Appl Microbiol Biotechnol (1989)30:120-124
@ Springer-Verlag1989
Production of extremelythermostablealkaline proteasefrom
Bacillas sp. no. AH-101
IIideto Takanli,Teruhiko Akiba,and Koki IIorikoshi
Thc lnstitutc of Physical and Chenlical Rcsearch,Hirosawa 2-1,Wako,Saitama 351-01,Japan
Summary. An alkalophilic Bacillus sp. no. AH-
101, which produced extremelythermostableal-
kaline protease,was isolatedamong 200 soil sam-
ples. The enzyme production reached its maxi-
mum level of 1500units/ml after about 24h in al-
kaline medium (pH 9.5). The enzyme was most
active toward casein at pH 12-13 and stable to
10min incubation at 60'C from pH 5-13. Cal-
cium ions were effective in stabilizing the eflzyme
especially at higher temperatures.The optimum
and stabletemperatureswere about 80'C and be-
low about 70oC respectivelyin the presenceof
5 mM calcium ions. The enzymewas completely
inactivated by phenylmethanesulphonyl fluoride,
but little affected by ethylenediaminetetraacetic
acid, urea, sodium dodecylbenzenesulphonate
and sodium dodecyl sulphate. The molecular
weight and sedimentationconstant were approxi-
mately 30000 and 3.0Srespectively,and the isoel-
ectric point was at pH 9.2. These results indicate
that no. AH-101 alkaline proteaseis more stable
againstboth temperatureand highly alkaline con-
ditions than any other proteaseso far reported.
lntroduction
Sincethe first report on alkaline proteasefrom al-
kalophilic Bacillussp. no. 227 was published(Ho-
rikoshi 797Ia), there have been extensivestudies
of the properties of alkaline proteasefrom other
strains of alkalophilic Bacillus such as NKS-21
(Tsuchidaet al. 1986),B-21-2(Fujiwara and Ya-
mamoto 1987) and B. thermoruberBi (Mana-
chini et al. 1988).An alkalophilicstrainof Strepto-
Offprint requestslo.' H. Takami
myces species also produces alkaline protease
(Nakanishi and Yamamoto 1974).All of these al-
kaline proteaseswere characlerizedby their activ-
ity and stability under highly alkaline conditions.
Streptomycesalkaline proteasehas been found to
be most active at around pH 13. No. 221 protease
was recognized to be stable not only at high pH
but also at a temperatureof 60'C. It has been
found that some enzymescan be used in laundry
detergents(Aunstrup et at.1972).
The purpose of this study is to explore new
types of alkaline proteasethat are stable not only
against high pH but also high temperature and
detergents.This paper deals with the isolation of
Bacillussp. no. AH-101, and propertiesof the en-
zyme.
Materials and methods
Isolation. The alkaline medium (A-II) and proceduresfor iso-
lation of alkalophilic bacteriapreviouslydescribed(Horikoshi
1971b)were employed in this study. Isolates were grown on
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.
Characterizationand identification of isolates. Microbiological
properties were investigated according to the methods de-
scribed in Aerobic sporeforming bacteria (Smith et aL 1952)
and Bergey'smanual of systematicbacteriology(Sneathet al.
1e86).
Cultiuationand media. The microorganismwas grown aerobi-
cally at 37'C in a 500mI flask containing50ml alkalineme-
dium (pH 9.5). The medium consistedof 2% soluble starch,
2.40/0cornsteepliquor, 1% soybeanmeat,0.7%K2HPO4,0.03%
MgSO+ '7H2O, 0.03o/o ZnCl2, 0.03% CaClz and 2% NaHCO:
(separatelyautoclavedas 20% solution). Large amounts of the
enzymewere produced using a 10-l jar fermentor containing
5 1 of the above alkaline medium (pH 9.5) at 37" C with agita-
tion at 300 rom and aeration of 1.5wm.
H. Takami et al.: Thermostablealkaline proteasefrom Bacillus sp, 121

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.

Table1. Purificationof alkalineprotease

Step Volumc Activity Protein Specific Recovery

(ml) (units./ml) (mg) activity (%)
(units,/mg-
protein)

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+.

