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@ Springer-Verlag1989
Summary. An alkalophilic Bacillus sp. no. AH- myces species also produces alkaline protease
101, which produced extremelythermostableal- (Nakanishi and Yamamoto 1974).All of these al-
kaline protease,was isolatedamong 200 soil sam- kaline proteaseswere characlerizedby their activ-
ples. The enzyme production reached its maxi- ity and stability under highly alkaline conditions.
mum level of 1500units/ml after about 24h in al- Streptomycesalkaline proteasehas been found to
kaline medium (pH 9.5). The enzyme was most be most active at around pH 13. No. 221 protease
active toward casein at pH 12-13 and stable to was recognized to be stable not only at high pH
10min incubation at 60'C from pH 5-13. Cal- but also at a temperatureof 60'C. It has been
cium ions were effective in stabilizing the eflzyme found that some enzymescan be used in laundry
especially at higher temperatures.The optimum detergents(Aunstrup et at.1972).
and stabletemperatureswere about 80'C and be- The purpose of this study is to explore new
low about 70oC respectivelyin the presenceof types of alkaline proteasethat are stable not only
5 mM calcium ions. The enzymewas completely against high pH but also high temperature and
inactivated by phenylmethanesulphonyl fluoride, detergents.This paper deals with the isolation of
but little affected by ethylenediaminetetraacetic Bacillussp. no. AH-101, and propertiesof the en-
acid, urea, sodium dodecylbenzenesulphonate zyme.
and sodium dodecyl sulphate. The molecular
weight and sedimentationconstant were approxi-
mately 30000 and 3.0Srespectively,and the isoel- Materials and methods
ectric point was at pH 9.2. These results indicate
that no. AH-101 alkaline proteaseis more stable Isolation. The alkaline medium (A-II) and proceduresfor iso-
againstboth temperatureand highly alkaline con- lation of alkalophilic bacteriapreviouslydescribed(Horikoshi
1971b)were employed in this study. Isolates were grown on
ditions than any other proteaseso far reported. A-II broth and the production of alkaline protease and its
thermostabilityat 70'C at pH 10.8were tested.Bacillussp. no.
AH-101 producing an extremely thermostable alkaline pro-
teasewas selectedfrom about 1000colonies.
Enzyme assay. Protease activity was assayedby the modified Purification of alkaline protease
method of Anson (Anson 1938).Enzymesolution (0.5ml) suit-
ably diluted was mixed with 2.5 ml of 0.6% Hammarstenca-
Culture fluid was added to 750 ml DEAE-Toyo-
sein (Merck & Co., Darmstadt,FRG) solution (pH 10'5,made
up with 50 mM glycine:NaCl: NaOH buffer). After incubation pearl (Toyo Soda Co., Tokyo, Japan),which had
for 20 min at 30'C, 2.5 ml of trichloroaceticacid (TCA) solu- been equilibratedwith 0.02M Na2CO3:NaHCO3
tion (consisting of 0.11 M TCA, 0.22M sodium acetate and buffer (pH 9.5), and was filtered after mixing for
0.33M acetic acid) was added to stop the reaction. The mix- 2 h. The no. AH-101 proteasewas not adsorbed
ture was further incubated at 30'C for 30min and then fil-
tered with Toyo Roshi filter paper no. 5C (Toyo Roshi Co., by DEAE-Toyopearl 650 M. The filtrate was dial-
Tokyo, Japan). To 0.5 ml of filtrate 2.5 ml of 0.5 M NazCO: ysed against tap water for 24 h then adjusted to
solution and 0.5 ml twofold-diluted Folin-Ciocalteaureagent pH 5.5 with acetic acid and loaded on a CM-
(Lowry et al. 1951) was added. After standing for 30 min at Toyopearl 650 M column (2.3 x 60 cm) equili-
room temperature,the absorbancewas measuredat 660 nm.
brated with 0.02 M acetate buffer (pH 5.5). The
One unit of proteaseactivity was de{ined as the amount of the
enzymeto produce 1 pg tyrosine./min. column was washedwith the samebuffer (pH 5.5)
and the enzyme was eluted with a linear NaCl
Protein contenf. Protein content was measuredat 280 nm, at (0.1-0.3 M) gradient in the buffer. Alkaline pro-
595 nm (Bradford 1976) using a protein assay kit (Bio-Rad teasewas purified threefold with an overall yield
Chemical Division, Calif, USA) and Lowry's method (Lowry
et al. 1951)with bovine plasma gamma globulin as standard
of 20%. Specific activity of the enzyme was 2427
protein. units/mg at pH 10.5 and 4080 units/mg at pH
12.5,which was about the optimum pH. The re-
Electrophoresisand detection of enzyme actiuity. Polyacrylam- sults of the purification are summarized in Table
ide gel slab electrophoresiswas carried out for determination
1. Figure 1 shows the single protein band which
of purity ofthe enzyme(Taber and Sherman1964).Separation
gel(7.5%)and stackinggel (a.8%)were preparedand 35.6mM had proteaseactivity.
