Description of Chryseobacterium fluminis sp. nov., a keratinolytic bacterium isolated from a freshwater river.

A Gram-stain-negative, aerobic, rod-shaped bacterial strain, designated MMS21-Ot14T, was isolated from a freshwater river, and shown to represent a novel species of the genus Chryseobacterium on the basis of the results from a polyphasic approach. The 16S rRNA gene sequence analysis revealed that MMS21-Ot14T represented a member of the genus Chryseobacterium of the family Weeksellaceae and was closely related to Chryseobacterium hagamense RHA2-9T (97.52 % sequence similarity), Chryseobacterium gwangjuense THG A18T (97.46 %) and Chryseobacterium gregarium P 461/12T (97.27 %). The optimal growth of MMS21-Ot14T occurred at 25-30 °C, pH 6.0-7.0 and in the absence of NaCl. MMS21-Ot14T was capable of hydrolysing casein, starch, DNA, Tween 20 and tyrosine. The strain also showed keratinolytic activity with keratin azure and decolourising activity with remazol brilliant blue R (RBBR), which indicated potential ability to degrade keratin and lignin. The main polar lipids of MMS21-Ot14T were phosphatidylethanolamine, unidentified aminophospholipids, unidentified aminolipids, an unidentified phospholipid and several unidentified lipids. The predominant fatty acids of MMS21-Ot14T were iso-C15 : 0 and iso-C17 : 0 3-OH, and the major isoprenoid quinone was menaquinone 6 (MK-6). The whole genome of MMS21-Ot14T was 5 062 016 bp in length with a DNA G+C content of 37.7 %. The average nucleotide identity and digital DNA-DNA hybridisation values between MMS21-Ot14T and phylogenetically related members of the genus Chryseobacterium were well below the threshold values for species delineation. It is evident from the results of this study that MMS21-Ot14T should be classified as representing a novel species of the genus Chryseobacterium, for which the name Chryseobacterium fluminis sp. nov. (type strain, MMS21-Ot14T = KCTC 92255T = LMG 32529T) is proposed.

Rapid and Sensitive Detection of Antibiotic Resistance Genes by Utilizing TALEs as a Diagnostic Probe with 2D-Nanosheet Graphene Oxide.

As antibiotic resistance has risen as one of the major health concerns associated with infectious diseases due to the reduced efficacy of antibiotics, rapid and sensitive detection of antibiotic resistance genes is critical for more effective and faster treatment of infectious diseases. A class of programmable DNA-binding domains called transcriptional activator-like effectors (TALEs) provides a novel scaffold for designing versatile DNA-binding proteins due to their modularity and predictability. Here, we developed a simple, rapid, and sensitive system for detecting antibiotic resistance genes by exploring the potential of TALE proteins for the creation of a sequence-specific DNA diagnostic along with 2D-nanosheet graphene oxide (GO). TALEs were engineered to directly recognize the specific double-stranded (ds) DNA sequences present in the tetracycline resistance gene (tetM), avoiding the need for dsDNA denaturation and renaturation. We take advantage of the GO as an effective signal quencher to quantum dot (QD)-labeled TALEs for creating a turn-on strategy. QD-labeled TALEs are adsorbed on the GO surface, which will bring QDs in close proximity to GO. Due to the fluorescence quenching ability of GO, QDs are expected to be quenched by GO via fluorescence resonance energy transfer (FRET). QD-labeled TALE binding to the target dsDNA would lead to the conformational change, which would result in dissociation from the GO surface, thereby restoring the fluorescence signal. Our sensing system was able to detect low concentrations of dsDNA sequences in the tetM gene after only 10-minute incubation with the DNA, providing a limit of detection as low as 1 fM of Staphylococcus aureus genomic DNA. This study demonstrated that our approach of utilizing TALEs as a new diagnostic probe along with GO as a sensing platform can provide a highly sensitive and rapid method for direct detection of the antibiotic resistance gene without requiring DNA amplification or labeling.

Immune Responses to Plant-Derived Recombinant Colorectal Cancer Glycoprotein EpCAM-FcK Fusion Protein in Mice.

