Bioscience, Biotechnology, and Biochemistry

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Site-directed Mutagenesis of Catalytic Active-site Residues of Taka-amylase A Tadashi Nagashima, Setsuzo Tada, Katsuhiko Kitamoto, Katsuya Gomi, Chieko Kumagai & Hiroko Toda To cite this article: Tadashi Nagashima, Setsuzo Tada, Katsuhiko Kitamoto, Katsuya Gomi, Chieko Kumagai & Hiroko Toda (1992) Site-directed Mutagenesis of Catalytic Active-site Residues of Taka-amylase A, Bioscience, Biotechnology, and Biochemistry, 56:2, 207-210, DOI: 10.1271/bbb.56.207 To link to this article: http://dx.doi.org/10.1271/bbb.56.207

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Biosci. Biotech. Biochem., 56 (2), 207-210, 1992

Site-directed Mutagenesis of Catalytic Active-site Residues of Taka-amylase A Tadashi NAGASHIMA,tt Setsuzo TADA,ttt Katsuhiko KITAMOTO,t Katsuya GOMI, Chieko KUMAGAI, and Hiroko TODA * National Research Institute of Brewing, 2-6-30, Takinogawa, Kita-ku, Tokyo 114, Japan Protein Research, Osaka University, Suita, Osaka 565, Japan Received June 17, 1991

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* Institute for

The cDNA encoding Taka-amylase A (EC.3.2.1.1, TAA) was isolated to identify functional amino acid residues of T AA by protein engineering. The putative catalytic active-site residues and the substrate binding residue of T AA were altered by site-directed mutagenesis: aspartic acid-206, glutamic acid-230, aspartic acid-297, and lysine-209 were replaced with asparagine or glutamic acid, glutamine or aspartic acid, asparagine or glutamic acid, and phenylalanine or arginine, respectively. Saccharomyces cerevisiae strain YPH 250 was transformed with the expression plasmids containing the altered cDNA of the TAA gene. All the transform ants with an expression vector containing the altered cDNA produced mutant TAAs that cross-reacted with the TAA antibody. The mutant TAA with alteration of Asp206, Glu230, or Asp297 in the putative catalytic site had no IX-amylase activity, while that with alteration of Lys209 in the putative binding site to Arg or Phe had reduced activity.

Taka-amylase A (TAA), an a-amylase (a-I,4-glucan-4glucanohydrolase, EC.3.2.1.l) produced by A. oryzae, is one of the best characterized proteins with respect to physical and chemical properties.!) T AA is a glycoprotein consisting of a single polypeptide chain of 478 amino acid residues 2 ) with a main-domain and C-domain in the three-dimensional structure. Glutamic acid (Glu230) and aspartic acid (Asp297) are located at the bottom of the cleft and are reported to be the catalytic residues. 3,4) Recently, we cloned genomic DNA encoding TAA and sequenced the nucleotides. 5 ) It was found that the gene consist of 2040 bp containing eight introns. The region of the eighth intron seems to distinguish the main-domain from C-domain. In addition to T AA, porcine pancreatic a-amylase (PPA) is also well characterized. 6 ) The enzyme is a single polypeptide chain of 496 amino acid residues and the amino acid sequence homology between T AA and PPA is only about 23%. However, the amino acid sequences at the active site between T AA and PP A are very similar. It is also known that the three-dimensional structures of T AA and PPA are very similar and the catalytic residue of PPA was proposed to be Asp 197 (corresponding to Asp297 in TAA) and Asp300 (corresponding to Asp206 in TAA).7) T AA is a good model protein for elucidation of the structure-function relationship of a-amylases. Therefore, we isolated cDNA encoding TAA using the cloned genomic DNA as a probe and prepared eight kinds of mutant T AAs at positions of the putative catalytically important or substrate binding residues, Asp206, Glu230, Asp297, and Lys209. We describe here cloning of the cDNA andcharacterization of the mutant T AA proteins.

