Surface Engineering of Escherichia coli to Display Its Phytase (AppA) and Functional Analysis of Enzyme Activities.

Escherichia coli phytase (AppA) is widely used as an exogenous enzyme in monogastric animal feed mainly because of its ability to degrade phytic acid or its salt (phytate), a natural source of phosphorus. Currently, successful recombinant production of soluble AppA has been achieved by gene overexpression using both bacterial and yeast systems. However, some methods for the biomembrane immobilization of phytases (including AppA), such as surface display on yeast cells and bacterial spores, have been investigated to avoid expensive enzyme purification processes. This study explored a homologous protein production approach for displaying AppA on the cell surface of E. coli by engineering its outer membrane (OM) for extracellular expression. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of total bacterial lysates and immunofluorescence microscopy of non-permeabilized cells revealed protein expression, whereas activity assays using whole cells or OM fractions indicated functional enzyme display, as evidenced by consistent hydrolytic rates on typical substrates (i.e., p-nitrophenyl phosphate and phytic acid). Furthermore, the in vitro results obtained using a simple method to simulate the gastrointestinal tract of poultry suggest that the whole-cell biocatalyst has potential as a feed additive. Overall, our findings support the notion that biomembrane-immobilized enzymes are reliable for the hydrolysis of poorly digestible substrates relevant to animal nutrition.

Characterization of recombinant phytase of Klebsiella sp. and the influence of novel 3-phytase on mineral solubility in broiler diets under an in vitro digestion assay.

Phytate (inositol hexaphosphate) is the major storage form of phosphorus (P) in nature, and phytases catalyze the hydrolysis of P from phytate and the formation of inositol phosphate isomers. In this study, a bacterium that produces phytase was isolated in a phytase screening medium. The bacterium was identified as Klebsiella sp. using phenotypic and molecular techniques. The PhyK phytase gene was successfully amplified from the genome, inserted into the pET-21a (+) vector, and expressed as a recombinant protein in E. Coli BL21. The efficiency of a laboratory phytase (Lab-Ph, PhyK phytase) was determined and compared with a commercial phytase (Com-Ph, Quantum Blue 40P phytase, AB Vista) under an in vitro digestion assay. The native signal peptide effectively facilitated the translocation of the protein to the periplasmic space of E. Coli BL21, resulting in the proper folding of the protein and the manifestation of desirable enzyme activity. The Lab-Ph displayed the temperature and pH optima at 50 °C and 5 respectively. In addition, the Lab-Ph was inactivated at 80 °C. Under an in vitro digestion assay condition, Lab-Ph improved the P solubility coefficient in broiler diets. In comparison, the Com-Ph significantly increased the P solubility coefficient even when compared with the Lab-Ph. In summary, this study has shown that Lab-Ph possesses the necessary biochemical properties to be used in various industrial applications. However, Lab-Ph is extremely sensitive to heat treatment. The Lab-Ph and Com-Ph under an in vitro digestion assay improved the solubility coefficient of P in the broiler diet.

Characterization of a putative metal-dependent PTP-like phosphatase from Lactobacillus helveticus 2126.

To date, there are very limited reports on sequence analysis and structure-based molecular modeling of phosphatases produced by probiotic bacteria. Therefore, a novel protein tyrosine-like phosphatase was characterized from L. helveticus 2126 in this study. The purified bacterial phosphatase was subjected to mass spectrometric analysis, and the identity of constructed sequence was analyzed using peptide mass fingerprint. The 3-D structure of protein was elucidated using homology modeling, while its stability was assessed using Ramachandran plot, VERIFY 3D, and PROCHECK. The bacterium produced an extracellular phosphatase of zone diameter 15 ± 0.8 mm on screening medium within 24 h of incubation. This bacterial phosphatase was highly specific towards sodium phytate as it yielded the lowest Km value of 299.50 ± 4.95 µM compared to other phosphorylated substrates. The activity was effectively stimulated in the presence of zinc, magnesium, and manganese ions thereby showing its PTP-like behavior. The phosphatase showed a molecular mass of 43 kDa, and the corresponding M/Z ratio data yielded 46% query coverage to Bacillus subtilis (3QY7). This showed a 61.1% sequence similarity to Ligilactobacillus ruminis (WP_046923835.1). The final sequence construct based on these bacteria showed a conserved motif "HCHILPGIDD" in their active site. In addition, homology modeling showed a distorted Tim barrel structure with a trinuclear metal center. The final model after energy minimization showed 90.9% of the residues in the favorable region of Ramachandran's plot. This structural information can be used in genetic engineering for improving the overall stability and catalytic efficiency of probiotic bacterial phosphatases.

