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Purpose: Chrysanthemum indicum Linne (CI) has been used as a traditional medicine. Although several biological effects of CI have been reported, the precise role and function of CI as nutri-cosmetic and cosmetic materials remain unclear. Therefore, we prepared water extract of Chrysanthemum indicum Linne (CIW). And nutri-cosmetic potential was discussed through evaluation of anti-oxidant, anti- adipogenic in 3T3-L1 cells and regulatory function of skin fibril- related genes in human skin fibroblast (HSF). Methods: The anti- oxidant activity of CIW was evaluated through metal chelating activity and reduction potential. The cytotoxicity of CIW in 3T3-L1 and HSF was evaluated by MTT assay. The anti-adipogenic effect of CIW was examined by Oil Red O (ORO) staining and microscopy observation in 3T3-L1 cells. The mRNA expression levels of skin fibril-related genes in HSF were analyzed by reverse transcription-polymer chain reaction (RT-PCR). Results: The anti-oxidant activity of CIW was increased in a dose dependent manner. The CIW treatment up to 100 µg/mL for 24 h did not affect to the viability of 3T3-L1 cells and HSF. Thus, up to 100 µg/mL of CIW was chosen in cell-based assay. The treatment of 10, 50, and 100 µg/mL CIW significantly inhibited 3T3- L1 adipogenesis by 5.17%, 72.80%, and 104.09%. In addition, up- regulated mRNA expression levels of skin fibril-related genes such as fibronectin and type Ⅰ collagen α2 (COL1A2) were observed in CIW- treated HSF. Conclusion: These results suggest that CIW has the potential as nutri-cosmetic and cosmetic ingredient which possess anti-oxidant and anti-adipogenic effects.

Two-component systems (TCSs) are an important signaling transduction pathway that adapt to changing environments. Commonly, a TCS comprises a sensor kinase that is usually an integral membrane histidine sensor kinase and a response regulator that mediates the cellular responses. Presently, however, we cloned a novel sensor kinase gene (tcsK) that is not adjacent to its cognate response regulator from Streptomyces acidiscabies that produces two secondary metabolites, thaxtomin A and WS5995B, and identified its functional involvement in the production of secondary metabolites and morphological differentiation. The elevated expression and disruption of the tcsK gene enhanced 7.1-fold and almost abolished WS5995B production in S. acidiscabies, respectively, but did not affect the production of thaxtomin A. In addition, spore formation of S. acidiscabies was decreased 120-fold by the disruption of tcsK, and the actinorhodin production of Streptomyces lividans TK24 was increased 5.7-fold by the high expression of tcsK. These results indicate that the novel unpaired tcsK gene may be related to the control of secondary metabolite production and spore formation in actinomycetes.

An effective transformation method for molecular genetic studies of Streptomyces cinnamoneus producing transglutaminase was established using intergeneric conjugal transfer based on the bacteriophage ϕC31 att/int system. The high efficiency of S. cinnamoneus transconjugation was obtained on mannitol soya flour medium containing 50 mM MgCl2, using 2.5 × 108Escherichia coli as donor and spores treated with heat treatment at 35°C for 10 min as host. By cloning and sequencing the attB integration site, a single attB site within an open reading frame coding for a pirin homologue was located in S. cinnamoneus genome. Its sequence exhibited the highest degree of sequence similarity with that of Streptomyces clavuligerus. These results provide sufficient efficiency to enable conjugal transfer of genetic elements for S. cinnamoneus, and also should facilitate molecular genetic studies for improvement in production ability of transglutaminase used in food industry.

To facilitate molecular studies of Streptomyces mobaraensis producing transglutaminase, an effective transformation method was established via intergeneric conjugal transfer using Escherichia coli ET12567 harboring the OC31-derived integration vector, pSET152. The highest frequency was attained on ISP4 medium containing 20 mM MgCl 2 , using 2.5 × 10 8 E. coli as donor and spore treated with heat treatment at 35°C for 10 min as host. The attB integration site in the S. mobaraensis genome was detected as a single attB site within an open reading frame coding for a pirin homolog. Its sequence showed the highest degree of homology with S. aureofaciens . Keywords: Streptomyces mobaraensis , conjugal transfer, integration site, exoconjugant, transglutaminase African Journal of Biotechnology , Vol 13(12), 1462-1466

