Phytase isozymes from Aspergillus niger NCIM 563 under solid state fermentation: Biochemical characterization and their correlation with submerged phytases

Aspergillus niger NCIM 563 produces dissimilar phytase isozymes under solid state and submerged fermentation conditions. Biochemical characterization and applications of phytase Phy III and Phy IV in SSF and their comparison with submerged fermentation Phy I and Phy III were studied. SSF phytases have a higher metabolic potential as compared to SmF. Phy I is tetramer and Phy II, III and IV are monomers. Phy I and IV have pH optima of 2.5 and Phy II and III have pH optima of 5.0 and 5.6, respectively. Phy I, III and IV exhibited very broad substrate specificity while Phy II was more specific for sodium phytate. SSF phytase is less thermostable as compared to SmF phytase. Phy I and II show homology with other known phytases while Phy III and IV show no homology with SmF phytases and any other known phytases from the literature suggesting their unique nature. This is the first report about differences among phytase produced under SSF and SmF by A. niger and this study provides basis for explanation of the stability and catalytic differences observed for these enzymes. Exclusive biochemical characteristics and multilevel application of SSF native phytases determine their efficacy and is exceptional.     

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. Received 07 June 1999/ Accepted in revised form 18 December 1999     

Phytase production by solid-state fermentation of groundnut oil cake by Aspergillus niger: A bioprocess optimization study for animal feedstock applications

This investigation deals with the use of agro-industrial waste, namely groundnut oil cake (GOC), for phytase production by the fungi Aspergillus niger NCIM 563. Plackett–Burman design (PBD) was used to evaluate the effect of 11 process variables and studies here showed that phytase production was significantly influenced by glucose, dextrin, distilled water, and MgSO₄ · 7H₂O. The use of response surface methodology (RSM) by Box–Behnken design (BBD) of experiments further enhanced the production by a remarkable 36.67-fold from the original finding of 15 IU/gds (grams of dry substrate) to 550 IU/gds. This is the highest solid-state fermentation (SSF) phytase production reported when compared to other microorganisms and in fact betters the best known by a factor of 2. Experiments carried out using dried fermented koji for phosphorus and mineral release and also thermal stability have shown the phytase to be as efficient as the liquid enzyme extract. Also, the enzyme, while exhibiting optimal activity under acidic conditions, was found to have significant activity in a broad range of pH values (1.5–6.5). The studies suggest the suitability of the koji supplemented with phytase produced in an SSF process by the “generally regarded as safe” (GRAS) microorganism A. niger as a cost-effective value-added livestock feed when compared to that obtained by submerged fermentation (SmF).     

Biochemical Characterization of Fungal Phytases (myo-Inositol Hexakisphosphate Phosphohydrolases): Catalytic Properties

Supplementation with phytase is an effective way to increase the availability of phosphorus in seed-based animal feed. The biochemical characteristics of an ideal phytase for this application are still largely unknown. To extend the biochemical characterization of wild-type phytases, the catalytic properties of a series of fungal phytases, as well as Escherichia coli phytase, were determined. The specific activities of the fungal phytases at 37°C ranged from 23 to 196 U · (mg of protein)⁻¹, and the pH optima ranged from 2.5 to 7.0. When excess phytase was used, all of the phytases were able to release five phosphate groups of phytic acid (myo-inositol hexakisphosphate), which left myo-inositol 2-monophosphate as the end product. A combination consisting of a phytase and Aspergillus niger pH 2.5 acid phosphatase was able to liberate all six phosphate groups. When substrate specificity was examined, the A. niger, Aspergillus terreus, and E. coli phytases were rather specific for phytic acid. On the other hand, the Aspergillus fumigatus, Emericella nidulans, and Myceliophthora thermophila phytases exhibited considerable activity with a broad range of phosphate compounds, including phenyl phosphate, p-nitrophenyl phosphate, sugar phosphates, α- and β-glycerophosphates, phosphoenolpyruvate, 3-phosphoglycerate, ADP, and ATP. Both phosphate liberation kinetics and a time course experiment in which high-performance liquid chromatography separation of the degradation intermediates was used showed that all of the myo-inositol phosphates from the hexakisphosphate to the bisphosphate were efficiently cleaved by A. fumigatus phytase. In contrast, phosphate liberation by A. niger or A. terreus phytase decreased with incubation time, and the myo-inositol tris- and bisphosphates accumulated, suggesting that these compounds are worse substrates than phytic acid is. To test whether broad substrate specificity may be advantageous for feed application, phosphate liberation kinetics were studied in vitro by using feed suspensions supplemented with 250 or 500 U of either A. fumigatus phytase or A. niger phytase (Natuphos) per kg of feed. Initially, phosphate liberation was linear and identical for the two phytases, but considerably more phosphate was liberated by the A. fumigatus phytase than by the A. niger phytase at later stages of incubation.     