Materials and Methods Strains. Aspergillus oryzae RIB40 was used for isolation of mRNA. Escherichia coli HB101 8 ) was used as a host in plasmid constructions and JM 109 9 ) as a host of M 13 derivative phages for site-directed mutagenesis. Saccharomyces cerevisiae YPH250 10 ) (MATa, trpl, ade2, ura3, his3, lys2, leu2) was used for mutant Taka-amylase A production. Media and culture conditions. A. oryzae was grown at 37°C in YPM-CD medium (3% maltose, 1% polypeptone, 0.5% yeast extract, 0.3% NaN0 3 , 0.2% KCI, 0.1% KH zP0 4 , 0.05% MgS0 4 '7H zO, and 0.001 % FeS0 4 ·7H zO). Yeast cells were grown with shaking at 30°C. Yeast transformants were selected on SD-A plates (0.67% Yeast Nitrogen Base wlo amino acids [Difco Laboratories, Inc.], 2% glucose, 20ppm adenine, 20 ppm L-histidine, 30 ppm L-Ieucine, 30 ppm L-Iysine, 20 ppm uracil) containing 2% agar. Yeast cells were grown at 30°C in YPD medium (1 % yeast extract [Difco], 2% polypeptone [Wako Junyaku] and 2% glucose). SD-A-WS plates (SD-A medium containing 0.5% wheat starch, and 2% agar) were used for halo formation. Construction of a eDNA library. A. oryzae (RIB 40) was grown in YPM-CD medium at 37°C for 48 hr with shaking. Then the culture broth was centrifuged to obtain wet mycelium, which was disrupted with a mortar and pestle in liquid nitrogen, and total RNAs were isolated from it by the guanidine thiocyanate methodY) Poly(A)+RNAs were obtained by chromatography on an oligo (dT)-cellulose column. Double-stranded cDNAs were synthesized llsihg the poly(A)+ RNAs with cDNA synthesis kit (Amersham). EcoRI linkers were ligated to each end of the cDNAs. The cDNAs with icoRI linkers were then inserted into the EcoRI site of Agt10 using a Lambda gtlO cloning kit (Stratagene). The DNA was packed using an iii vitro packaging kit (Stratagene), constructing a cDNA library consisting of approximately 1200 independent clones. cDNAs encoding TAA were .selected by plaque hybridization, using the genomic T AA probes. Construction of plasmid and transformation of E. coli and S. cerevisiae. DNA was manipulated by standard methods. 8 ) Restriction endonucleases, T4 DNA ligase, and alkaline phosphatase were purchased from Takara Shuzo ~o., Ltd., Toyobo Co., Ltd., and Boehringer Mannheim Co., Ltd., respectively. Plasmid preparation and analysis were done as described elsewhere. 8) The 1.8 kb EcoRI-EcoRI fragment containing the Taka-amylase A cDNA from pTcD-1 was isolated from agarose gel by a Geneclean kit

t To whom correspondence should be addressed. Present address: tt Shin Nihon Chemical Co., Ltd., 19-10, Shouwa-cho, Anjyo-shi 446, Japan. ttt Kirin Brewery Co., Ltd., Technical Center, 17-1, Namamugi l-chome, Tsurumi-ku, Yokohama-shi 230, Japan.

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(Funakoshi) and inserted at the EcoRI site of pYcDE-I containing the ADHl promoter and CYCl terminator, resulting in pYTA-I (Fig. I). E. coli strains HBlOi and JM109 were transformed by the standard method. 12 ) S. cerevisiae strain YPH2S0 was transformed by the lithium acetate method of Ito et al. 13 )

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Measurement of a-amylase activity. a-Amylase activity was measured by the modified blue value method of Adachi. 14) A soluble starch (Merck) solution was prepared by dissolving 1.0 g of starch in 100 ml of 0.04 M acetate buffer, pH S.O. A working iodine solution was prepared by diluting a stock solution (0.317 g iodine and 1.0 g potassium iodate/lliter of S% HCI solution) 10 times. A 2-ml solution of I % soluble starch was placed in a test tube and incubated at 40°C for S min, then 0.1 ml of enzyme solution was added. After the enzyme solution was reacted for 10 min, 20 III of the reaction mixture was added to 2 ml of the working iodine solution and the absorbance at 670 nm was measured. One unit of a-amylase was defined as the amount of the enzyme solution that hydrolyzed I ml of I % soluble starch in 30 min. The protein concentration was measured by the method of Bradford using bovine serum albumin as a standard protein. 15) A plate assay for a-amylase activity was done by a halo formation method using 12 vapor. 16 ) Maltosidase activity. Maltosidase activity was measured by liberated p-nitrophenol using p-nitrophenyl-a-maltoside as substrate. 1 7) One ml of enzyme solution and I ml of 0.2 M acetate buffer of pH S.3 were incubated at 37°C and the reaction was started by the addition of O.S% p-nitrophenyl-a-maltoside solution. After 30 min, S ml of S% sodium carbonate solution was added to stop the enzymatic reaction. The liberated p-nitrophenol was measured colorimetrically at 400 nm. One unit of maltosidase activity was defined as the amount liberating Illmol of p-nitrophenol from p-nitrophenyl-a-maltoside in 60 min. Oligonucleotide site-directed mutagenesis. Single-stranded DNA was isolated from E. coli JM109 containing pTcD-I using the helper phage M 13K07 by the manufacturer's suggested procedures. 8 ) The oligonucleoE

