Abstract:
The invention belongs to the field of biological engineering techniques and relates to a thermophilic alkaline phosphoesterase. The invention provides an amino acid sequence of a thermophilic alkaline phosphoesterase of the invention, as well as a DNA fragment encoding the amino acid sequence. The present invention also includes methods for cloning an expression vector containing the DNA fragment or a portion thereof and for producing the recombinant enzyme. The invention also relates to a method for tagging biological macromolecules utilizing the enzyme.

Description:
The present invention belongs to the biotechnology field and relates to a thermophilic alkaline phosphatase (or phosphoesterase). 
     The alkaline phosphatase is an important enzyme, which is widely distributed in various organisms, and participates in cellular phosphorus metabolism. The alkaline phosphatase is a non-specific phosphomonoesterase, which produces a phosphoserine intermediate and finally produces inorganic phosphorus and alcohol. The amino acid sequences and the corresponding genes of the alkaline phosphatase have been obtained from many prokaryote and eukaryote, such as  E.Coli, B. subtilis,  yeast, calf intestine, human placenta, etc. (J Bio Chem. 1991, 266: 1077-84). 
     The alkaline phosphatase is an important enzyme tool in the molecular biology study. It can be used in dephosphorization of the termini of DNA or RNA fragment in gene cloning, as a reagent for enzyme-linked assay in immunology research, and as a label for nucleic acid hybridization or detection of the PCR products. 
     Nucleic acid hybridization, one of the most extensive applied techniques in molecular biology, is a technique that detects the complementary nucleotide sequences by using the labeled DNA or RNA fragment as a probe. Typically, the label of the nucleotide probe is an isotope, such as  32 P or  35 S. Though the isotope label is very sensitive, its conventional biological and medical application and commercial kits are substantially restricted by the short half-life, the danger to the operator during the manipulating procedure, and the trouble of dealing with the isotopic wastes, etc. People have extensively studied the labeling of the nucleotide probe with the non-isotopic materials during the last decade (Mattews J. Anal Biochem.1988, 169:1-25). 
     For the time being, the common labels include enzyme, fluorescein, biotin, digoxin (Europe Patent EP 304934). The labeling methods can be divided into direct and indirect techniques based on whether the label can be detected directly or not after hybridization. The major indirect labels are haptens, such as biotin and digoxin; and the major direct labels are enzymes and fluoresceines. The alkaline phosphatase is the most extensively applied enzyme in both direct and indirect labeling methods. In the 1980&#39;s, the direct nucleotides labeling using the alkaline phosphatase was reported (Jablonski E: Nucleic Acid Res. 1986, 14: 6115˜6128). The enzymes described in the reports were mainly calf intestine alkaline phosphatase and  E.Coli  alkaline phosphatase. These alkaline phosphatases have a main drawback of being instable under high temperature, thus not suitable for hybridization in higher temperature. However, the hybridization under higher temperature is usually beneficial for reducing the background and enhancing the specificity. Additionally, these enzymes can not tolerate the strong hybridization and elution conditions, such as high concentration of SDS. Because of the poor thermostability, the oligonucleotides directly labeled by these alkaline phosphatases can not be used as the primers for the polymerase chain reaction (PCR). 
     The thermophilic bacteria are microorganisms that can live and grow at more than 55° C. Most enzymes in thermophilic bacteria, such as the thermophilic DNA polymerase used extensively in PCR, are thermophilic enzymes that have high application value. But there was no report or patent about the alkaline phosphatase from thermophilic bacteria before the present invention. 
     SUMMARY OF THE INVENTION 
     The present invention provides an alkaline phosphoesterase which has higher thermostability and is suitable for extensive use. 
     As used in this invention, the term “thermophilic alkaline phosphatase” (“FD-TAP” for short) means the enzyme with the following features or characteristics: its optimum reaction temperature is above 50° C., and its enzyme activity remains at least 70% after the incubation at 70° C. for 30 mins. As far as the same enzyme is concerned, the features or characteristics described above are observed under optimal preservation conditions and reaction systems. The features or characteristics might fluctuate as the conditions or reaction systems change. 
     The present invention provides a thermophilic alkaline phosphatase that is homologous or substantially homologous to the amino acid sequence shown in Table 1 (SEQ ID NO:2). 
     
       
         
               
             
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 The amino acid sequence of the thermophilic alkaline phosphatase 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   1 
                 
                   Met Lys Arg Arg Asp Ile Leu Lys Gly Gly Leu Ala Ala Gly Ala 
                 
               
               