Predicting vegetative inoculum performance to maximize phytase production in solid-state fermentation using response surface methodology

Microbial phytase is used to reduce the environmental loading of phosphorus from animal production facilities. The limiting factors in the use of this enzyme in animal feeds can be overcome by solid-state fermentation (SSF), which is a promising technology for commercial enzyme production with lower production costs. Inoculum quality and the influence of inoculum quality on phytase production are important factors which need in-depth investigation before scaling-up of high-yielding fermentation process. A full factorial experimental design for 240 h with sampling at every 24 h was used to determine the effects of the treatments, inoculum age (plate and liquid culture), media composition and the duration of SSF on the production of fungal biomass and phytase in SSF systems using Aspergillus niger. The optimal treatment combination for maximal phytase production was determined by statistically comparing all treatments at each sampling time. Both 7- and 14-day plate cultures and M1+ medium composition with 72-h-old liquid inoculum treatments resulted in optimal phytase production at 144 h of SSF, which was the shortest duration observed for maximal phytase production. This resulted in maximal phytase production with a mean of 884±121 U/g substrate, while the maximal phytase production observed at 216 h of SSF (mean phytase activity of 1008±121 U/g substrate), with the same treatment combinations, was not statistically significant from that at 144 h of SSF. Phytase production was strongly growth-associated with younger inocula. The significant treatment variables, age of liquid inoculum and the duration of SSF, were used to predict the system response for phytase production using response surface methodology. From the response surface model, the optimal response of the experiment was predicted and the reliability of the prediction was checked with the verification experiment. Journal of Industrial Microbiology & Biotechnology (2001) 26, 161–170. Received 06 June 2000/ Accepted in revised form 14 October 2000     

Reduction of phytic acid content in canola meal by Aspergillus ficuum in solid state fermentation process

Summary Solid state fermentation (SSF) of canola meal has been carried out to reduce its phytic acid content using Aspergillus ficuum NRRL 3135. In certain batches, a complete reduction of phytic acid content in canola meal was achieved in 48 h. A larger amount of biomass in the inoculum and older inoculum increased the rate of phytic acid hydrolysis. The optimum moisture content of the medium was found to be 67% for phytic acid hydrolysis in an SSF process. The substitution of water in the semi-solid medium with acetate buffer resulted in faster reduction of the phytic acid content. A 15% increase in the amount of protein after 120 h of incubation was observed in the treated meal. The crude phytase preparation extracted from the canola meal after it was treated in an SSF process was also used for reduction of the phytic acid content in new batches of canola meal both in semi-solid medium and in liquid medium. In the semi-solid medium, 58% of the phytic acid was hydrolysed at 45°C in 20 h, while 100% hydrolysis was recorded at 50°C in 12 h in the liquid medium. The SSF process seems to be beneficial for the upgrading of canola meal by reducing both its phytic acid content and increasing the amount of protein.Offprint requests to: Z. Duvnjak     

Engineering of Phytase for Improved Activity at Low pH

For industrial applications in animal feed, a phytase of interest must be optimally active in the pH range prevalent in the digestive tract. Therefore, the present investigation describes approaches to rationally engineer the pH activity profiles of Aspergillus fumigatus and consensus phytases. Decreasing the negative surface charge of the A. fumigatus Q27L phytase mutant by glycinamidylation of the surface carboxy groups (of Asp and Glu residues) lowered the pH optimum by ca. 0.5 unit but also resulted in 70 to 75% inactivation of the enzyme. Alternatively, detailed inspection of amino acid sequence alignments and of experimentally determined or homology modeled three-dimensional structures led to the identification of active-site amino acids that were considered to correlate with the activity maxima at low pH of A. niger NRRL 3135 phytase, A. niger pH 2.5 acid phosphatase, and Peniophora lycii phytase. Site-directed mutagenesis confirmed that, in A. fumigatus wild-type phytase, replacement of Gly-277 and Tyr-282 with the corresponding residues of A. niger phytase (Lys and His, respectively) gives rise to a second pH optimum at 2.8 to 3.4. In addition, the K68A single mutation (in both A. fumigatus and consensus phytase backbones), as well as the S140Y D141G double mutation (in A. fumigatus phytase backbones), decreased the pH optima with phytic acid as substrate by 0.5 to 1.0 unit, with either no change or even a slight increase in maximum specific activity. These findings significantly extend our tools for rationally designing an optimal phytase for a given purpose.     

