Simple and Rapid Determination of Phytase Activity

Engelen, Adrianus J; Heeft, Fred C van der; Randsdorp, Peter H G; Smit, E.L.C.

doi:10.1093/jaoac/77.3.760

Cited by 412 publications

(199 citation statements)

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“…Cell dry weight and enzyme activities were determined. Phytase activity was determined following the method described in Engelen et al (1994). One phytase activity unit was defined as the amount of enzyme that releases 1 mmol inorganic phosphate min À1 .…”

Section: Enzyme Characterizationmentioning

confidence: 99%

Coexpression of fungal phytase and xylanase utilizing the cis-acting hydrolase element in Pichia pastoris

Roongsawang

Promdonkoy

Wongwanichpokhin

et al. 2010

FEMS Yeast Research

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Plant-based animal feed contains antinutritive agents, necessitating the addition of digestive enzymes in commercial feeds. Enzyme additives are costly because they are currently produced separately from recombinant sources. The coexpression of digestive enzymes in a single recombinant cell system would thus be advantageous. A coexpression system for the extracellular production of phytase and xylanase was established in Pichia pastoris yeast. The genes for each enzyme were fused in-frame with the α-factor secretion signal and linked by the 2A-peptide-encoding sequence. Each enzyme was expressed extracellularly as individual functional proteins. The specific activities of 2A-expressed phytase (PhyA-2A) and 2A-expressed xylanase (XylB-2A) were 9.3 and 97.3 U mg(-1) , respectively. Optimal PhyA-2A activity was observed at 55 degreesC and pH 5.0. PhyA-2A also exhibited broad pH stability from 2.5 to 7.0 and retained approximately 70% activity after heating at 90 degreesC for 5 min. Meanwhile, XylB-2A exhibited optimal activity at 50 degreesC and pH 5.5 and showed pH stability from 5.0 to 8.0. It retained >50% activity after incubation at 50 degreesC for 10 min. These enzyme properties are similar to those of individually expressed recombinant enzymes. In vitro digestibility test showed that PhyA-2A and XylB-2A are as efficient as individually expressed enzymes for hydrolyzing phytate and crude fiber in feedstuff, respectively.

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Section: Enzyme Characterizationmentioning

confidence: 99%

Coexpression of fungal phytase and xylanase utilizing the cis-acting hydrolase element in Pichia pastoris

Roongsawang

Promdonkoy

Wongwanichpokhin

et al. 2010

FEMS Yeast Research

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“…Each point represents the average of duplicate determinations. Phytase activity was measured using the reaction mixture speci®ed inEngelen et al (1994) at 37°C. Relative activity of phytase at various temperatures was measured at pH 5.30.…”

mentioning

confidence: 99%

Dietary microbial phytase supplementation and the utilization of phosphorus, trace minerals and protein by rainbow trout [Oncorhynchus mykiss (Walbaum)] fed soybean meal-based diets

Sugiura

Gabaudan

Dong

et al. 2001

Aquaculture Research

184

158

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Effects of thermal and enzymatic treatments of soybean meal on apparent absorption of total phosphorus, phytate phosphorus, nitrogen (protein), ash, calcium, magnesium, copper, iron, manganese, strontium and zinc were examined using rainbow trout, Oncorhynchus mykiss (Walbaum), as the test species. Absorption of the test nutrients was estimated using yttrium as an inert non‐absorbable indicator. Thermal treatments (microwaving, dry roasting, steam heating, cooking) had no measurable effect on the apparent absorption of phosphorus and other minerals. Phytase supplementation increased the apparent absorption of phosphorus, nitrogen (protein), ash, calcium, magnesium, copper, iron, strontium and zinc in low‐ash diets containing soybean meal, but had little effect in high‐ash diets containing both soybean and fish meal. In low‐ash diets, the apparent absorption of phosphorus increased in accord with the level of phytase added to the diet, from 27% (no phytase added) up to 90% (phytase added, 4000 units kg−1 diet) or 93% (predigested with phytase, 200 units kg−1 soybean meal). In high‐ash diets, dietary acidification with citric acid decreased the effect of phytase, whereas in low‐ash diets, acidification markedly increased the effect of the enzyme. Excretion of phosphorus in the faeces of fish fed a low‐ash diet containing phytase‐treated soybean meal was 0.32 g per kg diet consumed, a 95%−98% reduction compared with phosphorus excretion by fish consuming commercial trout feeds.

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“…Prior to analysis, the tissues were milled to fine powder in an IKA TUBE Mill. Protein extraction, assaying and incubation for 1 h were performed as described already (Brinch-Pedersen et al, 2000;Engelen et al, 1994). Soluble inorganic phosphate (Pi) present in the extract already before phytase assaying was quantified by adding molybdovanadate reagent to aliquots of the soluble protein extracts before incubation.…”

Section: Analysis Of Phytase Activitymentioning

confidence: 99%

Barley HvPAPhy_a as transgene provides high and stable phytase activities in mature barley straw and in grains

Holme

Dionisio

Madsen

et al. 2016

Plant Biotechnology Journal

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SummaryThe phytase purple acid phosphatase (HvPAPhy_a) expressed during barley seed development was evaluated as transgene for overexpression in barley. The phytase was expressed constitutively driven by the cauliflower mosaic virus 35S‐promoter, and the phytase activity was measured in the mature grains, the green leaves and in the dry mature vegetative plant parts left after harvest of the grains. The T2‐generation of HvPAPhy_a transformed barley showed phytase activity increases up to 19‐fold (29 000 phytase units (FTU) per kg in mature grains). Moreover, also in green leaves and mature dry straw, phytase activities were increased significantly by 110‐fold (52 000 FTU/kg) and 57‐fold (51 000 FTU/kg), respectively. The HvPAPhy_a‐transformed barley plants with high phytase activities possess triple potential utilities for the improvement of phosphate bioavailability. First of all, the utilization of the mature grains as feed to increase the release of bio‐available phosphate and minerals bound to the phytate of the grains; secondly, the utilization of the powdered straw either directly or phytase extracted hereof as a supplement to high phytate feed or food; and finally, the use of the stubble to be ploughed into the soil for mobilizing phytate‐bound phosphate for plant growth.

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Simple and Rapid Determination of Phytase Activity

Abstract: A simple and rapid method is described for determining the enzymatic activity of microbial phytase. The method is based on the determination of inorganic orthophosphate released on hydrolysis of sodium phytate at pH 5.5.

Cited by 412 publications

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Coexpression of fungal phytase and xylanase utilizing the cis-acting hydrolase element in Pichia pastoris

Coexpression of fungal phytase and xylanase utilizing the cis-acting hydrolase element in Pichia pastoris

Dietary microbial phytase supplementation and the utilization of phosphorus, trace minerals and protein by rainbow trout [Oncorhynchus mykiss (Walbaum)] fed soybean meal-based diets

Barley HvPAPhy_a as transgene provides high and stable phytase activities in mature barley straw and in grains

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