                  16 
                   Leu Ala Leu Leu Pro Arg Gly His Thr Gln Gly  Ala Leu Gln Asn 
               
               
                  31 
                 Gln Pro Ser Leu Gly Arg Arg Tyr Arg Asn Leu Ile Val Phe yaI 
               
               
                  46 
                 Tyr Asp GIy Phe Ser Trp Glu Asp Tyr Ala Ile Ala Gln Ala Tyr 
               
               
                  61 
                 Ala Arg Arg Arg Gln Gly Arg Val Leu Ala Leu Glu Arg Leu Leu 
               
               
                  76 
                 Ala Arg Tyr Pro Asn Gly Leu Ile Asn Thr Tyr Ser Leu Thr Ser 
               
               
                  91 
                 Tyr Val Thr Glu Ser Ser Ala Ala GIy Asn Ala Phe Ser Cys Gly 
               
               
                 106 
                 Val Lys Thr Val Asn Gly Gly Leu Ala Ile His Ala Asp Gly Thr 
               
               
                 121 
                 Pro Leu Lys Pro Phe Phe Ala Ala Ala Lys Glu Ala Gly Lys Ala 
               
               
                 136 
                 Val Gly Leu Val Thr Thr Thr Thr Val Thr His Ala Thr Pro Ala 
               
               
                 151 
                 Ser Phe Val Val Ser Asn Pro Asp Arg Asn Ala Glu Glu Arg Ile 
               
               
                 166 
                 Ala Glu Gln Tyr Leu GIu Phe Gly Ala Glu Val Tyr Leu Gly Gly 
               
               
                 181 
                 Gly Asp Arg Phe Phe Asn Pro Ala Arg Arg Lys Asp Gly Lys Asp 
               
               
                 196 
                 Leu Tyr Ala Ala Phe Ala Ala Lys Gly Tyr Gly Val Val Arg Thr 
               
               
                 211 
                 Pro Glu Glu Leu Ala Arg Ser Asn Ala Thr Arg Leu Leu Gly Val 
               
               
                 226 
                 Phe Ala Asp GIy His Val Pro Tyr Glu Ile Asp Arg Arg Phe Gln 
               
               
                 241 
                 Gly Leu Gly Val Pro Ser Leu Lys Glu Met Val Gln Ala Ala Leu 
               
               
                 256 
                 Pro Arg Leu Ala Ala His Arg Gly Gly Phe Val Leu Gln Val Glu 
               
               
                 271 
                 Ala Gly Arg Ile Asp His Ala Asn His Leu Asn Asp Ala Gly Ala 
               
               
                 286 
                 Thr Leu Trp Asp Val Leu Ala Ala Asp Glu Val Leu Glu Leu Leu 
               
               
                 301 
                 Thr Ala Phe Val Asp Arg Asn Pro Asp Thr Leu Leu Leu Val Val 
               
               
                 316 
                 Ser Asp His Ala Thr Gly Val Gly Ala Leu Tyr Gly Ala Gly Arg 
               
               
                 331 
                 Ser Tyr Leu Glu Ser Ser Val Gly Ile Asp Leu Leu Gly Ala Gln 
               
               
                 346 
                 Lys Ala Ser Phe Glu Tyr Met Arg Arg Val Leu Gly Ser Ala Pro 
               
               
                 361 
                 Asp Ala Ala Gln Val Lys Glu Ala Tyr Gln Thr Leu Lys Gly Val 
               
               
                 376 
                 Ser Leu Thr Asp Glu Glu Ala Gln Met Val Val Arg Ala Ile Arg 
               
               
                 391 
                 Glu Arg Val Tyr Trp Pro Asp Ala Val Arg Gln Gly Jle Gln Pro 
               
               
                 406 
                 Glu Asn Thr Met Ala Trp Ala Met Val Gln Lys Asn Ala Ser Lys 
               
               
                 421 
                 Pro Asp Arg Pro Asn Ile Gty Trp Ser Ser Gly Gln His Thr Ala 
               
               
                 436 
                 Ser Pro Val Ile Leu Leu Leu Tyr Gly Gln Gly Leu Arg Phe Val 
               
               
                 451 
                 Gln Leu Gly Leu Val Asp Asn Thr His Val Phe Arg Leu Met Gly 
               
               
                 466 
                 Glu AIa Leu Asn Leu Arg Tyr Gln Asn Pro Val Met Ser Glu Glu 
               
               
                 481 
                 Glu Ala Leu Glu Ile Leu Lys Ala Arg Pro Gln Gly Met Arg His 
               
               
                 496 
                 Pro Glu Asp Val Trp Ala 
               
               
                   
               
             
          
         
       
     
     The signal peptide composed of 26 amino acid residues is underlined at the N-terminus of the amino acid sequence. 
     The present invention further provides DNA fragments which are homologous or substantially homologous to the nucleotide sequence as shown in Table 2 which encodes the enzyme of the invention. 
     
       
         
               
             
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 The nucleotide sequence of the thermophilic alkaline phosphatase gene 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                    1 
                 ATG AAG CGA AGG GAC ATC CTG AAA GGT GGC CTG GCT GCG GGG GCC 
               
