appA Gene Research Articles

Effect of Phytase Derived from the E. coli AppA Gene on Weaned Piglet Performance, Apparent Total Tract Digestibility and Bone Mineralization.

Simple SummaryPlants store phosphorus mainly in a form that is indigestible to pigs: phytate. Unabsorbed minerals excreted into the environment are considered to be environmental pollutants. Our study focuses on the effects of novel types of phytase enzymes in diets deficient in external phosphorus on the performance, digestibility of crude protein, dry matter and minerals, and bone mineralization of weaned piglets. Using novel phytases, we noted a positive impact on body weight gain, feed conversion ratio, the digestibility of phosphorus and calcium, and phosphorus and calcium content in bones. There was no significant improvement in the content of dry matter or digestion of crude protein. This study reveals novel phytases to be useful additives for weaned piglet diets because of their superior performance results and phosphorus absorption.The objective of this study was to assess the effect of novel appAT1 and appAT2 phytase inclusion at 250 phytase units (FTU)/kg on weaned piglet performance, the apparent total tract digestibility of P and Ca, and bone mineralization. Piglets (48 males) were randomly divided into four treatment groups: a positive control (PC), with recommended levels of phosphorus (P) and calcium (Ca), a negative control (NC) deficient in P and Ca, and two experimental groups with NC diets supplemented with phytase derived from the appA gene of Escherichia coli. Diets fed in a mashed form were divided into prestarter (0–21 days) and starter (22–42 days) periods. During the whole period of the study, the experimental diets improved (p < 0.05) the body weight gain (BWG) and feed conversion ratio (FCR) compared to the NC diet. The apparent total tract digestibility (ATTD) of the dry matter and crude protein was not significantly different among the diets. Phytase-supplemented diets improved the ATTD of P (p < 0.05) and the ATTD of Ca (p < 0.05). Bone ash content in the third metacarpal and P and Ca content were improved among the phytase supplemented diets compared to the NC diet.

Transgenic soybean expressing a thermostable phytase as substitution for feed additive phytase

Phytase is one of the most effective feed additives to increase the availability of phosphorus and minerals by catalyzing the hydrolysis of phytic acid. A modified appA gene (mappA) was transformed into soybean (Glycine max) under the control of a seed-specific promoter from common bean (Phaselous vulgaris). The soybean recombinant phytase showed optimal activity at pH 4.5 and 70 °C. A slight increase in enzyme activity occurred when the recombinant enzyme was pre-incubated with n-hexane. In addition, the phytase activity from our transgenic soybean does not reduce even after 2 hours of extraction with n-hexane at 55~65 °C. In conclusion, the oil extraction process using n-hexane does not inactivate the phytase expressed in the mAppA transgenic soybean, and the meal derived from the transgenic soybean processing can be used as feed supplement to livestock.

Genetically engineered rice with appA gene enhanced phosphorus and minerals

Phytic acid is the major source of phosphorus and other mineral bound compounds in many plant tissues especially seeds and bran of cereals. During germination, phytase enzyme degrades phytic acid and bound phosphate and minerals are released. The monogastric animal cannot digest phytate due to lack of the phytase enzyme. Considering that, we have generated low phytate rice by over expressing appA gene cloned from E. coli under the aleurone-specific promoter of maize zein gene. Molecular analysis confirmed the stable integration of transgene and plants were grown up to T3 generation. The T3 seeds showed around 45% decrease in seed phytate content with a fourfold increase of inorganic phosphorus (Pi) level. The enhanced iron and zinc was twofold and threefold respectively in polished seeds of transgenic plants. There was no change in germination behaviour and other morphological traits in transgenic seeds. Thus, this result provides evidence that tissue-specific expression of bacterial phytase can lead to the reduction of phytic acid in rice seeds without hampering its other physiological processes and phenotypic cost.

