Extracellular Phytase Production Research Articles

Screening and Characterization of Potential Fungi from Environmental Samples for Extracellular Phytase Production

Screening and Characterization of Potential Fungi from Environmental Samples for Extracellular Phytase Production

Isolation and identification of phytate-degrading yeast from traditional fermented food

Abstract. Suryani AE, Anggraeni AS, Istiqomah L, Damayanti E, Karimy MF. 2021. Isolation and identification of phytate-degrading yeast from traditional fermented food. Biodiversitas 22: 866-873. Application of phytase (myo-inositol hexakisphosphate phosphohydrolase) to catalyze the release of phosphate from phytates contained on grain-based feed has been used widely in poultry feed industry. In this study, yeast as phytase producer from traditional fermented food was isolated, screened and identified their morphological, biochemical, and molecular characteristics. Production of extracellular phytase from yeast was quantified using spectrophotometer. The results showed that among 8 yeast isolates that had phytase activity, there were two isolates with the highest phytase activity and specific activity which were TKd3 isolate (6.57 U/mL and 54.230 U/mg) and GF1 (6.07 U/mL and 53.68 U/mg). Morphological identification using Scanning Electron Microscope revealed that TKd3 cells isolated from soybean tempeh had an elongated oval cell structure, whereas the GF1 isolated from fresh gatot had a rounder cell structure. TKd3 isolate with accession number MW131530 had homology with Candida tropicalis ATCC 750 28S rRNA with 99.83% similarity and GF1 isolate with accession number MW131531 had homology with Candida tropicalis ATCC 750 28S rRNA with 100% similarity. It could be concluded that C. tropicalis yeast from traditional fermented food produced the extracellular phytase for further use of phytase in poultry feed additive.

A multistrategy approach for improving the expression of E. coli phytase in Pichia pastoris.

Phytase is an additive in animal feed that degrades phytic acid in plant material, reducing feeding costs, and pollution from fecal phosphorus excretion. A multistrategy approach was adopted to improve the expression of E. coli phytase in Pichia pastoris. We determined that the most suitable signal peptide for phytase secretion was an α-factor secretion signal with an initial enzyme activity of 153.51U/mL. Increasing the copy number of this gene to four increased phytase enzyme activity by 234.35%. PDI overexpression and Pep4 gene knockout increased extracellular phytase production by 35.33% and 26.64%, respectively. By combining favorable factors affecting phytase expression and secretion, the enzyme activity of the phytase-engineered strain was amplified 384.60% compared with that of the original strain. We also evaluated the potential for the industrial production of the engineered strain using a 50-L fed-batch fermenter and achieved a total activity of 30,246U/mL after 180h of fermentation.

Extracellular Phytase Production by the Wine Yeast S. cerevisiae (Finarome Strain) during Submerged Fermentation

One of the key steps in the production of phytases of microbial origin is selection of culture parameters, followed by isolation of the enzyme and evaluation of its catalytic activity. It was found that conditions for S. cerevisiae yeast culture, strain Finarome, giving the reduction in phytic acid concentration of more than 98% within 24 h of incubation were as follows: pH 5.5, 32 °C, continuous stirring at 80 rpm, the use of mannose as a carbon source and aspartic acid as a source of nitrogen. The highest catalytic activity of the isolated phytase was observed at 37 °C, pH 4.0 and using phytate as substrate at concentration of 5.0 mM. The presence of ethanol in the medium at a concentration of 12% v/v reduces the catalytic activity to above 60%. Properties of phytase derived from S. cerevisiae yeast culture, strain Finarome, indicate the possibility of its application in the form of a cell’s free crude protein isolate for the hydrolysis of phytic acid to improve the efficiency of alcoholic fermentation processes. Our results also suggest a possibility to use the strain under study to obtain a fusant derived with specialized distillery strains, capable of carrying out a highly efficient fermentation process combined with the utilization of phytates.