               
                 46 
                 CTG GCC CTC CTG CCC CGG GGC CAT ACC CAG GGG GCT CTG CAG AAC 
               
               
                 91 
                 CAG CCT TCC TTG GGA AGG CGG TAC CGC AAC CTC ATC GTC TTC GTC 
               
               
                 136 
                 TAC GAC GGG TTT TCC TGG GAG GAC TAC GCC ATC GCC CAG GCC TAC 
               
               
                 181 
                 GCC CGG AGG CGG CAG GGC CGG GTT CTC GCC CTG GAG CGC CTC CTC 
               
               
                 226 
                 GCC CGC TAC CCC AAC GGG CTC ATC AAC ACC TAC AGC CTC ACC AGC 
               
               
                 271 
                 TAC GTC ACC GAG TCC AGC GCC GCG GGG AAC GCC TTC TCC TGC GGG 
               
               
                 316 
                 GTG AAG ACG GTG AAC GGG GGG CTC GCC ATC CAC GCC GAC GGG ACC 
               
               
                 361 
                 CCC CTC AAG CCC TTC TTC GCC GCG GCC AAG GAG GCG GGG AAG GCC 
               
               
                 406 
                 GTG GGG CTC GTG ACC ACC ACC ACC GTC ACC CAC GCC ACC CCG GCG 
               
               
                 451 
                 AGC TTC GTG GTG TCC AAT CCC GAC CGG AAC GCC GAG GAG AGG ATC 
               
               
                 496 
                 GCC GAG CAG TAC CTG GAG TTC GGG GCC GAG GTG TAC CTT GGG GGC 
               
               
                 541 
                 GGG GAC CGC TTT TTC AAC CCC GCC AGG CGC AAG GAC GGG AAG GAC 
               
               
                 586 
                 CTC TAC GCC GCC TTC GCC GCC AAG GGG TAC GGG GTG GTG CGC ACC 
               
               
                 631 
                 CCC GAG GAG CTC GCC CGT TCC AAC GCC ACC CGG CTC CTG GGC GTC 
               
               
                 676 
                 TTC GCC GAC GGC CAC GTG CCC TAC GAG ATT GAC CGC CGC TTC CAG 
               
               
                 721 
                 GGC CTT GGG GTG CCG AGC CTC AAG GAA ATG GTC CAG GCC GCT TTG 
               
               
                 766 
                 CCC CGG CTT GCC GCC CAC CGC GGG GGC TTC GTC CTT CAG GTG GAA 
               
               
                 811 
                 GCG GGG CGG ATT GAC CAC GCC AAC CAT TTG AAC GAC GCC GGG GCC 
               
               
                 856 
                 ACC CTT TGG GAC GTG CTG GCG GCG GAC GAG GTC TTG GAG CTT CTC 
               
               
                 901 
                 ACC GCC TTC GTG GAC CGG AAC CCG GAC ACC CTC CTC CTC GTG GTC 
               
               
                 946 
                 TCG GAC CAC GCC ACC GGG GTG GGG GCC CTC TAC GGG GCG GGC CGG 
               
               
                 991 
                 AGC TAC CTG GAG AGC TCC GTG GGC ATT GAC CTC CTG GGG GCG CAA 
               
               
                 1036 
                 AAG GCC AGC TTT GAG TAC ATG CGC CGC GTC TTG GGC TCG GCC CCC 
               
               
                 1081 
                 GAT GCT GCC CAG GTG AAG GAG GCC TAC CAG ACC CTG AAG GGG GTC 
               
               
                 1126 
                 TCC CTC ACG GAC GAG GAG GCG CAG ATG GTG GTC CGG GCC ATC CGC 
               
               
                 1171 
                 GAG CGG GTC TAC TGG CCT GAT GCC GTG CGC CAG GGC ATC CAG CCC 
               
               
                 1216 
                 GAA AAC ACC ATG GCC TGG GCC ATG GTG CAG AAG AAC GCC AGC AAG 
               
               
                 1261 
                 CCC GAC CGG CCC AAC ATC GGC TGG AGC TCT GGG CAG CAC ACG GCG 
               
               
                 1306 
                 AGC CCC GTC ATC CTC CTC CTC TAC GGC CAG GGC CTG CGC TTC GTC 
               
               
                 1351 
                 CAG CTT GGC CTG GTG GAC AAC ACC CAC GTG TTC CGC CTG ATG GGC 
               
               
                 1396 
                 GAG GCC CTG AAC CTC CGC TAC CAG AAC CCG GTG ATG AGC GAG GAG 
               
               
                 1441 
                 GAG GCC CTG GAG ATC CTC AAG GCC AGG CCC CAG GGG ATG CGC CAC 
               
               
                 1486 
                 CCC GAG GAC GTC TGG GCC TAA 
               
               
                   
               
             
          
         
       
     
