Purification and properties of a phytase from Candida krusei WZ-001

A phytase from Candida krusei WZ-001 isolated from soil was purified to electrophoretic homogeneity by ion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. The phytase is composed of two different subunits with molecular masses of 116 kDa and 31 kDa on SDS-PAGE (or 120 kDa and 30 kDa on gel chromatography), with the larger subunit having a glycosylation rate of around 35%. The phytase has an optimum pH of 4.6, an optimum temperature of 40 degrees C and a pI value of 5.5. The phytase activity was stimulated by 2-mercapto-ethanol and dithiothreitol (DTT), and inhibited by Zn2+, Mg2+, iodoacetate, pI value of 5.5. The phytase activity was stimulated by 2-mercapto-ethanol ethanol and dithiothreitol (DTT), and inhibited by Zn2+, Mg2+, iodoacetate, p-chroloromercuribenzoate (pCMB) and phenylmethylsulfonyl fluoride (PMSF). The phytase displayed a broad substrate specificity and the K(m) for phytate was 0.03 mM. Phytate was sequentially hydrolyzed by the phytase. Furthermore, 1D and 2D NMR analyses and bioassay of myoinositol indicated that the end hydrolysis product of phytate was myoinositol 2-monophosphate.     

Purification and characterization of phytase from Klebsiella pneumoniae 9-3B

Phytase, an enzyme that catalyzes the hydrolysis of phytate, was purified from Klebsiella pneumoniae 9-3B. The isolate was preferentially selected in a medium which contains phytate as a sole carbon and phosphate source. Phytic acid was utilized for growth and consequently stimulated phytase production. Phytase production was detected throughout growth and the highest phytase production was observed at the onset of stationary phase. The purification scheme including ion exchange chromatography and gel filtration resulted in a 240 and 2077 fold purification of the enzyme with 2% and 15% recovery of the total activity for liberation of inorganic phosphate and inositol, respectively. The purified phytase was a monomeric protein with an estimated molecular weight of 45kDa based on size exclusion chromatography and SDS-PAGE analyses. The phytase has an optimum pH of 4.0 and optimum temperature of 50°C. The phytase activity was slightly stimulated by Ca(2+) and EDTA and inhibited by Zn(2+) and Fe(2+). The phytase exhibited broad substrate specificity and the K(m) value for phytate was 0.04mM. The enzyme completely hydrolyzed myo-inositol hexakisphosphate (phytate) to myo-inositol and inorganic phosphate. The properties of the enzyme prove that it is a good candidate for the hydrolysis of phytate for industrial applications.     

构巢曲霉产植酸酶的酶学特性分析

从构巢曲霉AnP-16菌株发酵液中纯化单一组分的耐热耐酸植酸酶,并对酶学特性进行分析,为其在食品工业中的应用提供理论依据。通过硫酸铵盐析、离子交换层析和疏水层析纯化植酸酶,SDS-PAGE电泳测定其分子量。结果表明,从该菌株发酵液中纯化到了单一组分的植酸酶,纯化倍数60.8倍、回收率41.6%,酶分子量约52 kDa。植酸酶最适作用温度和pH分别为55 ℃和pH4.0,在pH3.0~6.0范围酶活性较高,pH2.0~7.0下孵育3 h,仍能保持80%以上活性。植酸酶耐热性好,70 ℃孵育1 h仍能保持81%活性。Hg2+、Mn2+、Fe2+、Cu2+和Zn2+ 5 mmol/L浓度下对酶活性有明显抑制作用,但Ca2+和Mg2+在1和5 mmol/L浓度时均增强酶活性;有机溶剂甲醇和乙醇在2%浓度有激活作用;SDS抑制酶活性,但其它表面活性剂(Triton X-100和Tween 80)和有机溶剂(丙酮和异戊醇)对酶活性无明显影响。植酸酶有宽泛的底物特异性,但对植酸钠的催化活性最强,其K_m值为0.576 mmol/L。基于植酸酶耐酸耐热及宽广的底物催化活性,其有望应用于粮油食品加工领域,提高食品的营养价值。     