Bioprocess for the production of recombinant HAP phytase of the thermophilic mold Sporotrichum thermophile and its structural and biochemical characteristics

Thermophilc mold Sporotrichum thermophile secretes an acidstable and thermostable phytase, which finds application as a food and feed additive because of its adequate thermostability, acid stability, protease insensitivity and broad substrate spectrum. Low extracellular phytase production by the mold is a major bottleneck for its application on a commercial scale. We have successfully overcome this problem by constitutive secretary expression of codon optimized rStPhy under glyceraldehyde phosphate dehydrogenase (GAP) promoter in Pichia pastoris. A ∼41-fold improvement in rStPhy production has been achieved. Circular Dichroism (CD) spectra revealed that rStPhy is composed of 26.65% α-helices, 5.26% β-sheets and 68.09% random coils at pH 5.0 and 60°C, the optima for the enzyme activity. The melting temperature (Tm) of the enzyme is ∼73°C. The 3D structure of rStPhy displayed characteristic signature sequences (RHGXRXP and HD) of HAP phytase. The catalytically important amino acids (Arg74, His75, Arg78, His368 and Asp369) were identified by docking and site directed mutagenesis. Fluorescence quenching by N-bromosuccinimide (NBS) and CsCl exposed tryptophan residues surrounded by negative charges, which play a key role in maintaining structural integrity of rStPhy.

Effect of Different Cultural Conditions for Phytase Production by <i>Aspergillus niger</i> in Submerged Fermentation

In agriculture, phytase is one of the most important monogastric animal sources of nutrient components because it effectively catalyzes the release of phosphate from phytate and phosphorylated compounds. In present work, Aspergillus niger strain (own collection) was used. Various physical and chemical factors have been known to affect the growth and the production of phytase. The effect of carbon and nitrogen sources, temperature and pH for extra cellular phytase production was investigated. Maximal phytase activity of Aspergillus niger was detected in media with 1.0% sucrose as a carbon source. Among the inorganic and organic nitrogen sources, ammonium nitrate in concentration of 0.5% was found to be a favorable nitrogen source for phytase production in Aspergillus niger. Optimum temperature and pH for phytase production by Aspergillus niger were 30°C and 5.5.

Phytase-active lactic acid bacteria from sourdoughs: Isolation and identification

Whole-grain foods play an important role in human diet as they are relatively rich in minerals, however, the absorption of those minerals in human gut can be very low due to high content of the mineral binding phytate. Therefore, the object of this study was to identify phytase-active lactic acid bacteria (LAB) which could be used as a starter to increase mineral bioavailability in whole-meal bread. Hence, LAB isolates were isolated from Lithuanian sourdoughs, tested for phytase activity, and phytase active isolates were identified. Studies of phytase activity of the isolates were carried out at conditions optimal for leavening of bread dough (pH 5.5 and 30 °C). The phytase active isolates belonged to the species Lactobacillus panis, Lactobacillus reuteri, Lactobacillus fermentum, and Pediococcus pentosaceus. Phytase activities of the tested LAB isolates were both extra- and intra-cellular. The highest extracellular phytase production was found in L. panis with a volumetric phytase activity of 140 U/mL. Phytate degradation in whole-wheat dough fermented with L. panis or L. fermentum was 90% and 70%, respectively.

Optimization of five environmental factors to increase beta‐propeller phytase production in Pichia pastoris and impact on the physiological response of the host

Recently, we engineered Pichia pastoris Mut(s) strains to produce several beta-propeller phytases, one from Bacillus subtilis and the others designed by a structure-guided consensus approach. Furthermore, we demonstrated the ability of P. pastoris to produce and secrete these phytases in an active form in shake-flask cultures. In the present work, we used a design of experiments strategy (Simplex optimization method) to optimize five environmental factors that define the culture conditions in the induction step to increase beta-propeller phytase production in P. pastoris bioreactor cultures. With the optimization process, up to 347,682 U (82,814 U/L or 6.4 g/L culture medium) of phytase at 68 h of induction was achieved. In addition, the impact of the optimization process on the physiological response of the host was evaluated. The results indicate that the increase in extracellular phytase production through the optimization process was correlated with an increase in metabolic activity of P. pastoris, shown by an increase in oxygen demand and methanol consumption, that increase the specific growth rate. The increase in extracellular phytase production also occurred with a decrease in extracellular protease activity. Moreover, the optimized culture conditions increased the recombinant protein secretion by up to 88%, along with the extracellular phytase production efficiency per cell.