Effect of different phytases derived from E. coli AppA gene on the performance, bone mineralisation and nutrient digestibility of broiler chicken

AbstractThis study evaluated the effects of three different thermostable phytase variants, based on the AppA gene fromE. coli(AppAT1, AppAT2 and AppAT3) on growth performance, nutrient digestibility and bone mineralisation in broiler chickens at inclusion levels of 250 and 500 FTU/kg. The eight treatment groups included a positive control (PC) which was sufficient in Ca and P, a negative control (NC, the same basal formulation as the PC, but reduced in Ca and P), and NC supplemented with AppAT1 at 250 and 500 FTU/kg (AppAT1-250 and AppAT1-500), AppAT2 at 250 and 500 FTU/kg (AppAT2-250 and AppAT2-500) and with AppAT3 at 250 and 500 FTU/kg (AppAT3-250 and AppAT3-500). Over the entire feeding period, body weight (BW) and average daily gain (ADG) were significantly higher in the PC group, with all phytase supplemented groups being statistically the same, compared to the NC group. Feed conversion (FCR) for the PC-fed birds (1.479) was significantly (P&amp;lt;0.05) better compared to the NC birds (1.582) and those fed the AppAT3-250 diet (1.523). Reduced levels of Ca and P in the NC group led to significantly (P&amp;lt;0.05) lower tibia ash (40.9%) compared to the PC group (47.4%). Birds fed the phytase diets had significantly higher tibia ash compared to the NC birds, with those from the AppAT2-500 and AppAT3-500 groups being statistically the same as the PC group. Diets AppAT1-500, AppAT2-250, AppAT2-500 and AppAT3-500 significantly increased Ca digestibility compared to the NC. Apparent total track digestibility (ATTD) of P was improved for AppAT1-500 and AppAT2-250. The ATTD of Ca and P for all of the phytase supplemented groups reached the same level of the PC and AppAT1-500 group. It was concluded that adding any of the phytases tested, especially when included at 500 FTU/kg to a feed reduced in Ca and P, led to improved performance and bone mineralisation back to the same levels as seen for the Ca and P sufficient diet.

Ensifer meliloti overexpressing Escherichia coli phytase gene (appA) improves phosphorus (P) acquisition in maize plants.

The Escherichia coli phytase gene appA encoding enzyme AppA was cloned in a broad host range plasmid pBBR1MCS2 (lac promoter), termed pVA1, and transformed into the Ensifer meliloti 1020. Transformation of pVA1 in Ensifer meliloti {E. m (pVA1)} increased its phosphatase and phytase activity by ∼9- and ∼50-fold, respectively, compared to the transformants containing empty plasmid as control {E. m (pBBR1MCS2)}. The western blot experiments using rabbit anti-AppA antibody showed that AppA is translocated into the periplasm of the host after its expression. Ensifer meliloti harboring AppA protein {E. m (pVA1)} and {E. m (pBBR1MCS2)} could acidify the unbuffered phytate minimal media (pH 8.0) containing Ca-phytate or Na-phytate as sole organic P (Po) source to below pH 5.0 and released P. However, both {E. m (pVA1)} and {E. m (pBBR1MCS2)} neither dropped pH of the medium nor released P when the medium was buffered at pH 8.0 using Tris-Cl, indicating that acidification of medium was important for the enzymatic hydrolysis of phytate. Further experiments proved that maize plants inoculated with {E. m. (pVA1)} showed increase in growth under sterile semi solid agar (SSA) medium containing Na-phytate as sole P source. The present study could be helpful in generating better transgenic bioinoculants harboring phosphate mineralization properties that ultimately promote plant growth.

Optimization of Recombinant Expression of Synthetic Bacterial Phytase in Pichia pastoris Using Response Surface Methodology.

Background:Escherichia coli phytase is an acidic histidine phytase with great specific activity. Pichia pastoris is a powerful system for the heterologous expression of active and soluble proteins which can express recombinant proteins in high cell density fermenter without loss of product yield and efficiently secrete heterologous proteins into the media. Recombinant protein expression is influenced by expression conditions such as temperature, concentration of inducer, and pH. By optimization, the yield of expressed proteins can be increase. Response surface methodology (RSM) has been widely used for the optimization and studying of different parameters in biotechnological processes.Objectives:In this study, the expression of synthetic appA gene in P. pastoris was greatly improved by adjusting the expression condition.Materials and Methods:The appA gene with 410 amino acids was synthesized by P. pastoris codon preference and cloned in expression vector pPinkα-HC, under the control of AOX1 promoter, and it was transformed into P. pastoris GS115 by electroporation. Recombinant phytase was expressed in buffered methanol-complex medium (BMMY) and the expression was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and enzymatic assay. To achieve the highest level of expression, methanol concentration, pH and temperature were optimized via RSM. Finally, the optimum pH and temperature for recombinant phytase activity was determined.Results:Escherichia coli phytase was expressed in P. pastoris under different cultivation conditions (post-induction temperature, methanol concentration, and post-induction pH). The optimized conditions by RSM using face centered central composite design were 1% (v/v) methanol, pH = 5.8, and 24.5°C. Under the optimized conditions, appA was successfully expressed in P. pastoris and the maximum phytase activity was 237.2 U/mL after 72 hours of expression.Conclusions:By optimization of recombinant phytase expression in shake flask culture, we concluded that P. pastoris was a suitable host for high-level expression of phytase and it can possess high potential for industrial applications.