Global regulator for secondary metabolism, AfsR, is phosphorylated by the serine/threonine kinase AfsK. Phosphorylation of AfsR activates the transcription of afsS, resulting in the increased production of secondary metabolites in Streptomyces strains. We isolated an afsR homologue regulatory gene from Streptomyces acidiscabies ATCC 49003, which produces thaxtomin A and WS5995B as secondary metabolites. To examine the function of afsR in the production of secondary metabolites in S. acidiscabies, an intact 2,976 bp open reading frame of afsR was identified and characterized. In S. lividans TK 24 strain, the exconjugant harboring afsR high expression vector, began to generate actinorhodin at 36 h of culture, and the amount of accumulated actinorhodin became 10-fold higher than that of the exconjugant harboring the vector lacking the afsR gene. To clarify the in vivo function of afsR, an afsR-disrupted mutant was constructed and analyzed. No morphological difference was observed between the wild-type strain and the afsR disruptant, but production of thaxtomin A and WS5995B of the afsR disruptant were significantly decreased compared to those of the wild-type strains. Specially, WS5995B production was almost abolished by the disruption of only the afsR gene. These changes were restored to the original wild-type phenotype by the introduction of the intact afsR gene into the afsR disruptant, suggesting that the afsR gene participates in the production of secondary metabolites of S. acidiscabies.

The microbial transglutaminase (TGase) gene, stgA, was cloned from Streptomyces platensis YK-2, which was newly isolated from a forest soil sample collected in Daegu, Korea. The gene was expressed in several Streptomyces strains including the original strain as the host. The stgA consisted of an open reading frame of 1,257 bp encoding a putative signal peptide of 29 amino acids, a pro-domain of 57 amino acids, and a mature TGase of 332 amino acids, and also had the putative active site of TGase, YGCVG. For expression of the stgA gene, a high expression vector was constructed via the insertion of the entire stgA gene including its putative ribosomal binding site into pSET152ET, located immediately downstream of a strong constitutive ermE* promoter, and was introduced into several Streptomyces strains. The successful high expression and secretion of the correct StgA from streptomycetes were confirmed by electrophoretic sodium dodecyl sulfate polyacrylamide gel electrophoresis and N-terminal amino acid analysis.

Effective transformation procedure to facilitate molecular genetic studies of Streptomyces acidiscabies producing thaxtomin, which causes scab diseases in the economically important root and tuber crops was established via transconjugation from Escherichia coli ET12567 using an OC31-derived integration vector, pSET152, harboring the oriT and attP fragments. Greatest number of exconjugants was achieved on MS medium containing 50 mM MgCl2 without heat treatment of spores and E. coli cells. Additionally, the integration site, attB, of the genome of S. acidiscabies exhibited the highest degree of homology with S. avermitilis and its chromosomal location was found to exist as a single attB site within an open reading frame coding for a pirin homolog similar to those identified in other actinomycetes.

S-Adenosyl-L-methionine synthtase (SAM-s) catalyzes the biosynthesis of SAM from ATP and L-methionine. SAM plays important roles in the primary and secondary metabolism of cells. A metK encoding a SAM-s was searched from Streptomyces natalensis producing natamycin, a predominantly a strong antifungal agent, inhibiting the growth of both yeasts and molds and preventing the formation of aflatoxin in filamentous fungi. To obtain the metK of S. natalensis, PCR using primers designed from the two highly conserved regions for metK genes of Streptomyces strains was carried out, and an intact 1.2-kb metK gene of S. natalensis was cloned by genomic Southern hybridization with PCR product as a probe. To identify the function of the cloned metK gene, it was inserted into pSET152ET for its high expression in the Streptomyces strain, and then introduced into S. lividans TK24 as a host by transconjugation using E. coli ET12567(pUZ8002). The high expression of metK in S. lividans TK24 induced actinorhodin production on R5 solid medium, and its amount in R4 liquid medium was 10-fold higher than that by exconjugant including only pSET152ET.

0%, 3%, 5%, and 7% chrysanthemum powder was added to plain bread to investigate its effect on the quality and preservation of bread. The properties of dough, physical changes, and sensory evaluation of plain bread with chrysanthemum powder added were analyzed during storage at room temperature. Volumes of dough and bread were decreased by addition of chrysanthemum powder. Also, the pH and Hunter L (lightness) in bread with chrysanthemum powder were lower than those of the control bread, while its redness, yellowness, and hardness were higher. In sensory evaluation, when more than 5% chrysanthemum was added to bread, aftertaste and overall acceptability were rapidly decreased. In contrast, the addition of chrysanthemum minimized the drop of flavor caused by storage time. In conclusion, chrysanthemum powder added to plain bread improved flavor, which declines with storage time.

