Gene Cloning, Expression And Characterization Of Phytases From Bacteria Harbored In The Gut Of Batocera Horsfieldi Larvae

Phytase (EC 3.1.3.8 or EC 3.1.3.26) is a group of enzymes that initiate the stepwise hydrolysis of phytic acid to generate inorganic orthophosphate, lower myo-inositol phosphoric esters, and free myo-inositol. Recently, dietary phytase, as a novel animal feed additive and as a replacement for inorganic phosphorus, has been proved to be efficient in the improvement of phytate-phosphorus utilization and bioavailability of minerals to swine and poultry. In aquaculture, neutral phytases with appropriate properties are being developed. Insect digestive tract harbors abundant and diverse microorganisms. To our knowledge, no phytase has been identified in these symbiotes. This study aims to obtain novel phytases with application potentials from the microbes of neutral insect digestive tract.Twenty bacterial stains of various taxa isolated from the gut of Batocera horsfieldi were selected as microbial sources. By using degenerate PCR and TAIL-PCR techniques, four phytase-encoding genes were cloned from three strains, including phyA06 from Pseudomonas sp. TN06, phyH49 and phyB49 from Serratia sp. TN49 and phyA115 from Janthinobacterium sp. TN115. Of them, phyH49 encodes a histidine acid phosphatase (HAP), and phyA06, phyB49 and phyA115 encode threeβ-propeller phytases (BPPs). Sequence homology analysis revealed that the deduced amino acid sequences of these four phytases had highest identities of 47–64% with known proteins in the public databases and 39–51% identities among three BPPs, suggesting that these phytase genes from insect gut have high sequence novelty. Homology modeling indicated that all three BPPs have dual-domain structures and are distinct from most single-domain well-characterized BPPs. Serratia is a typical genus of Enterobacteriaceae. It is the first report of a BPP gene in Enterobacteriaceae and of more than one type of phytase in a single strain. Further phylogenetic analysis revealed that deduced PhyB49 is closely related with the dual-domain phytases of Pseudomonas spp. accreted with plant. Four phytase-encoding genes were recombined with pET-22b(+) and transformed into Escherichia coli BL21 (DE3) for expression, respectively. The recombinant proteins were purified to electrophoretic homogeneity by Ni2+-NTA metal chelating affinity chromatography and further characterized.PhyA06 from Pseudomonas sp. TN06 exhibited maximal activity at pH 7.0 and 55°C in the presence of 2 mM Ca²⁺ and retained > 50% activity at 30°C. PhyA06 was thermostable, and retained 70% of the initial activity after incubation at 65°C for 1 h. PhyA06 was highly resistant to trypsin, retaining almost all of the activity after 1-h treatment. These properties make PhyA06 favorable for potential application in aquaculture.PhyH49 from Serratia sp. TN49 had pH and temperature optima of 5.0 and 60oC, respectively. PhyB49 showed optimal activity at pH 7.5 and 45oC in the presence of 1 mM Ca²⁺. PhyH49 and PhyB49 retained about 30 and 60% of the maximal activity at 30°C, respectively. The presence of two types of phytases makes Serratia sp. TN49 more efficient in phytate-phosphorus utilization over a board pH range (2.0–9.0) and more adaptive to various environments. Furthermore, symbiotes harboring neutralβ-propeller phytase correspond to their adaptation to environment changes and evolution trend. The optimal activity of PhyA115 from Janthinobacterium sp. TN115 was found to occur at pH 8.5 and 45°C in the presence of 1 mM Ca²⁺. PhyA115 activity was found to be related with concentrations of Ca²⁺ and substrate phytate. When the concentrations of Ca²⁺ and phytate were similar, PhyA115 exhibited the maximal phytase activity. When the phytate concentration was low (0.1 mM) and Ca²⁺ was absent, PhyA115 had high phytase activity. Structure-function analysis revealed that the N-terminal incomplete domain has no phytase activity but can influence the pH optimum of PhyA115. This is the first time to report such a phenomenon. PhyA115 is not only an important material for basic research ofβ-propeller phytase but also a potential feed additive in aquaculture.It is the first study to explore the phytases from bacteria harbored in the gut of B. horsfieldi larvae. In this special environment, dual-domain BPP phytases are predominant phytate-degraders. These BPP phytases share low identities with known proteins and have some novel enzyme properties, and may represent good materials for basic research and industrial applications in future.