CO2-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16.

BACKGROUND: Existing plasmid systems offer a fundamental foundation for gene expression in Cupriavidus necator; however, their applicability is constrained by the limitations of conjugation. Low segregational stabilities and plasmid copy numbers, particularly in the absence of selection pressure, pose challenges. Phytases, recognized for their widespread application as supplements in animal feed to enhance phosphate availability, present an intriguing prospect for heterologous production in C. necator. The establishment of stable, high-copy number plasmid that can be electroporated would support the utilization of C. necator for the production of single-cell protein from CO2. RESULTS: In this study, we introduce a novel class of expression plasmids specifically designed for electroporation. These plasmids contain partitioning systems to boost segregation stability, eliminating the need for selection pressure. As a proof of concept, we successfully produced Escherichia coli derived AppA phytase in C. necator H16 PHB- 4 using these improved plasmids. Expression was directed by seven distinct promoters, encompassing the constitutive j5 promoter, hydrogenase promoters, and those governing the Calvin-Benson-Bassham cycle. The phytase activities observed in recombinant C. necator H16 strains ranged from 2 to 50 U/mg of total protein, contingent upon the choice of promoter and the mode of cell cultivation - heterotrophic or autotrophic. Further, an upscaling experiment conducted in a 1 l fed-batch gas fermentation system resulted in the attainment of the theoretical biomass. Phytase activity reached levels of up to 22 U/ml. CONCLUSION: The new expression system presented in this study offers a highly efficient platform for protein production and a wide array of synthetic biology applications. It incorporates robust promoters that exhibit either constitutive activity or can be selectively activated when cells transition from heterotrophic to autotrophic growth. This versatility makes it a powerful tool for tailored gene expression. Moreover, the potential to generate active phytases within C. necator H16 holds promising implications for the valorization of CO2 in the feed industry.

Harnessing alkaline-pH regulatable promoters for efficient methanol-free expression of enzymes of industrial interest in Komagataella Phaffii.

BACKGROUND: The yeast Komagataella phaffii has become a very popular host for heterologous protein expression, very often based on the use of the AOX1 promoter, which becomes activated when cells are grown with methanol as a carbon source. However, the use of methanol in industrial settings is not devoid of problems, and therefore, the search for alternative expression methods has become a priority in the last few years. RESULTS: We recently reported that moderate alkalinization of the medium triggers a fast and wide transcriptional response in K. phaffii. Here, we present the utilization of three alkaline pH-responsive promoters (pTSA1, pHSP12 and pPHO89) to drive the expression of a secreted phytase enzyme by simply shifting the pH of the medium to 8.0. These promoters offer a wide range of strengths, and the production of phytase could be modulated by adjusting the pH to specific values. The TSA1 and PHO89 promoters offered exquisite regulation, with virtually no enzyme production at acidic pH, while limitation of Pi in the medium further potentiated alkaline pH-driven phytase expression from the PHO89 promoter. An evolved strain based on this promoter was able to produce twice as much phytase as the reference pAOX1-based strain. Functional mapping of the TSA1 and HSP12 promoters suggests that both contain at least two alkaline pH-sensitive regulatory regions. CONCLUSIONS: Our work shows that the use of alkaline pH-regulatable promoters could be a useful alternative to methanol-based expression systems, offering advantages in terms of simplicity, safety and economy.

Dietary inclusion of phytase and stimbiotic decreases mortality and lameness in a wire ramp challenge model in broilers.

RESEARCH HIGHLIGHTS: Wire ramp model reproducibly induced lameness/BCO in broilers.Treatments did not affect growth, but phytase with stimbiotic significantly reduced BCO.Phytase increased circulating inositol, and wire flooring decreased bone inositol.

Statistical optimized production of Phytase from Hanseniaspora guilliermondii S1 and studies on purification, homology modelling and growth promotion effect.