238 PRODUCTION OF CLEAVAGE-RESISTANT PHYTASE TRANSGENIC PIGS BY HANDMADE CLONING

Rapidly developing and intensive animal husbandry of livestock is a major contributor to global environmental pollutions. Large quantities and high concentrations of manure waste that contains phytate phosphorus are generated. The use of phytase can effectively solve the problem of high phosphorus pollution in the fecal material of monogastric animals. Enviropigs, producing phytase in the salivary glands and secreting the enzyme in the saliva, were first generated at the University of Guelph (Guelph, ON, Canada) in 1999. However, phytase is easily inactivated in digestive processing. To address this problem, we improved the transgene construct and successfully generated phytase transgenic pigs by handmade cloning. The Escherichia coli periplasmic phosphoanhydride phosphohydrolase (appA) gene was subcloned. Using fragment substitution method, we designed a phytase gene that was insensitive to cleavage by pepsin and trypsin and had a higher affinity for the substrate. After codon optimization, the designed phytase gene was named Cafp and subcloned downstream of the pig parotid secretory protein (PSP) gene promoter. The tissue-specific vector p-PSP-Intron-Cafp was constructed and transferred into Landrace fetal fibroblasts by electroporation. The cell lines carrying Cafp were used as nuclear donors in handmade cloning. Cloned embryos were cultured in vitro to blastocysts and transferred to recipient sows. The presence of Cafp was tested by PCR and sequencing of cloned pigs. Phytase activity in saliva, feed, and feces was detected by the ammonium molybdate method with a slight modification. Immunohistochemistry (IHC) was used to determine tissue-specific expression. Three cell lines carrying Cafp were obtained. We generated 1027 blastocysts; 712 were of good quality and transferred to 6 recipients. Fourteen piglets were born, of which 6 survived. The PCR and sequencing results showed that 7 (3 live and 4 dead) of the 14 piglets carried Cafp. Phytase activity in the saliva of the 6 live cloned pigs was tested at 4 months of age and only 1 pig had 0.155 FTU mL–1 enzyme activity. The enzyme in the other 2 pigs may be inactivated in the transgenic parotid gland. Among all the transgenic pigs, the highest phosphorus digestion rate was 59.2% of intake, which represents a 25.4% decrease in fecal emissions compared with the average of controls. The IHC results on the 3 later dead, Cafp-positive pigs showed that the transgene was expressed only in parotids, confirming tissue-specific gene expression. In summary, cleavage-resistant phytase transgenic pigs were successfully produced through handmade cloning. The cloned pigs offer a unique biological approach to manage phosphorus nutrition and environmental pollution in animal husbandry.

A Phytase-Based Reporter System for Identification of Functional Secretion Signals in Bifidobacteria.

Health-promoting effects have been attributed to a number of Bifidobacterium sp. strains. These effects as well as the ability to colonise the host depend on secreted proteins. Moreover, rational design of protein secretion systems bears the potential for the generation of novel probiotic bifidobacteria with improved health-promoting or therapeutic properties. To date, there is only very limited data on secretion signals of bifidobacteria available. Using in silico analysis, we demonstrate that all bifidobacteria encode the major components of Sec-dependent secretion machineries but only B. longum strains harbour Tat protein translocation systems. A reporter plasmid for secretion signals in bifidobacteria was established by fusing the coding sequence of the signal peptide of a sialidase of Bifidobacterium bifidum S17 to the phytase gene appA of E. coli. The recombinant strain showed increased phytase activity in spent culture supernatants and reduced phytase levels in crude extracts compared to the control indicating efficient phytase secretion. The reporter plasmid was used to screen seven predicted signal peptides in B. bifidum S17 and B. longum E18. The tested signal peptides differed substantially in their efficacy to mediate protein secretion in different host strains. An efficient signal peptide was used for expression and secretion of a therapeutically relevant protein in B. bifidum S17. Expression of a secreted cytosine deaminase led to a 100-fold reduced sensitivity of B. bifidum S17 to 5-fluorocytosine compared to the non-secreted cytosine deaminase suggesting efficient conversion of 5-fluorocytosine to the cytotoxic cancer drug 5-fluorouracil by cytosine deaminase occurred outside the bacterial cell. Selection of appropriate signal peptides for defined protein secretion might improve therapeutic efficacy as well as probiotic properties of bifidobacteria.

