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Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Cloning, Expression, and Characterization of a Minor Alkaline Protease from Bacillus sp. No. AH-101 ab
b
Hideto Takami , Teruhiko Akiba & Koki Horikoshi
ab
a
Department of Bioengineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 227, Japan b
Microbiology Laboratory, The Institute ofPhysical and Chemical Research, 2–1, Hirosawa, Wako, Saitama 351–01, Japan Published online: 12 Jun 2014.
To cite this article: Hideto Takami, Teruhiko Akiba & Koki Horikoshi (1992) Cloning, Expression, and Characterization of a Minor Alkaline Protease from Bacillus sp. No. AH-101, Bioscience, Biotechnology, and Biochemistry, 56:3, 510-511, DOI: 10.1271/bbb.56.510 To link to this article: http://dx.doi.org/10.1271/bbb.56.510
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Biosci. Biotech. Biochem., 56 (3), 510-511, 1992
Note
Cloning, Expression, and Characterization of a Minor Alkaline Protease from Bacillus sp. No. AH-IOI Hideto TAKAMI, *,** Teruhiko AKIBA, ** and Koki HORIKOSHI*'**
*Department of Bioengineering, Faculty of Bioscience and Biotechnology,
Downloaded by [Virginia Commonwealth University Libraries] at 20:14 13 March 2015
Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 227, Japan **Microbiology Laboratory, The Institute ofPhysical and Chemical Research, 2-1, Hirosawa, Wako, Saitama 351-01, Japan Received June 20, 1991
An alkalophilic Bacillus sp. No. AH-101 produces an extracellular alkaline protease as a main product. The No. AH-101 alkaline protease was characterized in our previous papers. 1.2) On the other hand, it has been reported that most Bacilli produce and secrete three or more proteases at the early stationary phase of growth. 3) However, since the ratio of each protease amount produced varies with the genus Bacillus of strains,3.4) it is not easy to find lesser proteases produced in the culture broth. The shotgun cloning method was used to search for other proteases produced by Bacillus sp. No. AH-101 in this study. Here we report the obtaining of a minor alkaline protease and characterization of the enzymatic properties. Chromosomal DNA of Bacillus sp. No. AH-101 was digested with EcoRI and then DNA fragments longer than 1kb were separated by sucrose density gradient centrifugation. The cloning vector, pHY300PLK,5) was cleaved with EcoRI. The ligation and transformation to E. coli HB-101 were done by the standard methods. The transformants
were spread on LB agar plates (pH 8.0) containing 1% (wt/vol) skim milk (LBC agar plate). For E. coli HB-IOI carrying pHY300PLK and its derivatives, tetracycline was added to the media at a final concentration of 20 J-tg/ml. Protease activity of the transformants was detected as the formation of large halos around colonies on LBC agar plates. One ampicillin resistant (Amp'), tetracycline resistant (Tet') clone that formed a large clear halo on LBC agar plate was obtained. The plasmid isolated from the clone, pAH 10 IMP, contained an insert of approximately 1.4kb of DNA. The restriction map of pAHIOIMP is shown in Fig. 1. Southern blot analysis showed that the insert was derived from the chromosomal DNA of Bacillus sp. No. AH-101 (data not shown). E. coli HB-101 (pAHIOIMP) started to produce the alkaline protease at logarithmic phase on LB medium independent of the presence of skim milk. The protease was not secreted into the culture broth. The protease production by the transformant was 16000 units per 400-ml culture. Protease activity was assayed as described previously. I) The culture was harvested at the early stationary phase and resuspended in 0.02 M glycine-NaCI-NaOH buffer (pH 9.0). The supernatant fluid obtained from a sonicated lysate was heat-treated at 50°C for 10 min. After
Hae III
1
2
94.0k 67.0k
BanI
PvuI
43.0k
pAHIOIMP 6.3 kbp
36.8k -
HincH
_~
EcoRV
30.0k '28.8k 20.1k 14.4k
AccI
Fig. 2. SDS-PAGE of Minor Alkaline Protease Cloned in E. coli HB-l 0I (pAH 10 IMP) and Thermostable Alkaline Protease from Bacillus sp. No. AH-lOl.
NcoI
Fig. 1.
Restriction Map of Plasmid pAHlOIMP.
Amp', ampicillin resistance gene; Tet', tetracycline resistance gene. The thick line denotes the fragment from Bacillus sp. No. AH-IOI. The thin line denotes pHY300PLK.
Table I.
1: E. coli HB-IOI (pAHIOIMP). 2: Thermostable alkaline protease from Bacillus sp. No. AH-IOl.l) M.W.: Molecular mass markers: Phosphorylase b (94.0K), albumin (67.0 K), ovalbumin (43.0 K), carbonic anhydrase (30.0 K), trypsin inhibitor (20.1 K), IX-lactalbumin (14.4 K). K = kilodaltons.
Purification of a Minor Alkaline Protease Step
Sonication Heat-treatment (50°C) DEAE-Toyopearl Casein-Sepharose 6B
Volume (ml)
Activity (units/ml)
Protein (mg)
Specific activity (units/mg-protein)
Recovery
900 870 16 8
190 160 2010 2080
4300 2760 20 6
40 50 1560 2780
100 82
(%)
22 10
Offprint requests to: Hideto TAKAMI, Department oj Bioengineering, Faculty oj Bioscience and Biotechnology, Tokyo Institute oj Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 227, Japan.
