CELL DISRUPTION – CHEMICAL OR ENZYMATIC METHODS
Aim •
To distrupt the bacterial cells for isolation of the intracellular components like protein, nucleic acid and intracellular organelles.
Introduction
Single-cell Singl e-cell orga organisms nisms (microorganisms) (microorganisms) consist of a semipe semipermeabl rmeable, e, tough tough,, rigid rigid,, outer cell wall surrounding the protoplasmic (cytoplasmic) membrane and cytoplasm. The cytoplasm is made up of nucleic acids, proteins, carbohydrates, lipids, enzymes, inorganic ions, vitamins, pigments, inclusion bodies and about !" water. #n order to isolate and e$tract and of these substances from inside the cell, it is necessary to break the cell wall and protoplasmic membrane. #n some cases the cells may e$crete the desired substance% but, in most cases, the cell wall must be disrupted to release these substances. &ell 'isruption is the method or process for disrupting or lysing the cell inorder to release the contents contents out of the cell. &ell disruption disruption is done to release release the cell contents. contents. &ell ysis y sis or 'i 'isru srupt ptio ion n is do done ne fo forr is isol olati ating ng u ucl cleic eic ac acid idss (' ('* * + l lasm asmid ids) s),, r rot otei eins ns (#ntracellular roteins), rganelles, etc. &ell disruption categorized into physical, chemical and enzymatic methods based on the protocol and components used for cell disruption. #n chemical method, most widely used detergents are Tween !, tween !, triton $, S'S and &/*S. &/* S. #n enzymatic enzymatic method, lysozy lysozymes mes are used for bacterial bacterial cells, chitinase chitinase for yeast cells, pectinase for lysis of plant cell walls. The advantages of chemical and enzymatic methods are easy to perform and e$peditious. Principle
0nzymatic methods are easy and fast but cost for these methods are also high. #n this e$periment e$peri ment we use lysozyme for cell disruption. disruption. y ysozyme sozymes, s, also known as murami muramidase dase or -acetylmuramide glycanhydrolase, are glycoside hydrolases. These are enzymes (0& 1..2. 1. .2.23) 23) tha thatt dam damage age bac bacteri terial al cell wal walls ls by cata catalyz lyzing ing hy hydro droly lysis sis of 2,4 2,4-be -beta-l ta-link inkage agess between -acetylmuramic -acet ylmuramic acid and -acetyl-'-glucosamine residues in a peptidoglycan and between -acetyl-'-glucosamine residues. This dirupts the integrity of cell wall of bacteria and an d le lead ad to rel relea ease se of ce cell llul ular ar co cont nten ents ts.& .&hem hemic ical al ag agen ents ts can be us used ed fo forr ly lysin sing g ce cell ll membranes, chemical agents acts on the cell wall of bacteria causing it to rupture the cells and release the product out. 'etergents are used in cell lysis buffers they help to solubilize membrane proteins and lipids there by causing the cell to lyse and release its contents outside.
#n this e$perimen Triton 5 is used for cell disruption by chemical method. Triton 5-2!! is a nonionic surfactant that has a hydrophilic polyethylene o$ide chain (on average it has 6.7 ethylene o$ide units) and an aromatic hydrocarbon lipophilic or hydrophobic group. The polar head group of T52!! disrupts the hydrogen bonding in lipid bilayer as it becomes inserted in the lipid bilayer and ultimately demolishing the integrity of the of the lipid membrane, thus disrupting the cell to release the cellular contents.
Mteril Re!uired • • • • • • • • • • • • •
Tris /& a&l Triton 5 ysozyme *cetone 8acterial culture etri dishes 8 medium 9icrofuge tubes ippettes and micro tips #ce bo$ and #ce :ater bath 8S
Procedure
2. . 1. 4. 7. <. 3.
;row the bacterial cells overnight in 8 media Take 2 ml sample from the culture at 'reeze ?uickly on dry ice and leave for 1 min Thaw immediately at 4@&. =orte$ vigorously to mi$ well Aepeat the two previous steps for three more times (4 freeze-thaw-vorte$ cycles in
all) . Spin the tubes for 7 min at ma$imum speed in a microfuge 6. Separate the supernatant (contains soluble protein) from the pellet (contains insoluble protein) by pipetting out the supernatant to a clean tube 2!. To each supernatant, add 2 ml acetone and vorte$. >reeze or leave on ice for 27 min 22. Spin 7 min at maimum speed. Aemove the acetone by pipetting it out, being careful not to disturb the pellet 2. 'issolve the protein in 7!! u of 8S 21. 0stimate the protein concentration using owryBs method (Aefer protocol of protein estimation by owryBs method).
Preprtion o" Solution# L$#i# %u""er
!m9 Tris /&l p/ 3.7, 7! m9 a&l, !." triton 5-2!!, 2 mg+ml lysozyme P&S '"or ( litre) Re*ent
mount to dd'(+)
"inl concentrtion '(+,
a&l
g
213 m9
C&l
!. g
.3 m9
a/4
2.44 g
2! m9
C/4
!.4 g
2. m9
Note# •
Ceep water bath at the given temperature ready before start of the e$periment
•
Ceep ice bo$ and ice ready
•
ysis buffer (with Triton 5 and ysozyme ) lyses the cell membrane and cell wall respectively)
•
8S is normally used to dissolve protein. ther buffers like A#* buffer can also be used. :hen using 8S for dissolving protein, store the protein in 4 degree celcius
•
*cetone makes the protein to precipitate, by reducing the solubility in solution
•
>reeze thaw is done to rupture the bacterial cells completely, in this step sonication can also be done
Prctice -ue#tion#
(. :hat is the purpose of *cetone in this e$perimentD
. 'raw the chemical structure of cell wall.