Production of phytase in a low phosphate medium by a novel yeast Candida krusei

A yeast strain producing high levels of phytase was isolated from soil and identified as Candida krusei. The phytase was located on the yeast cell wall and was a glucanase-extractable protein. The phytase production was controlled by the phosphate concentration in the medium used. The maximum production of phytase occurred in a medium containing 0.5 mg of phosphorus per 100 ml, and most of the cells were ellipsoid-shaped and did not exhibit budding. Increasing the concentration of phosphorus in the medium to more than 5 mg of phosphorus per 100 ml caused inhibition of phytase production and 90% of the cells exhibited budding. On the other hand, transferring cells grown in the high-phosphate medium into a phosphate-free one derepressed the phytase production. For example, transferring cells grown in 2 mg of phosphorus per 100 ml into the phosphate-free medium, enhanced the total phytase activity up to 5.5-fold that in the medium containing 0.5 mg of phosphorus per 100 ml. The phytase showed two optimum pHs of 2.5 and 5.5, an optimum temperature of 40 degrees C and the K(m) value for Na-phytate was 0.03 mM. Using in vitro experiments that simulated the conditions of the digestive tract, 50-80% phosphorus was liberated from different plant samples (wheat bran, rice bran and feeds) by the strain.     

一种耐热性植酸酶的分离纯化及其酶学性质研究

采用半固态发酵方式培养泡盛曲霉(Aspergillus awamori)AS3.324,通过有机膜超滤、阴离子交换层析、凝胶层析后,得到纯化的植酸酶。酶学性质表明,其反应最适温度为50-55℃,最适pH为5.5,在37℃下以植酸钠为底物的Km值为1.03 nmol/L,Vmax为2.13μmol/(L.min)。EDTA基本不影响植酸酶活性;Ca2+,Mg2+,Mn2+对植酸酶活性有轻微的抑制作用;Fe2+,Zn2+对酶促反应有显著的抑制作用。对该酶的耐热性研究表明,经较高温度条件处理后,仍有较高残余酶活性,与当今商品化的植酸酶相比,有较强的耐热性。泡盛曲霉植酸酶作为动物饲料添加剂具有广泛的应用前景。     

PURIFICATION AND CHARACTERIZATION OF A PHYTASE FROM SPELT

Four soluble phytases were identified in germinating spelt. Although numerous purification strategies were applied, none of the four phytases could be purified to homogeneity. The purest phytase preparation, called D21, contained a phytase (major component) and an acid phosphatase (APH) (minor component). The phytase behaves like a monomeric protein of a molecular mass of about 68 kDa and shows a broad substrate specificity. Optimal pH for degradation of phytate was 6.0 and the optimal temperature 45C. Kinetic parameters for the hydrolysis of Na-phytate were K_M 400 μmoll^?1 and k_cat 368s^?1 at pH 6.0. The spelt phytase D21 degrades phytate stepwise.     

HYDROLYSIS OF PHYTATE IN BROWN RICE-ADDED BREAD BY ADDITION OF CRUDE AND PURIFIED ASPERGILLUS NIGER PHYTASE PREPARATIONS DURING BREAD MAKING

Brown rice-added breads were prepared with and without crude and purified Aspergillus niger phytase preparations. By adding 30% (w/w) brown rice flour and no phytase preparation, the phytate (myo-inositol hexaphosphate; IP₆) content of the bread was increased and the loaf volume was reduced. The direct addition of a food-additive grade phytase preparation (3,000 U) resulted in a significant decrease in the IP₆ content. However, it collapsed the bread crust. The crude phytase preparation had significant protease and amylase activities. A simple chromatographic method for the separation of phytase activity from the protease and amylase activities was developed. By using purified phytase, the IP₆ content was reduced without any adverse effects on the rising of the bread, which indicates that protease and amylase may be responsible for the collapse of the crust by the commercial phytase preparation.

PRACTICAL APPLICATIONS

Phytate in unrefined cereals and beans lowers bioavailability of minerals. On the other hand, these materials are rich in health-promoting components. To reduce the phytate in them during processing, some commercially available phytase preparations are now used. However, we found that such preparation contains significant amylase and protease activities, which may cause adverse effect on the appearance and textural properties of final products. To solve these problems, a simple and inexpensive procedure for separation of phyatase from amylase and protease was developed in the present study. The purified phytase can reduce phytate in brown rice-added bread without adverse effect on rising of bread. These results would encourage prepare the phytase preparation in high purity in an industrial scale to improve nutritional value of unrefined cereal- and bean-based functional foods.     