Epidermal cell adhesion molecule (EpCAM) is a tumor-associated antigen (TAA), which has been considered as a cancer vaccine candidate. The EpCAM protein fused to the fragment crystallizable region of immunoglobulin G (IgG) tagged with KDEL endoplasmic reticulum (ER) retention signal (EpCAM-FcK) has been successfully expressed in transgenic tobacco (Nicotiana tabacum cv. Xanthi) and purified from the plant leaf. In this study, we investigated the ability of the plant-derived EpCAM-FcK (EpCAM-FcKP) to elicit an immune response in vivo. The animal group injected with the EpCAM-FcKP showed a higher differentiated germinal center (GC) B cell population (~9%) compared with the animal group injected with the recombinant rhEpCAM-Fc chimera (EpCAM-FcM). The animal group injected with EpCAM-FcKP (~42%) had more differentiated T follicular helper cells (Tfh) than the animal group injected with EpCAM-FcM (~7%). This study demonstrated that the plant-derived EpCAM-FcK fusion antigenic protein induced a humoral immune response in mice.

Effect of an Endoplasmic Reticulum Retention Signal Tagged to Human Anti-Rabies mAb SO57 on Its Expression in Arabidopsis and Plant Growth.

Transgenic Arabidopsis thaliana expressing an anti-rabies monoclonal antibody (mAb), SO57, was obtained using Agrobacterium-mediated floral dip transformation. The endoplasmic reticulum (ER) retention signal Lys-Asp-Glu-Leu (KDEL) was tagged to the C-terminus of the anti-rabies mAb heavy chain to localize the mAb to the ER and enhance its accumulation. When the inaccurately folded proteins accumulated in the ER exceed its storage capacity, it results in stress that can affect plant development and growth. We generated T1 transformants and obtained homozygous T3 seeds from transgenic Arabidopsis to investigate the effect of KDEL on plant growth. The germination rate did not significantly differ between plants expressing mAb SO57 without KDEL (SO plant) and mAb SO57 with KDEL (SOK plant). The primary roots of SOK agar media grown plants were slightly shorter than those of SO plants. Transcriptomic analysis showed that expression of all 11 ER stress-related genes were not significantly changed in SOK plants relative to SO plants. SOK plants showed approximately three-fold higher mAb expression levels than those of SO plants. Consequently, the purified mAb amount per unit of SOK plant biomass was approximately three times higher than that of SO plants. A neutralization assay revealed that both plants exhibited efficient rapid fluorescent focus inhibition test values against the rabies virus relative to commercially available human rabies immunoglobulins. KDEL did not upregulate ER stress-related genes; therefore, the enhanced production of the mAb did not affect plant growth. Thus, KDEL fusion is recommended for enhancing mAb production in plant systems.

Application of Engineered Zinc Finger Proteins Immobilized on Paramagnetic Beads for Multiplexed Detection of Pathogenic DNA.

Micro-scale magnetic beads are widely used for isolation of proteins, DNA, and cells, leading to the development of in vitro diagnostics. Efficient isolation of target biomolecules is one of the keys to developing a simple and rapid point-of-care diagnostic. A zinc finger protein (ZFP) is a double-stranded (ds) DNA-binding domain, providing a useful scaffold for direct reading of the sequence information. Here, we utilized two engineered ZFPs (Stx2-268 and SEB-435) to detect the Shiga toxin (stx2) gene and the staphylococcal enterotoxin B (seb) gene present in foodborne pathogens, Escherichia coli O157 and Staphylococcus aureus, respectively. Engineered ZFPs are immobilized on a paramagnetic bead as a detection platform to efficiently isolate the target dsDNA-ZFP bound complex. The small paramagnetic beads provide a high surface area to volume ratio, allowing more ZFPs to be immobilized on the beads, which leads to increased target DNA detection. The fluorescence signal was measured upon ZFP binding to fluorophore-labeled target dsDNA. In this study, our system provided a detection limit of ≤ 60 fmol and demonstrated high specificity with multiplexing capability, suggesting a potential for development into a simple and reliable diagnostic for detecting multiple pathogens without target amplification.

Graphene Oxide-Based Simple and Rapid Detection of Antibiotic Resistance Gene via Quantum Dot-Labeled Zinc Finger Proteins.