Phytase production and decrease of phytic acid content in canola meal byAspergillus carbonarius in solid-state fermentation

Solid-state fermentation (SSF) usingAspergillus carbonarius with canola meal as a substrate showed that production of phytase was associated with growth; maximum activity was achieved after 72 h. Apparent 25% and 10% increases in the protein content of the canola meal were noticed after 48 h and 72 h, respectively but total carbohydrate concentration had fallen by 25% by the end of fermentation. The rate of decrease of phytic acid content was optimum with a moisture content between 53% and 60%; homogenization of the inoculum for 120 s led to the greatest biomass and lowest phytic acid content. Inoculation of sterile meal led to lower phytic acid contents than inoculation of non-sterile meal.The authors are with the Department of Chemical Engineering, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada     

Production profiles of phenolics from fungal tannic acid biodegradation in submerged and solid-state fermentation

Potent antioxidant phenolics are derived from tannin biodegradation. Understanding of biodegradation pathways through the identification of the intermediates molecules of great value like tannins is important to pursuit the production of bioactive monomers. Biodegradation of tannins remains poorly understood due to their chemical complexity and reactivity. Tannic acid biodegradation by Aspergillus niger GH1 in submerged fermentation (SF) and solid state fermentation (SSF) was evaluated by liquid chromatography coupled to mass spectrometry (LC–MS). Both cultures were kinetically monitored for the biodegradation profiles during 72 h. Differences in tannic acid composition were evidenced and the consumption of substrate and identification of biodegradation intermediates were achieved. The mechanism of tannic acid degradation by A. niger GH1 is by degradation of high molecular weight gallotannins and highly polymerized tannins to small molecules like gallic acid, digalloyl glucose and trigalloyl glucose. Important differences on time of substrate uptake and product release were revealed.     

Exopectinases produced by Aspergillus niger in solid-state and submerged fermentation: a comparative study

Exopectinase production by Aspergillus niger was compared in submerged fermentation (SmF) and solid-state fermentation (SSF). SSF was carried out using polyurethane foam (PUF) as the solid support. The purpose was to study the effect of sucrose addition (0 or 40 g/l) and water activity level (A _w=0.99 or 0.96) on the level of enzyme activity induced by 15 g/l of pectin. Mycelial growth, as well as extracellular protease production, was also monitored. Sucrose addition in SmF resulted in catabolite repression of exopectinase activity. However, in SSF, an enhancement of enzyme activity was observed. Protease levels were minimal in SSF experiments with sucrose and maximal in SmF without sucrose. Exopectinase yields (IU/g X) were negligible in SmF with sucrose. The high levels of exopectinase with sucrose and high A _w in SSF can be explained by a much higher level of biomass production without catabolite repression and with lower protease contamination. Journal of Industrial Microbiology & Biotechnology (2001) 26, 271–275. Received 05 July 2000/ Accepted in revised form 27 January 2001     

Solid-state fermentation of co-products from palm oil processing: Production of lipase and xylanase and effects on chemical composition

The palm oil industry generates large amounts of lignocellulosic co-products. Palm kernel cake (PKC) and palm pressed fibre (PPF) have nutritional limitations as ingredients in animal feed, and are therefore little used. Solid-state fermentation (SSF) is one alternative treatment to improve the nutritional value of these co-products and to increase their possible use, through the production of enzymes such as lipases and xylanases. These enzymes can reduce the contents of undesirable compounds, such as lipids, and degrade some components of the fibres to improve the digestibility of these co-products. The fungi Aspergillus niger, Aspergillus oryzae and Aspergillus awamori were able to grow in PKC/PPF (40/60 w/w) culture medium by SSF, and to produce xylanase and lipase. A. niger showed the highest lipase activity (20.7?U g^?1) at 72?h. A. awamori higher xylanase activity than the other fungi at all culture periods, reaching a maximum activity of 134.2?U g^?1 at 72?h. The unfermented co-products contained 7.49% lipids and 7.38% non-fibrous carbohydrates (NFC). Lipase produced by these fungi during SSF reduced the lipid content by 36%, 40% and 45% for A. oryzae, A. awamori and A. niger, respectively. The production of xylanases by SSF probably increased the NFC contents by up to 64%. Fermented solids with A. oryzae and A. awamori had the highest levels of NFC, 20.3% and 13.94%, respectively, which improved the nutritional value of these co-products.     

