s j

BJ 777,56(3),51C1 5H,1992
`

,V

C)loning,ExpressiOn,and(Characterization of a PIlinor Alkaline Protease from B
sp.Noo AII 101 `liJJys

1-IidctO TAKAMI, TCruhiko AKIBA, and Koki HoRIKOSHI

B s c
r c 2 c B j 7 1 1 , 5 6 ( 3 ) , 5 1 C 5H,1992

`
C)loning,IExpression,and(Characterization of a R4inor Alkaline Protease from B
sp.Noo AH 101 `lirryS

1-IidctO TAKAMI,*'**TCruhiko AKIBA,**and KokiI IoRIKOSHI*'**

* D `
r , 7 , r` 9B
g g , F 7 7 ` r l B S
4 6 ` 4 B 4
g , Z f 7 7
S / g , 2 5 9 N g r s
,
` ` ` ` `
f 07
lr, y /
,Kr7gl ' 227,
**17 , gLr
,7/7f/1Sr r PSC771 R`s`r ,2-, sltl , ,S
35,Iap
777
Received June 20, 1991

An alkalophilic Bttcillus sp. No. AH-101 produces an extracellular
alkaline protease as a main product. The No. AH-101 alkaline protease
was c ha r a c t e r i z e di n o u r p r e vio u s p a p e r s.l' 2 ) On th e o th e r h and, i t has
been reported that most Bacilll produce and secretethree or more proteases
at the earl\ slationary phase of growth.3) However, since the ratio of each
protease amount produced varies with the genus Bacillus of strains,3a) it
is not easy to find lesser proteases produced in the culture broth. The
shotgun cloning method was used to search for other proteasesproduced
by Bacilhts 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 I kb were separated
br s uc r o s e d e n s i t l ' g r ad ie n t ce n tr ifu g a tio n . T h e clo n ing vector,
pH\ -300 P L K . : ' u a s c l e a re d*ith Eco RL T h e lig a tio n a n d tr a n sformati on
t r- E. . r , r ' iH B - 1 0 1 u e r e C o n e b r th e sta n d a r dm e th o d s.T h e tr a nsformants
were spread on LB agar plates (pH 8.0) containing I % (wt/vol) skim milk
(LB C agar pl ate). For E . col i H B -101 carryi ng pH Y 3OOP LK and i ts
derivatives, tetracycline was added to the media at a final concentration
of 20pg/ml. Protease activity of the transformants was detected as lhe
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 l0l MP, contained an insert of approximately
l .4kb of D N A . The restri cti onmap of pA H l 0l MP i s show n i n Fi g. 1.
Southern blot analysis showed that the insert was derived from the
chromosomal DNA of -Bacil/assp. No. AH-101 (data not shown). E. coll
H B -101 (pA H l 01MP ) started to produce the al kal i ne p roteas e at
logarithmic phase on LB medium independent of the presence of skim
milk. The protease was not secretedinto the culture broth. The protease
production by the transformant was 16000 units per 400-m1 culture.
P roteaseacl i ri tl *as assayedas descrrbedpreri ousl l .rr
The culture u'as harvestedat the earl) stationary phaseand resuspended
in 0.02rr gllcine NaCl NaOH buffer (pH 9.0). The supernatant fluid
obtained from a sonicatedlysate was heat-treatedat 50"C for I 0 min. After

l IoW.2

94.Ok
1111::
67.Ok

43.Ok
36.8k

:k

EcoRV
20.lk

_ 14.4k

Fig. 2. SDS PAGE of Minor Alkalinc Protcasc Cloncd in E c

HB 101(pAH101MP)and ThCrmostablc Alkalinc Protcasc l lom B
``
sp No AH-101
Fig. I . R e stri cti o n Ma p of Plasm id pAH I 0l MP. 1l E`HB 101(pAH101 MP)2:Thcrmostablc alkalnc protcasc iom B s
-\mp'- ampicillin resistancegene; Tet', tetracycline resistancegene. The thick line sp No AH 1011'MW:Molccular mass markcrs Phospho lasc (940K),
denote: the fragmeni from Bacillus sp. No. AH-101. The thin line denotes albumin(670K),ovalbumin(430K),carbOnic anhydrasc(300K)trypSin inhibitor
pH\'_:t)t]PLK. (201K),
laCtalbumin(144K)K=kilodaltons

Table I. Punfication of a Minor Alkaline Protease

Volumc Activity Protcin Specilic activity Recovery

Step
(ml) (unitS
ml) (mg) (units/mg-protein) (%)

Sonication 900 190 4300 40 100

Heat-treatment (50"C) 870 160 2760 50 82
DEAE-Toyopearl 16 2010 20 1560 22
Casein-Sepharose6B 8 2080 6 2780 10

O ff p r i n t r e q u e s t s t o : H id e to T a r lu r , De p a r tm e n to f Bio e ngi neeri ng,Facul tyofB i osci enceandB i otechnol ogy,Tokyol n.tti tuteqfTechnol ogy ,4259
Nagatsuta, Miclori-ku, Yokohama, Kanagawa 227, Japan.
 Cloning of a Minor Alkaline Protease from Bacillus

A B

241

pH

C D
llX3

30 40 50 60 70 80 30 40 50 60 70 80
Temperature(C) Tempereture ( "C )

Fig. 3. Enzymatic Properties of Minor Alkaline Protease Cloned in E. coll (pAHl0lMP).