Streptomyces platensis YK-2, newly isolated from forest soil, produces transglutaminase (TGase), which catalyses an acyl transfer reaction between the primary grade amine and protein or γ-carboxyamide group of peptide bound glutamine residues. For a molecular genetic study of S. platensis, an effective transformation method was established by using a conjugal transfer of DNA from Escherichia coli to spores of actinomycetes. The highest transconjugation frequency of S. platensis was obtained on an MS medium containing 50 mM MgCl₂, using 5×10? E. coli as a DNA donor and 1×10? spores without heat treatment as a host. We also identified that S. platensis contains a single attB site within an ORF encoding a pirin-homolog, and that its attB site sequence shows high homology to that of S. logisporoflavus. In addition, it was confirmed by phenotypic analyses of exconjugants that the introduction of heterologous DNA into the attB site of the S. platensis chromosome does not affect its morphological differentiation and TGase production.

Four genes (bamA1, bamA2, bamA3 and bamA4) encoding homologues of the gamma-butyrolactone autoregulator receptor of Streptomyces were found and cloned from Amycolatopsis mediterranei, a typical non-Streptomyces actinomycetes and a producer of rifamycin, one of the major anti-tuberculosis drugs in clinical treatment. Transcriptional analysis demonstrated that bamA1 and bamA2 are transcribed in a growth-dependent manner, while bamA3 and bamA4 are constitutively transcribed during growth. Binding assays using (3)H-labeled autoregulator analogues as ligands confirmed that all of the recombinant BamA proteins expressed in Escherichia coli have clear binding activity toward several types of Streptomyces autoregulators. The ligand specificity of the recombinant BamA1 protein was identical to that of the crude cell-free lysates of A. mediterranei reported in our previous work. These results suggest that A. mediterranei, which is phylogenetically situated in a distal clade from the genus Streptomyces as non-Streptomyces actinomycetes, has an autoregulator-mediated signaling system.

A gene encoding a gamma-butyrolactone autoregulator receptor was cloned in to E. coli from Streptomyces ambofaciens producing spiramycin, a macrolide antibiotic used in both veterinary medicine and human medicine. A 714-bp intact receptor gene (saaR) was obtained by PCR and genomic Southern hybridization with the 100-bp PCR product as a probe. To clarify the in vivo function of saaR, a saaR-disrupted strain was constructed by means of homologous recombination, and phenotypes were compared with those of the wild-type strain. The number of saaR-disruptant spores was 4-fold less than that of the wild-type strain. In addition, saaR deletion from the S. ambofaciens chromosome resulted in complete loss of spiramycin production suggesting that saaR is a rare positive regulator, controlling both spiramycin biosynthesis and sporulation.

Natural marine products have recently become the focus of increased research interest, due to their potential pharmacological activities. Therefore, we have screened 50 varieties of marine seaweed and determined that the methanolic extracts from Plocamium telfairiae (PTE) exhibited a cytotoxic effect against HT-29 human colon carcinoma cells. In this study, we report on the cytotoxic activity and mechanism of PTE-induced apoptosis in HT-29 cells. The treatment of HT-29 cells with various PTE concentrations resulted in the inhibition of growth and the induction of apoptosis in a dose-dependent manner, as determined by the results of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay, a lactate dehydrogenase release assay, a morphological assay, and a colony formation assay. Interestingly, we also detected apoptotic bodies on Hoechst staining and attempted to determine whether the PTE-induced apoptosis involved the caspase pathway, using a caspase colorimetric assay. The activation of caspases-8, -9, -3, and -7 and the specific proteolytic cleavage of poly(ADP-ribose) polymerase were detected over the course of apoptosis induction. Our results showed that PTE may function as a chemopreventive and/or chemotherapeutic agent in colon carcinoma cells via the reduction of cell viability and the induction of apoptosis.

