BÁO cáo THỰC HÀNH môn CÔNG NGHỆ ENZYME

1







Báo cáo


GVHD: 
 Nhóm 6
1.  2008100148
2.  2008100070
3.  2008100001









2

Bài 1: SACCHAROMYCES CEREVISIAE



1. 

Saccharomyces cerevisiae
Saccharomycetaceae.Saccharomyces cerevisiae      
 7 nm.
              

 2 mm.
 35
0
 

0
C trong 10% glucose và 13
0

 39
0
C (Jermini and Schmidt-

H
3
PO
4
và pH = 1,8   
 



            





3

             
     


 

Pha lag:







Pha log:

X = X
0
.2
n

 X

0









4

   
xu

                

      

Pha suy vong:






2. 

5




:
Glucose: 50 g
MgSO
4
.7H
2
O: 2g
KH
2
PO
4
: 3g
Tryptone: 10g

Agar : 20g
pH : 5-6



6
 


O
C, 15 phút.

2.1.1 

Glucose: 10 g
MgSO
4
.7H
2
O: 0,4g
KH
2
PO
4
: 0,6g
Tryptone: 1g

Agar : 4g
pH : 5-6


2.1

Glucose: 10 g
MgSO
4
.7H
2
O: 0,4g
KH
2
PO

4
: 0,6g
Tryptone: 1g



7
pH : 5-6


2.1.

Glucose: 50 g
MgSO
4
.7H
2
O: 2g
KH
2
PO
4
: 3g
Tryptone: 10g
pH : 5-6
      

2.1. 
bông, bao gói
2.1. 


2.1. 

0

 .

2.2 



8



 

2.3 
0
C,15 phút 

2.4 Saccharomyces cerevisiae

0
Saccharomyces cerevisiae 




khoát.

2.5 Saccharomyces cerevisiae
       Saccharomyces cerevisiae    


   





9
2.6 



0

2.7 



 Saccharomyces cerevisiae hoà vào




2.8 
Saccharomyces cerevisiae 
kính.



2.9 
3 
- 




10
-  5-

- 
0

0
C hay 50
0






0

- 
0


- 


- Saccharomyces
cerevisiae.
- 



thái.
-  



121
0
C 




11

 



- 
 


4 

4.2 Saccharomyces cerevisiae  và








12



0



.


Saccharomyces cerevisiae 



13
4.3 

4.4 



14

Sacchromyces cerevisiae











           

5 - 
-          

6 
6.2 
- 

- 





15
-        
 



- 

- nhão.
- C
6.3 
- 
- 
- 





6.4 
- 




16


- 

- 






 


-Enzyme này còn dùng 
 
enzyme invertase.











17
BÀI 2. TÁCH CHIT VÀ XÁC NH HOT TÍNH ENZYME
INVERTASE TÁCH T SACCHAROMYCES CEREVISIAE

1. :



Invertase Saccharomyces 
 
 









18
Các protein e


protein enzyme l




             

           
glycerol,ethylacetate.
 


 Enzy


 

            







19





              





invertase.










20
2. 



a. 
Dch nm men nhân sinh khi em i ly tâm ln 1 (4500 vòng/phút) trong
10 phút, loi dch trong, thu hi sinh khi nm men.
Tip tc phi trn sinh khi nm men vi dung dch nc mui NaCl
(0,85%) theo   1/3 (w/w). Sau ó, tin hành ly tâm (4500 vòng/phút)


Thu sinh
khi nm
men

thu dd
enzyme thô
Xác nh
hot tính
enzyme
invertase

nh lng

ng kh
bng phng
pháp acid
dinitrosalicyli
-c (DNS)
Dng ng
chun và
xác nh
hàm lng
ng
Tính ht
tính
enzyme
invertase.
Phng
pháp t
phân

sóng siêu
âm



21
trong 10 phút  thu nhn sinh ki nm men Ra bng nc ct vô
trùng theo   1/3 (w/w) và gi li sinh khi nm men.
Bo qun sinh khi nm men  16-20
0
C (ngn mát ca  lnh).

