NGHIÊN CỨU THU NHẬN COLLAGEN TỪ GÂN VÀ DA BÒ BẰNG COLLAGENASE TỪ VI SINH VẬT VÀ THỰC VẬT, ĐỊNH HƯỚNG ỨNG DỤNG TRONG BĂNG GẠC ĐIỀU TRỊ VẾT THƯƠNG HỞ

SỞ GIÁO DỤC VÀ ĐÀO TẠO HÀ NỘI
TRƯỜNGTHPT NGUYỄN THỊ MINH KHAI - QUẬN BẮC TỪ LIÊM
**************
ĐỀ TÀI DỰ THI KHOA HỌC, KỸ THUẬT
DÀNH CHO HỌC SINH TRUNG HỌC CẤP THÀNH PHỐ
LẦN THỨ TƯ (NĂM HỌC 2014 - 2015).
Tên đề tài:

 !"# $
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NGƯỜI HƯỚNG DẪN
9:;8++<=>/?>
9@41<8345*A8'/0434545+06/4+
+78B/0424.CD(E
TÁC GIẢ:
FG4=/4+CD
HI'JJJKFLM45'N5,OP4+<=/4+(+>/
Hà Nội, tháng 11 năm 2014
J
MỤC LỤC
Trang
NQ:RS#
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NQ:T UV#  
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Z
NQ:UX [($U\
]
:J: +,^4_<1-*./0,8? >5`4 ]
:K: 5+/a48b,c/d,e/044,3/8fO*+g8++hIci*+,4+-4

8? >5`4>6`*j8+k451/e+,^4Lysinibacillus sphaericus
Z
l
:Z:
5+/a48b,*+,4+-48? >5`4>6`*jL. sphaericus Z
Jm
:]: 5+/a48b,*+,4+-4IF?*`>6`*jc,ck12nb> JK
:l: 5+/a48b,e+o4p45I+C48q*8? >5`48k>`4rOD*jL.
sphaericus Zn<8+8+/s*c,ck12nb>
Jl
:t: 5+/a48b,c/d,e/04I+C48q*8? >5`4*jn>125C4_u Jt
:v:
@_w45+/a48b,c/d,e/04I+x+hI8+?*;?D2458? >5`4
Jy
:y:
5+/a48b,*/4+6;8+`4rOD*j8+k45L. sphaericus Z12
nb>
Km
NQ:($
KZ
z=(\
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PHẦN I. LÝ DO CHỌN ĐỀ TÀI
F?45O+781-*./0,6/4++78Ff*8G4*+/s*e+/c/d,*F<8+?_04+4+C48G4
I+{,*+,-*_04+4+C4*|4*+L@45n>n?_}45+?~8n?4+/d,45,Oa44+C4e+A8
5/•I45LM/_04+cLh8*/sI8-4I+L@45I+AI8+€>_04+>4*?24F•*45q4*+M/
K
5/>48+Mch/*;?*CD.•nP8+<,:?;/1-*./0,6/4++788‚*/dD4p45b45nƒ45
.H4*F?45c/d,*F<8A81s**+L@45+„.28? >5`4:…nƒ45_p455;88‚_|6,45
8? >5`4c†I+C48q**+24+n;45eg8+*+LH84+}+u>*>46‡5/•II+‚45*+g8+

8? >5`4.2DD>,.24+1s**+L@455/oDcA45ei45,O8@ˆ,f*+/046‰?:‚.2
n?8? >5`4c~8_/0*.2.?;/+u>*>4D;8+45q4.2*+24+I+G48+g4+*F?45.HI
+;_Š12D;8+DA,8‚8+b84p458+g4+.2*p45*g4+c24+‹/12I+ƒ8+‹/8A8./a4
es*8A8D3*s_2?:? >5`4*j_ucLh88+b45D/4+.28‚*+24+I+G45G45Œ/1H/
8? >5`4*j8?445LM/g*5COI+o4b45D/P4n<8+*F?45•,A*FŠ4+c/d,*F<n?1-O
8+kOs,cLh845+/a48b,126…nƒ45*F?45c/d,*F<+@46?1H/8? >5`4*j.h412
8A:?8? >5`46o4ˆ,f*_Ž45I+L@45I+AI1-*.•+‚>+786‡.2DDf*8A88+b8
4p456/4++788k>8? >5`4*+M/5/>4I+C48q*e•?n2/4a4I+L@45I+AIcLh8
•,>4*CD+/044>O.28q*./a4es*8A86h/8? >5`4_Ž458? >5`4>6`.2D*p458A8
c?;48‚eg8+*+LH84+}F•*45q4*+M/5/>4+/0,6,f**+,126o4I+^D8? >5`4
8‚*g4++u>*>48>? +@4 _o?*?24c~8c/iD6/4++78*+C4*+/04D3/*FLM45:
? >5`4>6`*j1/e+,^48‚e+o4p458q*+G,+s*8A8.?;/8? >5`412*;/4+/d,1<
*Fg*Fa46h/8? >5`4:A88? >5`4>6`42O+G,+s*cd,8‚45,‹45•8*j1/6/4+
1-*5CO_04+8+?45LM/n?1-O1{4D>45Dw*c?;4IF?*`/48k>1/F,6 8‚e+o
4p45.2D.?•*Fw451s**+L@45:? >5`4>6`*jL. sphaericus91/e+,^4e+345
5CO_04+8+?45LM/98+L>cLh845+/a48b,Fw45F†/45?2/*+345*/48345_•
*FŠ4+*‘5`4`D†+‚>8? >5`4>6`*F?45+05`44+L45e+345D>45*FŠ4+*‘D†
+?A8k>1/F,6.CO.2Dw*L,c/iDe+/6…nƒ45`4rOD`*j1/e+,^442O*;?6o4
I+^D8? >5`4e+3458+b>8A8*A84+C45COD/P4n<8++?~8n<b45:a48;4+c‚
1/08I+•/+hIIF?*`>6`*j*+‘81-*4+Lnb>+>Oc,ck5/•II+C48q*8A8./a4es*
4w/I+C4*…8? >5`46>,e+/_‚6h/_<8q*_„/8? >5`4>6`6‡F•*45q4*+M/5/>4
*;?8A8c?;48? >5`445q4+u>*>4: 5+/a48b,`4rODciI+C48q*8A8I+ƒ
I+^D4+Ln>125C4_ucLh88345_•*-I*F,454+/d,8? >5`4+u>*>48+L>8‚
*;//0*>D:Š4+€45.•n?*Fa4cd*2/’5+/a48b,*+,4+-48? >5`4*j5C4
12n>_u_Ž458? >5`4>6`*j1/6/4+1-*12*+‘81-*c<4++LH45b45nƒ45*F?45
_p455;8c/d,*F<1s**+L@45+„“cLh8cdˆ,f**+‘8+/04:=ƒ8cg8+cd*2/.2*ŠD
F>•,O*FŠ4+*+,4+-48? >5`4*jI+C48q*5C412n>_u_Ž45`4rOD`c;*+/0,
6,f*8>?c@45/o4I+x+hI1H/c/d,e/04/0*>D*+C4*+/04D3/*FLM45_/s4
8A8I+ƒI+^D8+s_/s4*F„*+24+45,‹445,Oa4./0,8‚5/A*F<:
PHẦN II. TỔNG QUAN VẤN ĐỀ NGHIÊN CỨU VÀ ĐIỂM MỚI, SÁNG

