Further investigation will be needed to evaluate its performance under industrial conditions.

5. Conclusions

In this study, the diversity of phytate-utilizing bacteria in coastal seawater and sediment samples collected from Aoshan Bay, Qingdao, China, was investigated through enrichment cultivation and 16S rRNA gene amplicon sequencing. A total of 138 OTUs were identified, spanning 10 phyla, 15 classes, 37 orders, 55 families, and 70 genera. In addition, 27 culturable phytase-producing bacterial strains from 12 genera were isolated. Genome sequencing and gene annotation of three representative strains led to the identification of five extracellular phytase genes. Heterologous expression and biochemical characterization of the corresponding enzymes in E. coli revealed that all five are HAPhys with specific activities ranging from 0.18 to 2485.25 U/mg. Their optimal temperatures ranged from 50 to 60 °C, and their optimal pH values were between 4.0 and 5.0. Among these enzymes, phytase 3919 exhibited the highest specific activity (2485.25 U/mg), indicating promising potential for application as a feed additive. Overall, this study reveals the diversity of phytase-producing bacteria and their enzymes from coastal marine environments, contributing to a better understanding of marine phosphorus cycling and providing new candidates for industrial phytase development.

Data Availability Statement

The genome data of strain P1A3, P4A3, and PZQ1 have been submitted to the NCBI Genbank database under the accession numbers JBMUJK000000000.1, JBMUJL000000000.1, and JBKIIK000000000.1. It can be found here: https://www.ncbi.nlm.nih.gov/nuccore//.

CRediT authorship contribution statement

Xiao-Jie Yuan: Writing – original draft, Validation, Methodology, Investigation, Data curation, Conceptualization. Rui Liu: Validation, Investigation, Formal analysis, Conceptualization. Jian Li: Resources. Wen-Xiao Zhao: Methodology. Hui-Hui Fu: Supervision, Funding acquisition. Yan-Rong Zhou: Methodology, Funding acquisition. Mei-Ling Sun: Validation, Formal analysis, Data curation. Xiu-Lan Chen: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization. Yu-Qiang Zhang: Supervision, Conceptualization.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.