I

Bg BE

[ Sm!

S

!

!

'I

Taka-amylase A eDNA

ADH1

E

~

ADH1 E

terminator Yeast 211 m

TRP1

Fig. 1.

Construction of p YT A-I.

A eDNA clone of Taka-amylase A was subcloned into the EcoRI site of pUC118, resulting in pTcD-1. The enzyme cDNA was inserted at the EcoRI site of p Y cDE-I containing the ADHl promoter and CYCl terminator, resulting in pYTA-1. E, EcoRI; S, Sail; P, PstI; Sm, SmaI; Bg, BglII; B, BamHI.

Table I.

et al.

tide primers containing the desired changes for the mutagenesis were synthesized by the phosphoamidite method using a DNA synthesizer (Applied.Biosystems Model 381A) and these were purified through OPC cartridge (Applied Biosystems). Site-directed mutagenesis with the singlestranded Taka-amylase A cDNA and synthesized oligonucleotides listed in Table I was done with an oligonucleotide-directed in vitro mutagenesis system (Amersham Co., Ltd.) based on the method developed by Eckstein and coworkers. 18) The mutations were confirmed by restriction enzyme digestion for created restriction sites that are silent mutations with respect to amino acids (Table I). Expression of mutant enzymes and Western blotting. S. cerevlszae YPH2S0 was transformed with each of the yeast expression plasmids containing altered T AA cDNAs, p YT A-I containing wild type T AA cDNA, and pYcDE-1. The transformants were cultured first in SD-A medium, harvested by centrifugation, and inoculated into YPD medium. After two days of cultivation with shaking at 30°C, the culture supernatants were concentrated 20- to SO-fold by ultrafiltration (Kurabo Centricut) with a molecular weight cut-off of 10,000. SDS-PAGE was done with a 10-20% gradient gel (Daiichi Pure Chemicals Co., Ltd.). Resolved proteins in the gel were transferred to a PVDF membrane (Immobilon, Millipore) using a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad). The blotted membrane was soaked in 1% bovine serum albumin-PBS at 37°C for 2 hr to block the non-specific binding sites. The membrane was then dipped in a solution of diluted anti-Taka-amylase A polyclonal rabbit antibody and incubated at room temperature for 2 hr. After this was washed three times in Tween 20-PBS, peroxidase labelled donkey anti-rabbit F(ab')2 fragments (Amersham) were left to react with the membrane for 1 hr at room temperature. After being washed five more times with Tween 20-PBS, the membrane was stained with Immunostain HRP (Konica).

Results Cloning of T AA cD N A The cDNA library was screened by plaque hybridization with the genomic T AA probes and 6 positive clones were isolated. The restriction enzyme map of the isolated cDNA clones showed that all cDNAs have a BamHI site in the 3' flasking region. It suggested that they originated from amy B or amyC, since A. oryzae has three genes encoding TAA of which only the amyA gene has no BamHI site. 19 ) Among 6 clones, the longest cDNA was then subcloned into pUCl18 and designated pTcD-l. The nucleotide sequence of 5' and 3' flanking regions of the cDNA identified by the dideoxy sequencing method showed that the cDNA is identical with amyl reported by Wirsel et al. 20 ) and amyB and amyC by Gomi et al. 19 ) The cDNA was also inserted into a yeast expression vector, pYcDE-I, and T AA expression by S. cerevisiae was confirmed by halo formation and Western blotting after SDS-PAGE. Site-directed mutagenesis To analyze the roles of 4 amino acid residues reported