TẠO CỦA ĐỀ TÀI
F?45O+788? >5`4*+kOI+C46o4ˆ,f**j_uc†cLh883454+-4.2Dw*
.?;/*+,•8,•4512*+,•8*/aD>4*?24nx458+?8?445LM/:j4pDJ”yJ+>/
nu45*+,•8*/aD8? >5`4+u>*>4.?;/12*/4+e+/s*6o4ˆ,f**jn>_u1H/*a4
Z
*+L@45D;/.2•On`FD12•OI.>6*c†cLh86…nƒ45Fw45F†/8+?c/d,*F<8A8_f*
*+LM45cLM451/d4D3DdD:/044>O8? >5`4*/4+e+/s**j_ucLh86…nƒ45
*F?454524+834545+/0I*+^DD)4+Ln>12Fp4512c/d,*F<8A8_04+e+HI5C4
126ƒ4cLh8cA4+5/A+/0,•,o*F?45c/d,*F<I+ƒ8+‹/8A81s*_}456C,12Fw45:
? >5`48+/sDt–*+i*F7458k>5C4e+?o45vm–8f,*F•8n>12cLh8I+C4_•
8+kOs,„.HI+;_Š8k>n>:†8‚K”.?;/8? >5`4cLh88345_•8? >5`4+u>
*>4.?;/.2I+|_/s44+f*12*+LM45„*F?458A8D3./a4es*4+Ln>ˆL@455C4:
? >5`442O8‚e+o4p45.2D*p45*g4+c24+‹/12I+ƒ8+‹/8A8./a4es*„8A8
D3*s_2?:? >5`4>6`.2`4rOD`I+C48q*./a4es*I`I*/n`*Fa4_‚6h/8? >5`4
*;?8? >5`4+u>*>4:? >5`4>6`*j 1/ e+,^48‚*g4+L,1/0* .2  I+C48q*
8? >5`4+u>*>412e+345+u>*>48q*+G,+s*8A8.?;/8? >5`4124+/d,1<*Fg
*Fa46h/8? >5`4:? >5`4>6`*jClostridium histolyticumcLh845+/a48b,12
6o4ˆ,f**j4pDJ”ly8+?cs44>O:A88? >5`4>6`*jPseudomonas aeruginosa
—J”vl˜*j1/6/4+1-*_/i4Vibrio6I:12Achromobacter iophagus—J””t˜+G,
+s*cd,.21/6/4+1-*5CO_04+8+?45LM/1{4D>45Dw*c?;4IF?*`/48k>1/F,*
8‚e+o4p45.2DD„Fw451s*.?•*8k>1s*_}45: Lysinibacillus sphaericus .21/
e+,^48‚.h/12cLh8ˆ`D4+L*A84+C4n/0*D,™/6/4++78nx45*F?45e/iD6?A*
n<8+6•*ˆ,f*+,Os*6•*F•*:? >5`4>6`*jL. sphaericus8+L>cLh845+/a48b,
Fw45F†/45?2/*+345*/48345_•*FŠ4+*‘5`4`D†+‚>8? >5`4>6`*F?45+05`4
8k>1/e+,^442O4+L45e+345D>45c?;45`4D†+‚>8k>1/F,6:(+/6…nƒ45
`4rOD`*j1/e+,^442O*;?6o4I+^D8? >5`4D;8+45q4+u>*>46‡e+345
8+b>8A8*A84+C45COD/P4n<8++?~8n<b45:
Điểm mới, sáng tạo của đề tài: …nƒ4545,‹41-*./0,5/A*+24+Fšn‹/
n2?„/0*>D.25C412n>_uci*;?8? >5`4D;8+45q44+MI+C48q*_Ž45
8? >5`4>6`*jL. sphaericus.21/e+,^4e+3455CO_04+8+?45LM/8+L>8‚

45+/a48b,42?./a4•,>4cs4+?;**g4+8? >5`4>6`8k>1/e+,^442O.…nƒ45
*+aDDw*6•`4rOD`*j*+‘81-*Fš*/d46›48‚—nb>c,ck˜4C458>?e+o4p45
I+C48q*8? >5`4F•*45q4*+M/5/>4*+C4*+/041H/D3/*FLM45+@48?4cLM45
I+C48q*_Ž45+‚>+78:;?•,O*FŠ4+6o4ˆ,f*8? >5`4e+•Ieg413*Fx45c`D
.;/+/0,•,oe/4+*s8>?12>4*?24I+x+hI1H/c/d,e/04*F?454LH8*-4nƒ45
124C458>?5/A*F<8k>8A8I+ƒI+^D8+s_/s4:CO.2Dw*+LH4545+/a48b,
DH/*;//0*>D8‚•45+œ>e+?>+7812*+‘8*/P48‚*/dD4p45b45nƒ458>?:
PHẦN III. QUÁ TRÌNH NGHIÊN CỨU VÀ KẾT QUẢ
Nội dung nghiên cứu
• 5+/a48b,c/d,e/044,3/8fO*+g8++hIci*+,4+-48? >5`4>6`*j
8+k451/e+,^4L. sphaericusZ:
]
• 5+/a48b,*+,4+-48? >5`4>6`*j8+k451/e+,^4 L. sphaericus
Z:
• 5+/a48b,*+,4+-4IF?*`>6`*jc,ck12nb>:
• 5+/a4  8b,  e+o  4p45  I+C4  8q* 8? >5`4 8k>  `4rOD *+3 *j L.
sphaericusZ12n<8+8+/s*c,cknb>:
• 5+/a4 8b,  c/d,  e/04  I+C4 8p•*  5C4 12 n>  _u  _Ž45  `4rOD *j L.
sphaericusZ12c,cknb>:
• 5+/a48b,*A8+8+/s*8? >5`4>6`*j8+k45 L. sphaericus Z 12
IF?*`>6`*jc,cknb>:
• 5+/a48b,c/d,e/04*+g8++hI8+?*;?D2458? >5`4:
• ‘>8+748A8*+24+I+G4_|6,45*+g8++hI8+?D2458? >5`4nx45
*F?45c/d,*F<_}45:
• +…45+/0DD2458? >5`4*Fa4*+}c†5CO_}45*+‘845+/0D:
• žCOn‘45•,O*FŠ4+*+,4+-48? >5`4e+•Ieg413*Fx45c@45/o4:
III.1. Chuẩn bị vật liệu collagen
III.1.1. Dụng cụ, thiết bị và hóa chất
9ƒ458ƒ'8•8c?45JmmmD.4+‘>+wI4+‘>c‘45n>Ÿ5C4_u_Š4+*+kO*/4+
c‘45+™4+hIn<8+8? >5`4*+3:

