Международная научно
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практическая конференция
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54
HAYVONLARDAN AJRATILGAN IZOLYATLAR VA
B.SUBTILIS
SHTAMMLLARI Β
-GLYUKOZIDAZA GENLARINING PCR
AMPLIKATSIYASI
Safarov Husniddin, Qutliyeva G
o‘
zal, Turaeva Bakhora
Oʻzbekiston Fanlar akademiyasi mikrobiologiya ilmi
y tadqiqot instituti
Annotatsiya:
β
-glyukozidaza fermenti sellyuloza va boshqa glikanlarni
parchalaydigan muhim fermentlardan biridir. Ushbu fermentning asosiy vazifasi
β
-1,4-glikozid bo
g‘
larini gidroliz qilish orqali glyukoza molekulalarini ajratib
olis
hdir. β
-glyukozidaza fermenti k
o‘
plab mikroorganizmlar, shu jumladan
bakteriyalar va zamburu
g‘
lar tomonidan sintezlanadi. Ushbu fermentning genetik
asoslarini
o‘
rganish, biotexnologiya va sanoat sohalarida katta ahamiyatga ega
b
o‘
lib, sellyuloza asosidagi biomassa konversiyasini optimallashtirish imkonini
beradi. Tadqiqotimizda, Enterococcus faecium
,
Chryseobacterium anthropi
,
Proteus
vulgaris
,
Alcaligenes faecalis va Bacillus subtilis ning 7 ta shtammlarida
β
-glyukozidaza fermenti genining mavjudligi aniqlangan va ushbu genning PCR
amplifikatsiyasi orqali tasdiqlangan.
Kalit s
o‘
zlar:
B. subtilis, E.faecium, Ch.anthropi,
β
-glyukozidaza, sellyuloza,
β
-1,4-glyukozid, PCR, marker.
Аннотация
:
Фермент β
-
глюкозидаза является одним из важных
ферментов, расщепляющих целлюлозу и другие гликаны. Основная функция
этого фермента –
выделение молекул глюкозы путем гидролиза
β
-1,4-
гликозидных связей. Фермент β
-
глюкозидаза синтезируется многими
микроорганизмами, включая бактерии и грибы. Исследование генетической
основы этого фермента имеет большое значение в области биотехнологии
и промышленности и позволяет оптимизировать переработку биомассы на
основе целлюлозы. В нашем исследовании наличие гена фермента
β
-
глюкозидазы было обнаружено у 7 штаммов
Bacillus subtilis, Enterococcus
faecium
,
Chryseobacterium anthropi
,
Proteus vulgaris
и
Alcaligenes faecalis
и
подтверждено методом ПЦР
-
амплификации этого гена.
Ключевые слова:
B. subtilis, E. faecium, Ch. anthropi
, β
-
глюкозидаза,
целлюлоза, β
-1,4-
глюкозид, ПЦР, маркер.
Abstract:
The β
-glucosidase enzyme is one of the important enzymes that
breaks down cellulose and other glycans. The main function of this enzyme is to
isolate glucose molecules by hydrolyzing β
-1,4-
glycosidic bonds. β
-glucosidase
enzyme is synthesized by many microorganisms, including bacteria and fungi. The
study of the genetic basis of this enzyme is of great importance in the fields of
biotechnology and industry, and allows to optimize the conversion of cellulose-
based biomass. In our study, the presence of β
-glucosidase enzyme gene was
detected in 7 strains of Bacillus subtilis, Enterococcus faecium
,
Chryseobacterium
anthropi
,
Proteus vulgaris
and
Alcaligenes faecalis by PCR amplification of this gene.
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55
Keywords:
B. subtilis, E. faecium, Ch. anthropi, β
-glucosidase, cellulose,
β
-1,4-glucoside, PCR, marker.
Sellyuloza parchalanuvchi organizmlar uchta fermentdan iborat k
o‘
p
fermentli kompleksni
o‘
z ichiga olgan: ekzoglyukanaza (selobiogidrolazalar),
endoglyukanaza va
β
-glyukozidaza. Sellyulozaning t
o‘
liq gidrolizi uchun
bosqichma-bosqich ta
’
sir k
o‘
rsatadi [1]. Ushbu fermentativ tizimda
β
-glyukozidaza hal qiluvchi rol
o‘
ynaydi, bunda ular sellyuloza gidrolizining oraliq
mahsuloti b
o‘
lgan sellobiozani glyukozaga aylantirish orqali gidrolizning oxirgi
bosqichini yakunlaydi [2]. Kampos va sigir g
o‘
ngidan ajratilgan
B. subtilis
RA10
shtammida
β
-glyukozidaza
geni kuchaytirilib,
o‘
sish sharoitlari
optimallashtirilganda va b-glyukozidaza fermenti ishlab chiqarilishi
19,8 barobarga oshirilgan.
B. subtilis RA10
dan olinga
n β
-glyukozidaza faolligi
o‘rganilgan, 80 °C haroratda faolligi 78% ni, 50 °C haroratda 48 soat
inkubatsiyadan keyin ferment faolligining 68,32% gacha saqlab qolgan [3].