This investigation was performed to obtain a promising phytase enzyme producing yeast. In this regard, the PSM was used to isolate the phytase-producing Hanseniaspora guilliermondii S1 (MG663578) from sugarcane juice. The SSF optimum conditions for phytase generation were optimized using (OVAT) one-variable-at-a-time strategy using both Box-Behnken design and shake flask method (g/100 ml: 0.05 yeast extract, 0.15 Peptone, 0.05 malt extract 0.50 dextrose, pH 5.8 and 28ᵒC). The protein model developed was shown to be adequate for phytase production (91% accuracy), with the greatest phytase productivity in shake flask with substrate jack fruit seed powder being 395 ± 0.43 U/ml compared to 365U/ml for the BBD projected value. Crude Phytase was partially purified with a protein recovery of 43%, revealing a molecular weight of 120 kDa. It had an enzyme kinetic value of Km 3.3 mM and a Vmax of 19.1 mol/min. The 3D structure of PhyS1 amino acid sequences (PhyS1. B99990002) was simulated using Modeler 9.23, and the validated result revealed that 86.7% were in the favored region by Ramachandran plot. The SAVES server verified the 3D PDB file as satisfactory, and the model (in.pdb format) was uploaded in the PMDB database with the accession number ID: PM0082974. At the lab level, Hanseniaspora guilliermondii S1 (MG663578) producing phytase exhibited successful plant growth promotion activity in Ragi - CO 19 (Eleusine coracana L.) and Rice -Navarai - IR 64 (Oryza sativa L.). As a result, a phytase-based formulation for sustainable agriculture must be developed and tested on a large scale in diverse geographical areas of agricultural lands to determine its effect and potential on plant development.

Cyclic extraction of phosphate from soybean meal using immobilized Aspergillus oryzae SBS50 phytase.

Phytase enzyme found in plants, animals, and microorganisms is mainly involved in catalyzing the systematic removal of a phosphate group from phytic acid. Enzyme immobilization is one of the cost-effective methods for the wide usage of enzymes in the industrial sector. This paper reports the covalent immobilization of phytase on glutaraldehyde-activated aluminum oxide beads. The immobilization yield, efficiency, and activation energy were found to be 47.8%, 71.5%, and 15.78 J/mol, respectively. The bound enzyme displayed a shift in pH optima from 5.5 to 4.5, which is more beneficial to increase digestibility in comparison with the free enzyme. Immobilized phytase retained 42.60% of its activity after 1.0 h incubation at 80 °C, whereas free enzyme retained only 4.20% of its activity. Thermodynami increase in half-lives, D-values, enthalpy and free energy change after covalent immobilization could be credited to the enhanced stability. Immobilized phytase could be reused for five consecutive cycles retaining 51% of its initial activity with sodium phytate. The immobilized phytase was also found effective to hydrolyze the soybean meal, thus increasing the digestibility of poultry feed. The hydrolyzing reaction of soybean meal was carried out for six consecutive cycles and immobilized phytase retained nearly 50% of activity till the fifth cycle. The amount of phosphorus released after treatment with immobilized phytase was far higher than that from free phytase. Immobilization on this support is significant, as this support can sustain high mechanical resistance at high pH and temperature. This considerable stability and reusability of the bound enzyme may be advantageous for its industrial application.

Optimization of phytase production by Penicillium oxalicum in solid-state fermentation for potential as a feed additive.

The study aims to statistically optimize the phytase production by Penicillium oxalicum PBG30 in solid-state fermentation using wheat bran as substrate. Variables viz. pH, incubation days, MgSO4, and Tween-80 were the significant parameters identified through the Plackett-Burman design (PBD) that majorly influenced the phytase production. Further, central composite design (CCD) method of response surface methodology (RSM) defined the optimum values for these factors i.e., pH 7.0, 5 days of incubation, 0.75% of MgSO4, and 3.5% of Tween-80 that leads to maximum phytase production of 475.42 U/g DMR. Phytase production was also sustainable in flasks and trays of different sizes with phytase levels ranging from 394.95 to 475.42 U/g DMR. Enhancement in phytase production is 5.6-fold as compared to unoptimized conditions. The in-vitro dephytinization of feed showed an amelioration in the nutritive value by releasing inorganic phosphate and other nutrients in a time-dependent manner. The highest amount of inorganic phosphate (33.986 mg/g feed), reducing sugar (134.4 mg/g feed), and soluble protein (115.52 mg/g feed) was achieved at 37 °C with 200 U of phytase in 0.5 g feed for 48 h. This study reports the economical and large-scale production of phytase with applicability in enhancing feed nutrition.