9+/s*_<'*k.;4+?6+/_>—+-*˜DAOˆ>O*+<*—/0*>D˜DAO.O*CD—?F1> 
=)˜DAOc?I—=`**.`F?.`n?4+˜
9‚>8+f*'4LH88f*n,45n<8+>
K
N
]
mml=—Jv”mv5Ÿ˜>8`*/8>8/nml=
—ZmmKl5Ÿ˜lD=—JytJ5Ÿ˜8‹4vm
?
III.1.2. Tạo vật liệu collagen
-*./0,8? >5`4nx45*F?458A8*+g45+/0DcLh88+,^4_<4+L6>,'
9LH8J' …>6;8+n>Ÿ5C4_u_Ž454LH88f*6>,c‚_Ž458‹4vm
?
45CD5C4Ÿn>
*F?458‹4vm
?
:06/4+nƒ458ƒ12DAOˆ>O_Ž458‹4vm
?
9LH8K'5CDn>Ÿ5C4_uc†ˆ>O*F?45>
K
N
]
mml=„Iyv9”J„]
?

*F?45K]5/M*+>On<8+45CDKcs4Z.G4*F?45•,A*FŠ4+45CD
9LH8Z'?;/_}n<8+45CD12*+>O_Ž45>8`*/8>8/nml= lD=
9LH8]'O*CD.?;/_}8~4—I+G48? >5`4e+345_<8q*ŸI+G4IF?*`/4e+345
I+o/8? >5`4˜*+,cLh8+™4+hIn<8+8? >5`4*+3:
9LH8l'+,n<8+e`?8? >5`4*+312_o?•,o4*F?45*k.;4+]

?
cinx458+?
8A8*+g45+/0D:
l

Hình 1. Gân và da bò Hình 2. Keo collagen thô
III.2. Nghiên cứu điều kiện nuôi cấy thích hợp để thu nhận collagenase từ
chủng vi khuẩn Lysinibacillus sphaericus VN3
III.2.1. Lựa chọn môi trường thích hợp cho sinh trưởng và sinh collagenase
của chủng L. sphaericus VN3
Dụng cụ, thiết bị và hóa chất:
9ƒ458ƒ'_Š4+*>D5/A8n,45*g8+KlmD.—y8A/˜•,`8fOcG,*Fu4—J8A/˜
9+/s*_<'*kfD4,3/1/6/4+1-*—¡F/?8` b8˜4‹/e+…*Fx45—N+-*˜
DAO•,>45I+|—>_?D`nCO>4+>˜:
9‚>8+f*'8? >5`4—/5D>=)˜8A8+?A8+f*8‚*F?45*+24+I+G4D3/*FLM45
Thí nghiệm:
9+,^4_<D3/*FLM454,3/8fO'
=3/*FLM45.}45'8+,^4_<K.?;/8‚_|6,4512e+345_|6,458? >5`4
+24+I+G4+‚>8+f* 2D.Lh45—5Ÿ.˜
>?D`4 l
FOI*?4 Jm
>. l
? >5`4 Jm
LH88f* *H/JmmmD.
I v9vK
=3/*FLM45e+?A45N”8‚_|6,4512e+345_|6,458? >5`4
+24+I+G4+‚>8+f* 2D.Lh45—5Ÿ.˜
—
]
˜

K
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Kt]
(
K
N
]
KZy
(
K
N
]
ltl
=5
]
J
? >5`4 Jm
=,•/1/.Lh45 JD.
LH88f* JmmmD.
I ty9v
t
+,^4_<JmmD.n,45n<8+D,•/1/.Lh45
+24+I+G4+‚>8+f* F?45.Lh45—5Ÿ.˜
,
]
mt]
¡`
]
mJJ

=4.
K
mv”
•4
]
mJl
LH88f* JmmD.
+/>D3/*FLM4512?8A8_Š4+*>D5/A8D™/_Š4+KlD.1/s**a4D3/*FLM45
*Fa4*+24+_Š4+12*Fa45/fO_>?4•*_3456>,c‚c`De+…*Fx45„JKJ
?
*F?45
]mI+•*:,45n<8+D,•/1/.Lh45cLh8c‘45*F?45_Š4+*>D5/A8lmmD.12e+…
*Fx458x45D3/*FLM45:i45,w/D3/*FLM45c†e+…*Fx45_|6,451/.Lh45
*FLH8e+/8fO5/•45:
9fO5/•45'.fODw*1u45•,`8fO6/4+e+•/8+k451/e+,^4L. sphaericus Z
*j•45*+;8+45+/a458+,Oi412?D3/*FLM45.}45*F?45_Š4+*>D5/A8c†cLh8
ci45,w/:+,Oi48A8_Š4+*>D5/A8c†8fO8+k4512?DAO.q8*•8cwKKm
1u45ŸI+•*4,3/„Zv
?
*F?45K]5/M:
9(/iD*F>es*•,o'(+/es**+•84,3/8fO*/s4+24+cA4+5/ADb8cw6/4+*FL„45
_Ž458A8+c?n<8+4,3/8fO*Fa4DAO•,>45I+|„_LH86‚45tmm4D126‘
6/4+*|45+hI8? >5`4>6`:=3/*FLM458+?5/A*F<12+?;**g4+8? >5`4>6`
8>?4+f*6‡cLh8.‘>8+74cinx45*F?458A8*+g45+/0D*/sI*+`?:
A8+c?5/A*F<tmm4D'F…>8,1`*_Ž454LH88f*•ID/0458,1`*ˆ,•45
5/fO*+fD8+?+•*+s*4LH8nx45I/I`*_@DJD.4LH88f*12?8,1`*c~*12?
DAOch/D24+Š4++/04.a45/A*F<c?4+f4ŸcicL>1d5/A*F<m¢c|4LH8F>12
•ID/0458,1`*ˆ,•455/fO*+fD4+L*Fa4_@DJD.n<8+8G4c?cw+fII+ƒc~*
12?DAO5+/.;/5/A*F<+/i4*+<*Fa4D24+Š4+:
N+L@45I+AIˆA8c<4++?;**g4+8? >5`4>6`£v¤'