Evaluation of Phytase Production by Fish Gut Bacterium, Bacillus subtilis, for Processing of Ipomea aquatica Leaves as Probable Aquafeed Ingredient

Extracellular phytase production by Bacillus subtilis isolated from the gut of a minor carp, Labeo bata, has been investigated under solid-state fermentation using leaves from four low cost aquatic weeds (Ipomea aquatica, Pistia stratiotes, Lemna major, and Eichhornia crassipes) as substrates. Water spinach, I. aquatica, leaf meal supported maximum phytase activity (15.31 ± 0.38 U/g) and phytate degradation (66.79%). Incubation for 8 days at pH 7 and 35°C temperature revealed optimum phytase production. Maximum phytase production complied with 3.0-mL inoculum size (12.38 ± 0.05 U/g) and 60% initial moisture content (13.46 ± 0.08 U/g). Glucose (10 g/L) and ammonium sulfate (20.0 g/L) supplementation as additional carbon and nitrogen sources yielded maximum phytase production (12.24 ± 0.09 and 17.35 ± 0.06 U/g, respectively). Analysis of fermented leaf meal revealed that there was marginal increase (t-value significant at p < 0.05) in the contents of crude protein, lipid, minerals (Na, K, Ca, Mg, Zn, Fe, Cu, P), free amino acids, and fatty acids; along with reduction in the contents of the antinutritional factors—e.g., crude fiber, tannins, phytic acid, and trypsin inhibitor. The results indicate that there is ample scope for further research to appraise potential application of gut bacteria for sustainable utilization of aquatic weeds as aquafeed ingredients.

Optimization of cultivation conditions for the production of phytate-degrading enzymes by Enterobacter sakazakii ASUIA279 isolated from Malaysian maize root

The production of extracellular phytase by Enterobacter sakazakii ASUIA279 was optimized using response surface methodology with full-factorial faced centred central composite design. Two sets of experiments were carried out to optimize the five most profound factors of the cultivation conditions in order to maximize phytase production. Incubation temperature, initial pH of the media and percentage of rice bran supplemented into the media were optimized in Erlenmeyer flasks while agitation and aeration effect were controlled in a bioreactor. This design reduced the number of actual experiment performed to optimize phytase production and allowed the study of possible interactions among the factors. In the first set of experiments linear and quadratic effect of initial pH was determined to be the most significant factor affecting phytase production. In the bioreactor both linear effects of agitation and aeration, were identified to be highly significant (&gt; 99 %) in respect to phytase yields. Optimal phytase production was observed at a incubation temperature of 39.7 ºC, an initial pH of 7.1, supplementation with 13.6 % rice bran , 320 rpm of agitation and 0 vvm of aeration.&#x0D;

Isolation and identification of phytase-active yeasts from sourdoughs

Identification of high phytase-active yeasts is necessary in order to find prominent candidates for the production of wholemeal bread with high content of bioavailable minerals. Tested yeasts were isolated from Danish and Lithuanian sourdoughs, since high phytase-active yeasts adapted to grow in sourdough matrix would be a good choice for bread industry. Isolated species were; Saccharomyces cerevisiae, Pichia kudriavzevii, Pichia occidentalis, Candida humilis and Kazachstania exigua. Studies of phytase-positive isolates were carried out at conditions optimal for leavening of bread dough (pH 5.5 and 30 °C). All the tested yeasts isolated from sourdoughs exhibited phytase activities. The most prominent isolates for extracellular phytase production were found to be S. cerevisiae L1.12 with a specific extracellular activity of 10.6 U/1010 CFU, followed by S. cerevisiae L6.06 with a specific extracellular activity of 8.2 U/1010 CFU. Some other isolates of S. cerevisiae, one of C. humilis, and one of P. kudriavzevii also had high specific extracellular activities of c. 4–7 U/1010 CFU. These isolates may be potential candidates for improving mineral bioavailability in whole grain bread.