Degradation of phytate by Pichia kudriavzevii TY13 and Hanseniaspora guilliermondii TY14 in Tanzanian togwa

The fermented cereal-based gruel togwa is used as weaning food for children in Tanzania. Togwa is rich in minerals but these are often not available for uptake in the human intestine due to natural inhibitors, such as phytate (IP(6)). The yeasts Pichia kudriavzevii TY13, Hanseniaspora guilliermondii TY14 and TY20, isolated from Tanzanian togwa, and selected for high phytase activity in complex yeast medium YPD, were now studied regarding their ability to degrade IP(6) in maize-based model togwa. A modified constitutively high-phytase producing Saccharomyces cerevisiae BY80 and commercial Aspergillus ficuum phytase were included for comparison. In addition, a strain of Lactobacillus plantarum was included in the model-togwa set-up. All yeasts in the study grew and reached final cell density 1.5-2 log units higher than the start value. S. cerevisiae BY80 degraded 85% of the IP(6) in 48 h; the same degradation level as with A. ficuum phytase (89%). Of the togwa-isolated yeasts, P. kudriavzevii TY13 and H. guilliermondii TY14 showed strong phytate degradation in the model-togwa; 95% or more of the initial IP(6) was degraded after 48 h. This corresponds to a remaining level of 0.4 and 0.3μmol IP(6)/g dw. Co-inoculation with L. plantarum did not increase IP(6) degradation. Moreover, fermentation with P. kudriavzevii TY13 yielded a successive increase in inorganic phosphate (P(i)), from 0.7 to 5.4 mM, suggesting a phytase production in TY13 which is fairly insensitive to P(i) repression. The study shows that phytate in a model togwa is available for yeast phytase enzymes, and addresses the importance of strain selection for effectively degrading the phytate. Certain yeasts originating from togwa seem to have developed a natural high phytase production, and P. kudriavzevii TY13 and H. guilliermondii TY14 seem particularly well adapted to phytate degradation in togwa, and is our choice for further studies and strain improvement.     

Zinc and Phytate Distribution in Peas. Influence of Heat Treatment, Germination, pH, Substrate, and Phosphorus on Pea Phytate and Phytase

The largest proportions of zinc and phytate, 88.7 and 97.1%, respectively, were in the Garfield pea cotyledon; the greatest concentrations were in the germ. Cooking peas by two different methods resulted in 13% phytate reduction. Peas incubated 6.5 hr from 25 to 80°C yielded maximum phytate loss (25%) at 60°C due to phytase activated hydrolysis. Germination (10 d) decreased pea phytate 75% and increased phytase activity 12-fold. Semi-purified germinated pea phytase showed temperature optimum at 45°C, pH optimum of 5.2, 30% inhibition by 1 mM inorganic P, and substrate preference for pyrophosphate. Incubation of early germinated peas at optima pH and temperature is suggested for maximum phytate reduction.     

PURIFICATION AND CHARACTERIZATION OF CANOLA SEED (BRASSICA sp.) PHYTASE

Germinated Altex and Westar (Brassica napus) and Candle and Tobin (B. campestris) cultivars of Canola were screened for phytase activity. On the basis of this preliminary screening, 7-day germinated Altex seedlings were selected as a source for isolation and characterization of phytase. Partial purification of a crude extract (FI) by acetone precipitation resulted in an 8-fold increase in phytase activity. Ion-exchange chromatography of the partially purified preparation (FII) yielded two fractions (FIIIA and FIIIB) both of which demonstrated phytase and phosphatase activities. Further purification by gel filtration chromatography resulted in two fractions (FIVA1 and FIVA2) from fraction FIIIA and two fractions (FIVB1 and FIVB2) from fraction FIIIB. Fraction FIVB1 demonstrated both phytase and phosphatase activities, FIVA2 and FIVB2 demonstrated phosphatase activity but no phytase activity and FIVA1 showed phytase but no phosphatase activity. Fraction FIVB1, which showed highest phytase activity (5.3 IU/mg protein), had the following characteristics: temperature optimum of 50°C, pH optimum of 5.2, K_m of 0.36 mM and relative activity for pyrophosphate 232 times higher than for phytate.     

