免费文献传递   相关文献

Screening of high-yield hypocrellin A producing mutants from Shiraia sp.S8 by protoplast mutagenesis and ultraviolet irradiation

原生质体紫外诱变选育竹红菌甲素高产菌株



全 文 :!10
"!

#
2012
$
11
%
A
 
–
 
á
 
Ö
 
u
 
â
ChineseJournalofBioprocessEngineering
Vol.10No.6
Nov.2012
doi:10.3969/j.issn.1672-3678.2012.06.004
STUM
:2011-05-23
OGV!

ãäÿbdÄFçèéêë
(K513201011);
íÅhFèºFãäèéêë
(14317363);
ìíîÆhûig êë
(Bu132802);
ãähFA(óG_£ãäèéêë
WX.Y

ÇÈÉ
(1991—),
ø

PeÆPû

hý£¤A

£¤"ö

Êåô–A–¬­

ÔË)

– û
),
£¤Ì
,Email:jwwang@suda.edu.cn
&«ÃÙ0ö(ô)*+z,Ó-fzÆ
bcd

eff

ghi

jkl

5a./ Q/3

5a
215123)
7
 
i

ÀÁkíö‚‘’“”•–—¬‡òK&¬­

ÚÛܛ

¡–R¬
ShiraiaspS8
ª-«¬­

˜
™ÁšÞ,÷
(5mg/mL
›œò,S
10mg/mL
w,

â
30℃
žŸ¬ 
2h,
}ý¬ kíö
324×106
¡
/mL。
¡–R¬kíöâ¢ù
15W
‚£
30cm
ž¤¥‘¦

}ý‘’¬­
C6。
Җ—¬‡ò&îê
281mg/L,
LÎ-«¬­bKÌ
537%,
§¨©_Ã

ª(YKÇ«¬­l®¯Ád‹

jkl

–R¬

kíö‘’

–—¬ò


mnopq
:Q784    
6rsFt
:A    
6u?q
:1672-3678(2012)06-0018-06
ScreeningofhighyieldhypocrelinAproducingmutantsfrom
ShiraiaspS8byprotoplastmutagenesisandultravioletirradiation
PANWeisong,JIYuanyuan,YANGZhuying,WANGJianwen
(ColegeofPharmaceuticalSciences,SoochowUniversity,Suzhou215123,China)
Abstract:AShiraiaspS8mutantstrainwithhigheryieldofhypocrelinAwasscreened.Then,optimum
conditionsforprotoplastpreparation(324×106mL-1)weredetermined.After50hcultivationthehy
phawerecolectedanddigestedwiththemixingenzymesincludingcelulase(5mg/mL)andsnailase
(10mg/mL)for2hat30℃.TheC6mutantwithhigherhypocrelinAyieldwasobtainedviaultraviolet
iradiation(30cmfroma15WUVlamp)ontheisolatedprotoplasts.TheyieldofhyprocrelinA(281
mg/L)bymutantC6wasincreasedby537% thanthatofthestartstrainS8.Theproductionofthemu
tantwasverifiedtobegeneticalystable.
Keywords:Shiraiasp;protoplastmutation;hypocrelinA;screening
  
cdBf
(hypocrelin)
3&ÍÎGN3–

Ï
ÐcdBef
(hypocrelinA,

HA)、
7f

‡
f

qfo

ÁN3–

#ë7–_YOSª~Y
MúªÑ=

ÌU

cdBef

7f[:ÒÓÔ
Õ®¯

žÖ“

k†Ó×;Ö`

ØÑIÏÙ`G
Aڏ

Dn
HIV 1
Ý֓:k†%[
[1]。
cd
Bf6y±ÛBæYcÏB
(Shiraiabambusicola
Henn)、
cdB
(Hypocrelabambusae(BerketBr)
Sacc)
/EÜAB
Hypomyces(Fr)Tulsp。
cd
Bef+,x¸&mAcÏB

j$&mAcÏB
:×#u

nvԀÝ

6J•

¥òcdBe
fë7ޑ_^hÙÚA6

ÌZ[¶˜™xhY
ņ

4™©325f[cÏB

£¤û̔¼n
v™6cÏBYùG °|v™„…
[2],
ƒ„™c
dBefY¡¢âZN
[3],
2V™ßàá†vN
[4]
/5-¯NFXvYvw
[5]
AÖ%

