Characterisation of a soil MINPP phytase with remarkable long-term stability and activity from Acinetobacter sp.

Phylogenetic analysis, homology modelling and biochemical methods have been employed to characterize a phytase from a Gram-negative soil bacterium. Acinetobacter sp. AC1-2 phytase belongs to clade 2 of the histidine (acid) phytases, to the Multiple Inositol Polyphosphate Phosphatase (MINPP) subclass. The enzyme was extraordinarily stable in solution both at room temperature and 4°C, retaining near 100% activity over 755 days. It showed a broad pH activity profile from 2–8.5 with maxima at 3, 4.5–5 and 6. The enzyme showed Michaelis-Menten kinetics and substrate inhibition (Vmax, Km, and Ki, 228 U/mg, 0.65 mM and 2.23 mM, respectively). Homology modelling using the crystal structure of a homologous MINPP from a human gut commensal bacterium indicated the presence of a potentially stabilising polypeptide loop (a U-loop) straddling the active site. By employ of the enantiospecificity of Arabidopsis inositol tris/tetrakisphosphate kinase 1 for inositol pentakisphosphates, we show AC1-2 MINPP to possess D6-phytase activity, which allowed modelling of active site specificity pockets for InsP6 substrate. While phytase gene transcription was unaltered in rich media, it was repressed in minimal media with phytic acid and orthophosphate as phosphate sources. The results of this study reveal AC1-2 MINPP to possess desirable attributes relevant to biotechnological use.


Methods including
(cloning primers used) and Table S2  Genomic DNA extraction -Genomic DNA was isolated from a cell suspension grown from a single colony in LB. The cells were lysed with lysozyme. DNA was extracted with phenol-choroform, washed with 80% ethanol, air-dried and recovered in dH 2 O.
Gateway Cloning -Primer sets 1/3 were used to clone the gene without the signal peptide, with added attB sites and 3C protease cleavage site, from genomic DNA. The attB-PCR fragment was cloned into pDONR207™ with BP Clonase™ (Invitrogen) to generate the entry clone, BP reaction. This reaction used 2 μL TE buffer, 1 μL BP clonase, 1 μL Template DNA and 1 μL pDONR 207 and was incubated at 25 °C for 3hr 30mins. After which, the BP reaction mixture was incubated with 0.5 μL Proteinase K for 10 mins at 37 °C and then transformed into E. coli DH5α cells with the appropriate antibiotics. REV: 5ʹ-CAAGAAAGCTGGGTTTAA…TTGTATTTGATAGCACTGTTTC-3ʹ The cloned gene was transferred into the entry vector, pDONR207 using BP clonase to create the Gateway entry clone. This entry clone was transferred into the destination vector pDEST17, which contains a 6X His-tag, using LR clonase and transformed into the expression host Rosetta 2 pLysS.
Initial cloning with Primer Sets 1 and 2 (Table S1) yielded, on subsequent expression, mostly insoluble protein with low activity. Therefore, Primer Set 3 was designed to remove the first 19 amino acids corresponding to the signal peptide, SignalP 5.0 (Almagro Armenteros et al., 2019). Once purified, the resulting SDS PAGE band was sent for sequencing using Protein Mass Fingerprinting at the John Innes Centre (Norwich, UK) confirming the expression of the AC1-2 protein.
Successful transformations were confirmed using colony PCR and sequenced using internal attL primers, FOR 5'GCAGTTCCCTACTCTCGC 3' and REV 5' CATCAGAGATTTTGAGACAC 3' and a gene internal primer 5ʹ-TGA TTT AGA AGC AAT GAT G-3. The DH5α cells were grown overnight, and plasmid DNA obtained using the QIAprep Spin Miniprep Kit. The LR reaction was then used to create the expression clone, pDON7207 using LR Clonase™ (Invitrogen). This reaction used 2 μL TE buffer, 1 μL LR clonase, 1 μL pDONR 207 and 1 μL pDEST17 and was incubated at 25 °C for 3hr30mins. The LR reaction mix was then transformed into E. coli DH5α cells. Successful transformation was confirmed using colony PCR and the plasmid DNA was transformed again into the expression host Rosetta 2 pLysS (Novagen).
Transformation -DH5α cells were transformed by standard heat shock protocol.
Protein purification -Single colonies from the Rosetta 2 pLysS AC1-2 clone were inoculated into 100 mL LB, shaken overnight at 180 RPM and 37 °C, then used to inoculate 4X500 mL LB. At OD600 > 0.5, IPTG was added to 0.1 mM and the cultures incubated at 16 °C overnight. Cells were centrifuged at 4500 RPM at 20 °C using a Beckman JLA 8.1 rotor in a J-20 centrifuge, resuspended in 25 mL binding buffer containing cOmplete™ Protease Inhibitor Cocktail (LaRoche), Dnase, lysozyme and 50 μL EDTA (0.5 M pH 8). Cells were lysed by French Press, repeated 3 times, at 1000 psi, centrifuged for 45 minutes at 19,000 RPM at 4 °C in a Beckman JA-25.50 rotor in a J-20 centrifuge. The supernatant was passed through a 1 mL Histrap HP column on an ÄKTA pure protein purification. UV-absorbing fractions were pooled and concentrated to 1.5 mL using an Amicon® Ultra-15 centrifugal filter unit (30 kDA) cutoff). The concentrated protein was further purified on a HiLoad 16/600 Superdex 75 PG column. Fractions, selected by SDS PAGE, were pooled and concentrated to 1 mL (30 kDA cutoff). Protein was stored in 4 μM aliquots in different cryoprotectants: final concentration 25% (w/v) trehalose, 25% (w/v) trehalose and 1 mg/mL BSA, 25% (w/v) sucrose, 25% (w/v) sucrose and 1 mg/mL BSA, 25% (w/v) glycerol, 25% (w/v) glycerol and 1 mg/mL BSA, 1 mg/mL BSA, or gel filtration buffer in a 1:1 mixture. The protein was stored at -20 °C and thawed when necessary.
qPCR Primer Design -Primer efficiencies were calculated using a two-step qPCR protocol described below from a dilution series of cDNA