?;**g4+8? >5`4>6`cLh8ˆA8c<4+_Ž458A8+c?.Lh45`,8/4`5/o/I+‚45F>
*j8@8+f*8? >5`4•,>•,A*FŠ4+k1H/n<8+`4rOD`*+3:=w*c@41<+?;**g4+
`4rOD`cLh8c<4+45+œ>.2.Lh45`4rOD`8G4ci5/o/I+‚45F>J¥D?.`,8/4`
*j8@8+f*8? >5`4*F?45c/d,e/04*+g45+/0D:…nƒ454/4+OnF/4129`,8/4`
—I+>„8A84‹45cwmK¢m]¢mt¢my¢J¢JK¥D?.ŸD.˜ˆCOn‘45cLM458+,^4:
v
Hình 3. Đường chuẩn Leucine
Chuẩn bị các dung dịch:
0DlmD='I+>lmD.5‹D'KlD.F/6.JmmD= I¦y¢mlD.
KmD=¢]vD.4LH88f*¢J”y5>.
K
¢mJl5>.:
,45n<8+4/4+OnF/4]–'8C4]5I+>*F?45ZKD.4LH88f*12tyD.`*+>4?.
,45n<8+8@8+f*'8+b>J–—§Ÿ1˜8? >5`4*F?45c0D
?;**g4+8? >5`4>6`*F?45n<8+`4rOD`*+3cLh8ˆA8c<4+_Ž458A8+c?
.Lh45`,8/4`6/4+F>*F?45+™4+hII+o4b454+MI+L@45I+AI6?D2,*+`?
•,O*FŠ4+6>,'mlD.n<8+8@8+f*8? >5`4cLh8I+o4b451H/mlD.n<8+4,3/
1/e+,^4L. sphaericus Z8+b>`4rOD`*+3„Zv
?
*F?45K5/M:O*CDvmmm
1u45ŸI+•**F?45JlI+•*12*+,n<8+4|/:•*mlD.n<8+4|/12?•45`¨¨I`4n?¨
6>,c‚_|6,45mKD.4/4+OnF/4]–—§Ÿ1˜12c?cw+fI*+ƒA4+6A45„_LH8
6‚45lvm4D:
?;*cw8? >5`4>6`cLh8ˆA8c<4+*+`?8345*+b86>,'
?;*cw`4rOD`¦ —O©mv˜Ÿ—mv©mKl˜—,ŸD.˜
F?45c‚'
O'N+L@45*FŠ4+cLM458+,^4`,8/4`'O¦—Km”l©ˆ mml]t˜—¥D?.ŸD.˜¢
ˆ'5/A*F<
lvm4D
(s*•,o*+,cLh88+?*+fOD3/*FLM45.}458‚_|6,45J–8? >5`4

*+38+?e+o4p456/4+*FL„458>?4+f*—tmm¦Jvvv˜12N”.}458‚_|
6,45J–8? >5`48+?+?;**g4+8? >5`4>6`8>?4+f*c;*JKKyŸD.:žA8c<4+
c<4+*g4+8? >5`4>6`8+?*+fO*Fa4D3/*FLM45N”*+;8+8‚_|6,45J–
8? >5`48‚1u45I+C45/o/8? >5`41H/cLM45eg4+KJ8D—o45JŠ4+]12
l˜:+L1-O8‚*+i.‘>8+74D3/*FLM45N”_|6,45J–8? >5`4*+3ci4,3/
8fO8+k45L. sphaericus Z 8+?6/4+8? >5`4>6`:=3/*FLM45cLh8.‘>
8+74.2D3/*FLM454+C45/•45*;?6/4+e+•/8+?.a4D`48? >5`4>6`:
y
Bảng 1. Sinh trưởng và sinh tổng hợp collagenase của chủng L. sphaericus
VN3 trên các môi trường khác nhau
=3/*FLM45 (+o  4p45  6/4+  *FL„45
—tmm4D˜
?;*  *g4+  8? >5`4>6`
—ŸD.˜
 JJK 9
N” mlKy 9
 J–8? >5`4 Jvvv tZ]
N” J–8? >5`4 Jm]K JKKy

Hình 4. Hoạt tính phân giải collagen của Hình 5. Chủng VN3 sinh trưởng trên
chủng VN3 trên môi trường thạch ISP9 môi trường lỏng
III.2.2. Lựa chọn pH, nhiệt độ và nồng độ NaCl thích hợp cho sinh trưởng
và sinh collagenase của chủng L. sphaericus VN3
Dụng cụ, thiết bị và hóa chất:
9ƒ458ƒ'_Š4+*>D5/A8n,45*g8+KlmD.—t8A/˜•,`8fOcG,*Fu4—J8A/˜
9+/s*_<'*kfD4,3/1/6/4+1-*—¡F/?8` 4+˜4‹/e+…*Fx45—N+-*˜
9‚>8+f*'8A8+?A8+f*8‚*F?45*+24+I+G4D3/*FLM45N”
Thí nghiệm:
iˆA8c<4+e+o4p456/4+*FL„458k>1/e+,^4L. sphaericus VN3 „8A8
c/d,e/04e+A84+>,1/e+,^4cLh84,3/8fO*Fa4D3/*FLM45N”.}45„Zv

?

8‚_|6,45J–8? >5`4e+345+u>*>4124‹45cw>.*p45nG4*jJ9Jm–
+?~84,3/„4+/0*cw—]
?
¢Jm
?
¢Km
?
¢Zm
?
¢Zv
?
12]l
?
˜12Ie+A84+>,—*j
I]m9Jmm˜:U,>46A*6‘I+A**F/i412ˆA8c<4+e+o4p456/4+8? >5`4>6`8k>
1/e+,^4*Fa4D3/*FLM456>,J9Z452O4,3/8fO:
Bảng 2. Ảnh hưởng của pH, nhiệt độ và nồng độ NaCl đến sự sinh trưởng
và sinh collagenase của chủngL. sphaericus VN3
”
U,>•,A*FŠ4+*+g45+/0D8+?*+fO—o45KŠ4+t˜8+k451/e+,^4Z
.28+k45L>fDI+A**F/i4„4+/0*cw*jKm
?
cs4Zv
?
12I+A**F/i4*•*„Zv
?
:
&]

?
12Jm
?
8+k451/e+,^4e+345I+A**F/i4e+/4+/0*cw.a4*H/]l
?
8+k45
1/e+,^445j45I+A**F/i4:+k451/e+,^4Ze+345I+A**F/i4„I>ˆ/*„
I*jtcs4Jm8+k458‚e+o4p45I+A**F/i4I+A**F/i4*•*„e+?o45I*jvcs4
y:(+/I*p45.a4Jme+o4p456/4+*FL„458k>8+k45Z5/oDD;4+:&4‹45
cw>.*+fIe+o4p456/4+*FL„458k>8+k45*•*e+/4‹45cw–>.*F?45
D3/*FLM45*p45.a4e+o4p456/4+*FL„458k>8+k455/oDnG4:;/4‹45cw*Fa4
v–8+k45_<b88+s6/4+*FL„45:+L1-O.‘>8+74c/d,e/044,3/8fO 8+k451/
e+,^4L. sphaericusZ.2„Zv
?
>.ªl–12I¦v:
Jm
/d,e/044,3/8fO /4+*FL„45—tmm˜ ?;**g4+8? >5`4>6`—ŸD.˜
NaCl (%)
J ZyJK JK]m
K Zm”t JKZv
Z Zmlt JKZ]
] Km] JmKy
l Jyy yvl
t JZt tJK
v mm”K ]mJ
y myK 9
” m]K 9
Jm mZm] 9
Nhiệt độ (
o