Bone characteristics, pre-caecal phytate degradation, mineral digestibility and tissue expression were marginally affected by zinc level and source in phytase-supplemented diets in 21-day-old broiler chickens.

1. This study determined the effect of dietary Zn concentration and source in phytase-supplemented diets on bone mineralisation, gastrointestinal phytate breakdown, mRNA-level gene expression (in jejunum, liver and Pectoralis major muscle) and growth performance in broiler chickens.2. Male Cobb 500 broilers were housed in floor pens (d 0-d 21) to test seven treatments with six replicate pens (12 birds per pen). Diets were arranged in a 2 × 3 + 1-factorial arrangement. The experimental factors were Zn source (Zn-oxide (ZnO) or Zn-glycinate (ZnGly) and Zn supplementation level (10, 30 or 50 mg/kg of diet). A maize-soybean meal-based diet without supplementation and formulated to contain 28 mg Zn/kg (analysed to be 35 mg Zn/kg), served as a control.3. Zinc source and level did not influence (p > 0.05) bone ash concentration and quantity or mineral concentrations in bone ash. Tibia thickness was greater in the treatment ZnO10 than in the treatments ZnO30 and ZnGly50 (Zn level × Zn source: p = 0.036), but width and breaking strength were not affected.4. Pre-caecal P digestibility and concentrations of phytate breakdown products in the ileum, except for InsP5, were not affected by Zn source or level. Only the expression of EIF4EBP1 (eukaryotic translation initiation factor 4E-binding protein 1) and FBXO32 (F-box only protein 32) in Pectoralis major muscle was affected by source, where expression was increased in ZnO compared to ZnGly diets (p < 0.05).5. In conclusion, Zn level and source did not affect gastrointestinal phytate degradation and bone mineralisation in phytase-supplemented diets. The intrinsic Zn concentration appeared to be sufficient for maximum bone Zn deposition under the conditions of the present study but requires validation in longer-term trials.

Supplemental phosphorus can be completely replaced with microbial phytase in White Leghorn layer diets.

1. A study was conducted to assess the possibility of totally replacing supplemental phosphorus sources in White Leghorn (WL) layer diets (aged 28 to 45 weeks of age) with microbial phytase supplementation. One thousand commercial layers (HyLine White) of 28 weeks of age were housed in California cages fitted in open-sided poultry shed at the rate of 20 layers in each replicate. Ten replicates were randomly allotted to each treatment, and the respective diet was fed from 28 to 45 weeks of age.2. A control diet (CD) containing the recommended levels of non-phytate phosphorus (3.6 g/kg NPP) and four other test diets (2-5) having sub-optimal levels of NPP (2.4, 2.0, 1.6 and 1.2 g/kg), but with supplemental microbial phytase (600 FTU/kg) were prepared and fed for the trial duration.3. The layers fed with lower levels of NPP with phytase had the same laying performance as the group fed the CD. Egg production, feed efficiency, egg mass, shell defects, egg density, shell weight, shell thickness, ash content and breaking strength of the tibia and sternum were not affected by feeding the lowest concentration of NPP (1.2 g/kg) plus microbial phytase.4. Phytase supplementation in diets with sub-optimal levels of NPP (2.4, 2 and 1.6 g/kg) significantly improved the Haugh unit score compared to those fed the CD.5. It was concluded that supplemental phosphorus can be completely replaced with microbial phytase (600 FTU/kg) in a diet without affecting egg production, shell quality or bone mineral variables in WL layers (28 to 45 weeks).

Effects of graded levels of dietary microbial 6-phytase on performance, intestinal histomorphology, caecal microbial population and short-chain fatty acid composition of Lohmann white-classics.