The antimicrobial substance from Pyrola japonica were extracted and isolated. Eighty percent ethanol extract of dried Pyrola japonica was fractionated to hexane, diethyl ether, ethyl acetate and aqueous layer. The hexane-soluble fraction showed the highest inhibitory activity against Bacillus subtilis. Moreover the hexane layer was fractionated into 5 groups by silica gel column chromatography. From the results, group No. 2 ( fractions) showed the highest antimicrobial activity. The group was re-separated to 10 fractions by preparative thin layer chromatography and the peak I as active fraction was isolated by HPLC.

In previous studies we found that Morinda citrifolia (Noni) fruit extract up-regulated biosynthesis of type I collagen and glycosaminoglycans in primary cultures of normal human fibroblasts. The objective of this study was to identify the active ingredients in Noni fruit extract. An active single compound having a type I collagen-stimulating effect was isolated and identified as 1,4-dihydroxy-2-methoxy-7-methylanthraquinone by nuclear magnetic resonance, infrared, and mass analysis. It was revealed that anthraquinone showed significantly increased elaboration of procollagen type I C-terminal peptide and glycosaminoglycans and reduced expression of the collagenase matrix metalloproteinase-1 dose-dependently in human dermal fibroblasts. Furthermore, in a clinical trial, a nano-emulsion containing anthraquinone predominantly increased the dermal type I procollagen in nude mouse skin. These results suggest that anthraquinone derived from Noni extract is a good candidate for use as a new anti-wrinkle agent due to its strong induction of biosynthetic activity of extracellular matrix components.

An effective transformation procedure for Kitasatospora setae was established based on transconjugation from Escherichia coli ET12567 (pUZ8002) using a phi C31-derived integration vector, pSET152, containing oriT and attP fragments. While no transconjugation was observed under the standard transconjugation conditions for Streptomyces species, sufficient transconjugation (1 x 10(-6)) was achieved on ISP4 medium containing 30 mM MgCl(2) using a 25- to 125-fold excess of E. coli donor cells. In addition, the sequence and location of the chromosomal integration site attB of K. setae was identified for the first time in genera of non- Streptomyces actinomycetes. K. setae contains a single phi C31 attB site. Similar to the case of Streptomyces species, the attB site of K. setae is present within an ORF encoding a pirin-homolog, but the K. setae-attB sequence deviates slightly from the consensus sequence of Streptomyces attB sequences.

A metK gene encoding S-adenosyl-L-methionine synthetase was cloned from the non-Streptomyces actinomycetes, Actinoplanes teichomyceticus ATCC31121. In order to evaluate the effect of the metK expression on antibiotic production in actinomycetes, an expression vector harboring the metK gene was constructed and introduced into Streptomyces lividans TK24 and A. teichomyceticus, and the antibiotic production of the exconjugants was assessed. As a result, it was determined that the expression of metK induced 17-fold and 2.2-fold increases in actinorhodin production from S. lividans TK24 and teicoplanin production from A. teichomyceticus, respectively, compared with the control strains.

The relA gene encoding the highly phosphorylated guanine nucleotide, ppGpp synthetase, which performs a central role in triggering antibiotic biosynthesis and morphological differentiation in Streptomyces species, was cloned from the non-Streptomyces actinomycetes, Actinoplanes teichomyceticus ATCC31121, which produces teicoplanin, and relA antibiotic productionincreasing activity was confirmed. A 3.5-kb DNA fragment including an intact 2,466 bp open reading frame (ORF) evidencing high homology with the ppGpp synthetase (RelA) of Streptomyces species was acquired via polymerase chain reaction (PCR) using a newly designed primer based on the amino acid sequence of the previously studied RelA and Southern hybridization using the PCR product as a probe. S. lividans TK24 and A. teichomyceticus introduced with a high expression vector of the relA evidenced a 13- and 3-fold higher levels of actinorhodin and teicoplanin production, thus confirming for the first time that the high expression of relA derived from A. teichomyceticus is responsible for the induction of antibiotic production in Streptomyces species as well as in the non-Streptomyces actinomycetes, A. teichomyceticus.

To investigate the effects of PimM and PimR on the increase of natamycin production, exconjugants highly expressing pimM and pimR were constructed. Transcript amounts of pimM and pimR from the exconjugants for their over-expression were 2-fold higher than those of the control strain. Natamycin production of exconjugant for pimM also showed a 2.4-fold increase in liquid culture; however, no effect of pimR was observed. Therefore, promotion of pimM transcription only increases natamycin production.