内源性植酸酶对大豆乳、面团等食品中植酸的作用

天然植物食品中含有内源性植酸酶,一定条件下可以催化其中的植酸水解。实验表明,内源性植酸酶在对豆乳中的植酸进行水解时,45℃下24 h后植酸磷水解率达到了28.1%;内源性植酸酶在对面团中的植酸水解时,45℃下4 h后植酸磷水解率达到27.9%,而添加一定量酵母于面团中进行发酵处理,面团中植酸磷的水解率可以增至40.9%。所以,食品中内源性植酸酶对植酸的水解作用是存在的,但程度有限。     

Effects of Germination and Dietary Calcium on Zinc Bioavailability from Peas

Bioavailability of zinc was determined by rat weight gain and tibia Zn response from whole pea sprouts (WPS), heat treated peas (HTP) and purified diets (PD1 and 2) supplemented with Zn and phytate for similar phytate/Zn molar ratios (phy/Zn). With diets adequate Ca (0.75%), weight gains from WPS (phy/Zn = 24) and HTP (phy/Zn = 39) were equivalent or better to a nonphytate control (NPC). Tibia Zn decreased in order from: NPC, WPS, PD1, HTP and PD2. At the 1.5% Ca level reduced growth responses from WPS and HTP indicated interaction between Ca and phytate. Zn availability from peas may be superior to that from grains and other legumes previously reported. The decreased phytate and increased phytase resulting from germination may explain the enhanced responses from pea sprouts.     

Supplementation of alkaline phytase (Ds11) in whole-wheat bread reduces phytate content and improves mineral solubility

Abstract: In this study, alkaline phytase was added to whole‐wheat bread and the phytate content and mineral profiles were compared to commercially available acidic phytase. At neutral pH, some phytate (approximately 20%) was degraded by endogenous phytase in wheat flour, while 40% of phytate was hydrolyzed by alkaline phytase DS11 and a 35% reduction was observed with acidic phytase. Most of the enzymatic activity occurred during the proofing stage, and the rate of reaction depended on pH. DS11 phytase effectively degraded the phytate level within a 30 min treatment at pH 7; however, at least 60 min was needed with acidic phytase to achieve the same hydrolysis level. Mineral profiles were also dramatically affected by the phytate reduction. The biggest increase was observed in Fe²⁺ by the phytase treatment. The Fe²⁺ content increased 10‐fold at pH 7 and 8‐fold at pH 5 with alkaline phytase DS11. Alkaline phytase DS11 was shown to be effective at phytate reduction in whole‐wheat bread preparation. Additionally, phytate degradation enhanced the mineral availability of bread.     

Peniophora lycii phytase is stabile and degrades phytate and solubilises minerals in vitro during simulation of gastrointestinal digestion in the pig

BACKGROUND: Microbial phytases (EC 3.1.3) are widely used in diets for monogastric animals to hydrolyse phytate present in the feed and thereby increase phosphorus and mineral availability. Previous work has shown that phytate solubility is strongly affected by calcium in the feed and by pH in the gastrointestinal (GI) tract, which may have an effect on phytase efficacy. An in vitro model simulating the GI tract of pigs was used to study the survival of Peniophora lycii phytase and the effect of the phytase on phytate degradation, inositol phosphate formation and mineral solubilisation during in vitro digestion of a 30:70 soybean meal/maize meal blend with different calcium levels. RESULTS: The phytase retained 76 and 80% of its initial activity throughout the gastric in vitro digestion. Total phytate hydrolysis by P. lycii phytase was in the same range at total calcium levels of 1.2 and 6.2 mg g^?1 dry matter (DM), despite very large differences in phytate solubility at these calcium levels. However, at 11.2 and 21.2 mg Ca g^?1 DM, phytate hydrolysis was significantly lower. The amount of soluble mineral was generally increased by P. lycii phytase. CONCLUSION: Stability of P. lycii phytase during gastric digestion was not found to be critical for phytate hydrolysis. Furthermore, original phytate solubility was not an absolute requirement for phytate degradation; phytate solubility seemed to be in a steady state, allowing insoluble phytate to solubilise as soluble phytate was degraded. This is new and interesting knowledge that adds to the current understanding of phytate–phytase interaction. Copyright © 2007 Society of Chemical Industry     