Enterococcus faecalis (EF85)
va
E. faecium (EF83)
shtammlari makkaj
o‘
xori
o‘
simligini silos bostirishda foydalanilganda sut kislota miqdori sirka kislotaga
qaraganda sezilarli darajada oshganligi aniqlangan [4].
Pediococcus acidilactici
PA204
shatammining sellyulozani parchalashi va sut kislota hosil qilish qobiliyati
o‘
rganilgan. Tadqiqotda 5 L bioreaktorda fermentatsiya mahsuldorligi 92,01 g/L,
0,77 g/g ni tashlil qilganligi qayd etilgan [5].
Ushbu tadqiqodning maqsadi, tut ipak qurti
(Bombyx mori)
va ot
(Equus
caballus
) ovqat hazim qilish sistemasidan ajratib olingan, bakteriya
izolyatlarining sellyulozani gidrolizida qatnashadigan ferment genini
aniqlashdan iborat. Tadqiqotda hayvonlardan ajratib olingan
Bacillus subtilis,
Enterococcus faecium
,
Chryseobacterium anthropi
,
Proteus vulgaris
,
Alcaligenes
faecalis
va
β
-glyukozidaza (bglP) praymerlaridan foydalanildi.
Ozuqa muhiti tarkibi: Standart peptonli ozuqa muhiti (PB) (g/l): Pepton-10
g/l; glyukoza- 6 g/l; NaCl- 5 g/l; K
2
HPO
4
–
0,5 g/l; MgSO
4
-0,2 g/l, (pH-7,0).
Lizogenli bulon (LB): trypton 10g/l, yeast extract 5g/l, NaCl 5g/l, (pH 6,8-7,2).
De Man
–
Rogosa
–
Sharpe agar (MRS): pepton-1,0%, mol g
o‘
shti ekstrakti-1,0%,
xamirturush ekstrakti-0,4%, glyukoza-2,0%, natriy asetat uchgidrat-0,5%,
polisorbat 80
–
0,1% (Tween 80), kaliy gidrofosfat-0,2%, triammoniy sitrat-0,2%,
MgSO
4
·7H
2
O -0,02%, MnSO
4
·4H
2
O-0,005%, agar-1,0% (pH
–
6,2) [6].
Hayvonlardan ajratib olingan izolyatlar PB agarli ozuqa muhitida
(sterilizatsiya 121
o
C 20 daqiqa) 24 soat davomida 37
o
C haroratda
o‘
stirilib,
MALDI-TOF bilan idintifiatsiya qilindi (1-jadval). Bakteriya shtammlari suyuq
ozuqa muhitida
o‘
stirildi va epindoflarga 1.5 ml dan olinib 1400 rpm/min,
10 daqiqa davomida sentrifuga qilib ch
o‘
ktirildi. Lizis buffer, Proteinaza, RNaza,
Fenol: Xloroforim: izoamil spirti (25:24:1), isopropanol, Etanol 96% ushbu
ketma-ketlikda genom DNK ajratildi [7]. TE buferida eritilgan DNK
namunalarining konsentratsiyasi Implen NanoPhotometer® yordamida
o‘
lchandi (2-jadval).
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1-jadval
Ajratib olingan izolyatlarning MALDI-TOF yordamida idintifikatsiyasi
Spot
Ajratib olingan manba
Organizm
Score
A1
Ot s
o‘
lagi
Enterococcus faecium
1.80
A2
Tut ipak qurti lichinkasi
Chryseobacterium anthropi
1.75
A3
Tuzlangan karam
Pediococcus acidilactici
1.78
A4
Ot ichagi
Proteus vulgaris
2.40
A5
Ot oshqozoni
Alcaligenes faecalis
2.20
A6
Ot ingichka ichagi
Alcaligenes faecalis
2.33
2-jadval
Hayvonlardan ajratib olingan izolyatlar va
B.subtilis
shtammlarining
nuklein kislotalar konsentratsiyasi
Etidum bromid bilan b
o‘
yalgan 0.8% li agaroza gelida DNK namunalari
elektroforez (Bio-rad, USA) qilindi (1-rasm). Ushbu rasmdan shuni k
o‘
rishimiz
mumkinki,
B.subtilis ot 2
shtammida genom DNK dan tashqari tahminan 3000 juft
nukleotidli halqasimon plazmid ham ajralgan.
A
B
1-rasm. A)
B.subtilis
ning 7 ta shtammlari, B) 1)
E.faecium,
2)
Ch.anthropi,
3)
P.acidilactici,
4)
P.vulgaris,
5)
A.faecalis,
6)
A.faecalis
genom DNKsi 1kb markerda
k
o‘
rinishi.
№
Namunalar
DNK Kons
Ed
№
Namunalar
DNK Kons
Ed
1.
TE buferi
0,000
ng/ul
8
B.subtilis ot 1
271,20
ng/ul
2.