Phytase-active yeasts from grain-based food and beer

To screen yeast strains isolated from grain-based food and beer for phytase activity to identify high phytase-active strains. The screening of phytase-positive strains was carried out at conditions optimal for leavening of bread dough (pH 5·5 and 30°C), in order to identify strains that could be used for the baking industry. Two growth-based tests were used for the initial testing of phytase-active strains. Tested strains belonged to six species: Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces bayanus, Kazachstania exigua (former name Saccharomyces exiguus), Candida krusei (teleomorph Issachenkia orientalis) and Arxula adeninivorans. On the basis of initial testing results, 14 strains were selected for the further determination of extracellular and intracellular (cytoplasmic and/or cell-wall bound) phytase activities. The most prominent strains for extracellular phytase production were found to be S. pastorianus KVL008 (a lager beer strain), followed by S. cerevisiae KVL015 (an ale beer strain) and C. krusei P2 (isolated from sorghum beer). Intracellular phytase activities were relatively low in all tested strains. Herein, for the first time, beer-related strains of S. pastorianus and S. cerevisiae are reported as phytase-positive strains. The high level of extracellular phytase activity by the strains mentioned previously suggests them to be strains for the production of wholemeal bread with high content of bioavailable minerals.

Optimization of the extracellular production of a bacterial phytase with Escherichia coli by using different fed-batch fermentation strategies.

The extracellular production of Escherichia coli phytase was studied in fed-batch fermentations. Two different feeding strategies were compared: control by keeping the glucose concentration constant, and control by keeping a low constant oxygen level in the medium. For the feeding control based on glucose concentration, a recently developed rapid glucose controlling system was tested for the first time in bacterial cultivations and used to establish the fermentative production of extracellular phytase with E. coli. High activity levels (120 U ml(-1)) at short cultivation times (14 h) were obtained. Even higher activity levels - albeit at longer cultivation times - were reached by applying a feeding control, the main characteristic of which was a constant low oxygen concentration. The optimum oxygen level for the production of phytase was in the range of 5-10% saturation.

Solid-state fermentation for production of phytase by Rhizopus oligosporus.

Solid-state fermentation of coconut oil cake has been carried out with Rhizopus oligosporus for the production of phytase. Phytase is used commercially in the animal feed industry to improve animal performance because there is a substantial and growing interest among swine and poultry producers in the application of phytase to improve the nutritional quality in animal feeds. Demonstrated benefits include improved feed yield ratios and reduction in the environmental costs associated with the disposal of animal wastes. We report the production of extracellular phytase by R. oligosporus under solid-state fermentation using coconut oil cake as substrate. Maximal enzyme production (14.29 U/g of dry substrate) occurred at pH 5.3, 30 degrees C, and 54.5% moisture content after 96 h of incubation. The addition of extra nutrients to the substrate resulted in inhibition of product formation. The results indicate the scope for production of phytase using coconut oil cake as solid substrate without additional nutrients.

Microbial production of extra-cellular phytase using polystyrene as inert solid support

Aspergillus ficuum TUB F-1165 and Rhizopus oligosporus TUB F-1166 produced extra-cellular phytase during solid-state fermentation (SSF) using polystyrene as inert support. Maximal enzyme production (10.07 U/g dry substrate (U/gds) for A. ficuum and 4.52 U/gds for R. oligosporus) was observed when SSF was carried out with substrate pH 6.0 and moisture 58.3%, incubation temperature 30 °C, inoculum size of 1.3×10 7 spores/5 g substrate, for 72 h for A. ficuum and with substrate pH 7.0 and moisture 58.3%, incubation temperature 30 °C, inoculum size of 1×10 6 spores/5 g substrate for 96 h for R. oligosporus. Results indicated scope for production of phytase using polystyrene as inert support.