C)
]
?
 9 9
Jm 9 9
Km m”Z] lKt
Zm KJZ JmlZ
Zv ZyJK JKZ]
]l 9 9
pH
] 9 9
l 9 9
t m]K] Zyv
v ZtyZ JKZ]
y ZlJK JKJv
” Kyl JJmK
Jm mylJ 9
A B C
Hình 6. Chủng VN3 sinh trưởng trên môi trường ISP9 có nồng độ NaCl (A), pH
(B), nhiệt độ (C) khác nhau
III.3. Nghiên cứu thu nhận collagenase từ L. sphaericus VN3
III.3.1. Dụng cụ, thiết bị và hóa chất
9ƒ458ƒ'_Š4+*>D5/A8n,45*g8+JmmmD.—Z8A/˜•,`8fOcG,*Fu4—J8A/˜•45
45+/0DJyˆJymDD—l8A/˜
9+/s*_<'*kfD4,3/1/6/4+1-*4‹/e+…*Fx45DAO.q8|44+/0*—N24
U,•8˜
9‚>8+f*'8A8+?A8+f*8‚*F?45*+24+I+G4D3/*FLM45N”
III.3.2. Thí nghiệm
9 Nhân giống vi khuẩn cho lên men
+k451/e+,^4L. sphaericus Z *j•4545+/0D*+;8+45+/a45cLh8

+?;*+‚>_Ž458A8+8fO6>45•4545+/0DDH/4,3/K]5/MF‹/cLh88fO12?D3/
*FLM454,3/ „ Zv
? 
*F?45*+M/ 5/>4K] 5/M *Fa4DAO  .q8*•8cw KKm
1u45ŸI+•*:=-*cw5/•451/e+,^4c;*Jm
y
¡ŸD.:
9 Phương pháp lên men
fO5/•451/e+,^412?_Š4+*>D5/A8JmmmD..a4D`41H/*«.0Jm–
—1Ÿ1˜4,3/*Fa4DAO.q8*•8cwKKm1u45ŸI+•*„Zv
? 
*F?45*+M/5/>4Zm5/M:
=3/*FLM45.a4D`46…nƒ45.2N” J–8? >5`4*+3I¦v:F?45•,A*FŠ4+
.a4D`4.fOJmD.D{,c<4+e¬D™/Z5/MciˆA8c<4+6‘6/4+*FL„45—5/A*F<

tmm
˜12+?;**g4+8? >5`4>6`—ŸD.˜es*•,o*+,cLh88+?*+fO*j5/M*+bJK
8+k45_q*cG,6/4+8? >5`412+?;**g4+8>?4+f**;/K]5/M4+L1-O*+M/c/iD
*+g8++hI4+f*cinj45.a4D`412*+,8? >5`4>6`.2K]5/M—o45Z˜:
Bảng 3. Biến động quá trình lên men sinh collagenase bởi chủng L.
sphaericus VN3
+M/5/>4—/M˜ /4+*FL„45—tmm˜ ?;**g4+8? >5`4>6`—ŸD.˜
m 9 9
Z mltm 9
t mvyJ 9
” m”K] 9
JJ
JK Jm”l ]JJ
Jl JKlv tmZ
Jy KmJ” yJ”

KJ K”v] ”mt
K] ZymZ JKZ
Kv ZJlK JmKJ
Zm J”JJ y”J
III.3.3. Xác định hàm lượng protein theo phương pháp Bradford [4]
- Dụng cụ, thiết bị:
 =AOc?•,>45I+|—>_?D`nCO>4+>˜•45`¨¨I`4n?¨KD.—J]8A/˜
N/I`**`D>4JD.5/fO*+fD_Š4+*/>c‘458‹4:
- Hóa chất:
,45n<8+IF?*`/48+,^4—˜'8C4JmD5>._,D/4_Ž458C4I+C4*g8+
I+>*F?45JD.4LH88f*.q8cd,8+?*>4:/€„9Km
m
:(+/nx45I+>.?†45F>Jmm
.G4cLh8n,45n<8+8‚4‹45cwmJD5ŸD.:
,45n<8+*+,•8*+…F>n¨?Fn'n,45n<8+*+,•8*+…8‚*+24+I+G4*F?45J
.g*4+L6>,'??D>6/`F/ />4*.,`Klm8C4mJ512 .2D*>4*F?45lmD.
D`*+>4?.I+> >451H/4LH88f**+24+lmmD.cinLh8n,45n<8+—J˜c‘45
*F?458+>/D2,*•/8‚4qI:JmmD.ZN]yl–I+> >45*+24+lmmD.cLh8
n,45n<8+—K˜:(+/8G46…nƒ45+u>J—J˜12J—K˜F‹/.78.fOn<8+*F?45:
- Tiến hành:
+,^4_<Dw*.?;*y•45*+`?6•*+b*‘JKZ]ltvyciˆCOn‘45cLM45
8+,^4*+`?o45]12J•45`¨¨I`4n?¨8+b>D{,8G4I+C4*g8+:?cw+fI*+ƒ
8k>n,45n<8+„_LH86‚45l”l4D:
- Tính kết quả:
‡cLM45*,Os4*g4+5/€>4‹45cw>._,D/41H/D-*cw•,>45l”l:‘>12?
I+L@45*FŠ4+cLM45*,Os4*g4+5/€>4‹45cwIF?*`/41H/D-*cw•,>45l”l„
*Fa4*g4+cLh8+2D.Lh45IF?*`/4„*F?45D{,I+C4*g8+:
Bảng 4. Xây dựng đường chuẩn albumin

‹45cw>._,D/4

—D5ŸD.˜
,45n<8+
—D.˜
,45n<8+F>n¨?Fn
—D.˜
J mJKl mKl JvlD.
K mKl mKl JvlD.
Z ml mKl JvlD.
] mvl mKl JvlD.
l Jm mKl JvlD.
t Jl mKl JvlD.
v Km mKl JvlD.
JK
y m m KmD.