With the increasing rise of antibiotic-resistant pathogens, a simple and rapid detection of antibiotic resistance gene (ARG) is crucial to mitigate the spreading of antibiotic resistance. DNA-binding zinc finger proteins (ZFPs) can be engineered to recognize specific double-stranded (ds) DNA sequences in ARG. Here, we designed a simple and rapid method to detect ARG in bacteria utilizing engineered ZFPs and 2D nanosheet graphene oxide (GO) as a sensing platform. Our approach relies on the on and off effect of fluorescence signal in the presence and absence of target ARG, respectively. By taking advantage of the unique quenching capability of GO due to its electronic property, quantum dot (QD)-labeled ZFPs are adsorbed onto the GO sheets, and their fluorescence signal is quenched by proximal GO sheets through fluorescence resonance energy transfer (FRET). In the presence of target DNA, ZFP binding to the target DNA induces dissociation from GO, thereby restoring the fluorescence signal. Our system detects target DNA through restoration of QD emission as the restored signal increases directly with target DNA concentrations. Engineered ZFPs were able to detect specific dsDNA of the tetracycline resistance gene tetM with high specificity after only 10 min incubation on our GO-based sensing system. Our sensing system employed one-step FRET-based ZFP and GO combined technology to enable rapid and quantitative detection of ARG, providing a limit of detection as low as 1 nM. This study demonstrated the application of GO in conjunction with engineered DNA-binding domains for the direct detection of dsDNA with great potential as a rapid and reliable screening and detecton method against the growing threat of antibiotic resistant bacteria.

Pathogen-specific DNA sensing with engineered zinc finger proteins immobilized on a polymer chip.

A specific double-stranded DNA sensing system is of great interest for diagnostic and other biomedical applications. Zinc finger domains, which recognize double-stranded DNA, can be engineered to form custom DNA-binding proteins for the recognition of specific DNA sequences. As a proof of concept, a sequence-enabled reassembly of a TEM-1 ß-lactamase system (SEER-LAC) was previously demonstrated to develop zinc finger protein (ZFP) arrays for the detection of a double-stranded bacterial DNA sequence. Here, we implemented the SEER-LAC system to demonstrate the direct detection of pathogen-specific DNA sequences present in E. coli O157:H7 on a lab-on-a-chip. ZFPs custom-designed to detect Shiga toxin in E. coli O157:H7 were immobilized on a cyclic olefin copolymer (COC) chip, which can function as a non-PCR based molecular diagnostic device. Pathogen-specific double-stranded DNA was directly detected by using engineered ZFPs immobilized on the COC chip with high specificity, providing a detection limit of 10 fmol of target DNA in a colorimetric assay. Therefore, in this study, we demonstrated the great potential of ZFP arrays on the COC chip for further development of a simple and novel lab-on-a-chip technology for the detection of pathogens.

Washing-Free Electrochemical Detection of Amplified Double-Stranded DNAs Using a Zinc Finger Protein.

Recombinase polymerase amplification (RPA) has been combined with electrochemical detection for simple and rapid point-of-care testing. However, there are two major hindrances to this simple and rapid testing: (i) washing or purification steps are required to remove unbound labeled probes and interfering species in the sample; (ii) it is difficult to quantify double-stranded DNA (dsDNA) electrochemically by using biospecific affinity binding without dsDNA denaturation. In the present study, we describe a wash-free and rapid electrochemical method to detect RPA-amplified dsDNAs using a zinc finger protein, Zif268. Electrochemical detection is achieved using proximity-dependent electron mediation of ferrocenemethanol between a glucose-oxidase (GOx) label and an electrode, which differentiates the specifically electrode-bound and -unbound labels without a washing or purification step. RPA-amplified dsDNA containing a biotin-terminated forward primer is specifically bound to a neutravidin-modified electrode, and GOx-conjugated Zif268 is specifically bound to the dsDNA. The whole detection is performed within 17 min (15 min for the RPA reaction and <2 min for the electrochemical measurement). Electrochemical detection is carried out without an additional incubation period, because the specific binding between Zif268 and the dsDNA occurs during the RPA reaction. The detection method could discriminate between target template DNA of Piscirickettsia salmonis and nontarget DNAs (random sequence and calf thymus DNA). The detection limit for the target DNA is approximately 300 copies in 13.2 µL, indicating that the detection method is ultrasensitive. We believe that the method could offer a promising solution for simple and rapid point-of-care testing.

Engineering and Application of Zinc Finger Proteins and TALEs for Biomedical Research.

Engineered DNA-binding domains provide a powerful technology for numerous biomedical studies due to their ability to recognize specific DNA sequences. Zinc fingers (ZF) are one of the most common DNA-binding domains and have been extensively studied for a variety of applications, such as gene regulation, genome engineering and diagnostics. Another novel DNA-binding domain known as a transcriptional activator-like effector (TALE) has been more recently discovered, which has a previously undescribed DNA-binding mode. Due to their modular architecture and flexibility, TALEs have been rapidly developed into artificial gene targeting reagents. Here, we describe the methods used to design these DNA-binding proteins and their key applications in biomedical research.