NII-Electronic Library Service
Cloning of a Minor Alkaline Protease from Bacillus
A
B 100
~
~
~
Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Cloning, Expression, and Characterization of a Minor Alkaline Protease from Bacillus sp. No. AH-101 ab
b
Hideto Takami , Teruhiko Akiba & Koki Horikoshi
ab
a
Department of Bioengineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 227, Japan b
Microbiology Laboratory, The Institute ofPhysical and Chemical Research, 2–1, Hirosawa, Wako, Saitama 351–01, Japan Published online: 12 Jun 2014.
To cite this article: Hideto Takami, Teruhiko Akiba & Koki Horikoshi (1992) Cloning, Expression, and Characterization of a Minor Alkaline Protease from Bacillus sp. No. AH-101, Bioscience, Biotechnology, and Biochemistry, 56:3, 510-511, DOI: 10.1271/bbb.56.510 To link to this article: http://dx.doi.org/10.1271/bbb.56.510
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Biosci. Biotech. Biochem., 56 (3), 510-511, 1992
Note
Cloning, Expression, and Characterization of a Minor Alkaline Protease from Bacillus sp. No. AH-IOI Hideto TAKAMI, *,** Teruhiko AKIBA, ** and Koki HORIKOSHI*'**
*Department of Bioengineering, Faculty of Bioscience and Biotechnology,
Downloaded by [Virginia Commonwealth University Libraries] at 20:14 13 March 2015
Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 227, Japan **Microbiology Laboratory, The Institute ofPhysical and Chemical Research, 2-1, Hirosawa, Wako, Saitama 351-01, Japan Received June 20, 1991
An alkalophilic Bacillus sp. No. AH-101 produces an extracellular alkaline protease as a main product. The No. AH-101 alkaline protease was characterized in our previous papers. 1.2) On the other hand, it has been reported that most Bacilli produce and secrete three or more proteases at the early stationary phase of growth. 3) However, since the ratio of each protease amount produced varies with the genus Bacillus of strains,3.4) it is not easy to find lesser proteases produced in the culture broth. The shotgun cloning method was used to search for other proteases produced by Bacillus sp. No. AH-101 in this study. Here we report the obtaining of a minor alkaline protease and characterization of the enzymatic properties. Chromosomal DNA of Bacillus sp. No. AH-101 was digested with EcoRI and then DNA fragments longer than 1kb were separated by sucrose density gradient centrifugation. The cloning vector, pHY300PLK,5) was cleaved with EcoRI. The ligation and transformation to E. coli HB-101 were done by the standard methods. The transformants
were spread on LB agar plates (pH 8.0) containing 1% (wt/vol) skim milk (LBC agar plate). For E. coli HB-IOI carrying pHY300PLK and its derivatives, tetracycline was added to the media at a final concentration of 20 J-tg/ml. Protease activity of the transformants was detected as the formation of large halos around colonies on LBC agar plates. One ampicillin resistant (Amp'), tetracycline resistant (Tet') clone that formed a large clear halo on LBC agar plate was obtained. The plasmid isolated from the clone, pAH 10 IMP, contained an insert of approximately 1.4kb of DNA. The restriction map of pAHIOIMP is shown in Fig. 1. Southern blot analysis showed that the insert was derived from the chromosomal DNA of Bacillus sp. No. AH-101 (data not shown). E. coli HB-101 (pAHIOIMP) started to produce the alkaline protease at logarithmic phase on LB medium independent of the presence of skim milk. The protease was not secreted into the culture broth. The protease production by the transformant was 16000 units per 400-ml culture. Protease activity was assayed as described previously. I) The culture was harvested at the early stationary phase and resuspended in 0.02 M glycine-NaCI-NaOH buffer (pH 9.0). The supernatant fluid obtained from a sonicated lysate was heat-treated at 50°C for 10 min. After
Hae III
1
2
94.0k 67.0k
BanI
PvuI
43.0k
pAHIOIMP 6.3 kbp
36.8k -
HincH
_~
EcoRV
30.0k '28.8k 20.1k 14.4k
AccI
Fig. 2. SDS-PAGE of Minor Alkaline Protease Cloned in E. coli HB-l 0I (pAH 10 IMP) and Thermostable Alkaline Protease from Bacillus sp. No. AH-lOl.
NcoI
Fig. 1.
Restriction Map of Plasmid pAHlOIMP.
Amp', ampicillin resistance gene; Tet', tetracycline resistance gene. The thick line denotes the fragment from Bacillus sp. No. AH-IOI. The thin line denotes pHY300PLK.
Table I.
1: E. coli HB-IOI (pAHIOIMP). 2: Thermostable alkaline protease from Bacillus sp. No. AH-IOl.l) M.W.: Molecular mass markers: Phosphorylase b (94.0K), albumin (67.0 K), ovalbumin (43.0 K), carbonic anhydrase (30.0 K), trypsin inhibitor (20.1 K), IX-lactalbumin (14.4 K). K = kilodaltons.
Purification of a Minor Alkaline Protease Step
Sonication Heat-treatment (50°C) DEAE-Toyopearl Casein-Sepharose 6B
Volume (ml)
Activity (units/ml)
Protein (mg)
Specific activity (units/mg-protein)
Recovery
900 870 16 8
190 160 2010 2080
4300 2760 20 6
40 50 1560 2780
100 82
(%)
22 10
Offprint requests to: Hideto TAKAMI, Department oj Bioengineering, Faculty oj Bioscience and Biotechnology, Tokyo Institute oj Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 227, Japan.
NII-Electronic Library Service
Cloning of a Minor Alkaline Protease from Bacillus
A
B 100
~
~
~
