Phytase (P1259, Sigma-Aldrich, United States) was dissolved in 0.2 M NaOAc, pH 5.5, to give a concentration of 10 mg/ml. Actual Phytase concentration was quantified using a bicinchoninic acid protein determination kit (BCA1 and B9643, Sigma-Aldrich, United States) and a microplate reader (FLUOstar Omega, BMG Labtech, Germany). The Phytase working solution was prepared by adjusting the actual Phytase concentration to 4 mg/ml using the NaOAc buffer.
Phytase
Manufactured by Merck Group
Sourced in United States
Phytase is a lab equipment product manufactured by Merck Group. It is an enzyme that catalyzes the hydrolysis of phytic acid, which is the principal storage form of phosphorus in many plant tissues. Phytase plays a role in releasing phosphorus from phytic acid, making it available for use in various biological processes.
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5 protocols using phytase
1
Quantification and Preparation of Phytase
2
Inositol Phosphate Hydrolysis Assay
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Neutralised D. discoideum cell extract, or purified inositol phosphate, were incubated in 30 µl enzymatic reactions containing 5XBuffer (100 mM Hepes 6.8; 250 mM NaCl; 30 mM MgSO4; 5 mM DTT; 5 mM NaF), 2 µl of recombinant purified Ddp1 (10-2-ng) or Phytase (Sigma). Reactions were incubated at 37°C for 2 hr or overnight and stopped by the addition of 2 µl EDTA (100 mM). Acidic pyrophosphate hydrolysis was performed by incubating the cell extract at 90°C for 20 min prior to neutralisation. After this treatment the samples were neutralised using Potassium Carbonate as described above.
3
Fermentation of Quinoa with Lactobacillus plantarum
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Quinoa grains of Bolivian origin were purchased from Productos Alimenticios Andes Trópico, a commercial supplier, in Cochabamba, Bolivia, and at ICA Supermarket in Lund, Sweden, in 2017. Each batch was mixed and separated into portions of 500 g, vacuum‐packed, and stored under refrigeration (4°C) and in darkness to avoid mold growth. Lactobacillus plantarum 299v® (ProbiMage, Sweden) was used as a starter culture for the fermentation of quinoa. Wheat phytase (Enzyme Commission number 3.1.3.26, activity ≥ 0.01 unit/mg solid, Sigma‐Aldrich, St. Louis) was used as an exogenous source of phytase.
4
Detailed Biologically-Based Phosphorus Fractions
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Four P fractions, including CaCl2-P, citrate-P, enzyme-P, and HCl-P, were measured using the biologically based P extraction method according to DeLuca et al. (2015) (link). CaCl2 extractable P represents Pi that is easily available to plants, while enzyme extractable P represents available Po that is hydrolyzed by phytase and phosphatase. Citrate extractable P represents potential soluble Pi, which would be accessible to plants when soil organic acids are released into the soil. In addition, HCl extractable P represents recalcitrant Pi, which can be solubilized by proton excretion released by plant and microbes. Each of the P fractions was measured by shaking 0.5 g of fresh soil with 10 ml of extractant (10 mM CaCl2 for CaCl2-P, 0.2 U enzymes for enzyme-P, 10 mM citric acid for citrate-P, and 1 M HCl for HCl-P) in separate 15-ml centrifuge tubes on a reciprocal shaker at 200 rpm for 3 h. Extracts were centrifuged at 3000 × g for 5 min, and then all of the supernatant was determined by the malachite green method at 630 nm (Ohno and Zibilske, 1991 (link)) using a PowerWave-XS microplate spectrophotometer (Infinite M200 PRO, Switzerland). The enzyme extractant consisted of three enzymes: 0.5 U acid phosphomonoesterase (Sigma P3627), 0.5 U alkaline phosphomonoesterase (Sigma P5931), and 0.1 U phytase (Sigma P5931).
5
Transgenic Barley Cultivation for Poultry Feed
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Barley seed (cultivar Yangmai 3) was obtained from the plant genetic engineering laboratory of Shanghai Academy of Agricultural Sciences. The transgenic seedlings were transplanted to a paddy field in Baihe county, Qingpu district, Shanghai, China. Standard enzymes (phytase, β‐glucanase, and xylanase) and sodium phytate were purchased from Sigma Chemical Co., Ltd (St. Louis, MO, USA). All other chemicals were analytical grade and were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All primers were purchased from Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. (Shanghai, China). Agrobacterium tumefaciens strain EHA105 and Escherichia coli DH5α were obtained from the plant genetic engineering laboratory of Shanghai Academy of Agricultural Sciences. An RNA isolation kit was purchased from Fermentas International, Inc. (Burlington, ON, Canada), and a reverse Transcription System was purchased from Promega (Madison, WI). Twenty‐four 4‐week‐old black‐feathered laying hens (Xingyang) were obtained from the National Poultry Engineering Research Center of the Ministry of Agriculture (Shanghai, China).
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