The Establishment of Double-Transgenic Mice that Co-Express the appA and MxA Genes Mediated by Type A Spermatogonia In vivo

Type A spermatogonial stem cells are the only immortal diploid cells in the postnatal animal that undergo self-renewal through the lifetime of an animal and transmit genes to subsequent generations. In this paper, the generation and characterization of double-transgenic mice co-expressing the Escherichia coli appA gene and human MxA gene generated via the in vivo transfection of type A spermatogonial cells were reported for the first time. The dicistronic expression vector pcDNA-appA-MxA(AMP) and ExGen500 transfection reagent were injected into the testicular tissue of 7-d-old male ICR mice. The mice that underwent testis-mediated gene transfer were mated with wild-type female mice, and the integration and expression of the foreign genes in the offspring were evaluated. Transgenic mice that co-expressed appA and MxA showed a gene integration rate of 8.89% (16/180). The transgenic mice were environmentally friendly, as the amount of phosphorous remaining in the manure was reduced by as much as 11.1% by the appA gene (P<0.05); these animals also exhibited a strong anti-viral phenotype.

Modifying Thermostability of appA from Escherichia coli

In order to improve the thermostability of Escherichia coli AppA phytase, Error-prone PCR was used to randomize mutagenesis appA gene, and a gene mutation library was constructed. A mutant I408L was selected from the library by the method of high-throughput screening with 4-methyl-umbelliferylphosphate (4-MUP). The appA gene of the mutant was cloned and expressed in E. coli Origami (DE3). The recombinant protein was purified by Ni-affinity chromatography, and the enzymatic features were analyzed. The results indicated that AppA phytase activities of mutant I408L and wild-type (WT) strain remained at 51.3 and 28%, respectively, after treatment at 85°C for 5min. It means that the thermostability enhancement of AppA phytase I408L was 23.3% more as compared with WT. The K (m) of both phytase were 0.18 and 0.25mM, respectively, which indicated that the catalyzing efficiency of I408L was improved. AppA phytase of mutant I408L showed a significant enhancement against trypsin, which was nearly three times compared with WT. In addition, AppA phytase of mutant could be activated by Mg(2+) and Mn(2+); in contrast, it could be inhibited by Ca(2+), Co(2+), Cu(2+), and K(+) in varying degrees, and the enzymatic activity was almost lost the presence of Fe(3+) and Zn(2+). It appears that screening thermotolerant phytase of E. coli by high throughput screening with a fluorescence substrate is a fast, simple, and effective method. The mutant I408L obtained in this study could be used for the large-scale commercial production of phytase.

A new method for gene synthesis and its high-level expression

An optimized Citrobacter braakii phytase gene, appA-c, was chemically synthesized by oligonucleotides synthesis and over-lap PCR method. The appA-c gene encoding 423 amino acids was cloned into expression vector pPIC9 and transformed into methylotropic yeast Pichia pastoris. From about 2000 transformants, 400 transformants exhibiting phytase activity were obtained. One transformant showing the strongest phytase activity was selected for detailed analyses in 5 L bioreactor. Under control of the highly-inducible alcohol oxidase gene (AOX1) promoter, the transformant was able to secrete 3.85 mg/ml protein to the culture supernatant in about 110 h methanol induction, which comprises of 12,116 U ml − 1 phytase activity. Further characterization of the recombinant phytase was conducted. The optimal pH and temperature for this recombinant phytase was about 4.0 and 50 °C, respectively. Fe 3+, Zn 2+ and Cu 2+ could significantly inhibit the recombinant phytase enzyme activity. The specific activity of this recombinant enzyme was 3147 U mg − 1 . The K m and V max values for sodium phytate were determined to be 0.5 mM and 3085 U/mg, respectively. To our knowledge, this is the first report of a chemically synthesized C. braakii appA gene heterologous expression with the highest expression level and highest phytase activity achieved. The novel gene optimization and synthesis method can be applied to other related researches.