b. 
Phng pháp t phân (Làm trc 48 g theo phân công  ph lc)
Trong quá trình  phân nm men, h enzyme  phân có sn trong t bào
nm men c hot hóa. Chúng xúc tác phn ng phân gii các cht có trong thành
t bào nm men và gii phóng enzyme invertase ra ngoài t bào.
Cân 1g sinh khi nm men cho vào dung dch m acetate pH = 5.5 theo t
1/6 (w/w)  thc hin quá trình phân.  trong t m 45
0
C trong 50 gi. Sau
quá trình t phân, mu c ly tâm (5000 vòng/phút) trong 10 phút  tách b phn
không tan, phn dch lg thu c là ch phm invertase thô. S dng dch
enzyme này xác nh hot tính.
c. tase
- Hút 4ml dung dch m acetate pH = 4,5; 4,6 ml c ct và 0,4ml dch
cha enzyme cho vào bình tam giác 100ml.
- Thêm vào 1ml dung dch ng Saccharose ng  2%.
- Lc ,  yên cho phn ng xy ra  iu kin phòng trong 15 phút.
- un cách thy trong 2 phút và làm lnh di vòi nc  mu ty phân
dùng  xác nh hàm lng ng glucose bng phng pháp DNS,  ó suy ra s
mol Saccharose ã b thy phân.

d. Dng ng chun và xác nh hàm lng ng



22
- Cho vào 6 ng nghim sch vi các cht có th tích nh bng sau:
Hóa cht
ng 1
ng 2

ng 3
ng 4
ng 5
ng 6
ng mu
Glucose 1%
0
0,2
0,4
0,6
0,8
1,0
0
Nc ct
10
9,8
9,6
9,4
9,2
9
0
Mu thy phân
0
1
Ng  (%)
0
2
4
6
8

10

Hút 1ml t ng 1 n g 6 vào 6 ng nghim tng ng khác,
ng mu g nguyên
Thc th DNS
(ml)
3

o mt  quang  bc sóng 540 nm vi ng 1  chnh OD = 0.
V ng chun glucose vi trc tung là mt  quang (OD
540nm
), trc
hoành là ng  glucose.
Tìm phng trình biu din ng chun dng y = ax + b vi y =
OD
540nm
; x = [glucose] (%) và h s tng quan R
2
nh phn mm Excel.
Chú ý: tin hành các mu dng ng chun và mu thí nghim ng thi.
2.3.3. Tính ht tính enzyme invertase.
  phng trình  th ng chun tính c X (mg/ml) ng kh
trong dung dch ng pha lõang.
- Nhân vi h s pha lõang n  c lng ng trong 1 ml dung dch gc.
Chn h s pha lõang n sao cho OD nm trong gii hn ng chun.
- Tính hàm lng ng trong nguyên liu (mg/g) = X *n* V/m
Dùng giy nhôm bkín u các g nghim
t vào ni nc ang sôi trong 5 phút
Làm lnh n nhit  phòng
OD ( = 540 nm)

?
?
?
?
?
?



23
V = th tích dch ng gc (ml)
m = ki lng u cân (g)
Mt n v hot tính invertase là lng enzyme cn thit   thy
phân 1micromol ng Saccharose trong 1 phút  iu kin pH 4,5 và nhit 
30
o
C.
2. 

                  
glucose, fructose, investase.
             

cose và fructose, làm




                  

           


3. 





24

y=OD
x=[glucose]
0,207
2
0,223
4
0,307
6
0,373
8
0,454
10



25
4. - 

- aromyces cerevisiae.
- 
5. 
5.1. Ch X.V.n/m
-

 
1
cos
glu
nl
glu
m
V
m
eGlu 

=
Glu
m

-
 
nV
m
eGlu
nl

glu
.
cos 
v

Glu
m
=[Glu].V
nl
.n
- 
nl
nl
nl
glu
m
nVglu
m
m
.].[


m
nVX
gmg

)/( 

            