Hình 6. Đường chuẩn protein
‘>12?I+L@45*FŠ4+cLM458+,^4O¦JltlˆBmmlK*g4+cLh8+2D.Lh45
IF?*`/4*F?45n<8+4,3/8fO8+k45Z.2mJD5ŸD.*jc‚*g4+cLh8+?;**g4+
8? >5`4>6`F/a458k>8+k45Z'JKKyŸD5IF?*`/4.
III.4. Nghiên cứu thu nhận protease từ đu đủ và dứa
III.4.1. Dụng cụ, thiết bị và hóa chất
9ƒ458ƒ'FCOci.788•8c?45lmmD.JmmmD.
9+/s*_<'DAOˆ>O6/4+*•—N+/./I6=>.>O6/>˜C4+/D>nr,®KKm=>ˆKKm5
n¦m:JD5
9‚>8+f*'‹4vm
?
4LH88f*c0DF/6.ml=I¦l—8C4ZtZ]K5F/6
_>6`+u>*>4*F?45J.g*4LH88f*8+«4+I_Ž45n,45n<8+.Jm˜
9-*./0,'c,ckˆ>4+8,•45•,onb>
III.4.2. Thí nghiệm

Chiết enzym từ đu đủ và dứa:
H/Dƒ8cg8+F•*45q4*+M/5/>4I+C48q*8? >5`4*+3_Ž458A845,‹4`4rOD
Fš*/d46›48‚*;//0*>D`4rOD*jc,ck128,•45•,onb>6‡cLh8*+…
45+/0De+o4p45*+kOI+C41-*./0,8? >5`4: …>c,ck128,•45nb>_Ž45
4LH86;8+F…>.;/_Ž454LH88f*6>,c‚_Ž458‹4vm
?
:06/4+DAOˆ>O8A8
nƒ458ƒ_Ž458‹4vm
?
8+?12?_?ˆ8fO8x45nb>12c,ck_-*c¯4*F?45Zm
I+•*:C4c,ckˆ>4++?~88,•45•,onb>6>,c‚8+?12?DAOˆ>O6/4+*•
*+aDc0DF/6.ml=I¦l*+`?*«.0J'J—1gnƒ8‚Jmm5F>Dc,ck Jmm
D.c0D˜ˆ>O4+,OP4c|F>8•8c?45*+kO*/4+n,45*g8+JmmmD..78_Ž45FCO
JZ
*+,cLh8`4rOD*+3:žA8c<4++2D.Lh45IF?*`/4*|456•*+`?I+L@45I+AI
F>n¨?Fn12+?;*cwIF?*`>6`*+`?I+L@45I+AI46?48o/*/s4.

Hình 7. Chiết enzym từ đu đủ và dứa
Xác định hoạt tính protease theo phương pháp Anson cải tiến[1]:
+?IF?*`>6`*A8nƒ451H/8>6`/46o4I+^D*;?*+24+8‚8+b>8A8.?;/I`I*/n
45q4>8/n>D/4*F?45c‚*OF?6/4`8+/sDc>6•:žA8c<4+*OF?6/4`_Ž45I+o4b45
D2,1H/*+,•8*+…¨?./4*jc‚ˆA8c<4+cLh8+?;**g4+IF?*`>6`*+`?c<4+45+œ>'
?;**g4+IF?*`>6`cLh8_/i,*+<.26•D/8F?D?.*OF?6/4`6/4+F>n?*+kOI+C4
8>6`/4_„/JD5+™4+hI8+b>IF?*`>6`*F?45JI+•*„c/d,e/048+,^4—Zv
?

I¦vt˜:
ƒ458ƒ'•4545+/0DJyˆJym—t8A/˜Š4+c<4+Db8lmJmmKlmlmmD.
¡/44I/I`**`¡KJmm9JmmmD/8F?./*F`
Hóa chất:

,45n<8+>
K
N
]
 JŸJl='+u>*>4K”tv5>
K
N
]
:K
K
*F?454LH88f*
*+24+KlmD.:
,45n<8+(
K
N
]
JŸJl='+u>*>4m”mvK5(
K
N
]
*F?454LH88f**+24+Jmm
D.:
,45n<8+c0D?F`6`4JŸJl=I¦vt'*Fw4JvvD.n,45n<8+>
K
N
]
JŸJl
=12KZD.n,45n<8+(
K
N

]
JŸJl=:?128+«4+I¦vt:
,45n<8+8>6`/4J–'c,463/8A8+*+kOJ58>6`/4*F?45c0D?F`46`48+?cs4
*>4+?24*?24F‹/c<4+Db8.a4JmmD.:
,45n<8+Jm–'+u>*>4Jm5*F?454LH88f*8+?ckJmmD.:
,45n<8+>ml'+u>*>4Jm5>*F?454LH88f*8+?cklmmD.:
,45n<8+.mK'*Fw4]KlD..c-Dc~81H/4LH88+?ckKlmD.:
,45n<8+*OF?6/4`KmD=Ÿ'e+,fO45+/d4mJJy5*OF?6/4*F?45n,45n<8+
.mK1j>cklmD.:
,45n<8+*OF?6/4`8+,^4JD=Ÿ'I+>.?†45lD.*OF?6/4KmD=Ÿ*F?45.
mK*+24+JmmD.:
J]
Tiến hành:
Bảng 5. Xây dựng đường chuẩn tyrosine
°4545+/0D m J K Z ] l
,45n<8+*OF?6/4`8+,^4JD=Ÿ
—D.˜
m mK m] mt my J
,45n<8+.mK—D.˜ J my mt m] mK m
,45n<8+>ml—D.˜ K K K K K K
+,•8*+…¡?./4—D.˜ mt mt mt mt mt mt
Lh45*OF?6/4—¥D?.˜ m mK m] mt my J
q8cd,12ciOa4JmI+•*c?D-*cw•,>45„_LH86‚45ttm4D:‡c‹*+<_/i,
n/P46‘_/s4*+/a4D-*cw•,>45—±˜*+`?.Lh45*OF?6/4`„8A8•45—cLM45
8+,^4˜—Š4+y˜:
Hình 8. Đường chuẩn tyrosine
?;**g4+IF?*`>6`—ŸD.˜¦—ˆ::(˜Ÿ—*:1˜
ž'6•¥D?.*OF?6/4`6,OF>*jcLM458+,^4
'*|45*+i*g8++™4+hII+o4b45—JJD.˜
1'*+i*g8+n<8+.78c`DI+C4*g8+—JD.˜

e'cwI+>.?†45
*'*+M/5/>4I+o4b45
J¦J¥D?.*OF?6/4`ŸD.ŸI+•*+?~8J¥D?.ŸD5ŸI+•*
Bảng 6. Xác định lượng tyrosine trong dung dịch nghiên cứu
,45n<8++‚>8+f* +g45+/0D •/8+b45
>6`/4J–—D.˜ l l
Jm–—D.˜ m l
<8+`4rODD{,—D.˜ J m
q812kD{,„Zv
?
*F?45ZmI+•*
Jm–—D.˜ l m
<8+`4rODD{,—D.˜ m J
Jl
78.fOJD.n<8+.78*+‘8+/04I+o4b45D2,
<8+.78—D.˜ J J
,45 n<8+  >  ml
—D.˜
K K
+,•8*+…¡?./4—D.˜ mt mt
q8cd,12ciOa4JmI+•*c?D-*cw•,>45„_LH86‚45ttm4D
jes*•,o+2D.Lh45IF?*`/4—D5ŸD.˜12+?;**g4+IF?*`>6`—ŸD.˜*g4+cLh8
+?;**g4+F/a458k>`4rOD.2'6•c@41<+?;**g4+ŸD5IF?*`/4`4rOD:(s*•,o
cLh8*FŠ4+_2O*Fa4o45v:
Bảng 7. Hoạt tính protease của dịch chiết đu đủ và cuống dứa
-*./0, NF?*`/4*|456•
—D5ŸD.˜
?;*  *g4+  IF?*`>6`
—ŸD.˜
?;*  *g4+  IF?*`>6`