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.

A highly pH-stable phytase from Yersinia kristeensenii: Cloning, expression, and characterization

The gene appA, encoding a phytase from Yersinia kristeensenii, was cloned and heterologously expressed in Pichia pastoris. The open reading frame of appA comprised 1326 bp encoding a protein of 441 amino acids including a 24-amino acid signal peptide. The encoded phytase, APPA, was 87% identical to the phytase from Y. intermedia but was <52% identical to other histidine acid phosphatases. The purified recombinant phytase had an optimal activity at 55 °C and pH 4.5, and it exhibited enzymatic activity between pH 2.0 and 6.5, with a specific activity of 2656 U mg −1 at pH 4.5 and 37 °C. r-APPA retained more than 90% of its initial activity after being incubated under varying pH conditions (pH 1.5–11.0) at 37 °C for 3 h. r-APPA was resistant to heat inactivation, as it retained 46% of its initial activity after incubation at 80 °C for 10 min. r-APPA was effective for the hydrolysis of phytate phosphorus from soybean meal in vitro. Comparison of r-APPA with other well-known phytases suggests that the Y. kristeensenii phytase would be an attractive enzyme for feed industry use.

A novel phytase gene appA from Buttiauxella sp. GC21 isolated from grass carp intestine

A novel phytase gene, appA, was isolated by degenerate PCR and genomic library screening from Buttiauxella sp. GC21, a bacterium isolated from grass carp intestine. The full-length gene consists of an open reading frame of 1341 bp and encodes 446 amino acids, including 33 amino acid residues of a putative signal peptide. Containing the active site of typical histidine acid phosphatases, the amino acid sequence shows maximum identity (71%) to PhyA from Obesumbacterium proteus. The gene encoding the mature phytase was expressed in Escherichia coli and the purified recombinant APPA had a specific activity for sodium phytate of 1180 U mg−1 of protein. The optimum temperature was 55 °C and the optimum pH was 4.5. The Km value was 0.36 mM, with a Vmax of 1.39 mmol min−1 mg−1. This is the first report to detect and isolate a phytase from Buttiauxella sp.

Quantitative Conversion of Phytate to Inorganic Phosphorus in Soybean Seeds Expressing a Bacterial Phytase

Phytic acid (PA) contains the major portion of the phosphorus in the soybean (Glycine max) seed and chelates divalent cations. During germination, both minerals and phosphate are released upon phytase-catalyzed degradation of PA. We generated a soybean line (CAPPA) in which an Escherichia coli periplasmic phytase, the product of the appA gene, was expressed in the cytoplasm of developing cotyledons. CAPPA exhibited high levels of phytase expression, >or=90% reduction in seed PA, and concomitant increases in total free phosphate. These traits were stable, and, although resulted in a trend for reduced emergence and a statistically significant reduction in germination rates, had no effect on the number of seeds per plant or seed weight. Because phytate is not digested by monogastric animals, untreated soymeal does not provide monogastrics with sufficient phosphorus and minerals, and PA in the waste stream leads to phosphorus runoff. The expression of a cytoplasmic phytase in the CAPPA line therefore improves phosphorus availability and surpasses gains achieved by other reported transgenic and mutational strategies by combining in seeds both high phytase expression and significant increases in available phosphorus. Thus, in addition to its value as a high-phosphate meal source, soymeal from CAPPA could be used to convert PA of admixed meals, such as cornmeal, directly to utilizable inorganic phosphorus.

Toxicological evaluation of transgenic rice flour with an Escherichia coli phytase gene appA by sub‐chronic feeding study in Wistar rats

