Simultaneous degradation of phytic acid and starch by an industrial strain of Saccharomyces cerevisiae producing phytase and α-amylase

Phytase liberates inorganic phosphate from phytic acid (myo-inositol hexakisphosphate) which is the major phosphate reserve in plant-derived foods and feeds. An industrial strain of Saccharomyces cerevisiae expressing the Debaryomyces castellii phytase gene (phytDc) and D. occidentalis α-amylase gene (AMY) was developed. The phytDc and AMY genes were constitutively expressed under the ADC1 promoter in S. cerevisiae by using the δ-integration system, which contains DNA derived exclusively from yeast. The recombinant industrial strain secreted both phytase and α-amylase for the efficient degradation of phytic acid and starch as main components of plant seeds. This new strain hydrolyzed 90% of 0.5% (w/v) sodium phytate within 5 days of growth and utilized 100% of 2% (w/v) starch within 48 h simultaneously.     

The sweet potato sporamin promoter confers high-level phytase expression and improves organic phosphorus acquisition and tuber yield of transgenic potato

The sweet potato sporamin promoter was used to control the expression in transgenic potato of the E. coli appA gene, which encodes a bifunctional enzyme exhibiting both acid phosphatase and phytase activities. The sporamin promoter was highly active in leaves, stems and different size tubers of transgenic potato, with levels of phytase expression ranging from 3.8 to 7.4% of total soluble proteins. Phytase expression levels in transgenic potato tubers were stable over several cycles of propagation. Field tests showed that tuber size, number and yield increased in transgenic potato. Improved phosphorus (P) acquisition when phytate was provided as a sole P source and enhanced microtuber formation in cultured transgenic potato seedlings when phytate was provided as an additional P source were observed, which may account for the increase in leaf chloroplast accumulation (important for photosynthesis) and tuber yield of field-grown transgenic potato supplemented with organic fertilizers. Animal feeding tests indicated that the potato-produced phytase supplement was as effective as a commercially available microbial phytase in increasing the availability of phytate-P to weanling pigs. This study demonstrates that the sporamin promoter can effectively direct high-level recombinant protein expression in potato tubers. Moreover, overexpression of phytase in transgenic potato not only offers an ideal feed additive for improving phytate-P digestibility in monogastric animals but also improves tuber yield, enhances P acquisition from organic fertilizers, and has a potential for phytoremediation.     

黑曲霉N25植酸酶phyA基因的克隆及序列分析

通过对黑曲霉N2 5植酸酶phyA基因PCR扩增 ,获得了一条长约 1 6kb的特异性PCR产物 ,并进行了酶切鉴定。然后在pUC1 8质粒中构建了含有目的基因片段的克隆质粒pFNP 1。DNA序列测定表明 ,目的基因片段含有植酸酶phyA基因的完整序列 ,phyA基因全长1 50 6bp,其中包含一段长 1 0 2bp的内含子 ,编码 467个氨基酸 ,5’端有一段编码 1 9个氨基酸的信号肽序列。黑曲霉N2 5与产植酸酶酶活最高的天然黑曲霉标准菌株NRRL31 35的植酸酶phyA基因 (GenBankAccession :M94550 )相比较 ,其同源性为 96 746% ,编码的氨基酸序列同源性为 97 64%。将黑曲霉N2 5植酸酶phyA基因序列及其相应的氨基酸序列在国际基因库中注册 (注册号分别为 :AF2 1 881 3,AAF2 5481 1 ) ,此基因是目前中国在国际基因库中注册的第一个植酸酶phyA基因。     

Site-directed mutagenesis of Aspergillus niger NRRL 3135 phytase at residue 300 to enhance catalysis at pH 4.0

Increased phytase activity for Aspergillus niger NRRL 3135 phytaseA (phyA) at intermediate pH levels (3.0-5.0) was achieved by site-directed mutagenesis of its gene at amino acid residue 300. A single mutation, K300E, resulted in an increase of the hydrolysis of phytic acid of 56% and 19% at pH 4.0 and 5.0, respectively, at 37 degrees C. This amino acid residue has previously been identified as part of the substrate specificity site for phyA and a comparison of the amino acid sequences of other cloned fungal phytases indicated a correlation between a charged residue at this position and high specific activity for phytic acid hydrolysis. The substitution at this residue by either another basic (R), uncharged (T), or acidic amino acid (D) did not yield a recombinant enzyme with the same favorable properties. Therefore, we conclude that this residue is not only important for the catalytic function of phyA, but also essential for imparting a favorable pH environment for catalysis.     