  
g6BhYöa:f&_^cdBfYÙÚ
NA6

”qhhájcdBfEcÏÍFô{

6{Y25

t§Ž:f&nmAcÏYè¸Y
Z

 ¡\AHW¬­Y6^

\AHW_`V#
$Z[&BhYab

\AHW_`¬­–ÐZ[
˜β ˆ‰4â@[6]、Š¾4@[7]、ã䝯^§
4
[8]
g6BhYöa

f[\AHW_`¬­a
bcdBefg6BhY£¤‘÷ø

¤2V
cÏB\AHW†Ê£¤

Rtu\AHW]^_
`

ab6g6cdBfBh

/#4cdBfY
A–¬­25¡ÁHè¸/Êg1ª

1 
vw`xy
11 
ÄÅvw
111 

  
cÏBh

v™y?å]ççæuçc
Brach
ystachyumdensiflorum(Rendle)Keng
èMYcÏù
GÝBh
(ShiraiaspS8),
$UãÓA–BÁYÉ
;Ê é Ì s P t Ó A – U ó Y É

ê Z 4
CGMCC3984。
112 
„…
  
BWA‚‹W„…
:PDA
‹WÆ

  
RA„…
:PDA+06mol/LNaCl、50mmol/L
CaCl2。
  
BW×ń…

HJv½
,%):
>?Þ
07,
ˆ‰4
2,KH2PO4007,MgSO4·7H2O007。
  
¯W5-„…

HJv½
,%):
ƒš
20,
ˆ‰
4
2,KH2PO4 03,MgSO4015,VB10001,-.
Ý
05。
  
A넅

HJv½
,%):
ˆ‰4
01,
-.
Ý
025,KCl018,
ì«û
082,
ßà
15。
113 
+,¢=
  
EFf@
(BR)
íyãôŀ

îE@
(BR)
í
y
Biosharp
€

cdBef
(HA)
Q{$Uã
æFï9ùA¥æF£¤ïN3–ð¡

ij
k`Œ=4
06mol/L
Y
NaCl,
F¯4
02
mol/L、pH72
Y“«ºF¯

ñ[¢=*4°Ÿ
Žã6vwZ

114 
+,JÈ
  SW CJ 1FD
Ý#$Ö%&

íÅ$NòÊ:
ŀ
)、UV 2401PC
]^ Svªª³ã

é¡
Shimadzu
€
)、MLS 3780
gk´¿"Bñ

é¡
íò€
)、Agilent1260
¯Í ‡


Agilent
€

)、Alegra64R
ÝÇșó;
(Beckman
€
)、
CKX41
Ýó‚žÓô

é¡
Olympus)、PYX DHS
40X50 S ⅡÝë섅4((õ¼_£òÊ
ö

A

12 
ÄÅxy
121 
cdBf6J¯Œ"“
  
¢*™£‚YcÏBW÷
01g,
á
5mL
‡
øÔ¡
24h,
]X˜}³.Ñ

3D

Ñ¡¢¯

Œ
¦˜
10mL,
ò˜cdBef¡¢¯

¡¢¯Ð
045
μmÓù5Iu5ïÐ HPLCvw。A«CD4A
«K
SinoChromODS BP(46mm×250mm,5
μm),Kì45℃;ÅLÍV(e8)∶V({)=75∶25,
ÅM4
10mL/min,
N¯ú‚4
300nm,
°HJ4
20μL[9]。˜CD¾,cdBefYYħV4
235min。
122 
\AHWY†ÊâRA
  
OBW×ń…UçècÏB

&ë섅
4„…
50h,
C¢µJBW&™ó;U™ó×ÅB
W

¥[Aʺ{Ø7

Ñ

áèø†)u5zB
ïYÀû@ 
2mL,
ë씕@|
2h,
åï/
1500
r/min
™ó
10min,
¿óC¢9=

/
4000r/min
R
љó×Å\AHWD[ijk`Œ=Ø7

Ñ

]>ïÐ
75μmüÔu5,¥[×ýã½Æã½,
]X

Ñ

ãà6J

p
/mL)[10]。
123 
\AHW]^_`
  

15W
]^ÿh®…
30min,
K\AHW
>?&ijk`Œ=U&!]^ÿ
30cm
Év¥_
`
30~240s,
ÉÊdXïö÷˜µ£ä³"Ô&R
A„…

RAB#½$4
c,
t§"ÔÍtö÷´
½Y‘_`\AHW%4nÏ

ÌRAB#½$4
d,
xΓ%Ž
=(d-c)/d×100%
ãà

2 
‹Œ%)
21 
*.z}~«f*+z,Ó
  
„…
8d
YcÏBB&Ðu*™/‡øÔ¡
ï

/cdBefQ{%4^QnÏ

nÌ¡¢
¯°|
HPLC
vw

_£‘œ^
1。
$^

S_

cdBefYYħV4
235min,¯
òB&WU
cdBefJ4
151mg/L。
65BhÐ
28℃、
150r/min
JK„…
14d,
‘œ^
2。
$^

S_

O5-„…!