Oligonucleotides Used in Site-directed Mutagenesis

Mutation

Plasmid

Oligonucleotide

Restriction site

Asp206-Asn Asp206-Glu Lys209-Phe Lys209-Arg Glu230-Gln Glu230-Asp Asp297-Asn Asp297-Glu

pYTA-D206N pYTA-D206E pYTA-K209F pYTA-K209R pYTA-E230Q pYTA-E230D pYTA-D297N pYTA-D297E

3' -GAGGCATAGTTGTGTCATTTTGTGCACGTCTTCCTG-S' 3' -GAGGCATAGCTCTGTCATTTTGTGCACGTCTTCCTG-S' 3' -TAGCTGTGTCA TAAAGTGCACGTCTTCCTGAAG-S' 3' -T AGCTGTGTCATGTCGTGCACGTCTTCCTGAAG-S' 3' -CGTCCGCATATGACATAGCCGGTCCACGAGCTGCAC-S' 3' -CGTCCGCATATGACATAGCCGCTGCACGAGCTGCAC-S' 3' -GAGGACCCATGGAAGCAGCTCTTGGTGTTGTTGGGTGCC-S' 3' -GAGGACCCATGGAAGCAGCTCTTGGTGCTCTTGGGTGCC-S'

PmaCI PmaCI PmaCI PmaCI Accl Accl KpnI KpnI

The mismatches are shown by bold face characters. The underlinings of three bases show the converted amino acid and the underlinigs of six bases show the restriction site created containing silent mutations.

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kOa 1 2 3 4 5 6 7 8 9 10 11 12

94-

674330-

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Fig. 2. Western Blot Analysis of Wild-type and Mutant Taka-amylase A Secreted by Yeast Transformants. Lanes I and 12, native Taka-amylase A produced by A. oryzae (0.1 /lg); lane 2, the concentrated culture supernatant of yeast transformant harboring pYTA-I (wild-type); lane 3, pYcDE-l (yeast expression vector); lane 4, pYTA-D206N; lane 5, pYTA-D206E; lane 6, pYTA-K209F; lane 7, pYTA-K209R; lane 8, pYTAE230Q; lane 9, pYTA-E230D; lane 10, pYTA-D297N; lane 11, pYTA-D297E.

Table II. A

(X-Amylase and Maltosidase Activities of Mutant Taka-amylase

Mutant enzyme Wild-type Asp206-Asn Asp206-Glu Lys209-Phe Lys209-Arg Glu230-Gln Glu230-Asp Asp297-Asn Asp297-Glu

(X-Amylase activity

Maltosidase activity

0.35U/mg

2.9U/mg

N.D. N.D. N.D.

N.D. N.D. N.D.

0.18

5.9

N.D. N.D. N.D. N.D.

N.D. N.D. N.D. N.D.

The transformants were cultured as described in Materials and Methods and (X-amylase and maltosidase activities of the culture supernatants were analyzed. The protein ,concentration of the supernatants was measured and enzyme activity was expressed as U /mg protein. N.D., not detected.

mants were cultured as described in Materials and Methods and the culture supernatants were analyzed by SD8-PAGE. The gels were tested by Western blotting and the proteins reacting with the anti-Taka-amylase A antibody were detected in the culture media of the ten kinds of yeast transformants (Fig. 2). The transformants harboring the nine kinds of plasmids, except for pYcDE-l, secreted protein cross-reacting with the TAA antibody. All of the T AAs produced by yeast had a little larger molecular weight than native T AA. It is provable that they have more sugar chains.

Fig. 3.

Halo Formation on Wheat Starch Plates.

The yeast transform ants carrying one of the following plasm ids were grown on SD-A-WS plate at 30°C for 5 days. The plasmids are: (1) pYTA-l as a control; (2) pYTA-D206N; (3) pYTA-D206E; (4) pYTA-K209F; (5) pYTA-K209R; (6) pYTA-E230Q; (7) pYTA-E230D; (8) pYTA-D297N; (9) pYTA-D297E.