The Streptomyces butyrolactone autoregulator receptor protein (BarA) is a DNA-binding protein that regulates the biosynthesis of the antibiotic virginiamycin. In this study, BarA from S. virginiae was overexpressed in Escherichia coli, purified and crystallized. Crystals of purified protein have been grown that diffracted to beyond 3.0 A resolution at 100 K using synchrotron radiation. The protein crystals belonged to the hexagonal space group P6(5)22, with unit-cell parameters a = b = 128.0, c = 286.2 A. With four molecules per asymmetric unit, the crystal volume per unit protein mass (V(M)) was 3.2 A(3) Da(-1) and the solvent content was 62%.

Actinoplanes teichomyceticus produces teicoplanin, which is a glycopeptide antibiotic for Gram-positive pathogenic bacteria and methicillin-resistant Staphylococcus aureus (MRSA). For a molecular genetic study of A. teichomyceticus, an effective transformation method using the conjugal transfer of DNA from E. coli to spores of A. teichomyceticus was established for the first time, based on the bacteriophage varphiC31 att/int system, in the genus of Actinoplanes. The high frequency of transconjugation was obtained on MS medium containing 40 mM MgCl(2), using 1.25 x 10(8) E. coli donor cells and 10(5) spores without a heat treatment. In addition, by cloning and sequencing the attB site A. teichomyceticus was shown to contain a single attB site within an ORF coding for a pirin homolog. Also, its attB site sequence showed high homology to that of Streptomyces lividans, unlike the case of Kitasatospora setae despite being a non-Streptomyces actinomycete, which seems to be closely related to the high transconjugation frequency of A. teichomyceticus.

To facilitate molecular genetic studies of Streptomyces ambofaciens that produces spiramycin, a commercially important macrolide antibiotic used in human medicine against Gram-positive pathogenic bacteria, the conditions for the conjugal transfer of DNA from E. coli to S. ambofaciens were established using a bacteriophage phiC31 att/int system. The transconjugation efficiency of S. ambofaciens varied with the medium used; the highest frequency was obtained on AS-1 medium containing 10 mM MgCl(2) without heat treatment of the spores. In addition, by cloning and sequencing the attB site, we identified that S. ambofaciens contains a single attB site within an ORF coding for a pirin homolog, and its attB site sequence shows 100% nt identity to the sequence of S. coelicolor and S. lividans, which have the highest efficiency in transconjugation using the phiC31 att/int system.

The prior sequencing of the upstream region of the gamma-butyrolactone autoregulator receptor gene (sngR) in Streptomyces natalensis revealed the presence of a 972-bp gene encoding a BarX homologue (SngA), which acts as a pleiotropic regulator controlling secondary metabolism and morphological differentiation. In this study, we investigated the in vivo function of SngA in S. natalensis, by comparing the natamycin production, morphology, and transcription of genes related to natamycin biosynthesis in a wild-type strain and a sngA-deleted mutant. The disruption of sngA resulted in a decrease in natamycin production, and in the induction of pigment production that had not been previously observed from S. natalensis. On the other hand, the insertion of the intact sngA with its own promoter, into the wild-type strain, resulted in a 1.7-fold increase in natamycin production. Spore formation decreased in comparison to that of the wild-type strain when the sngA-deleted mutant was grown on YEME agar, MS medium, and ISP4 medium. All phenotypes were restored to the original wild-type phenotypes upon complementation with the intact sngA, suggesting that SngA has pleiotropic functions in controlling both morphological differentiation and secondary metabolite production.

γ-Butyrolactone autoregulator receptors of the genus Streptomyces have a common activity as DNA-binding transcriptional repressors, controlling secondary metabolism and/or morphological differentiation. A gene encoding a γ-butyrolactone autoregulator receptor was cloned from a bafilomycin B 1 producer, Kitasatospora setae , for the first time from a non- Streptomyces genus of actinomycetes, and its function was evaluated by in vitro and in vivo analyses. The gene fragment was initially cloned by PCR with primers designed from two highly conserved regions of Streptomyces autoregulator receptors (BarA, FarA, ScbR, and ArpA), followed by genomic Southern hybridization yielding a 7-kb BamHI fragment on which a 654-bp receptor gene ( ksbA ) was identified. The recombinant KsbA protein demonstrated clear binding activity toward 3 H-labeled autoregulators, especially toward [ 3 H]SCB1, confirming that ksbA encodes a real autoregulator receptor of K. setae . To clarify the in vivo function of ksbA , a ksbA -disrupted strain was constructed by means of homologous recombination after introducing a ksbA disruption construct via transconjugation from Escherichia coli . No difference in morphology was found between the wild-type strain and the ksbA disruptants. However, the ksbA disruptants started producing bafilomycin 18 h earlier than the wild-type strain and showed a 2.4-fold-higher accumulation of bafilomycin. The phenotype was restored to the original wild-type phenotype by complementation with intact ksbA , indicating that the autoregulator receptor protein of K. setae acts as a primary negative regulator of the biosynthesis of bafilomycin but plays no role in cytodifferentiation of K. setae . This indicates that, unlike the A-factor receptor of Streptomyces griseus , the autoregulator receptor ( ksbA ) of K. setae belongs to a family of autoregulator receptors which control secondary metabolism but play no role in morphological differentiation.