Optimum temperature and thermostability of the

enzyme Effect of surface actiue reagents and inhibitors

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

was examined by assayingresidual activity after

incubation at various temperatures at pH 10.8

(0.05M glycine:NaCl:NaOH buffer) for 10 min.

As shown in Fig. 5, the enzymewas stableat 30'-

70'C in the presenceof 5 mM Ca2+ and residual

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

67ok was measuredat pH 10.5.Symbols:V, absenceof Ca2+; O,

presenceof 5 mM Ca2+

1
43OK
1300k
-288K /

1201k /

/
/
1
11`44k /////
.///

Fig. 1 A-C. Polyacrylamide gel electrophoresis(PAGE). A.

Proteinstaining (PAGE). B. Activity staining(PAGE). C. Pro-

tein staining (SDS-PAGE). Molecular weight markers: phos-

phorylase b (94.0 K), albumin (67.0K), ovalbumin (43.0K), Fig. 3. Effect of pH on enzymeactivity. The substrate(casein)
carbonic anhydrase(30.0K) trypsin inhibitor (20.1K), a-lac- was dissolvedat eachpH and other conditions were the same
talbumin (1,4.4K\. K: kilodaltons as those of the standard method
 H. Takami et al.: Thermostablealkaline Droteasefrom Bacillus so. 123

followed by CM-Toyopearl 650 M column chro-

matography.The most important point for ion-ex-
changechromatographyis that NazCO3:NaHCO3
buffer (pH 9.5) was used when DEAE-Toyopearl
was equilibrated. By using this buffer, most other
proteins except no. AH-101 protease were re-

moved from the culture fluid. It seemsthat the

high isoelectricpoint (pH 9.2) of the enzymeal-

lowed this simple purification procedure. This

30 40 50 60 70 80 90
TemPerature("C)
Fig. 4. Effect of temperature on enzyme activity. The enzyme
reactionwas carried out at each temperaturefor 10 min at pH
10.5."Activity ratio" is the activity measureddivided by the
activityat 30'C. Symbols:O, absenceof Caz*; V, presence
of 5 mM Ca2+
will provide great advantagesfor massproduction
of the enzyme.The protein concentrationin purif-
ied enzyme preparations was measured by three
independent methods as described in Materials
and methods. As they gave almost the samevalue
(results not shown) it is unlikely that impurities
such as sugarsor nucleic acids are present.
The specific activity of no. AH-101 protease
toward casein was about 2500 units/mg protein.
This value is lower than those for no. 221 protease
(18000 units/mg protein) and 8-21-2 protease
(8200 units,/mg protein) under the same assay
conditions. Specific activities of alkaline pro-
teasesfrom B. subtilis were 2000-3000 units,/mg

protein (Tsuru et al. 1966,1967).The no. AH-101

protease was completely inactivated by PMSF,
which indicates that this enzyme is a serine pro-

tease.
The molecular weight of the eflzymeestimated

by the method of Schachman(Schachman 1957)

was about 30000. The SDS-PAGE method gave

about 29 000. The gel filtration method gave

72500, and it is possible that the reason for this

result is due to the high isoelectric point. The

same tendency of underestimating molecular
30 40 50 60 70 80 90 weight when using gel filtration methods has been
femperatureCC
)
reported (Horikoshi 1971a).The optimum pH to-
ward casein and pH stability were pH 12-13 and
Fig. 5. Effect of temperatureon enzymestability. The enzyme
was added to glycine:NaOH:NaCl buffer (pH 10.8), incu- approximately pH 5-13 respectively.The opti-
bated for 10 min, and the residual activities were measured. mum temperatureand thermostability in the pres-
Symbols:O, absenceof Ca2*; V, presenceof 5 mM Ca2+ enceof 5 mM Ca2+ were 80"C and below 70"C.
These values are higher than any other alkaline
proteasefrom alkalophilic Bacillus strains and we
concludethat the no. AH-101 proteaseis entirely
tion, but was completely inactivated by PMSF. different from any other alkaline protease so far
Ethylene diaminetetraacetic acid (EDTA) reported. To further investigatethe unique prop-
(5.0mM) and urea (8.0M) showed no inhibitory erties of thermostability and pH stability at the
effect on the enzyme. molecular level, the amino acid composition and
sequence of the protein and the cloning of the
proteasegene are now in progress.
Discussion Acknowledgements. We thank Mr. M. Chijimatsu of this Insti-
tute for his help with the ultracentrifugalanalysis.Thanks are
also due to Dr. R. Aono, YamanashiUniversity,and Miss Ale-
The protease from Bacillus sp. no. AH-101 was jandra Cruz V, FermentacionesMexicana, for their good sug-
easily purified from culture fluid by only two gestionsand encouragement.We thank Dr. Penelope Kulla-
steps,passing through DEAE-Toyopearl 650 M Ness for reading the manuscript and for many suggestions.
124 H. Takami et al.: Thermostablealkaline proteasefrom Bacillus sp.