Ultrasensitive Electrochemical Detection of miRNA-21 Using a Zinc Finger Protein Specific to DNA-RNA Hybrids.

Both high sensitivity and high specificity are crucial for detection of miRNAs that have emerged as important clinical biomarkers. Just Another Zinc finger proteins (JAZ, ZNF346) bind preferably (but nonsequence-specifically) to DNA-RNA hybrids over single-stranded RNAs, single-stranded DNAs, and double-stranded DNAs. We present an ultrasensitive and highly specific electrochemical method for miRNA-21 detection based on the selective binding of JAZ to the DNA-RNA hybrid formed between a DNA capture probe and a target miRNA-21. This enables us to use chemically stable DNA as a capture probe instead of RNA as well as to apply a standard sandwich-type assay format to miRNA detection. High signal amplification is obtained by (i) enzymatic amplification by alkaline phosphatase (ALP) coupled with (ii) electrochemical-chemical-chemical (ECC) redox cycling involving an ALP product (hydroquinone). Low nonspecific adsorption of ALP-conjugated JAZ is obtained using a polymeric self-assembled-monolayer-modified and casein-treated indium-tin oxide electrode. The detection method can discriminate between target miRNA-21 and nontarget nucleic acids (DNA-DNA hybrid, single-stranded DNA, miRNA-125b, miRNA-155, single-base mismatched miRNA, and three-base mismatched miRNA). The detection limits for miRNA-21 in buffer and 10-fold diluted serum are approximately 2 and 30 fM, respectively, indicating that the detection method is ultrasensitive. This detection method can be readily extended to multiplex detection of miRNAs with only one ALP-conjugated JAZ probe due to its nonsequence-specific binding character. We also believe that the method could offer a promising solution for point-of-care testing of miRNAs in body fluids.

Autophagy Has a Beneficial Role in Relieving Cigarette Smoke-Induced Apoptotic Death in Human Gingival Fibroblasts.

The deleterious role of cigarette smoke has long been documented in various human diseases including periodontal complications. In this report, we examined this adverse effect of cigarette smoke on human gingival fibroblasts (HGFs) which are critical not only in maintaining gingival tissue architecture but also in mediating immune responses. As well documented in other cell types, we also observed that cigarette smoke promoted cellular reactive oxygen species in HGFs. And we found that this cigarette smoke-induced oxidative stress reduced HGF viability through inducing apoptosis. Our results indicated that an increased Bax/Bcl-xL ratio and resulting caspase activation underlie the apoptotic death in HGFs exposed to cigarette smoke. Furthermore, we detected that cigarette smoke also triggered autophagy, an integrated cellular stress response. Interesting, a pharmacological suppression of the cigarette smoke-induced autophagy led to a further reduction in HGF viability while a pharmacological promotion of autophagy increased the viability of HGFs with cigarette smoke exposures. These findings suggest a protective role for autophagy in HGFs stressed with cigarette smoke, highlighting that modulation of autophagy can be a novel therapeutic target in periodontal complications with cigarette smoke.

Multiplexed detection of pathogen-specific DNA using engineered zinc finger proteins without target amplification.

Multiplexed detection of pathogen-specific DNA sequences in a simple and reliable way is in great demand for clinical and biomedical applications. However, there is still a lack of available DNA detection methods that are simple and pathogen-selective for point-of-care (POC) testing. Here, we report a novel zinc finger protein (ZFP)-based chemiluminescent method for direct detection of pathogenic double-stranded DNA (dsDNA) in a multiplexed platform. ZFPs are custom-designed to identify unique pathogenic DNA sequences. ZFP-based chemiluminescent detection of dsDNA provides sufficient sensitivity (≤50 fmol) and high specificity without target DNA amplification. Our study addresses the potential of developing a simple and selective pathogen detection method in a multiplexed fashion needed for POC application.

Sensitive and direct electrochemical detection of double-stranded DNA utilizing alkaline phosphatase-labelled zinc finger proteins.

Direct detection of double-stranded DNA (dsDNA) using zinc finger proteins (ZFPs) is of great importance in biomedical applications such as identifying pathogens and circulating DNAs. However, its sensitivity is still not sufficiently high because limited signalling labels can be conjugated or fused. Herein, we report sensitive and direct detection of dsDNA using (i) alkaline phosphatase (ALP) as a fast catalytic label conjugated to ZFPs along with (ii) electrochemical measurement of an ALP product (l-ascorbic acid) at the indium-tin oxide electrode with a high signal-to-background ratio. ALP is simply conjugated to a ZFP through lysine residues in a ZFP purification tag, a maltose binding protein (MBP). Sandwich-type electrochemical detection of dsDNA allows a detection limit of ca. 100 fM without using DNA amplification.