Metabolism of extracellular inositol hexaphosphate (phytate) by Saccharomyces cerevisiae

Iron and zinc deficiencies are global problems, frequently leading to severe illness in vulnerable human populations. Addition of phytases can improve the bioavailability of iron and zinc in food. Saccharomyces cerevisiae would be an ideal candidate as a bioavailability improving food additive if it demonstrates significant phytase activity. The purpose of the paper was to study yeast phytase activity to obtain information required to improve strains. All yeasts tested readily degraded extracellular inositol hexaphosphate (phytate; IP6) in media with IP6 as the sole phosphorous source. Phosphate (Pi) addition yielded repression consistent with the PHO system. However, repression of IP6-degrading enzymes was not only dependent on level of Pi, but also on pH and medium composition. In complex medium, containing Pi at a concentration previously suggested to yield full repression of the secretory acid phosphatases (SAPs; e.g., [Mol. Biol. Cell 11 (2000) 4309]), and at relatively high pH, repression of phytate-degrading enzymes was weak. The capacity to degrade phytate, irrespective of Pi addition or not, was highest at the pH most distant from the pH optimum of the SAPs [Microbiol. Res. 151 (1996) 291], suggesting that expression rather than enzyme activity was affected by pH. In synthetic medium, repression was strong and pH-independent (no IP6 degradation within the range tested). The distinct difference between media shows that, in addition to known regulatory role of Pi for the PHO system, additional factors may be involved. Using a deletion strain, we further demonstrate that the main secretory acid phosphatase Pho5p is not essential for intact phytate-degrading capacity and growth without Pi, neither is Pho3p. However, when constitutively overexpressing PHO5 an increased net phytase activity was obtained, in repressing and non-repressing conditions. This proves that, although redundant in a wild type, Pho5p can catalyze hydrolysis of IP6 and that at least one more enzyme is capable of effective hydrolysis of IP6 (sufficient to provide the cell with phosphorous at a rate yielding maximum growth). Finally, a bread dough experiment showed that the typical concentrations of Pi during leavening exceed levels shown to repress phytate degradation by a wild-type S. cerevisiae.     

Characterization of extra- and intracellular phytases from Rhizopus oligosporus used in tempeh production

Rhizopus oligosporus strain CT11K2, commonly used in tempeh (fermented soybean) production produced both extra- and intracellular phytases. The enzymes were isolated from growth media and the cultured mould and partially purified by acetone fractionation, gel filtration on Sephadex G-100 and DEAE-cellulose chromatography. Intracellular phytase activity was higher than that of the extracellular enzyme. Both enzymes showed maximum activity at pH 4.5 and 55 degrees C, suggesting relatively high thermostability. The enzymes were partially inhibited by high concentrations of substrate. The Km and Vmax values of the extracellular phytase were 0.15 mM and 0.076 mumol Pi per min per ml DEAE-cellulose purified enzyme, respectively, and for the intracellular phytase were 0.17 mM and 0.34 mumol Pi per min per ml enzyme, respectively. Extracellular phytases showed inactivation and activation energies for the hydrolysis of phytic acid of approximately 28,300 cal per mol and 6100 cal per mol, respectively, while inactivation and activation energies for the intracellular phytase were approximately 33,200 per mol and 9500 cal per mol, respectively.     

Monitoring the stepwise phytate degradation in the upper gastrointestinal tract of pigs