References Nakanishi T, Yamamoto T (1974)Action and specificity of a

Streptomycesalkalophilic proteinase. Agric Biol Chem
38:2391-2397
Anson ML (1938) The estimation of pepsin, papain, and ca-
SchachmannHK (1957)Ultracentrifugation,diffusion and vis-
thepsin with hemoglobin.J Gen Physiol 22:79-89
cometry. Methods Enzymol 4:32-103
Aunstrup K, Outtrup H, Andersen O, Dambmann C (1972)
Smith NR, Gordon RE, Clark FE (1952)Aerobic sporeform-
Proteasefrom alkalophilic Bacillus species.Ferment Tech-
ing bacteria. United States Department of Agriculture,
nol Today 299-305
Washington 25, D.C.
Bradford MM (1976) A rapid and sensitivemethod for the
Sneath PHA, Mair NS, Sharpe ME, Holt JG (1986) Bergey's
quantitation of microgram quantities of protein utilizing
manual of systematicbacteriology vol. 2. Williams and
the principle of protein-dye binding. Anal Biochem
Wilkins, Baltimore
72:248-254
Taber HW, Sherman F (1964) Spectrophotometricanalyzers
Fujiwara N, Yamamoto K (1987)Production of alkaline pro-
for disc electrophoresis:studiesof yeastcytochromec. Ann
teasein low-cost medium by alkalophilic Bacillus sp. and
NY Acad Sci 121:600-615
propertiesof the enzyme.J Ferment Technol 65:345-348
Tsuchida O, Yagima Y, Ishizuka T, Arai T, Yamada J, Takeu-
Horikoshi K (1971a)Production of alkaline enzymesby alka-
chi M, Ichishima E (1986)An alkaline proteinaseof an al-
lophilic microorganisms.Part I. Alkaline protease pro-
kalophilic Bacillus sp. Curr Microbiol 14 7 12
duced by Bacillus no. 227. Agric Biol Chem 35:1407-1414
Tsuru D, Kira H, Yamamoto T, Fukumoto J (1966)Studieson
Horikoshi K (1971b)Production of alkaline enzymesby alka-
bacterial protease. Part XVI. Purification, crystallization,
lophilic microorganisms.Part II. Alkaline amylase pro-
and some enzymaticpropertiesof alkaline proteaseof Ba-
duced by Bacillus no. A-40-2. Agric Biol Chem 35:1783-
cillus subtilis uar. amylosacchariticus. Agric Biol Chem
1791
31:330-335
Laemmli UK (1970)Cleavageof structureproteins during as-
Tsuru D, Kira H, Yamamoto T, Fukumoto J (1967)Studieson
sembly of the head of bacteriophageZa. Nature 277:680-
bacterialprotease.Part XVII. Somephysicochemicalprop-
68 5
erties and amino acid composition of alkaline proteaseof
Lowry OH, RosebroughNJ, Farr AL, Randall JR (1951)Pro-
Bacillus subtilis uar. amvlosaccharitics. Asric Biol Chem
tein measurementwith the Folin phenol reagent. J Biol
3 1 : 3 3 03 3 5
Chem 193:265-275
Manachini PL, Fortina MG, Parini C (1988)Thermostablea1-
kaline proteaseproduced by Bacillusthermoruber- a new
species of Bacillus. Appl Microbiol Biotechnol 28:409-
413 ReceivedMay 23, 1988,/AcceptedSeptember20, 1988