A Novel Phytase appA from <Emphasis Type="Italic">Citrobacter amalonaticus</Emphasis> CGMCC 1696: Gene Cloning and Overexpression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A novel phytase gene appA, with upstream and downstream sequences from Citrobacter amalonaticus CGMCC 1696, was cloned by degenerate polymerase chain reaction (PCR), and thermal asymmetric interlaced (TAIL) PCR and was overexpressed in Pichia pastoris. Sequence analysis revealed one open reading frame that consisted of 1311 bp encoding a 436–amino-acid protein, which had a deduced molecular mass of 46.3 kDa. The phytase appA belongs to the histidine acid phosphatase family and exhibits the highest identity (70.1%) with C. braakii phytase. The gene was overexpressed in P. pastoris. The secretion yield of recombinant appA protein was accumulated to approximately 4.2 mg·mL⁻¹, and the enzyme activity level reached 15,000 U·mL⁻¹, which is higher than any previous reports. r-appA was glycosylated, as shown by Endo H treatment. r-appA was purified and characterized. The specific activity of r-appA for sodium phytate was 3548 U·mg⁻¹. The optimum pH and temperature for enzyme activity were 4.5 and 55°C, respectively. r-appA was highly resistant to pepsin or trypsin treatment. This enzyme could be an economic and efficient alternative to the phytases currently used in the feed industry.     

Recombinant production of <Emphasis Type="Italic">Penicillium oxalicum</Emphasis> PJ3 phytase in <Emphasis Type="Italic">Pichia Pastoris</Emphasis>

The 1,332 bp phytase gene of Penicillium oxalicum PJ3 was inserted into the expression vector, pPICZαA and expressed in the methylotrophic yeast, Pichia pastoris as an active, extracellular phytase. The recombinant phytase reached a maximum yield of 12 U/ml of medium at 120 h of cultivation after methanol induction under shake-flask conditions. The enzyme was glycosylated, with a molecular mass of about 62.5 kDa. The Michaelis constant (K _m) and maximum reaction rate (V _max) for sodium phytate was 0.37 mM and 526.3 U/mg of protein, respectively. The optimal activity occurred at pH 4.5 and 55°C. Jaecheon Lee and Yunjaie Choi contributed equally to this work.     

Identification and characterization of a phytase of potential commercial interest

Phytases catalyse the hydrolytic degradation of phytic acid and its salts and are added to monogastric animal feed to ameliorate the negative environmental and nutritional consequences of dietary phytate. Screening of 58 microbial strains identified a phytase produced by Rhizopus oligosporus ATCC 22959 that displayed physicochemical characteristics likely to render it of potential industrial interest. The 124 kDa enzyme was purified to homogeneity by anion exchange chromatography, gel filtration and chromatofocusing. The monomeric glycosylated enzyme (30.5% total carbohydrate) displayed maximum activity at 65 °C and pH 5.0. It displayed a K_m of 10.4 μM, a V_max of 1.32 nmol s⁻¹ and a K_cat of 51 s⁻¹. It is acid tolerant, retaining full activity after incubation at pH 2.0 for 6 h. HPLC analysis indicated the enzyme’s ability to almost completely degrade phytate. Substrate specificity studies showed its ability to dephosphorylate several additional phosphorylated molecules. Activity was unaffected or moderately stimulated by a range of metal ions with only Ca²⁺ exerting a modest (13%) inhibitory effect. The enzyme is significantly more thermostable at 80 °C and retains a significantly greater proportion of maximal activity at physiological temperatures than do two commercial phytases tested for comparative purposes. This may render it of industrial interest.     