1. This study was conducted to determine the effects of graded levels of phytase on the performance, egg quality and gut health of white laying hens.2. Treatments consisted of a negative control (NC) diet containing 0.14% available phosphorus (avP), positive control (PC) diet containing 0.35% avP provided via dicalcium phosphate (DCP) and DCP replaced in the PC by with three graded levels of phytase derived from Komagataella phaffii at 500 (PC-500), 750 (PC-750) and 1000 (PC-1000) FTU/kg which provided 0.176%, 0.188% and 0.200% of avP, respectively.3. Egg production, feed intake, feed conversion ratio and jejunal morphometry were negatively affected in NC-fed birds (p < 0.05). Considering the whole period, birds fed a diet supplemented with graded levels of phytase shared the same egg production and feed intake levels with PC birds (p < 0.05). Feed conversion ratio was significantly lowered by 4.9%, 1.6% and 7.6% in hens fed on diets PC-500, PC-750 and PC-1000, respectively compared to those fed the PC (p < 0.05).4. Neither of the dietary treatments affected cracked eggs, dirty eggs, eggshell breaking strength and eggshell thickness. Dietary supplementation of phytase significantly increased villus surface area by 15%, 36% and 40% in PC-500, PC-750 and PC-1000 birds, respectively compared to PC (p < 0.05).5. A significant increase in lactobacillus count was observed in line with increasing the level of phytase (p < 0.05). Dietary treatments had no effect on the caecal coliform or aerobic populations. Furthermore, phytase supplementation significantly increased the concentrations of total caecal short-chain fatty acid (SCFA; p < 0.01).6. In conclusion, along with improving performance parameters, the inclusion of phytase in laying hen diets can ameliorate intestinal morphology and stimulate caecal microflora and increase SCFA concentrations.

Effects of phytase enzyme supplementation on growth performance, intestinal morphology and metabolism in Nile tilapia (Oreochromis niloticus).

Phytase is crucial in enhancing the bioavailability and release of phosphorus and other nutrients bound to phytic acid, making them more bioavailable for animal absorption. This study was carried out to inspect the effect of supplementing low phosphorus (P) diet with di-calcium phosphate (DCP) and liquid phytase enzyme (LP), which contains 1500 FTU/kg, on growth performance, intestinal morphometry, proximate body chemical composition, blood profile, immunity status, liver mitochondrial enzyme activities, the expression response and economic returns of Nile tilapia (Oreochromis niloticus). Three triplicate groups of fish (initial weight 5.405 ± 0.045 g, N = 90) were fed on three different diets for 90 days. The first was a control diet with zero DCP; the second was a control diet supplemented with 0.71% DCP; the third was a control diet supplemented with 0.03% LP. The groups were designated as CG, DCP and LP, respectively. Results showed that LP induced considerable improvements (p < 0.05) in FBW, body weight gain, weight gain rate, specific growth rate, HIS, viscero-somatic index, spleen-somatic index, feed conversion ratio, blood parameters and the histomorphometry assessment of intestinal villi absorptive capacity, compared with the other groups. Also, whole-body protein and lipid contents pointedly (p < 0.05) increased by LP, compared with the DCP group. A positive response (p < 0.05) to the phytase enzyme was noted in complexes I, III and IV of the mitochondrial liver complex enzyme activity. Likewise, the relative gene expression levels of (GHr-1, IGF-1, FAS and LPL) were notably (p < 0.05) upregulated by phytase enzyme, associated with DCP and control groups. Further, phytase recorded the highest total return and profit percentage. It can be concluded that Nile tilapia benefits from using phytase enzyme 1500 FTU/kg at 0.03% without adding DCP in terms of good performance and profits.

Isolation and in-vitro characterization of extracellular phytase producing bacterial isolates for potential application in poultry feed.

BACKGROUND: Phytase catalyses the breakdown of complex organic forms of phosphorous into simpler forms by sequential hydrolysis of phosphate ester bonds to liberate the inorganic phosphate. Supplementation of feeds with bacterial phytase therefore could enhance the bioavailability of phosphorus and micronutrients. Hence, the aim of this study was to isolate and characterize phytase producing bacteria from rhizosphere soil, fresh poultry excreta, and cattle shed to evaluate their potential in improving poultry feeds. Phytase producing bacteria were isolated using wheat bran extract medium. RESULTS: A total of 169 bacterial isolates were purified and screened for phytase activity. Out of these, 36 were confirmed as positive for phytase enzyme activity. The bacterial isolates were identified by cultural, morphological, and biochemical features. The isolates were also identified by using 16 S rRNA gene sequencing. The bacterial isolates (RS1, RS8, RS10 and RS15) were provided with gene bank database accession numbers of MZ407562, MZ407563, MZ407564 and MZ407565 respectively. All isolates increased phytase production when cultured in wheat bran extract medium (pH 6) supplemented with 1% (wt/v) galactose and 1% (wt/v) ammonium sulphate incubated at 50oC for 72 h. Proximate composition analysis after supplementation of phytase showed that phytase supplementation improved bioavailability of phosphorus, calcium, potassium and sodium in poultry feed. CONCLUSIONS: Overall, this study showed that the nutritional value of poultry feed can be improved using microbial phytase enzyme which reduces the cost of supplementation with inorganic phosphate.