Transcription activator like effector (TALE)-directed piggyBac transposition in human cells.

Insertional therapies have shown great potential for combating genetic disease and safer methods would undoubtedly broaden the variety of possible illness that can be treated. A major challenge that remains is reducing the risk of insertional mutagenesis due to random insertion by both viral and non-viral vectors. Targetable nucleases are capable of inducing double-stranded breaks to enhance homologous recombination for the introduction of transgenes at specific sequences. However, off-target DNA cleavages at unknown sites can lead to mutations that are difficult to detect. Alternatively, the piggyBac transposase is able perform all of the steps required for integration; therefore, cells confirmed to contain a single copy of a targeted transposon, for which its location is known, are likely to be devoid of aberrant genomic modifications. We aimed to retarget transposon insertions by comparing a series of novel hyperactive piggyBac constructs tethered to a custom transcription activator like effector DNA-binding domain designed to bind the first intron of the human CCR5 gene. Multiple targeting strategies were evaluated using combinations of both plasmid-DNA and transposase-protein relocalization to the target sequence. We demonstrated user-defined directed transposition to the CCR5 genomic safe harbor and isolated single-copy clones harboring targeted integrations.

Quantitative analysis of TALE-DNA interactions suggests polarity effects.

Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem 34-amino acid repeats recognize one base pair each and direct sequence-specific DNA binding through repeat variable di-residues (RVDs). We found that RVD choice can affect affinity by four orders of magnitude, with the relative RVD contribution in the order NG > HD ≈ NN >> NI > NK. The NN repeat preferred the base G over A, whereas the NK repeat bound G with 10(3)-fold lower affinity. We compared AvrBs3, a naturally occurring TALE that recognizes its target using some atypical RVD-base combinations, with a designed TALE that precisely matches 'standard' RVDs with the target bases. This comparison revealed unexpected differences in sensitivity to substitutions of the invariant 5'-T. Another surprising observation was that base mismatches at the 5' end of the target site had more disruptive effects on affinity than those at the 3' end, particularly in designed TALEs. These results provide evidence that TALE-DNA recognition exhibits a hitherto un-described polarity effect, in which the N-terminal repeats contribute more to affinity than C-terminal ones.

Probing membrane protein unfolding with pulse proteolysis.

Technical challenges have greatly impeded the investigation of membrane protein folding and unfolding. To develop a new tool that facilitates the study of membrane proteins, we tested pulse proteolysis as a probe for membrane protein unfolding. Pulse proteolysis is a method to monitor protein folding and unfolding, which exploits the significant difference in proteolytic susceptibility between folded and unfolded proteins. This method requires only a small amount of protein and, in many cases, may be used with unpurified proteins in cell lysates. To evaluate the effectiveness of pulse proteolysis as a probe for membrane protein unfolding, we chose Halobacterium halobium bacteriorhodopsin (bR) as a model system. The denaturation of bR in SDS has been investigated extensively by monitoring the change in the absorbance at 560 nm (A(560)). In this work, we demonstrate that denaturation of bR by SDS results in a significant increase in its susceptibility to proteolysis by subtilisin. When pulse proteolysis was applied to bR incubated in varying concentrations of SDS, the remaining intact protein determined by electrophoresis shows a cooperative transition. The midpoint of the cooperative transition (C(m)) shows excellent agreement with that determined by A(560). The C(m) values determined by pulse proteolysis for M56A and Y57A bRs are also consistent with the measurements made by A(560). Our results suggest that pulse proteolysis is a quantitative tool to probe membrane protein unfolding. Combining pulse proteolysis with Western blotting may allow the investigation of membrane protein unfolding in situ without overexpression or purification.

A zinc finger protein array for the visual detection of specific DNA sequences for diagnostic applications.