Root surface phosphatase activities and uptake of 32P-labelled inositol phosphate in field-collected gray birch and red maple roots

 This study examined select, naturally-occurring tree mycorrhizae for differences related to efficiency of organic phosphorus hydrolysis in forest soils. We investigated the activity of several phosphatases and root respiration in field-collected ectomycorrhizae of American beech and gray birch and VAM of red maple. Root materials were collected in the early and late growing season from a common soil type. American beech occurred in a late-successional stand, whereas gray birch and red maple grew in a mid-successional stand. All of the root types examined had phosphatase activities with p-nitrophenyl phosphate, bis-p-nitrophenyl phosphate and phytic acid and thus the potential to mineralize monoester and diester forms of organic phosphorus. Rates of hydrolysis at pH 5.0 were greatest with p-nitrophenyl phosphate. Although enzyme activity varied with season and ectomycorrhizal morphotype, VAM roots of red maple consistently had the lowest enzyme activities on a length and dry weight basis. Comparison of ³²P uptake from inositol phosphate by gray birch and red maple roots suggested that phosphomonoesterase activity was linked to P uptake from this source. Differences between species in oxygen consumption rates were less pronounced than those observed for enzymatic activities, suggesting similar short-term energy demands by the root types examined. The quantitative differences observed between plants growing on a common soil potentially relate to differences in host demand or reflect differences in basic morphology and/or physiology of associated mycobionts. Further study is necessary to understand the importance of these enzymes in the functional ecology of mycorrhizal fungi. Accepted: 20 December 1996     

Cloning of the thermostable phytase gene (phy) from Bacillus sp. DS11 and its overexpression in Escherichia coli

Phytase hydrolyzes phytate to release inorganic phosphate, which would decrease the addition of phosphorus to feedstuffs for monogastric animals and thus reduce environmental pollution. The gene encoding phytase from Bacillus sp. DS11 was cloned in Escherichia coli and its sequence determined. A 560-bp DNA fragment was used as a probe to screen the genomic library. It was obtained through PCR of Bacillus sp. DS11 chromosomal DNA and two oligonucleotide primers based on N-terminal amino acid sequences of the purified protein and the cyanogen bromide-cleaved 21-kDa fragment. The phy cloned was encoded by a 2.2-kb fragment. This gene comprises 1152 nucleotides and encodes a polypeptide of 383 amino acids with a deduced molecular mass of 41 808 Da. Phytase was produced to 20% content of total soluble proteins in E. coli BL21 (DE3) using the pET22b(+) vector with the inducible T7 promoter. This is the first nucleic sequence report on phytase from a bacterial strain.     

Performance and total-tract digestibility responses to exogenous xylanase and phytase in diets for growing pigs

Sixty individually housed male pigs (Large White X Landrace, average weight 19.7±0.56 kg) were used in a completely randomised block design having five wheat-based dietary treatments (n=12 pigs per treatment) to study the interactive effects between added xylanase (XYL) and phytase (PHY), either alone or in combination, and days on trial on coefficient of total-tract digestibility (CTTAD) and performance. The diets used were: (i) positive control (PC); (ii) negative control with reduced energy and mineral content (NC); (iii) NC with added XYL (NCX); (iv) NC with added PHY (NCP); and (v) NC with added XYL plus PHY (NCXP). The performance response and CTTAD of dietary components were measured 3 and 7 weeks after commencement of feeding, with the experiment finishing when pigs reached approximately 65 kg live weight.Pigs fed the NC diet decreased average daily gain (ADG) by 13% (P<0.01) and increased feed conversion ratio (FCR) by 11% (P<0.01) compared to pigs fed the PC diet. Addition of PHY in the NC diet increased ADG by 9% (P<0.05) and decreased FCR by 5% (P=0.065), whilst addition of XYL in the NC diet had no effect on the performance indices. Combined addition of XYL and PHY in the NC diet did not improve performance of pigs for the first 21 days but improved FCR by 8% (P=0.008) during the days 22–49. Digestibility measurements showed that combined use of XYL and PHY in the NC diet improved CTTAD of dry matter (DM, P<0.05), gross energy (GE, P<0.05) and crude protein (CP, P<0.01) only during the days 22–49. Independent supplementation of PHY in the NC diet tended to improve CTTAD of GE (P=0.09) only during the days 22–49. In contrast, supplementation of PHY immediately improved CTTAD of P and maintained it to similar levels at 49 days. Results suggested that supplementation of PHY plus XYL in combination in the NC diet improved FCR and CTTAD of DM, GE, CP and P but the beneficial effects other than CTTAD of P were derived mainly during days 22–49.