Beta-lactamase is an enzyme produced by bacteria that provides resistance to beta-lactam antibiotics like penicillin. It does this by hydrolyzing the beta-lactam ring structure in these antibiotics, rendering them ineffective. The document discusses the classification, mechanism of action, and applications of beta-lactamase. It describes how beta-lactamase is used to inactivate beta-lactam antibiotics in blood cultures and antibiotic assays to prevent false negative results. Protocols are provided for using beta-lactamase in blood cultures and aminoglycoside antibiotic assays.

1. 1
BETA LACTAMASE
GUIDEBETA LACTAMASE
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2. 1
WHAT IS BETA-LACTAMASE?
Beta-lactamase enzymes can be classified under two classifica�on schemes: the molecular
classifica�on and the func�onal classifica�on. The molecular classifica�on, of which is a
more classical approach, is dependent upon the primary structure of the beta-lactamase
enzyme, effec�vely dividing beta-lactamase into class A, B, C, and D enzymes . The
func�onal classifica�on scheme relies more on the phenotypic expressions of beta
-lactamase enzyme’s substrate and inhibitor profiles.
The manipula�on of bacteria in biotechnology
in order to extract the an�bio�c-resistant genes
for other uses has yielded enzymes targeted
towards specific substances to be of use in other
research se�ngs. Beta-lactamase, also known
as penicillinase, are one of such enzymes
targeted to break down penicillin. Found in
bacterial plasmid as a form of protec�on against
beta- lactam an�bio�cs, beta-lactamase a�acks
the beta-lactam ring found within penicillin’s
structure, destroying the cyclic amide ring via
hydrolysis. Beta-lactamase enzymes are respon-
sible for the resistance to beta-lactam class
an�bio�cs.
PRODUCT INFORMATION
Specifica�on
Product Number
CAS #:
Chemical
Name:
Appearance:
Solubility:
Storage
Temperature:
Size:
Structure
L-1163
9073-60-3
penicillin amido
-β-lactam hydrolase
Freeze dried
powder
Soluble in Water
+4°C
1 vial
Specifica�on Structure
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3. 2
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Table 1. Ambler Classification Scheme of Beta Lactamases
Bush-Jacoby Group
(2009)
Bush-Jacoby
Medeiros Group
(2005)
Molecular Class
(Subclass)
Dis�nc�ve subsrate(s) CA or
TZB
EDTA
Defining Characteris�cs
Represen�ve
Enzymes
1 1 C Cephalosporins No No
Greater hydrolysis of
cephalosporins than
benzylpenicillin; hydrolyzes
cephamycins
E. coli AmpC,
P99, ACT-1, CMY-
2, FOX-1, MIR-1
1e NI* C Cephalosporins No No
Increased hydrolysis of
ce�azidime and o�en other
oxyimino-β-lactams
GC1, CMY-37
2a 2a A Penicillins Yes No
Greater hydrolysis of
benzylpenicillin than
cephalosporins
PC1
2b 2b A
Penicillins, early
cephalosporins
Yes No
Similar hydrolysis of
benzylpenicillin and
cephalosporins
TEM-1, TEM-2,
SHV-1
2be 2be A
Extended-spectrum
cephalosporins,
monobactams
Yes No
Increased hydrolysis of
oxyimino-β-lactams
(cefotaxime, ce�azidime,
ce�riaxone, cefepime,
aztreonam)
TEM-3, SHV-2,
CTX-M-15, PER-1,
VEB-1
2br 2br A Penicillins No No
Resistance to clavulanic acid,
sulbactam, and tazobactam
TEM-30, SHV-10
2ber NI* A
Extended-spectrum
cephalosporins,
monobactams
No No
Increased hydrolysis of
oxyimino-β-lactams combined
with resistance to clavulanic
acid, sulbactam, and tazobactam
TEM-50
2c 2c A Carbenicillin Yes No
Increased hydrolysis of
carbenicillin
PSE-1, CARB-3
2ce NI* A
Carbenicillin,
cefepime
Yes No
Increased hydrolysis of
carbenicillin, cefepime, and
cefpirome
RTG-4
2d 2d D Cloxacillin Variable No
Increased hydrolysis of
cloxacillin or oxacillin
OXA-1, OXA-10
2de NI* D
Extended-spectrum
cephalosporins
Variable No
Hydrolyzes cloxacillin or oxacillin
and oxyimino-β-lactams
OXA-11, OXA-15
2df NI* D Carbapenems Variable No
Hydrolyzes cloxacillin or oxacillin
and carbapenems
OXA-23, OXA-48
2e 2e A
Extended-spectrum
cephalosporins
Yes No
Hydrolyzes cephalosporins.
Inhibited by clavulanic acid but
not aztreonam
CepA
2f 2f A Carbapenems Variable No
Increased hydrolysis of
carbapenems, oxyimino-β-
lactams, cephamycins
KPC-2, IMI-1,
SME-1
3a 3 B (B1) Carbapenems No Yes
Broad-spectrum hydrolysis
including carbapenems but not
monobactams
IMP-1, VIM-1,
CcrA, IND-1
B (B3)
L1, CAU-1, GOB-
1, FEZ-1
3b 3 B (B2) Carbapenems No Yes
Preferen�al hydrolysis of
carbapenems
CphA, S�-1
NI 4 Unknown
Inhibited By