AbstractBACKGROUND: Phytase is increasingly used as an ingredient in swine and poultry feeds to improve the bioavailability of phytate‐bound phosphorus and reduce the supplement of elemental phosphorus in feeds. Recently, a transgenic rise (TGR) with phytase appA gene from E. coli has been developed in Taiwan. To assure the food safety of this TGR, we performed a sub‐chronic whole‐food feeding study with Wistar rats.RESULTS: Weaned feeding male and female rats were divided into TGR and non‐TGR (NTGR) groups (20 rats per group), and fed diets containing 76.8% rice flour of TGR and NTGR, respectively. After 93 days feeding, although some hematological findings and blood chemistry values differed significantly between the TGR and NTGR groups, all values were still within the normal range for rats of this age and sex. No adverse effects of TGR were observed in terms of animal behavior, weight gain, or feed utilization rate. Necropsy at the end of the experiment indicated that neither pathological lesions nor histopathological abnormalities were present in organs such as liver, kidney, intestines and testes of rats in TGR and NTGR groups. In addition, no significant differences (P &gt; 0.05) were observed in relative organ weights, hemograms and blood indices of rats between the two groups.CONCLUSION: These results demonstrated that in a 93‐day feeding study with Wistar rats a diet containing 76.8% of TGR flour having a phytase activity of about 1500 U kg−1 body weight per day has no adverse effects. Copyright © 2007 Society of Chemical Industry

A Novel Phytase appA from Citrobacter amalonaticus CGMCC 1696: Gene Cloning and Overexpression in Pichia pastoris

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 x 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 degrees 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.

Conserved Role of the Linker α-Helix of the Bacterial Disulfide Isomerase DsbC in the Avoidance of Misoxidation by DsbB

In the bacterial periplasm the co-existence of a catalyst of disulfide bond formation (DsbA) that is maintained in an oxidized state and of a reduced enzyme that catalyzes the rearrangement of mispaired cysteine residues (DsbC) is important for the folding of proteins containing multiple disulfide bonds. The kinetic partitioning of the DsbA/DsbB and DsbC/DsbD pathways partly depends on the ability of DsbB to oxidize DsbA at rates >1000 times greater than DsbC. We show that the resistance of DsbC to oxidation by DsbB is abolished by deletions of one or more amino acids within the alpha-helix that connects the N-terminal dimerization domain with the C-terminal thioredoxin domain. As a result, mutant DsbC carrying alpha-helix deletions could catalyze disulfide bond formation and complemented the phenotypes of dsbA cells. Examination of DsbC homologues from Haemophilus influenzae, Pseudomonas aeruginosa, Erwinia chrysanthemi, Yersinia pseudotuberculosis, Vibrio cholerae (30-70% sequence identity with the Escherichia coli enzyme) revealed that the mechanism responsible for avoiding oxidation by DsbB is a general property of DsbC family enzymes. In addition we found that deletions in the linker region reduced, but did not abolish, the ability of DsbC to assist the formation of active vtPA and phytase in vivo, in a DsbD-dependent manner, revealing that interactions between DsbD and DsbC are also conserved.

A Pichia pastoris fermentation strategy for enhancing the heterologous expression of an Escherichia coli phytase

The Escherichia coli phytase gene appA was highly expressed in the methylotrophic yeast Pichia pastoris under the control of the AOX1 promoter. Replacement of culture medium with fresh medium in order to remove repressing glycerol and metabolic wastes prior to methanol induction significantly improved phytase expression. The phytase activity level was enhanced from 118 to 204 U/ml at the flask scale and 1880–4946 U/ml for high cell-density fermentation, respectively, by appropriately modifying the medium composition and fermentation strategy. Most of the protein in the culture supernatant was recombinant phytase, the enzyme characteristics of which were similar to native E. coli phytase.

Safety evaluation of a phytase, expressed in Schizosaccharomyces pombe, intended for use in animal feed

BD006 phytase is the product of the Escherichia coli B appA gene expressed in Schizosaccharomyces pombe strain ASP595-1. This enzyme preparation is intended for use in animal feed applications where it improves the bioavailability of phosphate for monogastric animals. BD006 phytase was tested as an unrefined (DV006U) and a refined (DV006R) preparation for its effects on genotoxicity and in acute, inhalation and subchronic toxicity studies. Dosages ranged from 5000 μg/plate for in vitro toxicity studies to a high of 2000 X for oral in vivo toxicity studies. The highest oral dose tested (2000 X) is 2000 times the highest expected consumption of product by poultry or swine ( X=50 units of phytase per kg bwt/day). There was no genotoxicity or any in vivo toxicity reported which could be directly related to systemic effects of the product. Other additional safety studies conducted were devoid of any relevant toxicity. The historic safety profile of the production organism S. pombe, and the results of the toxicity studies presented herein, attest to the safety of BD006 phytase for use in animal feed applications.