ʧ

cÏBA‚l˜g 

*HJ
91 
!

# ÇÈÉA

\AHW]^_`abcdBefg6Bh
l182g/L,
!
10
ʧcdBf6Jl˜3g

4
183mg/L。
n
1 
*+z,Ӊ-²›Ö/0oŠn0
Fig.1 HPLCchromatogramonhypocrelin
n
2 ShiraiaspS8
‰«1`*+z,zf1
Fig.2 BiomassandhypocrelinAproduction
ofShiraiaspS8
22 
&«Ãى‚ƒ
221 
\AHWY÷2
  
tužÓôx£¤\AHWY÷2uâ

^
3。
$^

S_

„…
50h
;B
(^ 3(a));
áè@¯@|
1h
;<

B&2T(
2D÷26\AHW
(^ 3(b));
@|˜
2h
ï

B
&W¡À‚

÷26hJ\AHW

Ð"BïY
75μmüÔu5ï)9\AHW(^ 3(c)),S/°
|ïUYã½

_`ERA_£

(a)
@|±T
            (b)
@|
1h          (c)
@|
2h
n
3 

S8
‰&«ÃÙ23ü
Fig.3 ReleaseofprotoplastsfromShiraiaspS8
222 
B*n\AHW6JYrs
  
v¥¢„…§V4
30~70h
YB&O
10
mg/mL
EFf@/
10mg/mL
îE@˜XY23@
 
、30℃
CD¾@|
1h,
:[×ýã½Æ°|ã
½

‘œ^
4。
$^

S_

B*4
50h
§\AH
W6Jl˜3g

n
4 
z4ž*.z&«ÃÙf1‰Ÿ 
Fig.4 Efectsofcultivationtimeonprotoplast
productionofShiraiaspS8
223 
@|W Y:Œ
  
/€tä³YEFf@/îE@˜X@|W
 

×ÅB*4
50h
YB&W

O
30℃
CD¾”•
@|
1h,
/:Œ3OYÇZW 

‘œ^
5。
$
^

S_

îE@Yä³n\AHW6Jrs~C
ž

îE@䳡g

\AHW6J(áá+ð

ïU_£:[
5mg/mL
EFf@/
10mg/mL
îE
@˜XY23@¯

224 
@|§Vn\AHW6JYrs
  
:[
5mg/mL
EFf@/
10mg/mL
îE@˜X
Y23@

×Å
50h
B*YB&W

O
30℃
CD¾v
¥”•@|
05~3h,
ª9@|§Vn\AHW6J
Yrs

‘œ^
6。
$^

S_

@|2T,-

 ¡
@|§VY.‚

\AHW6J=>áá

@|l˜
2h
§\AHW6J3g
(324×106
p
/mL)。
02
A
 
–
 
á
 
Ö
 
u
 
â
  
!
10
"
 
n5 
¿šÙ5ùž*.z&«ÃÙf1‰Ÿ 
Fig.5 Efectsofenzymesonprotoplast
productionofShiraiaspS8
n
6 
¿š6òž*.z&«ÃÙf1‰Ÿ 
Fig.6 Efectsofenzymolysistimeonprotoplast
productionofShiraiaspS8
225 
@|ì³n\AHW6JYrs
  
:[EFf@
(5mg/mL)
/îE@
(10
mg/mL)
˜XY23@

×Å
50h
B*YB&W

v
¥O
26~34℃
CD¾@|
2h,
ª9@|ì³nAHW6JYrs

‘œ^
7。
$^

S_

@|
ì³n\AHW6JrsCž

O@|ì³4
30℃
§\AHW6Jl˜3g

#u
30℃
§

\AHW
6J @|ì³¢g(^¾}+ð

n
7 
¿šË̞*.z&«ÃÙf1‰Ÿ 
Fig.7 Efectsofenzymolysistemperatureon
ShiraiaspS8protoplastproduction
23 
&«ÃÙ0ö(%7ô
231 
_`=JY:Œ
  