Application of the fission yeast Schizosaccharomyces pombe in human nutrition.

Fission yeast Schizosaccharomyces pombe (S. pombe) is renowned as a powerful genetic model for deciphering cellular and molecular biological phenomena, including cell division, chromosomal events, stress responses, and human carcinogenesis. Traditionally, Africans use S. pombe to ferment the beer called 'Pombe', which continues to be consumed in many parts of Africa. Although not as widely utilized as the baker's yeast Saccharomyces cerevisiae, S. pombe has secured several niches in the food industry for human nutrition because of its unique metabolism. This review will explore three specific facets of human nutrition where S. pombe has made a significant impact: namely, in wine fermentation, animal husbandry and neutraceutical supplementation coenzyme Q10 production. Discussions focus on the current gaps in these areas, and the potential research advances useful for addressing future challenges. Overall, gaining a better understanding of S. pombe metabolism will strengthen production in these areas and potentially spearhead novel future applications.

Digestion and utilization of plant-based diets by transgenic pigs secreting ß-glucanase, xylanase, and phytase in their salivary glands.

Novel transgenic (TG) pigs co-expressing three microbial enzymes, ß-glucanase, xylanase, and phytase, in their salivary glands were previously generated, which exhibited reduced phosphorus and nitrogen emissions and improved growth performances. In the present study, we attempted to explore the age-related change of the TG enzymic activity, the residual activity of the enzymes in the simulated gastrointestinal tract, and the effect of the transgenes on the digestion of nitrogen and phosphorus content in the fiber-rich, plant-based diets. Results showed that all the three enzymes were stably expressed over the growing and finishing periods in the F2 generation TG pigs. In simulated gastric juice, all the three enzymes exhibited excellent gastrointestinal environment adaptability. The apparent total tract digestibility of phosphorus was increased by 69.05% and 499.64%, while fecal phosphate outputs were decreased by 56.66% and 37.32%, in the TG pigs compared with the wild-type littermates fed with low non-starch polysaccharides diets and high fiber diets, respectively. Over half of available phosphorus and water-soluble phosphorus in fecal phosphorus were reduced. We also found the performance of phosphorus, calcium, and nitrogen retention rates were significantly improved, resulting in faster growth performance in TG pigs. The results indicate that TG pigs can effectively digest the high-fiber diets and exhibit good growth performance compared with wild type pigs.

Immobilized phytases: an overview of different strategies, support material, and their applications in improving food and feed nutrition.

Phytases are the most widely used food and feed enzymes, which aid in nutritional improvement by reducing anti-nutritional factor. Despite the benefits, enzymes usage in the industry is restricted by several factors such as their short life-span and poor reusability, which result in high costs for large-scale utilization at commercial scale. Furthermore, under pelleting conditions such as high temperatures, pH, and other factors, the enzyme becomes inactive due to lesser stability. Immobilization of phytases has been suggested as a way to overcome these limitations with improved performance. Matrices used to immobilize phytases include inorganic (Hydroxypatite, zeolite, and silica), organic (Polyacrylamide, epoxy resins, alginate, chitosan, and starch agar), soluble matrix (Polyvinyl alcohol), and nanomaterials including nanoparticles, nanofibers, nanotubes. Several surface analysis methods, including thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC), and FTIR analysis, have been used to characterize immobilized phytase. Immobilized phytases have been used in a broad range of biotechnological applications such as animal feed, biodegradation of food phytates, preparations of myo-inositol phosphates, and sulfoxidation by vanadate-substituted peroxidase. This article provides information on different matrices used for phytase immobilization from the last two decades, including the process of immobilization and support material, surface analysis techniques, and multifarious biotechnological applications of the immobilized phytases.