Production of high activity thermostable phytase from thermotolerant Aspergillus niger in solid state fermentation

The thermotolerant fungus, Aspergillus niger NCIM 563, was used for production of extracellular phytase on agricultural residues: wheat bran, mustard cake, cowpea meal, groundnut cake, coconut cake, cotton cake and black bean flour in solid state fermentation (SSF). Maximum enzyme activity (108 U g−1 dry mouldy bran, DMB) was obtained with cowpea meal. During the fermentation phytic acid was hydrolysed completely with a corresponding increase in biomass and phytase activity within 7 days. Phosphate in the form of KH2PO4 (10 mg per 100 g of agriculture residue) increased phytase activity. Among various surfactants added to SSF, Trition X-100 (0.5%) exhibited a 30% increase in phytase activity. The optimum pH and temperature of the crude enzyme were 5.0 and 50°C respectively. Phytase activity (86%) was retained in buffer of pH 3.5 for 24 h. The enzyme retained 75% of its activity on incubation at 55°C for 1 h. In the presence of 1 mM K+ and Zn2+, 95% and 55% of the activity were retained. Scanning electron microscopy showed a high density growth of fungal mycelia on wheat bran particles during SSF. Journal of Industrial Microbiology & Biotechnology (2000) 24, 237–243.

High-level Expression of a Recombinant Thermostable Phytase in Bacillus subtilis.

An efficient expression system was developed in Bacillus subtilis for the large scale production of phytase. The phytase gene with a native promoter derived from Bacillus amyloliquefaciens was cloned in the Bacillus expression vector pJH27 under a strong BJ27 promoter and its expression was optimized. The expression of the phytase gene occurred during late exponential growth and the extracellular phytase production was 2.0 units/ml, which constituted over 90% of the total protein. The yield was 100-fold higher than wild type Bacillus amyloliquefaciens DS11.

Lactobacillus amylovorus as a phytase producer in submerged culture

Nineteen strains of lactic acid producing bacteria of the genera Lactobacillus and Streptococcus collected from different culture collections were screened for the production of extracellular phytase. A number of them exhibited the enzyme activity in the fermentation medium but Lactobacillus amylovorus B4552 produced the maximum amounts of phytase ranging from 125 to 146 units ml-1 extracellularly under the submerged cultivation conditions. The optimum glucose and inorganic phosphate levels for highest phytase production were found to be 1% and 24% mg, respectively. Lactobacillus amylovorus has potential in improving nutritional qualities of cereal and pulse-based food fermentations.

Survey of microorganism for the production of extracellular phytase.

A culture enrichment technique was used to isolate phytase-producing microorganisms. Also, microorganisms from various culture collections were tested for their phytase-producing ability. A number of the Aspergillus niger group produced extracellular phytase which dephosphorylated calcium phytate in acidic solution. A soil isolate, A. ficuum NRRL 3135, produced the most active phytase in a cornstarch-based medium. Production of phytase was strongly repressed by inorganic phosphates and required a high carbon to phosphorus ratio in the medium.

Survey of Microorganisms for the Production of Extracellular Phytase

A culture enrichment technique was used to isolate phytase-producing microorganisms. Also, microorganisms from various culture collections were tested for their phytase-producing ability. A number of the Aspergillus niger group produced extracellular phytase which dephosphorylated calcium phytate in acidic solution. A soil isolate, A. ficuum NRRL 3135, produced the most active phytase in a cornstarch-based medium. Production of phytase was strongly repressed by inorganic phosphates and required a high carbon to phosphorus ratio in the medium.