:[]^En\AHW°|€t§VYÏ-

\AHWY“%­©]^

ñO

$^

S_

 
¡]^EÏ-§VY.‚

“%ŽCž¡g

£É
ʧVl˜
3min
;<§

“%ŽS/l˜
95%
/
9
,4min
/ï\AHWed· 

65Bh\A
HWO
30s
˜
120s
§V-/“%ŽžŸ¢g

w
Co.,-]^EnÌrsxh

O_£Uv¥,

30、60、90、120s
ÉÊïYRAB#

ò˜YB
hv¥/
A(n)、B(n)、C(n)、D(n)
°|êZ

n
8 
0Ý896òž*.z&«ÃÙ
:;µ‰Ÿ 
Fig.8 Efectsofdiferentmutationtimeon
deathrateofShiraiasp.S8protoplasts
232 
\AHW_`hY±ö
  
0žÇZ#1€tBhOA‚UÍ2§

t.
á†3¸YBhŒXYB#S/*!Y‚O.4

€tá†3¸YBhŒXYB#€#5¼yY


Bh-VŒX0žE
[8]。
O0žE¯¢U

,6â65BhŒX0žEYB#

‘œ^
9。
$^

S_

K,a6YB#
(^ 9(a))
ЯW5-
10d
×ÅBW

²~cdBef6J

±öIs,
¢™
40
h_`Bh

cdBf6J`N¶·”67
€6cdBf˜3gY
27mg/L。
KåBhYA‚
­©

cdBef6JE0žÇZ°|_`hY±
}öa

_`ïYBh:YB^6cdBef6
J¡¢

:YÇq67€6cdBef
(^ 9(b)),
tu€tBh
01g
BW‡ø¡¢¯Y²~S/5
^

]^E_`nBhG¨Yrs[: ;G

O
BÁabÖ%U_^ŒöbÁ±å#£¤Y.p
],"ö

233 
_`hYXö
  
бöò˜YG¨

6J¡¢Y_`Bh:
12 
!

# ÇÈÉA

\AHW]^_`abcdBefg6Bh
  (a)
0žÇZ
              (b)01g
BW‡ø¡¢¯
n
9 
*.zö(Æ%Fig.9 ComparisonbetweenmutantandparentstrainsofShiraiaspS8
A1、B2、B3、C5、C6、D1、D3,
KÌçÁ˜¯W5-„
…

&JKO
28℃、150r/min、
ƯJ
50mL
C
D¾„…
10d,
×ÅBWŒ*£‚

]
121
"“t
u
HPLC
“:¯ŒcdBef6J

DpBh]
X

˜
,¯
òYcdBf6J‘œ¢*%

_£‘
œB
1。
¡
1 
*.zö(Æ%Table1 ComparisonofHAproductionbetween
mutatedandoriginalstrains
BhêZ
ρ(HA)/
(mg·L-1)
¡gŽ


65B
183 0
A1 200 82
B2 217 186
B3 202 107
C5 196 73
C6 281 537
D1 213 165
D3 240 315
  
$B

S/y6

”±öò˜YBh
HA
6J
*:.Œâ³Y¡¢

ÌU
C6
BhB^3O
,HA
6Jl˜
281mg/L,
â65BhͲ¡g
537%,
BhA‚­©Ž:ˆ8

S/Z[&5-A6c
dBef

24 
ö(Æ>?腣@Ñ
  
Köaò˜YBh
C6
9U†Û

Ñ

ЯW
J~5-„…

tu
HPLC
¯Œ6J

‘œB
2。
$B

S_
:C6
BhYcdBf6J~`Œ

á†
`ŒGú)

¡
2 C6
zÆ?’è…£@ыŒ
Table2 VerificationofmutationstrainC6
Û½
ρ(HA)/
(mg·L-1)
`NŽ


C6 281 0
1 282 05
2 289 29
3 275 -20
4 274 -23
5 283 08
3 
‹
 
)
  
tu_£ò˜YcÏB\AHW†Ê3OC
D

B*4
50h
YcÏB&W

:[
5mg/mL
EF
f@/
10mg/mL
îE@

7HJä³

Y23@
 

O
30℃
CD¾@|
2h。
\AHW6JS/l
˜
324×106
p
/mL,
RAŽS/l˜
30%。
°.
}f[]^_`¬­n\AHW°|ÉÊ

Ðu±
ö/Xö

ò˜.hG¨¡¢YcÏ_`Bh
C6,
Ðu¯W5-

ÌcdBef6J²65Bh¡g
537%,
á†`ŒGú)

