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Cite this: Anal. Methods, 2012, 4, 2574

www.rsc.org/methods TECHNICAL NOTE

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An improved protocol for rapid extraction of membrane enzymes from Grampositive bacteria

Pooja Attri, Drukshakshi Jodha, Jasbir Singh and Suman Dhanda*

Received 24th December 2011, Accepted 3rd May 2012

DOI: 10.1039/c2ay05931b

A simple and reproducible method for extraction of membrane enzymes fromGram positive bacteria in

active form has been developed. The method employs the incubation of cells with lysozyme and

sonication in the presence of salt and detergent followed by repeated freezing and thawing to disrupt the

lipid–protein interactions. Although developed for Dipeptidylpeptidase-III as a demonstration, this

protocol has been used for the successful extraction of some synthetic substrate hydrolyzing activities

from three different Gram positive bacteria with yield of more than 90% for most studied activities.

This method can be routinely used for a wide range of applications and it can also be used to extract and

solubilize other enzymes/proteins only by manipulating detergent, salt and their concentration. It is

simple, straightforward, reproducible, cost effective and a less time consuming protocol for extracting

and solubilizing membrane enzymes.

Introduction

Membrane associated proteins of microorganisms often present

a challenge as well as opportunity, in the quest for better thera-

peutic and prophylactic interactions against infectious diseases,

as membrane associated proteins are important for pathogenicity

of bacteria. Pediococcus acidilactici is an important microor-

ganism with wide applications in the food industry. It is generally

regarded as safe (GRAS) but its vancomycin resistant strains

may cause blood septicemia.1 Dipeptidylpeptidase-III (DPP-III)

removes dipeptide moieties from amino-terminus of oligopep-

tides.2,3 It has been purified from different species but its func-

tions are not well studied.4 As the DPP-III is thought to be

involved in enkephalin degradation with micromolar affinity3,5 it

has emerged as an important target for developing inhibitors

which could act as analgesics for patients of neuropathic and

cancer pain.6 DPP-III is unexplored in microbes so, in order to

understand the functional significance of DPP-III, studies were

planned for purification and characterization of DPP-III from

Pediococcus acidilactici, a microbe in the dairy industry. More-

over it is easier to develop the knock out in microbes which help

to know the particular function. Although DPP-III is a cytosolic

enzyme in mammals,7 studies in our laboratory revealed that it is

located in membranes in Pediococcus acidilactici. Study of

membrane proteins is one of major difficulty because of prob-

lems encountered in extraction while maintaining solubility and

activity. To date, several methods for extracting membrane

proteins have been described.8–13 All these have their own

Department of Biochemistry, Kurukshetra University, Kurukshetra, India.E-mail: dhanda.suman999@gmail.com; Fax: +91-1744-238277; Tel: +91-1744-238196 ext. 2751

2574 | Anal. Methods, 2012, 4, 2574–2577

inherent advantages and disadvantages and none benefit from

the existence of internationally recognized standards for

universally applicable extraction of membrane proteins.

In the present technical note, a simple, straightforward,

reproducible and less time consuming protocol is reported for

extracting and solubilizing membrane enzymes. As a test, this

protocol has also been successfully used for extraction of

aminopeptidase B, DPP-II, cathepsin C, proline iminopeptidase,

Ala-Ala-bNA, Leu-Ala-bNA, Glu-Ala-bNA, Ile-bNA, Ser-Met-

bNA, Phe-bNA, Trp-bNA, Val-bNA, Tyr-bNA and Ser-bNA

hydrolyzing activities in three different Gram positive lactic

acid bacteria (LAB) viz. Pediococcus acidilactici, Lactobaccillus

plantarum, Lactobacillus brevis.

Experimental

Bacterial strains

All the bacterial strains (Pediococcus acidilactici NCDC 252,

Lactobaccillus plantarum NCDC 020 and Lactobacillus brevis

NCDC 001) were purchased from National Dairy Research

Institute (NDRI), Karnal, India.

Chemicals and reagents

All the bacteria were grown at their respective temperatures in

Man Rogosa Sharpe (MRS) purchased from Himedia chemical

laboratory. All the chemicals used for the experiments were of

the highest possible quality. Lysozyme, sodium phosphate, SDS,

Tween-20, Tween-80 and TritonX-100, ethanol, butanol, CoCl2,

CaCl2, EDTA, NaCl was purchased from Himedia chemical

Laboratory, Bombay. Fast Garnet GBC, Arg-4mbNA from

Sigma Aldrich.

This journal is ª The Royal Society of Chemistry 2012

Fig. 1 Extraction scheme for isolation of membrane enzymes.