     As used herein, the phrase “DNA fragment(s)” described above includes single- or double-stranded DNA. 
     Based on the specific circumstance, the term “homologous” means that (1) a DNA fragment has the identical nucleotide sequence when comparing with another DNA fragment; or (2) a protein has the identical amino acid sequence when comparing with another protein. 
     The term “substantially homologous” means that: (1) compared with another DNA fragment, a DNA fragment has enough identical nucleotide sequence so that the translated protein has the same or similar features or characteristics; (2) compared with another protein, a protein has enough identical amino acid sequence, so that both proteins have the same or similar features or characteristics. 
     There are various organisms, such as prokaryote, yeast and mammals, which can be used as the resources for the thermophilic alkaline phosphatase or its DNA. Preferably, said organism is a prokaryote, especially the thermophilic bacteria, such as the commercially available bacteria  Thermus thermophilus.    
     One can make the DNA fragment encoding the thermophilic alkaline phosphatase partially deleted by using the genetic engineering techniques. Serial deletions can be made from 5′ terminus to 3′ terminus or from 3′ terminus to 5′ terminus of the DNA fragment. Alternatively, the DNA fragment can be deleted sequentially from both ends to the center. In addition, the middle part of the DNA fragment can be deleted and the two end parts can then be ligated together. The shortest DNA fragment is composed of only 60 bases after deletion. Generally, the polypeptide encoded by the deleted DNA fragment retains the features or characteristics of the thermophilic alkaline phosphatase. 
     The present invention also provides a recombinant vector which comprises one or more copies of the DNA fragment (or the deleted DNA fragment) of the invention. Said vector can be used to express the thermophilic alkaline phosphatase in host cells. 
     The vector includes eucaryotic vector and prokaryotic vector, preferably the prokaryotic vector so as to facilitate the amplification in prokaryote. The prokaryotic vector includes bacteriophage λ (such as λgtt11, λDash, λZapII, etc.) and plasmid (such as pBR322, pUC series, pBluescript, etc.). Plasmid is preferred. The above vectors are commercially available. 
     The present invention also provides a microorganism transformed by the recombinant vector of the invention. Gram-negative bacteria, especially  E.Coli,  are first recommended to be used as the host cells. 
     The recombinant vector of the invention can be obtained by using the following protocol: 
     (1) isolating the chromosome DNA from the prokaryotic organism, and digesting said DNA with an appropriate restriction endonuclease; 
     (2) integrating the digested DNA into a vector, using said recombinant vector to transform an appropriate host, and then constructing the gene library; 
     (3) screening the gene library of step (2) using an appropriate method; 
     (4) analyzing the positive clone screened out in step (3). 
     The chromosome DNA can be isolated by treating the prokaryote cells with lysozyme and then adding proteinase K. 
     The DNA can be digested using an appropriate restriction endonuclease according to the conventional molecular biology methods known in the art. The digested DNA is ligated into an appropriate clone vector, and the recombinant vector is used to transform appropriate organism to construct the gene library. The detailed description about these protocols can be found in laboratory manuals of gene engineering (Sambrook J, et al. In: Molecular Cloning, A Laboratory Manual. 2 ed., CSH Press, 1989). 
     The gene can be isolated by screening a library using the following methods: (A) hybridization using oligonucleotide probe; (B) polymerase chain reaction (PCR); (C) screening with a specific antibody; and (D) screening based on enzymatic activity. In situ hybridization with nucleic acid or oligonucleotide probes is a common method, however, screening based on enzymatic activity is preferred in the invention because it is easy to detect the activity of the thermophilic alkaline phosphatase. Moreover, the positive clones, which express the thermophilic alkaline phosphatase, can be screened in situ from colonies based on its thermostability. 
     The inserted DNA fragments in the positive clones can be bi-directionally sequenced by Sanger dideoxy-mediated chain termination method (conventional radioactive isotope manual sequencing or automatic sequencing by the automatic sequencing apparatus). The result is shown in FIG.  1 . 
     The present invention also includes a method for producing the thermophilic alkaline phosphatase that is homologous or substantially homologous to the amino acid sequence shown in FIG. 1, which comprises: 
     (1) transforming the appropriate host cells with the DNA fragment (or partially delected DNA fragment) encoding the enzyme of the invention or with a recombinant vector containing said DNA fragment; 
     (2) culturing the transformed host cells in the appropriate medium; 
     (3) isolating and purifying the protein from the cultured medium or the host cells. 
     The recombinant thermophilic alkaline phosphatase can be expressed under the control of any appropriate promoters and translation regulatory elements. The suitable hosts include the prokaryote, yeast, mammal cells, insect and plant, etc. The prokaryote is preferred and  E.Coli  is more preferred. The selection of vector depends on the particular host. In  E.Coli,  plasmids, such as pJLA503 (Lehauder B, et al. Gene, 1987; 53: 279-283) and pET series (a product available from Stratagene), are commonly used as the expression vector. Typically, the culture medium for  E.Coli  is abundant medium, such as 2×YT, etc. Based on the different vectors, the proteinase expression can be induced by changing the temperature or using a chemical method (such as using ITPG). 
     The thermophilic alkaline phosphatase can be isolated and purified from the cultured cells or medium. If the expressed enzyme protein is present inside the cells, the cells are centrifuged, then lysated by ultrasonic wave, lysozyme, or frozen-thaw cycles. The raw products can be obtained by centrifuging and filtering the cell lysate. If the enzyme protein is secreted into the medium, the enzyme can be obtained by centrifuging and removing the cells, and then purifying from the supernatant. There are many methods for the purification of the enzyme, such as salting-out, ultra-filtration, dialysis, ion-exchange chromatography, HPLC, etc. During the purification of the enzymes, the contaminated proteins can be eliminated effectively by incubating in an elevated temperature (such as 60° C.) for a period of time, which makes the purification procedure easier and more convenient. 
     The thermophilic alkaline phosphatase is useful for the labeling of nucleotides, proteins, or other biomacromolecules, and dephosphorizing the termini of DNA or RNA fragments in gene cloning. The primary use is to label nucleic acids or oligonucleotides directly. There are three main applications of labeled nucleic acids or oligonucleotides: (1) they can be used as the probes for nucleic acid hybridization and foot printing assay, including Southern blot, Northern blot, Slot blot, dot blot, Southern-western blot, hybridization in situ, etc.; (2) they can be used as the primers for nucleic acid amplification in vitro; (3) they can be used for DNA sequence analysis. The linkage between the enzyme protein and nucleic acids or oligonucleotides is a covalent bond established chemically or physically. The terminal group of nucleic acids or oligonucleotides can be linked to the amino group or hydrosulfide group of enzyme protein by a linker arm. (Ruth et al: DNA 1985; 4:93). The detection methods can be chemical, physical or biological methods. Depending on the solid-phase hybridization or the liquid-phase hybridization, the color visualization method using BCIP/NBT as a substrate or the chemiluminescent method using AMPPD as a substrate (Schaap A, et al. Clin Chem 1989, 35: 1863-1864) are preferred for the solid-phase hybridization; and pNPP is preferred as the substrate for liquid-phase hybridization. A quantitative assay may also be performed. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIGS. 1A,  1 B,  1 C, and  1 D show the nucleotide sequence (SEQ ID NO:3) of the thermophilic alkaline phosphatase gene and its deduced amino acid sequence (SEQ ID NO:2). The sequence of amino acid expressed by three letters is listed under the DNA sequence. The underlined N-terminus of the amino acid sequence is a signal peptide composed of 26 amino acid residues. 
     FIG. 2 shows the map of high expression plasmid pTAP503 containing FD-TAP gene. 
     FIG. 3 shows the optimum temperature for FD-TAP. 
     FIG. 4 shows the thermostability of FD-TAP. The enzyme activity was measured at 70° C. after incubating the enzyme solution at 95° C. for a different period of time. 
     FIG. 5 shows the effect of pH on FD-TAP activity. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The presetn invention is further elucidated by the examples, which are provided to describe the specific embodiments of the invention but are not to be construed as limiting the invention in any way. 