F/a45—ŸD5˜
,•45nb> mKJ Kyyl JKvZy
,ck mZKK Klyy v”]]
III.5. Nghiên cứu khả năng phân cắt collagen của enzym từ L. sphaericus
VN3, dịch chiết đu đủ và dứa
V.1. Dụng cụ, thiết bị và hóa chất
9ƒ458ƒ'cœ>I`*F/8+b>D3/*FLM45N” J–8? >5`4—Kcœ>˜nƒ458ƒcƒ8.™
—J8A/˜
9+/s*_<'¡/44I/I`**`¡KJmm9JmmmD/8F?./*F`*k.;4+—?6+/_>+-*˜*k4,3/
1/6/4+1-*—¡F/?8` b8˜
9‚>8+f*'*+24+I+G4D3/*FLM45N” J–8? >5`4e+345+u>*>4
V.2. Thí nghiệm
- +,^4_<D3/*FLM45*+;8+ e+?A45N” J–8? >5`4e+345+u>*>4c|cœ>
I`*F/ci45,w/cƒ8.™4+}n<8+8+/s*`4rODc,ck
12nb>n<8+.a4D`48+k45Zci*F?4545p4DA*
*k.;4+K5/M8+?`4rODe+,s8+*A46>,c‚.fOF>ci
*F?45*k4,3/1/6/4+1-*Zv
?
:U,>46A*12c?1u45
I+C4  5/o/  8? >5`4  6>,  J9Z  452O:  •/  8+b45  .2
8? >5`4>6`8+,^4—/5D>˜124LH88f*:(s*•,o8+?
*+fO`4rOD*j8+k45Z12n<8+8+/s*`4rODnb>
c,ck8‚e+o4p45I+C48q*8? >5`4e+o4p45I+C4
5/o/8k>8+k45Z²n<8+8+/s*`4rODnb>²
n<8+8+/s*`4rODc,ck:
Jt
Hình 9. Hoạt tính phân giải
collagen của chủng VN3 và
dịch chiết đu đủ, dứa
III.6. Nghiên cứu điều kiện phân cắt collagen từ da và gân bò

VI.6.1. Dụng cụ, thiết bị và hóa chất
9ƒ458ƒ'_Š4+*>D5/A8JmmD.—Km8A/˜
9+/s*_<'*k4,3/1/6/4+1-*Zv
?
—¡F/?8` b8˜
9‚>8+f*'n<8+e`?8? >5`4*+3—*j5C412n>_u˜
VI.6.2. Thí nghiệm
Fw4n<8+4,3/8fO8+k45Z—c†.?;/6/4+e+•/˜+>O8‚*+i57/.2n<8+`4rOD
*+3n<8+8+/s*`4rODc,cknb>1H/n<8+e`?8? >5`4*+3*+`?8A8*«.0J'JK'J
Z'J]'J:Y„Zv
?
+245452O•,>46A*6‘n<8++‚>e`?8? >5`4*+3_Ž45Dq*
*+LM45*F?45J*,G4:(s*•,o8+?*+fO*«.0I+•/*Fw4`4rOD'8? >5`4*+3¦]'J
*+g8++hI8+?I+C48q*8? >5`4:&8A8*«.08u4.;/*F?45•,A*FŠ4+k8‚*+in?
.Lh45n<8+g*4a4e+345*+fOF-6‘n<8++‚>„D{,8‚*«.0J'J8? >5`4*FL@45
.a4*F?45•,A*FŠ4+k*;?*+24+Dw*e+•/:


Hình 10. Thủy phân collagen thô từ gân, da bò bởi enzym từ dứa (A), đu đủ (B)
và chủng VN3 (C).
Fa48@6„8+74cLh8*«.0I+•/*Fw4]'J*/s4+24+45+/a48b,c/d,e/04
I+C48q*8? >5`4*+3*j5C412n>_u*F?45v452Oci*ŠDF>8345*+b8I+•/*Fw4
`4rOD128? >5`4*+g8++hI:+,^4_<8A8D{,*+kOI+C48? >5`4*j5C412n>
_u*F?45_Š4+*>D5/A8JmmD.4+L6>,'
={,J'<8+8+/s*`4rODc,ck 8? >5`4*j5C4n>_u*«.0]'J
={,K'<8+8+/s*`4rODnb> 8? >5`4*j5C4n>_u*«.0]'J
={,Z'<8+`4rOD*+38+k45Z 8? >5`4*j5C4n>_u*«.0]'J
={,]'<8+`4rOD*+38+k45Z n<8+8+/s*`4rODc,ck 8? >5`4*j
5C4n>_u*«.0K'K'J
={,l'<8+`4rOD*+38+k45Z n<8+8+/s*`4rODnb> 8? >5`4*j5C4

n>_u*«.0K'K'J
={,t'<8+8? >5`4>6`8+,^4—JKlŸD.˜ 8? >5`4*j5C4n>_u*«.0]'J
Jv
={,v'LH88f* 8? >5`4*j5C4n>_u*«.0]'J
={,y'<8+`4rOD*+38+k45Z J–8? >5`4/5D>
F?45c‚D{,tvy.2D{,c•/8+b45:U,>46A*6‘n<8++‚>12ˆA8c<4+
+2D.Lh45IF?*`/4*F?45I+G4n<8+8k>+™4+hI+245452O:(s*•,oc?5/A*F<
l”l4D8+?*+fOD{,l8‚*«.0n<8+`4rOD*j8+k45Z'n<8+8+/s*`4rOD
nb>'8? >5`4*+3¦K'K'J8+?5/A*F<8>?4+f*12*+M/5/>4I+C48q*]452O.2
*+g8++hI4+f*+2D.Lh45IF?*`/4*|456•c;*8>?4+f*.2mJtK]D5ŸD.n<8+
I+o4b456>,*+M/5/>442O+2D.Lh45IF?*`/4e+345*p45.a44€>:={,]5‹D
n<8+Z'n<8+8+/s*c,ck'8? >5`4*+3¦K'K'J8‚*+M/5/>4I+C48q**+g8++hI
.2Z452OIF?*`/4*|456•*F?45n<8+I+o4b45.2mJlyD5ŸD.:={,Z6…nƒ45
n<8+nb>es*+hI1H/`4rOD*j8+k45ZcLh8.‘>8+74n?45,‹4*+,`4rOD
.28,•45•,onb>9I+ƒI+^D8k>8+s_/s4nb>:+M1-O8‚*+i4C458>?e+o
4p458q*8? >5`4F•*45q4*+M/5/>4n?IF?*`>6`*jnb>I+C48q*8A8./a4es*4w/
I+C4*…8? >5`46>,e+/_‚6h/_<8q**;/4+/d,1<*Fgn?*A8nƒ458k>8? >5`4>6`
*j8+k45Z:a48;4+c‚1/0842O8u45/•I*-4nƒ45124C458>?5/A*F<8k>
45,‹4I+ƒI+^D42O5‚II+G45/oD34+/PDD3/*FLM45n?1/08*-I*F,45
8,•45nb>*+o/_}6>,•,A*FŠ4+8+s_/s4:
={,l.‘>8+74cLh8nx45ci*;?+™4+hII+C48q*8? >5`4„„I—ltvy˜
124+/0*cw—ZmZv]m
?
˜e+A84+>,4+ŽD*ŠDF>c/d,e/04*+g8++hI—Š4+JJ˜:
(s*•,oc?5/A*F<l”l4DciˆA8c<4++2D.Lh45IF?*`/4*+,cLh8*F?45n<8+
I+o4b458+?*+fOI+C48q*„4+/0*cwZm
?
12I¦v8+?es*•,o8>?4+f*c;*
mKZ]D5ŸD.:jc‚.‘>8+74cLh8c/d,e/04I+C48q*8? >5`4*jn>125C4_u
.2'4+/0*cwZm