4. MECHANISM OF β-LACTAM DRUGS
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Beta-lactam an�bio�cs are similar in structure of the terminal amino acid residues of the small
proteins comprising the pep�doglycan matrix, and thus act as antagonis�c drugs by binding to
the ac�ve site. The nature of the binding between beta lactam an�bio�cs and the penicillin-bind-
ing protein ac�ve sites are both irreversible and inhibitory, disrup�ng the forma�on of the
pep�doglycan layer. This inhibi�on causes the bacterial cell to shed its cell wall, forming a
spheroplast that is fragile to its environment. The bacterial cell, suscep�ble to death due to the
lack of protec�on, fails in its a�empt of undergoing binary fission.
Beta-lactam an�bio�cs, such as cephams and penicillin deriva�ves, contain a four-member cyclic
amide ring, which inhibits the pep�doglycan layer synthesis in bacteria. A woven complex of
sugar and protein pieces, these first class bacteriocidal drugs destroy the integrity of the
pep�doglycan layer. This method of bacterial cellular death is especially effec�ve to Gram-
posi�ve bacteria due to the pep�doglycan layer providing the outermost resistance to
encapsulated bacterial innards. In binary fission, the process which bacterial cells undergo for
reproduc�ve means, the bacterial cell furrows to form a cell plate comprising of the cell wall and,
consequently, the pep�doglycan layer as the cell divides into two daughter cells. The bacteria rely
on penicillin-binding proteins to facilitate the synthesis of a new cellular wall in a process called
'transpep�da�on'.

5. PENICILLIN RESISTANCE
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Various strains of bacteria have now developed
a form of resistance to beta-lactam an�bio�cs.
Beta-lactamase hydrolyzes the beta-lactam
ring, which characterizes beta-lactam an�biot-
ics, adding a hydroxyl group to the structure. By
adding the hydroxyl group, the beta-lactam
ring’s structure is destroyed, and the an�bio�c
is rendered useless. The gene expression for
beta-lactamase may be induced by repeated
exposure to beta-lactam an�bio�cs, increasing
the selec�ve pressure for bacteria to obtain the
enzyme.
In order to combat bacterial an�bio�c resistance, beta-lactam an�bio�cs can be administered
with beta-lactamase inhibitors such as augmen�n, which is an an�bio�c comprised of amoxicillin
(found within the beta-lactam an�bio�c family) and clavulanic acid. Clavulanic acid acts by using
its structure, of which is analogous to beta-lactam molecules, as a site for beta-lactamase to bind
to instead of the beta-lactam ring located on the an�bio�c. This allows for beta-lactamase to
hydrolyze the beta-lactam ring in clavulanic acid, keeping the integrity of the beta-lactam
an�bio�c’s structure and allowing beta-lactam an�bio�cs to inhibit pep�doglycan synthesis.
Carbapenems also inhibit class A beta-lactamase enzymes through hydrolysis, and are very
effec�ve against extended spectrum beta-lactamases.
BETA LACTAMASE APPLICATIONS
Beta-lactamase is especially effec-
�ve in body fluids and thus have 2
main applica�ons in clinical labora-
tory se�ngs.
Bacterial infec�ons are diagnosed by preparing a
blood culture. The results of these blood cultures
o�en assist in customizing an�microbrial therapy.
An�bio�cs, however, may alter the results of the
blood cultures, yielding false nega�ve results which
may hinder the crea�on of an effec�ve an�microbrial
therapy treatment. Beta-lactamase can be added into
these blood cultures in order to prevent false
nega�ve responses.
BLOOD CULTURES