E.faecium
69,00
ng/ul
9
B.subtilis ot 2
207,40
ng/ul
3.
Ch.anthropi
61,45
ng/ul
10
B.subtilis ot 3
272,70
ng/ul
4.
P.acidilactici
30,65
ng/ul
11
B.subtilis ot 4
91,10
ng/ul
5.
P.vulgaris
102,75
ng/ul
12
B.subtilis ot 5
290,90
ng/ul
6.
A.faecalis
475,85
ng/ul
13
B.subtilis ot 6
204,40
ng/ul
7.
A.faecalis
7,15
ng/ul
14
B.subtilis ot 7
150,0
ng/ul
3000
1000
3000
1000
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Sellyulozani parchalashda qatnashadigan
β
-glyukozidaza genlariga
praymer dizayn qilindi (2-rasm). Ushbu praymer Integrated DNA Technologies
(IDT) firmasi tomonidan sintez qilindi [8].
2-rasm.
β
-glyukozidaza (4290 bp) genida dizayn qilingan praymerlarning joylashgan
o‘
rni
Praymer
Sekvinslangan nukleotidlar
Gen nomi
PCR mahsuloti
o‘
lchami
bglP-F
5
՛
TGC TGA GTG CGG TCT TTG AT 3
՛
β
-glyukozidaza
991 bp
bglP-R
5
՛
AGG CCG ATCA TCG CGT AAA T 3
՛
GenPak® PCR MasterMix yordamida ajratib olingan DNK namunalariga
PCR amplifikatsiyasi amalga oshirildi. Har bir PCR reaksiyasi 20 mkl hajmda
tayyorlandi, bunda 10 mkl masterMix, 8.2 mkl distillangan suv (dis H2O), 0.4 mkl
Forward praymer (Praymer-F), 0.4 mkl Reverse praymer (Praymer-R) va 1 mkl
DNK namunasi ishlatildi. Denaturatsiya 94°C da 5 daqiqa, 58°C da 50 soniya, va
72°C da 2 daqiqa davomida 30 sikl baja
rildi. Amplifikatsiyadan s
o‘
ng, PCR
mahsulotlari 0.8% li agaroza gelida k
o‘
rildi. (2-rasm).
A
B
4-rasm.
β
-glyukozidaza genning PCR amplikatsiyasi:
A)
Otlardan ajratilgan izolyatlar
B)
B.subtilis
ning 7 ta shtammlari
Tadqiqot natijalari shuni k
o‘
rsatdiki,
E.faecium, Ch.anthropi, P.acidilactici,
P.vulgaris
, va
A.faecalis
shtammlarida sellyulozani parchalashda qatnashadigan
β
-glyukozidaza fermenti geni mavjud. Ushbu gen uchun dizayn qilingan
1 kb 1 2 3 4 5 6 M
3000
1000
1 kb 1 2 3 4 5 6 7 M
3000
1000
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bglP paymeri 991 bp uzunlikda b
o‘
lib, PCR amplifikatsiya mahsuloti ham aynan
shu
o‘
lchamda ekanligi aniqlandi. Tajribada olingan natijalar asosida, turli
manbalardan ajratib olingan bakteriyalarning sellyulozani parchalashda ishtirok
etuvchi β
-glyukozidaza genlarining PCR amplifikatsiyasi usuli samarali skrining
imkoniyatlarini yaratadi.
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bottleneck in industrial use of lignocellulosic materials. Biomolecules.
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31.
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glucose-
tolerant β
-glucosidase as supplementation to enhance the hydrolysis of
sugarcane bagasse at high glucose concentration. Biotechnol Biofuels.
2015;8(1):202.
3. Tiwari R, Singh PK, Singh S, Nain PKS, Nain L, Shukla P. Bioprospecting of
novel thermostable
β
-glucosidase from
Bacillus subtilis
RA10 and its application
in biomass hydrolysis. Biotechnol Biofuels. 2017 Oct 30;10:246. doi:
10.1186/s13068-017-0932-8. PMID: 29093750; PMCID: PMC5663093.
4. Zhao J, Shao T, Chen S, Tao X, Li J. Characterization and identification of
cellulase-producing Enterococcus species isolated from Tibetan yak (Bos
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33484057.
5. Zhang Z, Li Y, Zhang J, Peng N, Liang Y, Zhao S. High-Titer Lactic Acid
Production by
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Batch Simultaneous Saccharification and Fermentation. Microorganisms. 2020
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PMCID: PMC7600695.
6. De Man, J.C.; Rogosa, M.; Sharpe, M.E. (1960). "A Medium for the
Cultivation of Lactobacilli". Journal of Applied Bacteriology. 23 (1): 130
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7. Francisco S.S., Liselott S.S., Antonio B., Daniel J.L., Roger K., Edward R. B.
Moore & Margarita Gomila Culture Collection University of Gothenburg (CCUG). A
protocol for extraction and purification of high
–
quality and quantity bacterial
DNA applicable for genome sequencing: a modified version of the Marmur
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Method
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Protocol
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10.1038/protex.2018.084.
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