Purification and biochemical characterization of phytase from Bacillus cereus isolated from gastrointestinal tract of African giant snail (Achatina fulica).

Phytases are specialized enzymes meant for phytic acid degradation. They possess ability to prevent phytic acid indigestion, including its attendant environmental pollution. This study was aimed at investigating biochemical properties of purified phytase of B. cereus isolated from Achatina fulica. Phytase produced from Bacillus cereus that exhibited optimal phytate degrading-ability of all the bacteria isolated was purified in a three-step purification. The biochemical properties of the purified enzyme were also determined. The phytase homogeny of approximately 45 kDa exhibited 12.8-purification fold and 1.6% yield with optima phytate degrading efficiency and maximum stability at pH 7 and 50 °C. Remaining activity of 52 and 47% obtained between 60 and 70 °C after 2 h further established thermostability of the purified phytase. Mg2+ and Zn2+ enhanced phytate hydrolysis by the enzyme, while Na+ showed mild inhibition but Hg2+ severely inhibited the enzymatic activity. Km and Vmax were estimated to be 0.11 mM and 55.6 µmol/min/mL, displaying enzyme-high substrate affinity and catalytic efficiency, respectively. Phytase purified from Bacillus cereus, isolated from African giant snails, has shown excellent characteristics suitable for phytic acid hydrolysis and could be employed in industrial and biotechnological applications.

Spectroscopic Investigation of Phosphorus Mineralization as Affected by the Calcite-Water Interfacial Chemistry.

The mineralization and bioavailability of phytic acid, the predominant organic phosphorus (OP) species in many soils, have generally been rendered limited due to its interaction with soil minerals. In particularly calcareous and neutral to slightly alkaline soils, phytic acid is known to actively react with calcite, although how this interaction affects phytic acid mineralization is still unknown. This study, therefore, investigated the mechanisms regarding how the calcite-water interface influences phytic acid mineralization by phytase, at pHs 6 and 8 using in situ spectroscopic techniques including solution nuclear magnetic resonance and attenuated total reflection Fourier transform infrared spectroscopy. The findings indicated a pH-specific effect of the calcite-water interface. Inhibited phytase activity and thus impaired phytic acid mineralization were induced by calcite at pH 6, while the opposite effect was observed at pH 8. How the interaction between phytic acid and calcite and between phytase and calcite differed between the two pH values contributed to the pH-specific effect. The results demonstrate the importance of soil pH, enzyme-, and OP-clay mineral interactions in controlling the mineralization and transformation of OP and, consequently, the release of phosphate in soils. The findings can also provide implications for the management of calcite-rich and limed soils.

Phytate and Arsenic Enhance Each Other's Uptake in As-hyperaccumulator Pteris vittata: Root Exudation of Phytate and Phytase, and Plant Uptake of Phytate-P.

Phytate as a root exudate is rare in plants as it mainly serves as a P storage in the seeds; however, As-hyperaccumulator Pteris vittata effectively secretes phytate and utilizes phytate-P, especially under As exposure. This study investigated the effects of As on its phytate and phytase exudation and the impacts of As and/or phytate on each other's uptake in P. vittata through two hydroponic experiments. Under 10-100 µM arsenate (AsV), the exudation of phytate and phytase by P. vittata was increased by 50-72% to 20.4-23.4 µmol h-1 g-1 and by 28-104% to 18.6-29.5 nmol h-1 plant-1, but they were undetected in non-hyperaccumulator Pteris ensiformis at 10 µM AsV. Furthermore, compared to 500 µM phytate, the phytate concentration in the growth media was reduced by 69% to 155 µM, whereas the P and As contents in P. vittata fronds and roots were enhanced by 68-134% and 44-81% to 2423-2954 and 82-407 mg kg-1 under 500 µM phytate plus 50 µM AsV. The increased P/As uptake in P. vittata was probably attributed to 3.0-4.5-fold increase in expressions of P transporters PvPht1;3-1;4. Besides, under As exposure, plant P may be converted to phytate in P. vittata roots, thereby increasing phytate's contents by 84% to 840 mg kg-1. Overall, our results suggest that As-induced phytate/phytase exudation and phytate-P uptake stimulate its growth and As hyperaccumulation by P. vittata.