6. AMINOGLYCOSIDE
ANTIBIOTIC ASSAY
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Assays are performed on poten�ally nephrotoxic
aminoglycosidic an�bio�cs by tradi�onal means. Beta-lactam
an�bio�cs are commonly prescribed alongside aminoglyco-
side an�bio�cs during treatment; however, beta- lactam an�-
bio�cs complicate assays. To simplify the assay performed,
beta-lactam an�bio�cs can be inac�vated u�lizing beta-
lactamase. Incuba�on of the serum will allow beta-lactamase
enough �me to hydrolyze the beta-lactam an�bio�cs located
within the sample, which can then be tested for complete
an�bio�c inac�va�on.
BLOOD CULTURE
PROTOCOL
Blood cultures are a means of assessing the
contents of blood within a pa�ent, screening
for any possible infec�ons in order to devise
an an�-microbrial treatment. Beta-lactam
an�bio�cs may cause false nega�ve results;
therefore, inac�va�ng beta-lactam
an�bio�cs by using beta-lactamase can be a
solu�on to purifying a blood culture.
Assemble a mixture of one vial of
beta-lactamase in 5 ml sterile water.
Add 1 ml of the beta-lactamase solu�on
to 100 ml of the cultured medium.
Incubate the culture medium and
nutrient broth at 37°C for 18-24 hours or
according to preferred protocol. The
dilu�on of the an�bio�cs in the culture
medium ensures effec�ve inac�va�on of
beta-lactam an�bio�cs when coupled
with long incuba�on �me.
AMINOGLYCOSIDE ANTIBIOTIC
ASSAY METHODOLOGY
Assemble a mixture of one vial of
beta-lactamase in 5 ml sterile water.
Add 0.2 ml of the enzyme solu�on to 1.0 ml
serum or cerebrospinal fluid.
Incubate the mixture at room temperature
for 5 minutes.
Use the incubated sample in the assay
method of preference.
OPTIONAL: If the sample is too small to allow
for accurate enzyme dilution, the beta-
lactamase can be incorporated into the assay
medium.
A�er autoclaving and addi�on of test
organism, add 0.2 ml beta-lactamase
solu�on to 10 ml tryp�case soy agar.
Mix thoroughly and pour suspension into
petri dishes or glass tubes.
Storage at 4°C is possible for
up to one week, without significant loss of
beta-lactamase ac�vity.
Beta-lactamase is a freeze-dried product which contains buffer
salts and zinc. It is soluble in water and is prepared into vials.
Store beta-lactamase vials in 4°C.
STORAGE AND HANDLING

7. 6
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Weigh 10 g of gela�n and dissolve the gela�n into 1000 ml of purified water. Heat to above
45°C in order to dissolve the gela�n fully and cool the mixture to room temperature. If the
procedure will be done over the span of mul�ple days, then make a fresh batch daily. Do not
use mixture from a prior day.
Mix thoroughly and pour suspension into
petri dishes or glass tubes.
Weigh 1.86 g of Ethylenediaminetetraace�c acid (EDTA) into a 200 ml volumetric flask and
dissolve in purified water and bring the solu�on up to the mark. Make a new batch daily
Weigh 1.5 g of Benzylpenicillin into a 800 ml beaker and dissolve in 200 ml of purified water.
Add 350 ml of gela�n solu�on and 200 ml of EDTA solu�on. Adjust the pH to 6.8-6.9 with 1.0
M NaOH. Transfer to a 1000 ml volumetric flask and bring the solu�on to the mark with
purified water. Make a new batch daily. This solu�on should be sufficient for 25 assays.
Pipe�e 50 ml of 0.1 M standard solu�on into a 500 ml volumetric flask and bring the solu�on
to the mark. Make a new batch daily.
Make the beta-lactamase enzyme solu�on so that there is a 10 mg/ml concentrate solu�on.
Set the auto-�trator up according to the �trator opera�ng instruc�ons.
Flush the �trant lines and bure�e assembly with fresh NaOH solu�on (10 mM).
Pre-incubate the substrate to 25°C in the water bath.
Measure 40 ml of substrate solu�on into the reac�on vessel and �trate to pH 7.0.
Add 0.1 ml of the enzyme solu�on prepared in step 5.
Start the �trator and follow the rate of addi�on of �trant over a 3 minute period. The rate
should be between 1.0 and 3.0 ml/minute; otherwise, adjust the sample volume accordingly.
Calculate the volume of �trant per minute used to maintain the pH at 7.0.
Repeat the above procedure un�l each vial has been assayed once. Assay at least ten vials
per batch.
BETA-LACTAMASE ASSAY

8. REFERENCES
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9. REFERENCES CONTINUED
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San Diego, CA 92121
Your Loyal Companion in ScienceTM
12. Neu HC. Effect of Beta-Lactamase Loca�on in Escherichia Coli on Penicillin Synergy. Appl Microbiol.
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