For cells of Brevibacterium sp. under conditions of biotin limitation, the efflux of metabolites through the cell membrane was affected by temperature. After the temperature shift-up from 30 degrees C to 38 degrees C, both the specific production rate of glutamic acid and the excretion of intracellular materials, such as glucose-6-phosphate and nucleotides, were increased simultaneously. For the production of glutamic acid, not only the yield but also the specific production rate was increased by the temperature shift-up.

With primers designed for the conserved region of the gamma-butyrolactone autoregulator receptor proteins from Streptomyces species, PCR using the Streptomyces clavuligerus genome DNA as a template gave a clear band of 100 bp, the sequence of which revealed high similarity to the expected region of a receptor gene. By Southern blot and colony hybridization with the 100-bp insert as a probe, plasmid pSCA, harboring a 4.2 kb- SalI fragment, was obtained. Sequence analysis on the insert revealed a 702-bp ORF encoding a protein with a moderate similarity (identity, 33-43%; similarity, 51-62%) to known gamma-butyrolactone autoregulator receptor proteins from Streptomyces sp. The ORF was named scaR ( S. clavuligerus autoregulator receptor). The scaR/pET-3d plasmid was constructed for overexpression of the recombinant ScaR protein (rScaR) in Escherichia coli, and the rScaR protein was purified to homogeneity by DEAE-ion-exchange HPLC. The molecular mass of the purified rScaR protein was determined to be 27 kDa as determined by SDS-PAGE, and 54 kDa by gel filtration HPLC under nondenatured conditions at a low protein concentration, indicating that the majority of the native ScaR is present in the form of a dimer, although rScaR tended to aggregate into a higher molecular form of 230 kDa at a high protein concentration. A binding assay with tritium-labeled autoregulators indicated that IM-2 type compounds with a long C2 side chain were the most effective ligands for rScaR, demonstrating for the first time that the beta-lactam producer S. clavuligerus contains a gene for the gamma-butyrolactone autoregulator receptor.

The presence of gamma-butyrolactone autoregulators and their receptor proteins were investigated in five representative strains of non- Streptomyces actinomycetes producing commercially important secondary metabolites. Ethyl acetate extracts of culture were assayed using wild-type S. virginiae for virginiae butanolide, S. lavendulae FRI-5 for IM-2, and S. griseus HH1 for A-factor. Actinoplanes teichomyceticus and Amycolatopsis mediterranei were shown to produce autoregulators. Corresponding autoregulator-binding activities were found in the crude cell-free lysates of these strains, using the binding assay with tritium-labeled autoregulator analogues as ligands, which suggests that non- Streptomyces actinomycetes might have autoregulator-dependent signaling cascades.

A novel fungal strain Aspergillus sp. L117 that produced acid-stable and thermostable phytase was isolated on basis of the clearing zone on PSM plate and the ability of Na-phytate hydrolysis. The phytase of isolate showed a 3-fold higher activity than that of A. ficuun NRRL3135. The Aspergillus sp. L117 produced maximal level of phytase at initial pH of 5.0 and 30℃. The optimal pH and temperature for phytase activity were 5.5 and 50℃, respectively. The phytase showed totally stable activity after 20 min of exposure between 30 and 90℃, and even at 100℃. The highest level of residual phytase activity was obtained at pH 5.5, and still retained the stability at the broadest pH ranges (2.0 to 7.0) of all the aforementioned phytases. Storage stability of phytase was preserved over 96% of initial activities for 60 days at 4, -20, and -70℃ and to retain even 70% of the initial activity at room temperature.