     The following abbreviations are used in the examples: 
     TE: 10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH8.0 
     TH: 0.3% peptone, 0.3% yeast extract, 0.2% NaCl, pH7.0 
     LB: 1% peptone, 0.5% yeast extract, 1% NaCl, pH7.0 
       2 ×YT: 1.6% peptone, 1% yeast extract, 0.5% NaCl, pH7.0 
     FD-TAP: The thermophilic alkaline phosphatase derived from Thermus sp. FD3041. 
     EXAMPLE 1 
     Isolation of Chromosome DNA from Thermus sp. FD3041 
     Thermus sp. FD3041 (commercially available from Fu Hua Co., Ltd., Shanghai, China) was cultured in 200 ml of TH liquid medium at 70° C. The bacteria cells were harvested by centrifugation, suspended in 12 ml of TE buffer solution supplemented with 1 ml of TE buffer containing 10 mg/ml lysozyme, and then incubated in a water bath for 2 hours at 37° C. 1.5 ml of TE buffer containing 10% sodium dodecyl sarcosinate (Sarcosyl) and 1 mg/ml proteinase K was added and the resultant mixture was incubated at 37° C. for 1 hour. The mixture was extracted twice with phenol, and extracted twice with chloroform/isoamyl alcohol (24:1). 1/10 volume of 3 mol/L NaAc was added into the water phase. DNA was precipitated with 2 volumes of ethanol. The flocculent precipitate was reeled up with a glass stick, vacuum dried, and then dissolved in 3 nm TE buffer. 50 ul RNase A (10 mg/ml) was added. The chromosome DNA was extracted once with chloroform, precipitated with ethanol, and then dissolved in TE buffer. 
     EXAMPLE 2 
     Cloning of the DNA Fragment that Encodes the Thermostabe Alkaline Phosphatase 
     20 ug of chromosome DNA of Thermus sp. FD3041 was partially digested with enzyme Sau3AI. The 3-10 Kb DNA fragments were recovered by using low melting-point agarose electrophoresis. The two bases of the cohesive ends were partially filled in by using Klenow fragment and dGTP and DATP, so as to avoid self-ligation. The vector pUC118 was digested completely with enzyme Sal I. The larger fragment was recovered and the two bases of the cohesive ends were filled in using Klenow fragment and dCTP and dTTP. After filling-in, the cohesive ends of the chromosome DNA and the vector DNA were ligated together. After ligation with T4 ligase, the ligated DNA was used to transform  E.Coli  TG1. The white recombinant transformants were picked on LB plates containing ITPG, X-gal and ampicillin (100 ug/ml). Totally, 12,000 transformants were obtained, 85% of which contained 3-10 kb inserted fragments as confirmed by identifying the extracted plasmid. Thus, the chromosome gene library of Thermus sp. FD3041 was constructed. 
     The gene library was screened in situ by using the alkaline phosphatase color visualization method. The colonies were transferred onto a 3 mm filter paper. The paper was soaked in lysis buffer (1 mol/L diethanolamine, 1% SDS) and incubated at 85° C. for 10 mins, and then soaked in reaction buffer (6 mol/L pNPP, 1 mol/L diethanolamine, 1% SDS) at 70° C. for 10 mins. The positive colonies were those which turned yellow. After screening, five positive clones were isolated. For one clone (pTAP362), the physical map was constructed and TAP activity was tested for partially deleted plasmids. The FD-TAP was located in a 2 kb DNA fragment. 
     The DNA sequence was determined by using Sanger dideoxy-mediated chain-termination method, and a nucleotide sequence of 2030 bp was obtained (FIG.  1 ). According to computer analysis, the FD-TAP gene was 1506 bp in length with 68.2% of G+C% and encoded a proenzyme of 501 amino acid residues. For the third base of the codons, the G+C% was 92.7%, which was consistent with the characteristics of thermophilic bacteria gene. The 26 amino acid residues at the N-terminus of the proenzyme formed a signal peptide sequence and the mature enzyme was composed of 475 amino acid residues. FIG. 1 shows the DNA sequence of the FD-TAP gene and the amino acid sequence of its coded protein. 
     EXAMPLE 3 
     Subcloning and High Expression of the FD-TAP Gene 
     Primers were designed according to the sequences at the start codon and stop codon of FD-TAP gene. Nde I and BamH I site were introduced to the 5′ end of the primers, respectively. The sequence of the mature FD-TAP gene was amplified by PCR, using pTAP118B plasmid as template. After digestion, the gene was cloned into the high expression vector pJLA503, and vector pJLA503 was used to transform  E.Coli  strain Mph44, which was defective in phoA gene. On the LB plate containing ampicillin, the recombinant transformants were screened in situ by using color visualization method. 50% of the transformants were positive for FD-TAP expression. For the recombinant plasmid in one clone (pTAP 503, FIG.  2 ), its DNA sequence was determined and the results showed that there was no mutation in the gene. 
       E.Coli  Mph44 (pTAP503) was cultured in liquid medium at 30° C. and then induced at 42° C. for 10 hours. SDS-PAGE results showed an expressed enzyme of about 53 KDa. The recombinant protein was about 10% of the total proteins. 
     EXAMPLE 4 
     Isolation and Purification of the Recombinant FD-TAP Protein 
       E.coli  Mph44 strain (pTAP503) was inoculated into 2×YT medium containing ampicillin 100 ug/ml. The bacteria was cultured in a shaker overnight at 30° C. to form a stock culture. This stock culture (2% of the final volume) was transferred into 2×YT medium and cultured in a shaker at 30° C. until the A 600 , was 0.4-0.6, and then further cultured at 42° C. for 10 hours. bacteria cells were harvested by centrifugation and suspended in Buffer A (50 mmol/L Tris pH 8.8, glycerol, 10 mmol/L β-mercaptoethanol). The cells were lysed by supersonication in ice-water bath (total time=400 seconds, pulse time=1 second, interval time=1 second, output power=25%). The lysate was centrifuged for 15 minutes at 15,000 rpm. The precipitate was discarded and the supernatant was collected. PEI was slowly added into the supernatant so that the final concentration PEI was 0.04% to remove the nucleic acids. After further centrifugation for 15 min at 15,000 rpm, the precipitate was discarded. To the supernatant, NaCl was added so that the final concentration of NaCl was 0.8 mmol/L. The supernatant was denatured by incubating in water bath at 70° C. for 30 minutes, centrifuged again for 15 minutes at 15,000 rpm to get rid of the non-thermotolerante contaminant proteins and the supernatant was collected. The solid ammonium sulfate was gradually added into the supernatant to reach the saturation concentration of 60% with stirring for 1 hour at 4° C. The supernatant was centrifuged for 20 minutes at 12,000 rpm. The supernatant was discarded and the precipitate was dissolved in ⅛ volume of Buffer B (10 mmol/L Na 2 HPO 4 —NaH 2 PO 4 , pH 6.8, glycerol, 10 mmol/L β-mercaptoethanol), and then dialyzed against the same buffer at 4° C. for desalting. After dialysis, the protein sample was subject to ion-exchange chromatography using CM sepharose Flast Flow column. After the sample was loaded, the column was eluted with Buffer B until A280 nm absorbance was back to basal level. The elution was further performed with NaCl linear gradient solution (0˜0.5 mol/L). The eluate fractions were collected with 1.5 ml per tube. The purity of the protein was analyzed using SDS-PAGE. The purified protein peak fractions were pooled, lyophilized and stored at −20° C. 
     EXAMPLE 5 
     The Enzyme Activity and Characteristics of FD-TAP Protein 
     10 ul of enzyme solution was added into 1000 ul reaction system (6 mmol/L pNPP, 1 mol/L diethanolamine, pH 11.6). After incubating at 70° C. for 10 minutes, 990 μl of trichloroacetic acid was added to stop the reaction. The absorbance at 405 nm (OD 405 ) of the resultant product was determined on UV260 apparatus. The unit of enzyme activity was defined as follows: one unit was defined as the amount of enzyme required to produce 1 μmol/L NPP per minute at 70° C., pH 11.6. Enzyme unit=A405×2/(18.8×10), in which 2 stands for total reaction volume, 10 for reaction time and the molar extinction coefficient of NPP at 405 nm is 18.8×10 6 . 
     Some enzymological properties of FD-TAP: 
     Optimum reaction temperature: 70° C. 
     Thermo-tolerance: The enzyme was solved in a system (50 mmol/L Tris, pH 8.8, 25° C.). After incubating at 95° C. for 30 minutes, the enzyme activity remained more than 90% (FIG.  4 ). 
     Optimum pH: pH 12 (FIG.  5 ). 
     EXAMPLE 6 
     Partial Deletion of FD-TAP Gene 
     The primers were designed according to the sequences at different positions of FD-TAP gene. The DNA fragments with different sizes were amplified: 79→1506, 79→1416, 79→960, 271→480, 271→330. NdeI site was introduced to the 5′ end of upstream primers, stop codon and BamH I site were introduced to downstream primers. The desired fragments were amplified by PCR, using pTAP118B plasmid as template. These amplified DNA fragments were cloned into high expression vector pJLA503, which was used to transform  E.Coli  Mph44. The transformants were screened to obtain the positive colonies containing the DNA fragments mentioned above. Expression of the protein was induced and the recombinant polypeptides were isolated and purified. The enzymological properties of said polypeptieds were studied. The results showed that these polypeptides had properties and characteristics similar to those of the intact FD-TAP. 
     