  
\AHW_`¬­OcdBef6J¡¢9
[:x)Yœ

j_`ïBhG¨Y`N[: 
;G

öaYÖ%Jxh

t§OBhA‚:#

W
XµZG"êl:CžYˆ8

;ïY£¤U¤
22
A
 
–
 
á
 
Ö
 
u
 
â
  
!
10
"
 
K°.}ƒ„cdBefYA–3XW@/_
’¬­

p[v¦A–F¬­n3XW@UYF²
@A°|Bl<=

tuA–ÇZÈA6¬­Yƒ
„

4cdBefA–¬­A6¡þ/1ª

Q·6r

[1] 
a>f

?Á@

AB

A

ô[BcÏY£¤°V
[J].
C
(†‡+ô
,2011,7(7):160163.
[2] 
DE

FG@

cÏYv™„…£¤°V
[J].
B–£¤

2005,3(1):5357.
[3] 
HåI

JKL

M”N

A

cÏUcdBefYv™âZ
N
[J].
çèÊÖhFF÷

yåæFO
,2004,18(2):9194.
[4] 
¶P

.XQ

âR

A

ÛècÏB€tßàá†vNY
RAPD
vw
[J].
ؘFâZ[A–F
,2009,28(5):
889895.
[5] 
SßT

UV

º„W

A

cÏB5-¯NFXv£¤
[J].
PeU+FïF÷
,2011,30(6):5860.
[6] 
ÔXL

‰ˆd

aY

A

ZG¹\AHW_`ab βˆ‰
4â@g6Bh
[J].
A–ÖâF÷
,2009,25(12):
19211926.
[7] 
Aó 

AÛç

ÔV

A

\AHW]^_`abŠ¾4
@g6Bh
[J].
Uã0v
,2010(9):7376.
[8] 
JXL

[KÓ

i\à

A

ãä\AHW]^_`ab
¯^§4g6Bh
[J].
ÖgÓA–
,2011,41(2):5156.
[9] LiangXH,CaiYJ,LiaoXR,etal.Isolationandidentificationof
anewhypocrelinAproducingstrainShiraiasp.SUPERH168
[J].MicrobiolRes,2009,164:917.
[10] 
]E^



.hABY\AHW†ÊERAuâ£
¤
[J].
_?ë¶hFF÷

yåæFO
,2006,34(2):
櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒
106109.
,-./
½¾ÃABCDsE#$F%G
2012
$

%
21
é
,《AngewandteChemieInternationalEdition》
5B™UãæFïA––Ê£¤ñÔìW
£¤˜/S`£¤˜3%Xœ
“GeneticalyEncodedCyclopropeneDirectsRapid,PhotoclickChemistryMediated
ProteinLabelinginMammalianCels”。
ü3ó£¤tuðVØab¦

_^™[:ªÈc GY@Êå½
«W‡de½«OfgL–UYØêb

ªÏCD¾

ÎhÈ`3™W‡de½«Y>?Hâ¿v
¦’iN3–5AWáXÇZ

AXjª G€

”q_^™§‘SmYnfgL–®¯>?Îh
ÈYQ$

HI†JKÂÇLùab^_«fMNÓgh#$O¶4%G
kåNÖhFlÊVÿ!€ôOãäyåæFçYhÄmT¾

tuØ]˜¬­£56.ÁóÝ
]˜01-.

S[&EFf785-

ÍFg)5BO
2012
$Y
《PNAS》
9

ü£¤nÑO-.Uxè™ÎhG&>?m¤YBêVO¬­

t§‘3EFf@ YvIBl

m
¤/EFf@Y¯^y˜Æ

XÚO-.®¯Bê¨ñ™ûÖÓÝEF¿W

EF¿WYBlJ~7û
£¤ò˜h\¡g

ü]˜-.€g}|S
qnÑ|i™
]˜-.ù“¢çf[‘’ÝEFfY…Ÿ


ŠU°

32 
!

# ÇÈÉA

\AHW]^_`abcdBefg6Bh