C

C

L

L

O

O

N

N

I

I

N

N

G

G

A

A

N

N

D

D

P

P

U

U

R

R

I

I

F

F

I

I

C

C

A

A

T

T

I

I

O

O

N

N

O

O

F

F

A

A

T

T

E

E

T

T

R

R

A

A

M

M

E

E

R

R

I

I

C

C

O

O

X

X

I

I

D

D

O

O

R

R

E

E

D

D

U

U

C

C

T

T

A

A

S

S

E

E

F

F

R

R

O

O

M

M

A

A

R

R

T

T

H

H

R

R

O

O

B

B

A

A

C

C

T

T

E

E

R

R

N

N

I

I

C

C

O

O

T

T

I

I

N

N

O

O

V

V

O

O

R

R

A

A

N

N

S

S

p

p

A

A

O

O

1

1

1. „Alexandru Ioan Cuza” Universi ty, Facul ty of Biology, Department of Molecular and Experimental Biology, Iasi , Romania

2. Inst i tute for Biochemistry and Molecular Biology, Center for Biochemistry and Molecular Cel l Research, Albert Ludwigs Universi ty, Freiburg, Germany

* marius .mihasan@uaic.ro

Marius Mihăşan

1

, Vlad Artenie

1*

, Roderich Brandsch

2

The soil bacterium Arthrobacter nicotinovorans carries the pAO1 catabolic megaplasmid which enables

it to grow on nicotine (Igloi and Brandsch, 2003). Besides the well-characterized pathway for nicotine

degradation (Brandsch, 2006), pAO1 carries a gene cluster of a hypothetical pathway for carbohydrate

utilization. This cluster consists of ORFs of a transcriptional regulator, of a sugar ABC-transporter, and

of several putative dehydrogenases and oxidoreductases. Previously, we established that the pAO1 orf39

gene encodes an aldehyde-dehydrogenase (Mihasan, 2010) and orf40 encodes an sugar dehydrogenase.

Here we focus on further characterization the ORF40 protein and elucidation of its possible role in the

cell. By cloning the gene in the plasmid vector pH6EX3, we were able to express it as a recombinant

His-tagged protein and to easily purify it to homogenity.

GntR

ATP-b Perm Perm

PSBP

ORF38

ORF39

OR

ORF40

ORF42

ORF37

ORF41

1 kB

ABC type transporter system

Isloation and cloning of orf40. The orf40 was isolated by PCR using the primers in table 1 and a

suspension of Arthrobacter nicotinovorans cells as template. Directional cloning (Sambrook J, Fritsch

EF, Maniatis T,1989) of the fragment containing the orf40 in the pH6EX3 vector was achieved by

using HindIII şi XhaI (NEB, U.K) enzymes and Rapid DNA ligation Kit, Roche). Transformed E. coli

XL1 Blue competent cells were selected on plates containing ampiciline (50 microg/ml) and the

recombinant plasmid was checked for the presence of insert by restriction enzyme digestion.

Protein expresion was achived using auto-inducible medium as described elsewhere. (Mihasan,

Ungureanu & Artenie, 2007) Protein purification was done using standard IMAC techniques

(Ausubel M Frederick et al., 2002) on Fast-Flow Ni-chelating Sepharose (Amersham Biosciences,

Sweden). Native molecular weight determination was done using gel permeation chromatography on

an HiLoad 16/60 Superdex 200 column connected to an AKTA Basic FPLC system. Protein

concentration was assayed using the dye-binding method of Bradford (Bradford,1976). SDS-PAGE

was performed using the discontinuous system of Laemlli fallowing the procedure described by

Sambrook, 1989.

I n t r o d u c t i o n

M e t h o d s

P u r i f i c a t i o n a n d n a t i v e M W d e t e r m i n a t i o n

The ORF40 was cloned, expressed and purified to homogeneity. It consists of an novel sugar-oxidoreductase of 47 kDa, which is an tetramer as assayed by gel-permeation chromatography. The brown color of the

purified enzyme preparations indicated that it contains an co-factor. Mass spectroscopy indicated that the enzyme contains 2 atoms of Zn per molecule of monomer, classifying the enzyme as a alcohol dehydrogenase.

In order to fully establish the biotechnological potential of this enzyme, serious efforts are undertaken to develop a suitable method for assaying the enzyme activity and to further characterise the enzyme (heat stability,

pH stability, Km, Kcat,).

C o n c l u s i o n s

1 Ausubel M Frederick, Brent Roger, Kingston E Robert , Moore D David,Seidman J G,Smith A John, Struhl Kevin (2002): Short protocols in molecular biology, , a-5-a ed., vol. , John Wiley & Sons.

2. Bradford, M (1976): A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem, 72(), 248-254,

3. Brandsch Roderich. (2006): Microbiology and biochemistry of nicotine degradation. Appl. Microbiol. Biotechnol, 69(), 493-498,

4. Igloi GL,, Brandsch R, (2003): Sequence of the 165-kilobase catabolic plasmid pAO1 from Arthrobacter nicotinovorans and identification of a pAO1-dependent nicotine uptake system. J Bacteriol, 185(6),

1976-1986,

5. Mihasan M., Ungureanu E., Artenie V. (2007): Optimum parameters for overexpression of recombinat proteins from tac promotors on autoinducible medium. Roumanian Biotechnological letters, 12(6),

3473-3482,

6. Mihasan M, (2010) - In-silico evidence of a pAO1 encoded tagatose-derivate catabolic pathway in Arthrobacter nicotinovorans Biologia sect. Cellular and Molecular Biology, vol. 65 (5). p.760-768

7. Sambrook J, Fritsch EF, Maniatis T (1989): Molecular cloning - a laboratory manual, vol. III, Cold Spring Harbour Laboratory Press.

B i b l i o g r a p h y

Table 1. Oligo-nucleotides used for the isolation and cloning of orf40

Figure 2. Orf40 encoded protein was purified to homogenity.

M –Molecular Weight Marker Sigma Wide Range

1,2,3 – Purified protein 5, 10, 15 microg respectivelly)

M

1

2

3

55

45

kDA

C o f a c t o r

Figure 3. Determination of native molecular mass of ORF40 protein. 1.6 mg

purified ORF40 protein was injected on a HiLoad 16/60 Superdex 200 previosly calibrated using Blue-Dextran,

Feritine (440 kDa), Catalase (232 Kda), Aldolase (158 kDA), Albumine (67 kDa), Ovalbumine (43 kDa),

Ribonuclease (17.4 kDa).

Void Volume

ORF40 - 163 kDa

47 kDa

Shoulder at 405 nm

Figure 4. UV-Vis spectrum of the

purified ORF40 protein

Figure 1. General organisation of the putative carbohydrate

utilisation gene cluster from pAO1

Acknowledgements. This work was supported by CNCSIS-UEFISCSU, project number PN II- RU 337/2010.

www.bio.uaic.ro/cercetare/contracte/PD337-Mihasan/pd337.html

Table 2. Zn content in the analyzed samples