       
         
           
             3 
           
           
             1 
             1506 
             DNA 
             Thermus 
             
               CDS 
               (1)...(1506) 
               encodes a thermophilic alkaline phosphoesterase 
             
           
            1
atgaagcgaa gggacatcct gaaaggtggc ctggctgcgg gggccctggc cctcctgccc     60
cggggccata cccagggggc tctgcagaac cagccttcct tgggaaggcg gtaccgcaac    120
ctcatcgtct tcgtctacga cgggttttcc tgggaggact acgccatcgc ccaggcctac    180
gcccggaggc ggcagggccg ggttctcgcc ctggagcgcc tcctcgcccg ctaccccaac    240
gggctcatca acacctacag cctcaccagc tacgtcaccg agtccagcgc cgcggggaac    300
gccttctcct gcggggtgaa gacggtgaac ggggggctcg ccatccacgc cgacgggacc    360
cccctcaagc ccttcttcgc cgcggccaag gaggcgggga aggccgtggg gctcgtgacc    420
accaccaccg tcacccacgc caccccggcg agcttcgtgg tgtccaatcc cgaccggaac    480
gccgaggaga ggatcgccga gcagtacctg gagttcgggg ccgaggtgta ccttgggggc    540
ggggaccgct ttttcaaccc cgccaggcgc aaggacggga aggacctcta cgccgccttc    600
gccgccaagg ggtacggggt ggtgcgcacc cccgaggagc tcgcccgttc caacgccacc    660
cggctcctgg gcgtcttcgc cgacggccac gtgccctacg agattgaccg ccgcttccag    720
ggccttgggg tgccgagcct caaggaaatg gtccaggccg ctttgccccg gcttgccgcc    780
caccgcgggg gcttcgtcct tcaggtggaa gcggggcgga ttgaccacgc caaccatttg    840
aacgacgccg gggccaccct ttgggacgtg ctggcggcgg acgaggtctt ggagcttctc    900
accgccttcg tggaccggaa cccggacacc ctcctcctcg tggtctcgga ccacgccacc    960
ggggtggggg ccctctacgg ggcgggccgg agctacctgg agagctccgt gggcattgac   1020
ctcctggggg cgcaaaaggc cagctttgag tacatgcgcc gcgtcttggg ctcggccccc   1080
gatgctgccc aggtgaagga ggcctaccag accctgaagg gggtctccct cacggacgag   1140
gaggcgcaga tggtggtccg ggccatccgc gagcgggtct actggcctga tgccgtgcgc   1200
cagggcatcc agcccgaaaa caccatggcc tgggccatgg tgcagaagaa cgccagcaag   1260
cccgaccggc ccaacatcgg ctggagctct gggcagcaca cggcgagccc cgtcatcctc   1320
ctcctctacg gccagggcct gcgcttcgtc cagcttggcc tggtggacaa cacccacgtg   1380
ttccgcctga tgggcgaggc cctgaacctc cgctaccaga acccggtgat gagcgaggag   1440
gaggccctgg agatcctcaa ggccaggccc caggggatgc gccaccccga ggacgtctgg   1500
gcctaa                                                              1506
 