to be catalytically important, eight kinds of codon conversion (Asp206-Asn, Asp206-Glu, Glu230-Gln, Glu230Asp, Asp297-Asn, Asp297-Glu, Lys209-Phe, and Lys209Arg) were done by the oligonucleotide-directed in vitro mutagenesis method. As shown in Table I, to introduce two to four base changes necessary for the desired codon conversion, 33-39 mer oligonucleotides were synthesized and used as probes of site-directed mutagenesis. The resulting double-stranded DNA was inserted to a yeast expression vector (pYcDE-l), as well as wild-type cDNA. The eight expression plasmids were designated p YTAD206N, pYTA-D206E, pYTA-K209F, pYTA-K209R, pYTA-E230Q, pYTA-E230D, pYTA-D297N, and pYTAD297E, according to the conversions described above. Western blotting analysis of wild-type and mutant Takaamylase A S. cerevisiae YPH250 was transformed with the eight expression plasmids containing mutated cDNAs, pYTA-l containing wild-type cDNA, and pYcDE-l. The transfor-

Assay of mutant Taka-amylase A produced by S. cerevisiae The eight kinds of mutant TAA and wild-type TAA (as a control) by the transformants were assayed both by halo formation on the SD-A-WS plates and by measuring the a-amylase activity and maltosidase activity of the concentrated culture supernatants (Fig. 3 and Table II). The transformant harboring pYTA-K209R formed as large and clear halo as pYTA-1 and a small and unclear halo was detected around the transformant harboring pYTA-K209F. No halo was detected around the other transformants harboring pYTA-D206N, pYTA-D206E, pYTA-E230Q, pYTA-E230D, pYTA-D297N, and pYTAD297E. We analyzed the a-amylase and maltosidase activities of the culture broth. The mutant enzyme Lys209-Arg had about half of the wild-type a-amylase activity but the maltosidase activity of this mutant enzyme showed about 2-fold increase. a-Amylase and maltosidase activities of Lys209-Phe were not detected. The activities of the other mutant T AAs were also not detected, even using 50-fold concentrated culture broth, indicating that Asp206, Glu230, and Asp297 are indispensable for the catalytic activity.

Discussion A TAA cDNA from A. oryzae was cloned and expressed by S. cerevisiae, and using the cDNA, eight kinds of mutated cDNA were constructed by site-directed mutagenesis. The positions of three residues which are well conserved in the amino acid sequences of the a-amylases have been identified by X-ray crystallographic method:

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Asp206, Glu230, and Asp297 were at the end of the 4th p-strand, at the end of the 5th p-strand, and at the loop region between the 7th p-strand and the 7th a-helix in the (pa)8 barrel structure, respectively. On a model fitting of an amylose chain in the catalytic site of the enzyme, Glu230 and Asp297, which are at the bottom of the cleft, are placed about 3 A from the glucosidic oxygen between the 4th and 5th site glucoses. 3 ) The three carboxylic acids, Asp206, Glu230, and Asp297 in the catalytic active site of T AA, were altered at each residue to their corresponding amide or the other carboxylic acid, and Lys209, which is positioned in the substrate binding residue, was replaced by phenylalanine or arginine. These altered TAA cDNAs were expressed and mutant T AAs were secreted by S. cerevisiae as a host. No a-amylase activities of the secreted mutant enzymes, except for alteration of Lys209, were detected. This strongly suggests that the carboxylic acid residues Asp206, Glu230, and Asp297 are the catalytic residues of T AA, supporting the prediction of the catalytic residues by crystallographic studies of T AA. 4) Recently, Takase et a/. 2 l) reported that in site-directed mutagenesis of the a-amylase of Bacillus subtilis (BSA), Asp176 (corresponding to Asp297 in TAA and Asp197 in PP A), Glu208 (corresponding to Glu 230 in T AA), and Asp269 (corresponding to Asp206 in T AA and Asp300 in PPA) were replaced with their corresponding amide, resulting in complete loss of their enzyme activities. It strongly indicates that these three carboxylic acid residues in BSA are situated in the catalytic active site and associated with the expression of the catalytic activity. The results of TAA reported here are consistent with those of BSA. a-Amylase and maltosidase activities of the mutant enzyme Lys209-Arg were about half and 2-fold compared with those of wild-type enzyme, respectively, but the other mutant enzyme, Lys209-Phe, showed only a very little a-amylase activity on a halo formation plate. Ikenaka 17) previously reported that maltosidase activity of the modified TAA with p-phenylazobenzoyl (PhAB) chloride increases more than the original activity, but that a-amylase activity markedly decreases. It is suggested that the site of combination of the PhAB residue seems to be an 8-amino group of the special lysine residue situated in the vicinity of the active site. The result of the mutant enzyme Lys209 strongly supports this report of Ikenaka. Although alteration of the substrate-binding residues of other a-amylases has not yet been done, Muraki et a/. 22 ) reported replacement of the Arg115, which is the substrate-binding residue of human lysozyme, by sitedirected mutagenesis. The conversions of Argl15 to Lys or His (at acidic pH) affected the activity only a little, but the conversions of Arg115 to His (at neutral and alkaline pH), GIn or Glu did not reduce the activity. The results with