The visual detection of specific double-stranded DNA sequences possesses great potential for the development of diagnostics. Zinc finger domains provide a powerful scaffold for creating custom DNA-binding proteins that recognize specific DNA sequences. We previously demonstrated sequence-enabled reassembly of TEM-1 ß-lactamase (SEER-LAC), a system consisting of two inactive fragments of ß-lactamase each linked to engineered zinc finger proteins (ZFPs). Here the SEER-LAC system was applied to develop ZFP arrays that function as simple devices to identify bacterial double-stranded DNA sequences. The ZFP arrays provided a quantitative assay with a detection limit of 50 fmol of target DNA. The method could distinguish target DNA from non-target DNA within 5 min. The ZFP arrays provided sufficient sensitivity and high specificity to recognize specific DNA sequences. These results suggest that ZFP arrays have the potential to be developed into a simple and rapid point-of-care (POC) diagnostic for the multiplexed detection of pathogens.

Determining protein stability in cell lysates by pulse proteolysis and Western blotting.

Proteins require proper conformational energetics to fold and to function correctly. Despite the importance of having information on conformational energetics, the investigation of thermodynamic stability has been limited to proteins, which can be easily expressed and purified. Many biologically important proteins are not suitable for conventional biophysical investigation because of the difficulty of expression and purification. As an effort to overcome this limitation, we have developed a method to determine the thermodynamic stability of low abundant proteins in cell lysates. Previously, it was demonstrated that protein stability can be determined quantitatively by measuring the fraction of folded proteins with a pulse of proteolysis (Pulse proteolysis). Here, we show that thermodynamic stability of low abundant proteins can be determined reliably in cell lysates by combining pulse proteolysis with quantitative Western blotting (Pulse and Western). To demonstrate the reliability of this method, we determined the thermodynamic stability of recombinant human H-ras added to lysates of E. coli and human Jurkat T cells. Comparison with the thermodynamic stability determined with pure H-ras revealed that Pulse and Western is a reliable way to monitor protein stability in cell lysates and the stability of H-ras is not affected by other proteins present in cell lysates. This method allows the investigation of conformational energetics of proteins in cell lysates without cloning, purification, or labeling.

Assembly of mutations for improving thermostability of Escherichia coli AppA2 phytase.

We previously identified a number of mutations in Escherichia coli AppA2 phytase for enhancing its thermostability. The objective of the present study was to determine if these mutations (K46E, K65E, G103S, D112N, D144N, S209G, V227A, and G344D) could be sequentially added to further improve the thermostability of AppA2. Compared with the wild-type enzyme, two variants (D144N/V227A and D144N/V227A/G344D) out of the eight resulting mutants showed 15% enhancement in thermostability (as measured by residual activity after being heated at 80 degrees C for 10 min) and 4 to 5 degrees C increases in the melting temperatures (T (m)). Based on the structural predictions with a highly homologous AppA phytase, the substitution D144N introduces a side-chain-side-chain hydrogen bond, thereby stabilizing the loop region (Gln137-Asn144), and the V227A substitution might eliminate structural hindrance between Val222 and Val227 that face each other in the beta-hairpin structure. In addition, overall catalytic efficiency (k (cat)/K (m)) of the two mutants was also improved (P < 0.05) compared to the wild type. However, no further improvement in thermostability was observed by adding other mutations to D144N/V227A/G344D, which might result from unfavorable electrostatic interactions or structural perturbation. In conclusion, our results underscore the potential as well as difficulty of predicting synergistic effects of multiple mutations on thermostability within phytase.

Enhancing thermostability of Escherichia coli phytase AppA2 by error-prone PCR.

Phytases are used to improve phosphorus nutrition of food animals and reduce their phosphorus excretion to the environment. Due to favorable properties, Escherichia coli AppA2 phytase is of particular interest for biotechnological applications. Directed evolution was applied in the present study to improve AppA2 phytase thermostability for lowering its heat inactivation during feed pelleting (60-80 degrees C). After a mutant library of AppA2 was generated by error-prone polymerase chain reaction, variants were expressed initially in Saccharomyces cerevisiae for screening and then in Pichia pastoris for characterizing thermostability. Compared with the wild-type enzyme, two variants (K46E and K65E/K97M/S209G) showed over 20% improvement in thermostability (80 degrees C for 10 min), and 6-7 degrees C increases in melting temperatures (T (m)). Structural predictions suggest that substitutions of K46E and K65E might introduce additional hydrogen bonds with adjacent residues, improving the enzyme thermostability by stabilizing local interactions. Overall catalytic efficiency (k (cat) / K (m)) of K46E and K65E/K97M/S209G was improved by 56% and 152% than that of wild type at pH 3.5, respectively. Thus, the catalytic efficiency of these enzymes was not inversely related to their thermostability.