           
             2 
             501 
             PRT 
             Thermus sp. FD3041 
             
               SIGNAL 
               (1)...(26) 
               Signal peptide 
             
           
            2
Met Lys Arg Arg Asp Ile Leu Lys Gly Gly Leu Ala Ala Gly Ala Leu
    -25                 -20                 -15
Ala Leu Leu Pro Arg Gly His Thr Gln Gly Ala Leu Gln Asn Gln Pro
-10                 -5                   1               5
Ser Leu Gly Arg Arg Tyr Arg Asn Leu Ile Val Phe Val Tyr Asp Gly
            10                  15                  20
Phe Ser Trp Glu Asp Tyr Ala Ile Ala Gln Ala Tyr Ala Arg Arg Arg
        25                  30                  35
Gln Gly Arg Val Leu Ala Leu Glu Arg Leu Leu Ala Arg Tyr Pro Asn
    40                  45                  50
Gly Leu Ile Asn Thr Tyr Ser Leu Thr Ser Tyr Val Thr Glu Ser Ser
55                  60                  65                  70
Ala Ala Gly Asn Ala Phe Ser Cys Gly Val Lys Thr Val Asn Gly Gly
                75                  80                  85
Leu Ala Ile His Ala Asp Gly Thr Pro Leu Lys Pro Phe Phe Ala Ala
            90                  95                  100
Ala Lys Glu Ala Gly Lys Ala Val Gly Leu Val Thr Thr Thr Thr Val
        105                 110                 115
Thr His Ala Thr Pro Ala Ser Phe Val Val Ser Asn Pro Asp Arg Asn
    120                 125                 130
Ala Glu Glu Arg Ile Ala Glu Gln Tyr Leu Glu Phe Gly Ala Glu Val
135                 140                 145                 150
Tyr Leu Gly Gly Gly Asp Arg Phe Phe Asn Pro Ala Arg Arg Lys Asp
                155                 160                 165
Gly Lys Asp Leu Tyr Ala Ala Phe Ala Ala Lys Gly Tyr Gly Val Val
            170                 175                 180
Arg Thr Pro Glu Glu Leu Ala Arg Ser Asn Ala Thr Arg Leu Leu Gly
        185                 190                 195
Val Phe Ala Asp Gly His Val Pro Tyr Glu Ile Asp Arg Arg Phe Gln
    200                 205                 210
Gly Leu Gly Val Pro Ser Leu Lys Glu Met Val Gln Ala Ala Leu Pro
215                 220                 225                 230
Arg Leu Ala Ala His Arg Gly Gly Phe Val Leu Gln Val Glu Ala Gly
                235                 240                 245
Arg Ile Asp His Ala Asn His Leu Asn Asp Ala Gly Ala Thr Leu Trp
            250                 255                 260
Asp Val Leu Ala Ala Asp Glu Val Leu Glu Leu Leu Thr Ala Phe Val
        265                 270                 275
Asp Arg Asn Pro Asp Thr Leu Leu Leu Val Val Ser Asp His Ala Thr
    280                 285                 290
Gly Val Gly Ala Leu Tyr Gly Ala Gly Arg Ser Tyr Leu Glu Ser Ser
295                 300                 305                 310
Val Gly Ile Asp Leu Leu Gly Ala Gln Lys Ala Ser Phe Glu Tyr Met
                315                 320                 325
Arg Arg Val Leu Gly Ser Ala Pro Asp Ala Ala Gln Val Lys Glu Ala
            330                 335                 340
Tyr Gln Thr Leu Lys Gly Val Ser Leu Thr Asp Glu Glu Ala Gln Met
        345                 350                 355
Val Val Arg Ala Ile Arg Glu Arg Val Tyr Trp Pro Asp Ala Val Arg
    360                 365                 370
Gln Gly Ile Gln Pro Glu Asn Thr Met Ala Trp Ala Met Val Gln Lys
375                 380                 385                 390
Asn Ala Ser Lys Pro Asp Arg Pro Asn Ile Gly Trp Ser Ser Gly Gln
                395                 400                 405
His Thr Ala Ser Pro Val Ile Leu Leu Leu Tyr Gly Gln Gly Leu Arg
            410                 415                 420
Phe Val Gln Leu Gly Leu Val Asp Asn Thr His Val Phe Arg Leu Met
        425                 430                 435
Gly Glu Ala Leu Asn Leu Arg Tyr Gln Asn Pro Val Met Ser Glu Glu
    440                 445                 450
Glu Ala Leu Glu Ile Leu Lys Ala Arg Pro Gln Gly Met Arg His Pro
455                 460                 465                 470
Glu Asp Val Trp Ala
                475
 
           
             3 
             2030 
             DNA 
             Thermus sp. FD3041 
             
               CDS 
               (82)...(1587) 
               encodes a thermophilic alkaline phosphoesterase 
             