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General method

All the bacteria were grown in Man Rogosa Sharpe (MRS)

medium for 36 h and bacterial cells were harvested by centrifu-

gation at 9300g at 4 �C for 10 min and suspended in buffer A

(50 mM sodium phosphate, pH 8.0). Lysozyme (500 000 units

per g wet weight) was added to the cell suspension and stirred for

1 h at 37 �C. Lysozyme treated cells were harvested by centri-

fugation at 9300g at 4 �C for 10 min and resuspended in buffer A.

These cells were subjected to sonication for 1 min at 7 kHz

frequency while keeping in an ice bath. The homogenate was

centrifuged at 16 000g for 60 min. The activity was checked for

both DPP-III and aminopeptidase B in sonicate and supernatant

using synthetic substrates Arg-Arg-4mbNA and Arg-4mbNA at

pH 8.5 and 7.5, respectively, as described by Dhanda et al.4 and

Bogra et al.14All other enzymes were assayed in 50 mMTris–HCl

buffer, pH 7.4 using a similar protocol. One unit of enzyme

activity was defined as the amount of enzyme which releases one

nanomole of 4-methoxy-b-naphthylamine/b-naphthylamine per

min from substrate under assay conditions. Different detergents

(SDS, Tween-20, Tween-80 and TritonX-100) and organic/

inorganic chemicals (ethanol, butanol, CoCl2, CaCl2, EDTA,

NaCl) at 0.5% w/v or v/v concentration were tried for solubili-

zation of DPP-III and aminopeptidase B. Then, pH of sonica-

tion buffer, NaCl concentration and time of sonication were

standardized. It has become evident that both TritonX-100 and

NaCl facilitate the solubilization of the enzymes. Hence, the

following protocol for extracting membrane enzymes was

developed: The cells obtained after lysozyme treatment, were

suspended in 50 mM sodium phosphate buffer, pH 8.0 con-

taining 300 mM NaCl and 1% TritonX-100 (buffer B), and

subjected to sonication for 1 min at 7 kHz frequency while

keeping in ice bath. This homogenate was centrifuged at

16,000g for 60 min to get the supernatant (S1). The sonicate

(pellet of the above step) was washed and resuspended in buffer

B and immediately transferred to �20 �C for 30 min and then

homogenized at 37 �C for 10 min. This step was repeated twice

and the supernatant (S2) obtained after centrifugation (16,000g

for 30 min) was pooled with S1. This scheme of extraction is

shown in Fig. 1. The same protocol was also used for extracting

DPP-II, cathepsin C, proline iminopeptidase and Ala-Ala-bNA,

Leu-Ala-bNA, Glu-Ala-bNA, Ile-bNA, Ser-Met-bNA, Phe-

bNA, Trp-bNA, Val-bNA, Tyr-bNA and Ser-bNA hydrolyzing

activities from Pediococcus acidilactici, Lactobacillus plantarum

and Lactobacillus brevis.

Results and discussion

This is the first report that reveals the presence of DPP-III and

aminopeptidase B in bacterial membranes and their successful

extraction. The total activity in sonicated pellet was used as

a reference because lysozyme treatment and simple sonication

did not solubilize any of the membrane enzymes. Results of

studies with different detergents and organic/inorganic chemicals

used for extraction are summarized in Table 1. Presence of Tri-

tonX-100 in extraction buffer at 1% was found to be most suit-

able for solubilization of DPP-III and aminopeptidase

B. TritonX-100 was selected for extraction of enzyme as

higher activity was obtained with this detergent. Moreover

This journal is ª The Royal Society of Chemistry 2012

TritonX-100 is very often used for membrane protein solubili-

zation. Decrease in activity above 1% TritonX-100 is presumably

because of substrate dilution kinetics. Amongst studied salts,

NaCl resulted in enzymes solubilization. Then, pH of sonication

buffer, NaCl concentration and time of sonication were stan-

dardized and found to be 8.0, 300 mM and 1 min respectively.

Therefore, NaCl, TritonX-100 were incorporated in sonication

buffer.

This single step resulted in 73% and 65% solubilization of

DPP-III and aminopeptidase B respectively in Supernatant

(S1). Repeated freezing and thawing is known to weaken the

lipid–protein interactions and thereby disrupt membrane

structure, thus it might help in the extraction of membrane

proteins. As a test, the same protocol was also used for

extracting other synthetic substrate hydrolyzing enzymes from

three different LAB. The results are revealed in Fig. 2. This

protocol resulted in more than 90% yield for studied enzymes.