2.6-lutidine,18.2mM glycine buffer (pH 8.3) was used as the
electrode buffer. Sodium dodecyl sulphate-polyacrylamide
electrophoresis(SDS-PAGE) was also performed for the de- Sedimentationconstant,molecular weight and
termination of molecular weight with 720/ogels (Laemmli
1970).No. AH-101 proteasecontaining 2 mM phenylmethane
isoelectricpoint
sulphonyl fluoride (PMSF) as the final concentrationwas heat
treated with sample application buffer containing 8% SDS at The purified enzymegave only one peak by ultra-
80"C for 10min. Low Molecular Weight Calibration Kit centrifugal analysisat 60000 rpm. The sedimenta-
(PharmaciaAB, Uppsala, Sweden)was used as the SDS mo-
lecular weight marker. Gel electrophoresiswas run at a regul-
tion constant of the enzyme was about 3.0S.The
ated current of 30mA per gel slab for 2h at 4'C. Staining molecular weight calculated by the method of
enzyme activity was carried out with the following method. Schachman(Schachman1957)was about 30000
The polyacrylamide gel was attached to a 70/oagaroseplate while the SDS-PAGE method gave about 29000
containing 1%milk caseinto blot the enzyme.After incubating (Fig. 1). The isoelectricpoint was estimatedusing
for 30min at 37"C, the agaroseplate was steepedin 0.11M
TCA solution. The enzyme activity was detectedon the aga- pH gradientpolyacrylamidegels to be pH 9.2.
rose plate as a clear zone causedby digestion of casein.
The isoelectricpoint was determined by using Serva pH
gradient polyacrylamidegels,pH 3-10 (ServaFeinbiochemica Effect of pH on stability and actiuity of the
Heidelberg,FRG). The following proteins were used as mark-
ers of isoelectricpoint: cytochromec (pI 9.6), whale myoglo-
enzyme
bin (pI 8.1), horse myoglobin (pI 7.0), human carbonic anhy-
drase B (pI 6.5), bovine serum albumin (pI 6.0). Stability of the enzyme was investigated at var-
ious pH's in the presenceand absenceof 5 mM
CaClz. The enzyme solutions were incubated at
Results 60'C for 10 min at a rangeof pH values and re-
sidual activity was measuredat pH 10.5.The buf-
Characteristicsof Bacillus sp. no. AH-101 fer systemsused were: 0.02 M acetatebuffer (pH
4-6); 0.02 M 3-[n-morpholino]propanesulphonic
Strain no. AH-101 was aerobic, spore-forming, acid buffer (pH 7-g)' 0.02M glY-
gram-positive,motile, rod-shaped, catalase-pro- cine:NaCl:NaOH buffer (pH 9-11.5); and
ducing, oxidase-producing,and citrate-utilizing in 0.02M KCI:NaOH buffer (pH 12-13).As shown
Koser medium. The GC content of the DNA was in Fig. 2, the enzyme was almost 100% stable at
43.2 molo/uIt is clear that the bacterium should pH 5-13 independently of the presenceof cal-
belong to the genus Bacillus. The temperature for cium ions. Figure 3 shows the effect of pH on the
growth was 20'C-55"C and the pH for growth protease activity. The pH was adjusted with the
was pH 7-11. abovebuffer systemsand the enzymewas assayed
1 22 H. Takami et al.: Thermostablealkaline proteasefrom Bacillus sp.
Culture fluid
DEAE-Toyopearl
CM-Toyopearl
at 30" C. The proteasewas most active toward ca- activity of about 60% was retained at 80'C. Sta-
sein at a pH range of 12-13. bility dropped off at a somewhat lower tempera-
ture ((60'C) in the absenceof Ca2+.
The optimum temperature was around 80'C and The enzymewas slightly inhibited in 0.2%sodium
70"C in the presenceand absenceof 5 mM Ca2+ sulphonate(DSB) and SDS solu-
dodecylbenzene
respectively.The activity at 80"C in the presence
of 5 mM Ca2* was 16-fold higher than at 30'C
used for the standardassay,and that at 70'C in
the absenceof 5mM Ca2+ was about 11-fold
higher (Fig. a). The thermostability of the enzyme
C M.W
Fig. 2. Effect of pH on enzymestability.The reaction mixtures
940k were incubated at 60'C for 10 min and the remaining activity
1
43OK
1300k
-288K /
1201k /
/
/
1
11`44k /////
.///
tease.
The molecular weight of the eflzymeestimated