           
            3
gccttcccag ggtcacgggg tcattatccc ctgaccttcc cctgacttgc gctccttact     60
ttgaactcgg aggtgagaag c atg aag cga agg gac atc ctg aaa ggt ggc      111
                        Met Lys Arg Arg Asp Ile Leu Lys Gly Gly
                         1               5                   10
ctg gct gcg ggg gcc ctg gcc ctc ctg ccc cgg ggc cat acc cag ggg      159
Leu Ala Ala Gly Ala Leu Ala Leu Leu Pro Arg Gly His Thr Gln Gly
                 15                  20                  25
gct ctg cag aac cag cct tcc ttg gga agg cgg tac cgc aac ctc atc      207
Ala Leu Gln Asn Gln Pro Ser Leu Gly Arg Arg Tyr Arg Asn Leu Ile
             30                  35                  40
gtc ttc gtc tac gac ggg ttt tcc tgg gag gac tac gcc atc gcc cag      255
Val Phe Val Tyr Asp Gly Phe Ser Trp Glu Asp Tyr Ala Ile Ala Gln
         45                  50                  55
gcc tac gcc cgg agg cgg cag ggc cgg gtt ctc gcc ctg gag cgc ctc      303
Ala Tyr Ala Arg Arg Arg Gln Gly Arg Val Leu Ala Leu Glu Arg Leu
     60                  65                  70
ctc gcc cgc tac ccc aac ggg ctc atc aac acc tac agc ctc acc agc      351
Leu Ala Arg Tyr Pro Asn Gly Leu Ile Asn Thr Tyr Ser Leu Thr Ser
 75                  80                  85                  90
tac gtc acc gag tcc agc gcc gcg ggg aac gcc ttc tcc tgc ggg gtg      399
Tyr Val Thr Glu Ser Ser Ala Ala Gly Asn Ala Phe Ser Cys Gly Val
                 95                 100                 105
aag acg gtg aac ggg ggg ctc gcc atc cac gcc gac ggg acc ccc ctc      447
Lys Thr Val Asn Gly Gly Leu Ala Ile His Ala Asp Gly Thr Pro Leu
            110                 115                 120
aag ccc ttc ttc gcc gcg gcc aag gag gcg ggg aag gcc gtg ggg ctc      495
Lys Pro Phe Phe Ala Ala Ala Lys Glu Ala Gly Lys Ala Val Gly Leu
        125                 130                 135
gtg acc acc acc acc gtc acc cac gcc acc ccg gcg agc ttc gtg gtg      543
Val Thr Thr Thr Thr Val Thr His Ala Thr Pro Ala Ser Phe Val Val
    140                 145                 150
tcc aat ccc gac cgg aac gcc gag gag agg atc gcc gag cag tac ctg      591
Ser Asn Pro Asp Arg Asn Ala Glu Glu Arg Ile Ala Glu Gln Tyr Leu
155                 160                 165                 170
gag ttc ggg gcc gag gtg tac ctt ggg ggc ggg gac cgc ttt ttc aac      639
Glu Phe Gly Ala Glu Val Tyr Leu Gly Gly Gly Asp Arg Phe Phe Asn
                175                 180                 185
ccc gcc agg cgc aag gac ggg aag gac ctc tac gcc gcc ttc gcc gcc      687
Pro Ala Arg Arg Lys Asp Gly Lys Asp Leu Tyr Ala Ala Phe Ala Ala
            190                 195                 200
aag ggg tac ggg gtg gtg cgc acc ccc gag gag ctc gcc cgt tcc aac      735
Lys Gly Tyr Gly Val Val Arg Thr Pro Glu Glu Leu Ala Arg Ser Asn
        205                 210                 215
gcc acc cgg ctc ctg ggc gtc ttc gcc gac ggc cac gtg ccc tac gag      783
Ala Thr Arg Leu Leu Gly Val Phe Ala Asp Gly His Val Pro Tyr Glu
    220                 225                 230
att gac cgc cgc ttc cag ggc ctt ggg gtg ccg agc ctc aag gaa atg      831
Ile Asp Arg Arg Phe Gln Gly Leu Gly Val Pro Ser Leu Lys Glu Met
235                 240                 245                 250
gtc cag gcc gct ttg ccc cgg ctt gcc gcc cac cgc ggg ggc ttc gtc      879
Val Gln Ala Ala Leu Pro Arg Leu Ala Ala His Arg Gly Gly Phe Val
                255                 260                 265
ctt cag gtg gaa gcg ggg cgg att gac cac gcc aac cat ttg aac gac      927
Leu Gln Val Glu Ala Gly Arg Ile Asp His Ala Asn His Leu Asn Asp
            270                 275                 280
gcc ggg gcc acc ctt tgg gac gtg ctg gcg gcg gac gag gtc ttg gag      975
Ala Gly Ala Thr Leu Trp Asp Val Leu Ala Ala Asp Glu Val Leu Glu
        285                 290                 295
ctt ctc acc gcc ttc gtg gac cgg aac ccg gac acc ctc ctc ctc gtg     1023
Leu Leu Thr Ala Phe Val Asp Arg Asn Pro Asp Thr Leu Leu Leu Val
    300                 305                 310
gtc tcg gac cac gcc acc ggg gtg ggg gcc ctc tac ggg gcg ggc cgg     1071
Val Ser Asp His Ala Thr Gly Val Gly Ala Leu Tyr Gly Ala Gly Arg
315                 320                 325                 330
agc tac ctg gag agc tcc gtg ggc att gac ctc ctg ggg gcg caa aag     1119
Ser Tyr Leu Glu Ser Ser Val Gly Ile Asp Leu Leu Gly Ala Gln Lys
                335                 340                 345
gcc agc ttt gag tac atg cgc cgc gtc ttg ggc tcg gcc ccc gat gct     1167
Ala Ser Phe Glu Tyr Met Arg Arg Val Leu Gly Ser Ala Pro Asp Ala
            350                 355                 360
gcc cag gtg aag gag gcc tac cag acc ctg aag ggg gtc tcc ctc acg     1215
Ala Gln Val Lys Glu Ala Tyr Gln Thr Leu Lys Gly Val Ser Leu Thr
        365                 370                 375
gac gag gag gcg cag atg gtg gtc cgg gcc atc cgc gag cgg gtc tac     1263
Asp Glu Glu Ala Gln Met Val Val Arg Ala Ile Arg Glu Arg Val Tyr
    380                 385                 390
tgg cct gat gcc gtg cgc cag ggc atc cag ccc gaa aac acc atg gcc     1311
Trp Pro Asp Ala Val Arg Gln Gly Ile Gln Pro Glu Asn Thr Met Ala
395                 400                 405                 410
tgg gcc atg gtg cag aag aac gcc agc aag ccc gac cgg ccc aac atc     1359
Trp Ala Met Val Gln Lys Asn Ala Ser Lys Pro Asp Arg Pro Asn Ile
                415                 420                 425
ggc tgg agc tct ggg cag cac acg gcg agc ccc gtc atc ctc ctc ctc     1407
Gly Trp Ser Ser Gly Gln His Thr Ala Ser Pro Val Ile Leu Leu Leu
            430                 435                 440
tac ggc cag ggc ctg cgc ttc gtc cag ctt ggc ctg gtg gac aac acc     1455
Tyr Gly Gln Gly Leu Arg Phe Val Gln Leu Gly Leu Val Asp Asn Thr
        445                 450                 455
cac gtg ttc cgc ctg atg ggc gag gcc ctg aac ctc cgc tac cag aac     1503
His Val Phe Arg Leu Met Gly Glu Ala Leu Asn Leu Arg Tyr Gln Asn
    460                 465                 470
ccg gtg atg agc gag gag gag gcc ctg gag atc ctc aag gcc agg ccc     1551
Pro Val Met Ser Glu Glu Glu Ala Leu Glu Ile Leu Lys Ala Arg Pro
475                 480                 485                 490
cag ggg atg cgc cac ccc gag gac gtc tgg gcc taa gggcgggtcg          1597
Gln Gly Met Arg His Pro Glu Asp Val Trp Ala  *
                495                 500
cgggatcggc cggggccggt tggggtccgt gggagccggg cttttggctt cctgggcggg   1657
aaccttgccc ccgccgaggc agggccgccc cgccaccagg aggtaggcct cctgagccgc   1717
ctcggccaaa agggcgttca cctggcccag gaggtcccgg tagcggcggg cgagggggtt   1777
ttgggggacg atccccatcc ccacctcgtt ggagacggcg atgaccctct tgccgctttc   1837
ctccaccgcg cttaggaagc gcctcgcctc caagaggggg tccaggcccc gttccatcag   1897
gttggcgaac ccagagggtg aggcagtcca ccaccacggt ggggtggcgg gccctcttta   1957
gggcccccgg gaggtccagg ggctcctcca gggtctccca ggtggggggg cgctcctcct   2017
ggtgggcggc gga                                                      2030