Simplification of the Biomass to Ethanol Conversion Process by Using the Whole Medium of Filamentous Fungi Cultivated Under Solid-State Fermentation

A novel simplified configuration is proposed for the conversion of biomass to ethanol using whole medium enzymatic cocktails (WM) and enzymatic extracts (EE) from different filamentous fungi (Trichoderma reesei, Aspergillus niger, and Aspergillus oryzae) cultivated under solid-state fermentation (SSF) for the hydrolysis of steam-exploded sugarcane bagasse (SESB). The hydrolyzed material derived from the saccharification of SESB using the combinations A. niger WM + T. reesei EE, A. oryzae WM + A. niger EE, and A. niger EE + T. reesei WM resulted in the best biomass conversion yields (66, 65, and 64 % of the theoretical reducing sugar yields, respectively). The best ethanol production (84 % of the theoretical yield) was obtained using the material hydrolyzed by a combination of A. oryzae WM + A. niger EE. The enzymatic conversion of SESB using on-site produced enzymes from the whole SSF cultivation medium, followed by an ethanol production step, is a potential configuration for the biomass to ethanol conversion process. This novel simplified configuration would enable the use of a single reactor system, avoiding the need for additional separation steps.     

Solid-state fermentation of industrial solid wastes from the fruits of milk thistle Silybum marianum for feed quality improvement

The industrial solid wastes generated during the production of silymarin from the fruits of milk thistle Silybum marianum was used as the substrate. Preparation and evaluation of the feeds produced by solid-state fermentation (SSF) of the industrial solid wastes was carried out. The protein content of the fermented feed (FF) from a combination of Aspergillus niger and Candida tropicalis was the highest among the examined strains. The optimal process parameters for protein enrichment with SSF using A. niger and C. tropicalis included incubation temperature of 30.8 °C, fermentation time of 87.0 h, and initial moisture content of 59.7 %. Under these conditions, the value additions of FF occurred. The fiber of FF was decreased by 25.07 %, while the digestibility of protein, protein content, and the ratio of total essential amino acids to total amino acids were increased by 79.85, 16.22, and 8.21 %, respectively. The analysis indicated that FF contained 1.44 mg/kg flavonoids and 0.5 mg/kg silybin, which significantly increased by 2.42 and 1.63 times, respectively than those in unfermented substrates. FF recorded reduced molecular weight of proteins from 20.1 to 44.3 kDa to below 14.3 kDa. The results of feeding trial of FF replacement with soybean meal in broilers diets for 8 weeks showed that FF significantly improved carcass characteristics including abdominal fat rate, serum biochemical parameters including aspartate transaminase, blood urea nitrogen and high density lipoprotein cholesterol, and immune responses of broilers. A potential feed quality improvement was achieved through mixed strains SSF of industrial solid wastes of S. marianum fruits.     

Production of cellulosic ethanol and enzyme from waste fiber sludge using SSF,recycling of hydrolytic enzymes and yeast,and recombinant cellulase-producing Aspergillus niger

Bioethanol and enzymes were produced from fiber sludges through sequential microbial cultivations. After a first simultaneous saccharification and fermentation (SSF) with yeast, the bioethanol concentrations of sulfate and sulfite fiber sludges were 45.6 and 64.7 g/L, respectively. The second SSF, which included fresh fiber sludges and recycled yeast and enzymes from the first SSF, resulted in ethanol concentrations of 38.3 g/L for sulfate fiber sludge and 24.4 g/L for sulfite fiber sludge. Aspergillus niger carrying the endoglucanase-encoding Cel7B gene of Trichoderma reesei was grown in the spent fiber sludge hydrolysates. The cellulase activities obtained with spent hydrolysates of sulfate and sulfite fiber sludges were 2,700 and 2,900 nkat/mL, respectively. The high cellulase activities produced by using stillage and the significant ethanol concentrations produced in the second SSF suggest that onsite enzyme production and recycling of enzyme are realistic concepts that warrant further attention.     