The degradation and formation of inositol phosphates as affected by microbial phytase and gastrointestinal enzyme activities during the passage of phytate through the stomach and small intestine were studied in two experiments with four barrows and three collection periods. The degradation and formation of inositol phosphates were measured at the duodenal and ileal sites using Cr‐NDR, TiO₂ and Co‐EDTA as indigestible markers. In experiment 1, the effect of graded doses of Aspergillus niger phytase (0, 150 and 900 FTU Natuphos^® kg^?1), added to a maize–soybean meal‐based diet with very low intrinsic phytase activity on the degradation of phytate and the formation of inositol phosphates during digestion in the stomach and small intestine was investigated. In experiment 2, three different mixtures of inositol phosphates, produced by Aspergillus niger phytase, containing mainly high, intermediate and low phosphorylated inositol phosphates, were added to the same maize–soybean meal‐based diet as used in experiment I. The fate of the inositol phosphates during digestion in the stomach and small intestine was studied. Experiment 1 showed that the extent of phytate degradation was dependent of the graded dietary phytase activities. At high phytase activity (900 FTU kg^?1 of diet), strong phytate degradation occurred and the once hydrolysed phytate was rapidly dephosphorylated to lower inositol phosphates (mainly inositol di‐ and triphosphates). Intermediate inositol phosphates, such as inositol tetraphosphates, were quantitatively unimportant in duodenal and ileal digesta. At a phytase activity of 150 FTU kg^?1 of diet, a broader spectrum of intermediate inositol phosphates was determined, which was probably due to a slower breakdown of phytate. Experiment 2 showed as a predominant result that lower inositol phosphates such InsP₄ and InsP₃ were degraded, whereas InsP₂ accumulated in the duodenal and ileal digesta. No substantial disappearance of phytate from the stomach and small intestine was found when high concentrations of soluble phytate were added to the diet, which indicates that no substantial phytate absorption occurs in the upper part of the pig gut. Copyright © 2005 Society of Chemical Industry     

枯草芽孢杆菌B.subtilis CNMC-0014深层摇瓶发酵生产中性蛋白酶 -培养基组分及培养条件对酶生物合成的影响

应用统计学方法优化了B.sustilis CNMC-0014中性蛋白酶产生的培养基组分.单因素试验研究发现,在测试的6种碳源中,以甘油和玉米粉对产酶影响显著,酶活力分别达到了3955.16±2.15U/ml和3939.15± 1.87U/ml.氮源试验中发现,大豆粉对产酶影响最为显著,酶活力达到4318.12±5.66U/ml.通过极差分析与正交分析优化了培养基配比,优化方案为:2%玉米粉,1%甘油,3%玉米粉和3%麸皮.方差分析结果发现,甘油和玉米粉对产酶影响显著(p＜0.05),大豆粉对产酶极显著(p＜0.01), 麸皮对产酶影响不显著(p＞0.05).同时,还 研究了金属离子、溶氧、起始pH值、菌龄以及接种量对产酶的影响,研究结果表明,Ca 2 、Mg2 、Na 、Zn 、Mn2 可以在不同程度对产酶有激活作用,特别是Ca 2 ,酶活力达到了4552.97U/ml,而Fe3 、Fe2 、K 、Ag 对产酶有强烈的抑制作用,特别是Ag (酶活力仅为987.46 U/ml),最适摇瓶装量为50ml/300ml,最佳起始pH值为7.5,最佳菌龄与接种量分别为24h和3%.     

STEREOSPECIFICITY OF MY0-INOSITOL HEXAKISPHOSPHATE DEPHOSPHORYLATION BY PHYTATE-DEGRADING ENZYMES OF CEREALS

Using a combination of High‐Performance Ion Chromatography analysis and kinetic studies the stereospedfidty of myo‐inositol hexakisphosphate dephosphorylation by the spelt phytase D21, the rye phytase, the barley phytases P1 and P2, and the oat phytase was established. The data demonstrate that all the cereal phytate‐degrading enzymes under investigation dephosphorylate myo‐inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D‐Ins(l,2, 3,5, 6)P₅, D‐Ins(l,2, 5,6)P₄, D‐Ins(l,2, 6)P₃, D‐Ins(l,2)P₂ to finally Ins(2)P (notation 4/3/5/6/1).     

一株产植酸酶酵母菌株的分类鉴定与诱变育种

根据酵母形态学特征、生理生化特性和ITS区序列的测定结果把产植酸酶酵母RCEF4907菌株分类鉴定为东方伊萨酵母Issatchenkia orientalis。利用亚硝基胍、紫外线、亚硝酸和氯化锂4种诱变剂对酵母RCEF4907菌株进行复合诱变处理,将经过处理的菌悬液用植酸钙琼脂平板进行初筛,然后取发酵上清液在37℃,pH2.5的条件下,利用钒钼酸铵比色法测定植酸酶活力进行复筛。经过多次诱变处理后,最终从大量的突变菌株中筛选出高产植酸酶的G3297突变株,其植酸酶活性达到409 U/mL,是出发菌株RCEF4907的5.76倍。