This can be further improved by manipulating salt and deter-

gent concentration. Lactobacillus plantarum has the largest

genome size among all the lactic acid bacteria, and thus

a complex proteolytic system and maximum number of

hydrolytic activities. Lactobacillus brevis has a relatively weak

proteolytic system and thus fewer activities are reported15

whereas Pediococcus acidilactici is poorly studied with respect

to its proteolytic system.

This protocol has also been assessed and compared with

previously described methods for DPP-III and aminopeptidase

B8–13 and the results are shown in Fig. 3 This newly developed

method resulted in even better extraction. Detergent extraction

combined with ultracentrifugation is by far the most commonly

used method for membrane protein isolation16–18 however; this is

a multi step process involving mechanical disruption of cells

followed by lengthy centrifugation prior to solubilization of the

proteins in detergent. The developed protocol resulted in better

solubilization as compared to the above method. The available

protocols using EDTA and CaCl2 did not work for DPP-III and

Anal. Methods, 2012, 4, 2574–2577 | 2575

Table 1 Effect of detergents/chemicals on solubilization of DPP-III and Aminopeptidase B

Detergents/chemicals (0.5% v/v and w/v)

% Activity � SD

DPP-III Aminopeptidase B

Pellets Supernatant Pellets Supernatant

Control 100 � 0.1 Nil 100 � 0.11 NilEthanol 0.87 � 0.1 0.52 � 0.13 1.53 � 0.03 0.61 � 0.05Butanol 0.44 � 0.22 Nil 0.34 � 0.30 0.03 � 0.01CoCl2 14.91 � 0.42 3.5 � 0.93 10.7 � 0.3 12.88 � 0.42CaCl2 12.71 � 0.16 8.3 � 0.13 2.45 � 0.82 4.29 � 0.56NaCl 53.21 � 0.06 39.45 � 0.05 61.93 � 0.27 41.18 � 0.30EDTA 1.31 � 0.32 Nil 6.44 � 0.1 11.65 � 0.43SDS 8.77 � 0.50 0.87 � 0.81 3.98 � 0.62 3.0 � 0.79Tween-20 12.28 � 0.21 40.35 � 0.64 13.19 � 0.18 22.69 � 0.44Tween-80 14.47 � 0.2 30.26 � 0.60 18.0 � 0.1 20.8 � 0.51TritonX-100 (0.5%) 52.84 � 0.02 51.68 � 0.29 58.49 � 0.23 49.61 � 0.42TritonX-100 (1%) 35.26 � 0.33 60.38 � 0.44 44.83 � 0.47 63.39 � 0.64

Values are a mean of three different experiments � SD.

Fig. 2 Percent solubility of different synthetic substrate hydrolyzing

activities in three bacteria. Total activity in the pellet was taken as

control. Ile-bNA hydrolyzing activity (A); aminopeptidase B (B);

Ala3Ala3bNA hydrolyzing activity (C); Leu-Ala-bNA hydrolyzing

activity (D); DPP-III (E); Val-bNA hydrolyzing activity (F); cathepsin C

(G); Glu-Ala-bNA (H); Ser-Met-bNA (I); Phe-bNA (J); Trp-bNA (K);

Tyr-bNA (L); and Ser-bNA hydrolyzing activities (M); DPP-II (N);

proline-iminopeptidase (O).

Fig. 3 Percent solubility of DPP-III and aminopeptidase B using

different extraction protocols. Protocol explained in this paper (A);

Cousminer et al.8 (B); Zia et al.9 (C); Progulske et al.10 (D); Chapalma-

dugu et al.11 (E); Belur et al.12 (F); Salva et al.13 (G).

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aminopeptidase-B, probably because both are metalloenzymes

and CaCl2 is their inhibitor.5

Conclusion

The present protocol is simple and straightforward for the

extraction of membrane enzyme(s). The application of method

has been studied for several hydrolytic enzymes from different

Gram positive bacteria. This method has obvious advantages

over other conventional protocols. First it is cost effective and

requires no sophisticated instruments. Second, sonication in the

presence of salt and detergent followed by repeated freezing and

thawing makes the current protocol very useful. Further, this

protocol eliminates the need for lengthy centrifugation/ultra-

centrifugation. Besides, this method may be used to extract and

solubilize other enzymes/proteins only by manipulating deter-

gent, salt and their concentration.

2576 | Anal. Methods, 2012, 4, 2574–2577

Acknowledgements

DPP-III substrate is a generous gift of Dr Rajvir Dahiya and all

other synthetic substrates used in the present study are generous

gifts from Dr Hari Singh, Professor Emeritus, Department of

Biochemistry, K. U. Kurukshetra, India.

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