Screening of phytase producers and optimization of culture conditions for submerged fermentation

Phytase (myo-inositol-hexakisphosphate phosphohydrolase) is an enzyme, which breaks down phytate to inositol and orthophosphoric acid. Phytase has been used as feed additive, and in some medical applications for years. To date, phytase production has been usually performed as a solid-state fermentation with small production volumes. Therefore, the aim of this study was to increase the phytase activity in submerged fermentations by screening several microorganism strains based on the literature to select the most productive phytase producer and optimizing growth parameters such as temperature, pH, and aeration level using response surface methodology (RSM). As a result, among the four different microorganisms evaluated, Aspergillus ficuum (NRRL 3135) was selected as the most productive strain. Optimum temperature, pH, and aeration values were determined as 33 °C, 4.5, and 0.9 vvm, respectively, for A. ficuum in 2-l batch submerged phytase productions. Under these conditions, phytase activity was measured as 2.27 U/ml. Therefore, this is a unique study showing the production of phytase with A. ficuum successfully in submerged fermentation as opposed to the traditional solid-state fermentation.     

植酸酶对草鱼和新吉富罗非鱼消化酶活性的影响

植酸酶能水解植酸络合物,释放被植酸束缚的各种营养因子,因此能有效解除植酸与内源性消化酶的结合,促进消化酶的作用。本实验在全植物性饲料中添加植酸酶,研究其对草鱼(Ctenopharyngodon idellus)和新吉富罗非鱼(Oreochromis niloticus)淀粉酶及蛋白酶比活力的影响。以全植物性饲料为阴性对照组,添加磷酸氢钙(dibasic calcium phosphate,DCP)实验组为阳性对照组,另设4个不同梯度的植酸酶实验组(250 U/kg、500 U/kg、1 000 U/kg和2 000 U/kg)。实验选取健壮、规格齐整平均体质量为(12.59±0.09)g的草鱼和平均体质量为(9.59±0.12)g的新吉富罗非鱼,分别随机分为6个组,每组5个平行,每个平行20尾鱼。养殖8周后,草鱼平均体质量(18.29±0.63)g,新吉富罗非鱼平均体质量为(24.68±1.34)g,抽样取出胃、肠和肝胰脏用来分析淀粉酶和蛋白酶比活力。结果表明,植酸酶对无胃鱼草鱼和有胃鱼罗非鱼淀粉酶及蛋白酶比活力都有显著的促进作用。相比较而言,植酸酶对罗非鱼的应用效果较明显,低剂量就能显著提高其淀粉酶及蛋白酶比活力(P<0.05)。当植酸酶添加量达到1 000 U/kg时,草鱼和罗非鱼淀粉酶及蛋白酶比活力均达到峰值,此时,罗非鱼淀粉酶和蛋白酶比活力与阳性对照组无显著差异(P>0.05),而草鱼肝胰脏蛋白酶比活力显著高于阳性对照组(P<0.05)。植酸酶2 000 U/kg实验组,罗非鱼淀粉酶和蛋白酶比活力与1 000 U/kg植酸酶实验组无显著差异(P>0.05),但草鱼肝胰脏蛋白酶比活力显著低于1 000 U/kg植酸酶实验组(P<0.05)。因此,本实验条件下,植酸酶在草鱼和新吉富罗非鱼全植物性蛋白质配合饲料中的适宜添加量均为1 000 U/kg,生产实践中可通过添加植酸酶部分替代无机磷源。     

High level phytase production by <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 563 in solid state culture: response surface optimization,up-scaling,and its partial characterization

Phytase production by Aspergillus niger NCIM 563 was optimized by using wheat bran in solid state fermentation (SSF). An integrated statistical optimization approach involving the combination of Placket–Burman design (PBD) and Box–Behnken design (BBD) was employed. PBD was used to evaluate the effect of 11 variables related to phytase production, and five statistically significant variables, namely, glucose, dextrin, NaNO₃, distilled water, and MgSO₄·7H₂O, were selected for further optimization studies. The levels of five variables for maximum phytase production were determined by a BBD. Phytase production improved from 50 IU/g dry moldy bran (DMB) to 154 IU/g DMB indicating 3.08-fold increase after optimization. A simultaneous reduction in fermentation time from 7 to 4 days shows a high productivity of 38,500 IU/kg/day. Scaling up the process in trays gave reproducible phytase production overcoming industrial constraints of practicability and economics. The culture extract also had 133.2, 41.58, and 310.34 IU/g DMB of xylanase, cellulase, and amylase activities, respectively. The partially purified phytase was optimally active at 55°C and pH 6.0. The enzyme retained ca. 75% activity over a wide pH range 2.0–9.5. It also released more inorganic phosphorus from soybean meal in a broad pH range from 2.5 to 6.5 under emulated gastric conditions. Molecular weight of phytase on Sephacryl S-200 was approximately 87 kDa. The K _m and V _max observed were 0.156 mM and 220 μm/min/mg. The SSF phytase from A. niger NCIM 563 offers an economical production capability and its wide pH stability shows its suitability for use in poultry feed.     