1. :hat are the natural sources of yoszymeD
4. :hat are the detergents that can be used for cell lysisD
7. /ow does Triton 5 disrupt the cell membrane
E/lution #c0eme S. No 1 2 " 4 %
Description Completion of all steps Observation absorbance reading correlated it! sonication time Completion of calculation and grap! #nser to t!e practice $uestions MC&'(iva
Maximum Marks
Marks Obtained
4 2 1 1 2
)otal Marks
1*
O%#er/tion
;ive your observation with the help of following points. E bserve the pellet obtained at the end of the procedure E &alculate the amount of protein using owryBs method with standards of previous e$periment E lot the graph for the same
Re#ult nd Interprettion (:rite in your own word)
Cell di#ruption %$ c0emicl1en2$mtic met0od3 4cult$ u#e (. 5uideline "or conductin* t0e l%3 &ell disruption by chemical+enzyme method is an individual e$periment done by students in microfuge tubes. 0ach students have to perform the e$periment and estimate the protein concentration based on previous owryBs standard values and they have to make graph for the results. ysis buffer and 8S can be prepared in common for all batches (237 students). The culture should be made ready the previous day with appropriate bacterial sample ( E.coli). The students have to perform owryBs estimation only for their samples, the standard values can be obtained from previous e$periments to determine the concentration. 6. Mteril re!uired "or conductin* t0e prcticl3 (27 students+individual) Per l% #e##ion '8, S. No. Mteril Re!uired 4or (7 #tudent# #tudent#)
2
8 or nutrient 8roth
7!ml
2!! ml
Tris /&l
gms
4 gms
1
a&l
4gms
gms
4
triton 5-2!!
ml
4 ml
7
lysozyme
2mg
mg
<
a&l
gms
gms
3
C&l
4 gm
4 gms
a/4
2 gm
gm
6
C/4
2 gm
gm
2!
9icrofuge stand
4
22
&onical flask
2
2
9icrofuge tube ml
1!
21
2ml pipette
1
<
24
!! ul pipette
2
27
*cetone
27
1!
S. No.
Mteril Re!uired
Per l% #e##ion '8, #tudent#)
4or totl 9 l% #e##ion
2
8 or nutrient 8roth
2!! ml
1!! ml
1!!gm
il
Tris /&l
4gms
! ml
!! gms
nil
1
a&l
gms
gms
17!gms
nil
4
triton 5-2!!
4 ml
4ml
il
A/il%le in #toc:
Need to purc0#e
7
lysozyme
mg
< mg
nil
2! mg
<
a&l
gms
4! gms
17! gms
il
3
C&l
4 gms
4 gms
27!
1!
a/4
gm
gm
2
il
6
C/4
gm
gm
2!
7
2!
9icrofuge stand
4
4
2!
7
22
&onical flask
4
4
2
9icrofuge tube ml
4!!
2!
il
21
2ml pipette
1
<
-
24
!! ul pipette
<
-
27
*cetone
1 ml
! ml
.7
o need
Prctice -ue#tion# (. ;0t i# t0e purpo#e o" Acetone in t0i# e
*cetone discourage the dispersion of protein molecules in the media. Thus, the solubility of proteins can be lowered and precipitation can be induced by lowering the effective dielectric constant of the media. This is commonly achieved by adding a water-soluble solvent such as acetone to an a?ueous solution of protein
6. Dr> t0e c0emicl #tructure o" cell >ll.
8. ;0t re t0e nturl #ource# o" L$o#2$me=
ysozyme is abundant in a number of secretions, such as tears, saliva, human milk, and mucus. #t is also present in cytoplasmic granules of the macrophages and the polymorphonuclear neutrophils (9s). arge amounts of lysozyme can be found in egg white
?. ;0t re t0e C0emicl# nd en2$me# t0t cn %e u#ed "or cell l$#i#=
&hemicalsF Tween !, Tween !, S'S, NP 40 Enzymes: Lysozyme, Pectinase, Hyaluronase, Chitinase
7. Ho> doe# Triton + di#rupt t0e cell mem%rne=
Triton 5-2!! is a nonionic surfactant that has a hydrophilic polyethylene o$ide chain and an aromatic hydrocarbon lipophilic or hydrophobic group. The polar head group of T-52!! disrupts the hydrogen bonding in lipid bilayer as it becomes inserted in the lipid bilayer and ultimately demolishing the integrity of the of the lipid membrane
4eed %c: "orm
Title o" t0e e
@enue 3
4cult$ Inc0r*e3
2. :hether you received the e$pected outcome o
Ges
. *ble to complete the e$periment in the stipulated time
Ges
o
1. :hether you learn the principle and procedure of the e$periment
Ges
o
4. :hether all the e?uiments are in working conditions
Ges
o
*ny other suggestionsF