?
I¦v:
 
Hình 11. Phân cắt collagen thô bằng enzym
<8+6>,I+C48q*8‚n;45e`?4+H*cLh8nx45ci*;?D2458? >5`4
e/iD*F>_Ž458A8+8+ƒIo4+nLH/eg4++/i41/c/04*…•,•*8+?*+fO8? >5`4
*FLH8e+/_<I+C48q*_„/`4rOD8‚n;458A86h/n2/c>4ˆ`44+>,*+24+.LH/
Jy
8? >5`46>,*A8nƒ458k>`4rOD_<8q*F>*+24+8A8c?;445q4—Š4+JK˜:+L
1-O1/08nx45`4rOD*+3*j8+k45Zes*+hI1H/`4rOD*+3*jnb>c,ck8‚
e+o4p45I+C48q*8? >5`4*+3*j5C412n>_ucCO.2es*•,o6@_w_>4cG,*;?
8@6„8+?4+€45_LH845+/a48b,*/sI*+`?4+ŽD*;?D2458? >5`48‚*+ib45
nƒ45*F?45*+‘8*s:
A B
Hình 12. Ảnh chụp dưới kính hiển vi điện tử quét collagen trước (A) và sau
(B) khi phân cắt bằng enzym (x 5000)
III.7. Sơ bộ nghiên cứu điều kiện phù hợp cho tạo màng collagen
III.7.1. Dụng cụ, thiết bị, hóa chất
ƒ458ƒ'*fDD/8>c‚45_Š>6A8+—mZ*M˜cŒ>*+kO*/4+—J8+/s8˜FCO_w*—J8A/˜
*+Š>/4?ˆ—J8A/˜
+/s*_<'*k8fO13*Fx45—N+AI˜
‚>8+f*'n,45n<8+,45n<8+>l'+u>*>4Jm5>*F?454LH88f*
8+?cklmD.:
,45n<8+.K'*Fw4]KlD..c-Dc~81H/4LH88+?ckKlD.:
III7.2. Thí nghiệm:
C412n>_u6>,e+/ˆ>OcLh845CD*F?45n,45n<8+8‚l–>8`*/8>8/n12_o?
•,o4*F?45*k.;4+cinx458+?8A8*+g45+/0D:>,e+/_<*+kOI+C4_„/`4rOD
„*Fa4*+,cLh8n;45n<8+e`?4+H*:/d,8+«4+I8k>n<8+1dKZ]ltvy
_Ž458A8n,45n<8+>l+?~8.K:U,•*n<8+8‚Ie+A84+>,.a4
5/fOD/8>cie+3*‘4+/a4*F?45*k8fO13*Fx4512•,>46A*e+o4p45*;?D245

cA4+5/A8oD•,>4Db8cw*;?D245:(s*•,o*FŠ4+_2O*Fa4o45y:
Bảng 8. Khả năng tạo màng collagen của dịch sau thủy phân có pH khác
nhau
I K Z ] l t 7 y
;?  *+24+
D245
(+345
*;?D245
(+345
*;?
(+345
*;?
*;?
D245
*;?
D245
*;?
D245
*;?
cLh8
J”
D245 D245 F- F- D245
=245
*F?45
9 9 9 H* =245
*F?45
=245
*F?45
8‚  6h/
*Fq45

=245  8‚
cwn>/
9 9 9 e+345
n>/
‚  cw
n>/
‚  cw
n>/
‚  cw
n>/  g*
+@4
=2454+›4
D<4
9 9 9 9 +›4
D<4
+›4
D<4
(+345
D<4
1‚4
8ƒ8
(s*.,-4'*;/Iv*;?D245*•*4+f*D245*;?*+24+*F?45D<48‚cwn>/e+3
4+>4++@44a48‚*+i*A8+e+}/*fDD/8>6HD+@4n<8+„I>ˆ/*+@4:+L1-O
.28? >5`46>,I+C48q*_Ž45`4rOD*j8+k45Z12nb>8‚*+i*;?cLh8
*+24+D245*;/I¦v1f4cdc~*F>.26o4I+^DD245+LH45*H/b45nƒ45
*F?45c/d,*F<4a48G4I+o/6;8+:LH845+/a48b,*/sI*+`?.2*/4+6;8+`4rOD
6>,c‚nx458+?I+C48q*e`?8? >5`4*+3:

Km

Hình 13. Thí nghiệm tạo màng collagen ở pH khác nhau trên tấm mica
III.8. Nghiên cứu tinh sạch enzym từ chủng L. sphaericus VN3 và dứa
III.8.1. Dụng cụ, thiết bị và hóa chất
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III.8.2. Thí nghiệm
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+hI4+f*D2„c‚*+,cLh84+/d,es**k>1H/+?;**g4+`4rOD8>?:>,e+/_|
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(s*•,o*+,cLh8*Fa4_o45”12Jm8+?*+fO+2D.Lh45—
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tm–4‹45cw_†?+u>:;/c‚.Lh45IF?*`/4es**k>4+/d,4+f*+?;**g4+`4rOD
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Bảng 9. Hàm lượng protein và hoạt tính collagenase trong dịch tủa
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0.136 491.596 21.82 78975.82 218.17
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0.366 1324.395 25.18 91147.98 251.79
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0.727 2633.079 29.58 107074.17 295.79
70
1.435 5193.795 35.93 130053.39 359.26
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0.809 2927.675 27.88 100931.21 278.82
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Bảng 10. Hàm lượng protein và hoạt tính protease trong dịch tủa
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PHẦN VI. KẾT LUẬN
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TÀI LIỆU THAM KHẢO
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8+>F>8*`F/6*/86?ăD`>*6>DI.`*F`>*`nĐ/*+n/ăă`F`4*I.>4*IF?*`>6`6African J of
Biotechnol:JJvt'J]myy9J]ml:
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