Lys209 of TAA are very similar to that of Argl15 of human lysozyme. We consider that this residue, Lys209, is structurally required not for specific hydrogen bonding interaction with a sugar residue but for the positive charge character in the construction of a sub site in T AA. We report here that the three carboxylic acids, Asp206, Glu230, and Asp297 participate in an important functional catalytic reaction, and that the Lys209 is crucial as the substrate-binding residue. However, further studies should be done such as kinetic characterization and the crystallographic analysis of complexes of the substrate and the mutant enzymes. These studies using the mutant enzymes are in progress. Acknowledgments. We thank Dr. Y. Ohya of Tokyo University for his kind gift of S. cerevisiae strain YPH250 and Y. Hata for helpful discussions.

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9) 10) 11) 12) 13) 14) 15) 16) 17) 18) 19)

20) 21) 22)

T. Takagi, H. Toda, and T. Isemura, in "The Enzymes," Vol. 5, ed. by P. D. Boyer, 3rd Ed., Academic Press, Inc., New York, 1971, pp. 235-271. H. Toda, K. Kondo, and K. Narita, Proc. Jpn. Acad., 58B, 208-212 (1982). Y. Matsuura, M. Kusunoki, W. Harada, and M. Kakudo, J. Biochem., 95, 697-702 (1984). T. Matsuura, M. Kusunoki, and M. Kakudo, Denpun Kagaku, 38, 137-139 (1991). S. Tada, Y. Iimura, K. Gomi, K. Takahashi, S. Hara, and K. Yoshizawa, Agric. Bioi. Chem., 53, 593-599 (1989). L. Pasero, Y. Mazzei-Pierron, B. Abadie, Y. Chicheportiche, and G. Marchis-Mouren, Biochim. Biophys. Acta, 869, 147-157 (1986). G. Buisson, E. Duee, R. Haser, and F. Payan, EMBO J., 6, 3909-3916 (1987). T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory, New York, 1982. J. Messing, Gene, 33, 103-119 (1985). R. S. Sikorski and P. Hieter, Genetics, 122, 19-27 (1989). H. Okayama, M. Kawaichi, M. Brownstein, F. Lee, T. Yokota, and K. Arai, Methods in Enzymol., 154, 3-28 (1987). D. Hanahan, J. Mol. Bioi., 166, 557-580 (1983). H. Ito, Y. Fukuda, K. Murata, and A. Kimura, J. Bacteriol., 153, 163-168 (1983). T. Adachi, J. Ferment. Technol., 32, 43-44 (1954). J. F. Bradford, Anal. Biochem., 72, 248-254 (1976). M. Emori, T. Tojo, and B. Maruo, Agric. Bioi. Chem., 52,399-406 (1988). T. Ikenaka, J. Biochem., 46, 297-304 (1959). J. W. Taylor, J. Ott, and F. Eckstein, Nucl. Acids Res., 13, 8765-8785 (1985). S. Hara, K. Kitamoto, and K. Gomi, in "Aspergillus," ed. by J. W. Bennett and M. A. Klich, Butterworth, Stoneham, M. A., 1992, pp. 133-153. S. Wirse1, A. Lachmund, G. Wildhardt, and E. Ruttkowski, Mol. Microbiol., 3, 3-14 (1989). K. Takase, T. Matsumoto, H. Mizuno, and K. Yamane, 2nd Int. Conference of Protein Engineering Abstracts, PE89 (1989). M. Muraki, M. Morikawa, Y. Jigami, and H. Tanaka, Eur. J. Biochem., 179, 573-579 (1989).

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Site-directed mutagenesis of catalytic active-site residues of Taka-amylase A.

The cDNA encoding Taka-amylase A (EC.3.2.1.1, TAA) was isolated to identify functional amino acid residues of TAA by protein engineering. The putative...
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