Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-05-04
2003-11-18
Saidha, Tekchand (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S195000, C435S194000, C435S252300, C435S320100, C536S023200
Reexamination Certificate
active
06649390
ABSTRACT:
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'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 G
Ji Chaoneng
Mao Yumin
Sheng Xiaoyu
Yuan Youzhong
Zhou Zongxiang
Baker & Botts L.L.P.
Fudan University
Saidha Tekchand
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