Expression of an Aspergillus niger Phytase Gene (phyA) in Saccharomyces cerevisiae

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals and reduces phosphorus pollution of animal waste. Our objectives were to express an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae and to determine the effects of glycosylation on the phytase’s activity and thermostability. A 1.4-kb DNA fragment containing the coding region of the phyA gene was inserted into the expression vector pYES2 and was expressed in S. cerevisiae as an active, extracellular phytase. The yield of total extracellular phytase activity was affected by the signal peptide and the medium composition. The expressed phytase had two pH optima (2 to 2.5 and 5 to 5.5) and a temperature optimum between 55 and 60°C, and it cross-reacted with a rabbit polyclonal antibody against the wild-type enzyme. Due to the heavy glycosylation, the expressed phytase had a molecular size of approximately 120 kDa and appeared to be more thermostable than the commercial enzyme. Deglycosylation of the phytase resulted in losses of 9% of its activity and 40% of its thermostability. The recombinant phytase was effective in hydrolyzing phytate phosphorus from corn or soybean meal in vitro. In conclusion, the phyA gene was expressed as an active, extracellular phytase in S. cerevisiae, and its thermostability was affected by glycosylation.     

Production of phytase under solid-state fermentation using Rhizopus oryzae: novel strain improvement approach and studies on purification and characterization

Present study introduces linseed oil cake as a novel substrate for phytase production by Rhizopus oryzae. Statistical approach was employed to optimize various medium components under solid state fermentation (SSF). An overall 8.41-fold increase in phytase production was achieved at the optimum concentrations (w/w, mannitol, 2.05%; ammonium sulfate, 2.84% and phosphate, 0.38%). Further enhancement by 59% was observed due to a novel strain improvement approach. Purified phytase (～34 kDa) showed optimal temperature of 45 °C, dual pH optima at 1.5 and 5.5 and possesses high catalytic efficiency (2.38×10(6) M(-1) s(-1)). Characterization study demonstrates the phytase as highly thermostable and resistant to proteolysis, heavy metal ions, etc. Furthermore, an improved HPLC method was introduced to confirm the ability of phytase to degrade phytic acid completely and was found to be an efficient method.     

Optimization of Fermentation Conditions for Phytase Production by Two Strains of Bacillus licheniformis (LF1 and LH1) Isolated from the Intestine of Rohu, Labeo rohita (Hamilton)

The culture conditions for extracellular production of phytase by two strains of Bacillus licheniformis (LF1 and LH1) isolated from the proximal and distal intestine of rohu (Labeo rohita) were optimized to obtain maximum level of phytase. Both the strains were cultured TSA broth for 24 h at 37 ± 2 °C, when average viable count of 9.75 × 10⁷ cells ml^?1 culture broth was obtained. This was used as the inoculum for the production medium. Sesame (Sesamum indicum) oilseed meal was used as the source of phytic acid (substrate). The effects of moisture, pH, temperature, fermentation period, inoculum size, different nitrogen sources, vitamins and surfactants on phytase production by these two strains were evaluated. Phytase yield was highest (1.87 U in LF1 and 1.57 U in LH1) in solid-state fermentation. Enzyme production in both the isolates increased in an optimum pH range of 5.5–6.5. Minimum phytase production was observed at 50 °C, while maximum production was obtained at 40 °C. To standardize the fermentation period for phytase production, production rate was measured at 12-h intervals up to 120 h. Enzyme production increased for 72 h of fermentation in both strains, and decreased thereafter. The enzyme production increased with increased inoculum size up to 3.0 percentage points for the strain LF1 and up to 2.0 % for the strains LH1. Ammonium sulphate as the nitrogen source was most effective in LF1, while beef extract proved useful to maximize enzyme production by LH1.