A: Effect of pH on enzlme activity. B: Effect of pH on enzyme stability. The reaction mixtures $ere incubated at 45'C and the remaining activity was measured at pH
1 0 . 5 . Sl mb o l s: 1 .0 .0 2 rr a cetate buffer ( pH a 6) ; O,0.02u 3- [r - m or phol i no]pr opanes ul phoni c ac i d ( M OPS) buffer ( pH 6.5 8.5) ; a,0.02M gl y c i ne -N a C l N a O H
buffer (pH 8.5 l2): !. 0.02u KCI NaOH buffer (pH 12-l3). C: Effect of temperature on enz!me activity. The enzyme reaction was done at iach temperature for
20min at pH i0.5. "Activity ratio" is the activity tneasured divided by the activity at l0'C. D: Effect of temperature on enzyme stability. The *1. added to
gilcine NaOH NaCI buffer (pH 10.8), incubated for l0min, and the residual activities were measured. Slmbol: O. absenceof Cla2+; O, presenceof "nry."
5mu Ca2*

Table II. Comparison of Two Alkaline Proteasesfrom Bacil/as sp. No SDS_PAGE.

AH-10 1 The enzymatic properties $ere investi-satedas described previously.l)
A s show n i n Fi g. 3, the enzyme ras al mosl l 00oo srabl e ar pH G l l .5
(Enzyrnatic properties) Protcasc Thcrmostablc and most active toward casein at pH 10.5.The optimum Lcmperarure\\'as
cloncd protcascl.2) 65'C and 50'C i n the presenceand absenceof5m\r C a:- res pec ti reh
Molecular weight 36800 28800 (Fig. 3). The enzyme activity at 65'C in the presenceol _im\r Ca:- *as
Optimum pH 105 12--13 five-fold higher than at 30'C (Fig. 3). The enzymewas stablear j0,C-55,C
pH stability 6 115 _ h0
1 2 5 i n the presenceof 5mM C a2+ and 30% of acti vi ty $as rerai nedat 65"C .
Optimum Temperature ('C) 50 70 The enzyme was completely inactivated by I mu phenvlmethanesulfonyl
Temperature stability ('C) 45 60 fluoride (PMSF). No inhibitory effect of iodoacetate 15mrr) which is a
Specific activity (units/mg-protein) 2780 2500 thiol reagent was observedon enzyme activin but p-chloromercuribenzoic
(Effects of r'arious inhibitors) (Rc dual acivity%) acid (PCMB) inactivated the enzyme acrivit) ar 5mrr. The enzyme was
PM S F ( 1 . 0 m r r ) affecled by 5mu ethylenediaminetetraacetic acid (EDTA). These results
0 0
are summari zedand comparedw i th thoseof A H -l 0l thermost abl eal k al i ne
E DT A ( 5 . 0 m r Q 65 93
proteasein Table II. There was a great differencebetweenthe cloned minor
PCM B ( 5 . 0 m a ) 22 94
protease and major protease from Batillus sp. No. AH-101 in enzymatic
Iodoacetate (5.0 mu) 99 94
properties. In addition, this cloned protease did no1 crossreact $ith the
antiserum against the major protease from Bacillus sp. No. -{H-101. On
the other hand, for the major protease,the thermostable alkaline prorease
denatured protein was removed, the supernatant fluid was put on a genehas been cloned (unpublished).These two proteasesenescloned from
DEAE- T o y o p e a r l c o l u m n ( 2 6 x7 0 m m ) e q u ilib r a te dwith 0 .0 2u gl yci ne Bacillus sp. No. AH-l0l seem to be useful for studling rhe origins of
NaOH buffer (pH 9.0). After the column was washed with the same thermostability and pH stability under highly alkaiine conditions.
buffer, the enzyme was eluted with a linear NaCl (0.1-0.3u) gradient
in the buffer. The eluents was dialyzed against 0.021r borate buffer (pH References
10. 5) f o r l 2 h a n d p u t o n a ca se in - Se p h a r o se6 8 co lu m n ( l0x20mm)
l) H. Takami, T. Akiba, and K. Horikoshi. .|ppl. Microbiol. Biotechnol.,
equilibrated with 0.02u borate buffer. Casein-Sepharose68 was made
30. 120-124 i l 989).
ofepoxy-activaled Sepharose68 lPharmacia AB). The enzyme was eluted
2) H. Takami, T. Akiba, and K. Horikoshi, I ppl. Microbiol. Biotechnol..
with 0.05u borate buffer. Five mu MgSOo and 2mrra2-mercaptoethanol
33, 519-s23 (1990).
were added to all buffers. Alkaline protease was purified seventy-fold
3) F. G. Priest, Bacteriolocol Retiev,s, Sept., 711 753 (1917).
with an overall yield of l0%. Specific activity of the enzyme was 2780
4) H. Uehara. Y. Yoneda. K. Yamane, and B. Murao, J. Bac'teriol.
units/mg-protein at pH 10.5, which was the optimum pH. The results
rr9, 82-91 (1974).
of the purification are summarized in Table I. Figure 2 shows the single
5) J pn. J. Genet., 61.5 I 5-, i 18 ( I 986).
H . Ishi w a and H . S hi bahara-S one,
protein band the molecular weight of which was estimated at 36800 by