Transcript Document

ENANTIOSELECTIVE MICROBIAL ENZYMES
Indian Institute of Integrative Medicine
Canal Road, Jammu Tawi - 180,001, India
Tel.: +91-191-2546368; Fax No. 91-191-2543829
OH
~1500 MICROORGANISMS SCREENED FOR ENANTIO-SPECIFIC
Arylalkyl Carbinols
OH
AcO
ESTER HYDROLASES.
H
H
R
10 SHORTLISTED & 4 STUDIED IN DETAIL
H3CO
N
(IDENTIFIED BY 16S RIBOTYPING)
(Enzyme activity)
Arthrobacter sps, RRL-1:
(1.7U/mg wet cell mass)
Trichosporon sps. RRLY-15 :
(0.02U/mg wet cell mass)
Bacillus subtilis
RRL-BB1 :
COLLECTION OF ENVIOURNMENTAL SAMPLES FOR ISOLATION
OF MICROORGANISMS FROM NW HIMALAYAS FOR NOVEL
ENANTIOSELECTIVE ENZYMES
COOMe
AcO
N
H
(0.003mg wet cell mass)
BROAD SUBSTRATE SPECIFICITY
(S)
1 2
1
1 0
0 . 8
8
0 . 6
6
0 . 4
4
0 . 2
2
0
0
3
6
9
12
T i me ( h r )
15
18
PMP
EE 99%
OH
HO
2- benzyl –1,3-propanediol
AcO
H
H
H
S
N
EE 76%
Wet cell mass (g/l)
Enzyme activity
(U/mg wet cell mass)
1. 2
H
O
MODERATE TO HIGH ENANTIOSELECTIVITY
PROTEIN ENGINEERING (Stability & desired enantioselectivity)
H
O
OR
1 6
1 4
-lactams
(intermediat
es of
Phenyl
isoserine)
N
HYPEREXPRESSION & SECRETION
1. 4
PMP
RRL-1/ RRLBB1
EE 99%
Indoline 2-carboxylate
CLONING OF ESTER HYDROLASE GENES.
1. 6
O
EE 99%
(0.06U/ mg wet cell mass)
Bacillus pumilus DBRL-191:
EE 98%
EE 70-99%
N
O
O
PMP
PMP
EE=98%
EE 99%
SUBSTRATE PROFILE OF RRL-1 & RRLBB1
2 1
Gr o wt h and enz yme act ivit y o f A r t hr o b act er sp ecies
t r i but yr i n
1
t r i acet i n
2
3
cel l mass
AGAR DIFFUSION ASSAY FOR ESTEROLYTIC
AND LIPOLYTIC ACTIVITY
4
kD
2
3
4
5
180
116
120
84
100
58
42
36
22
% Relative activity
BBE
1
80
60
40
20
18
0
C2
C4
C6
C8
C12
C16
Triacylglycerols
0.7% AGAROSE GEL SHOWING INSERTS
PHYSICAL MAP OF POSITIVE
FROM CLONES pBS1 AND pBS21
CLONE (pBS21) OBTAINED
LANE 1. HIND 111 / pUC19
AFTER DELETION OF 1.7 KB
LANE 2. HIND 111 / pBS1 WITH 0.9 KB INSERT WITHIN pBS2.1
LANE 3. HIND 111 /pBS21 WITH 4.5 KB INSERT
LANE 4. DNA MARKER
1
2
11% SDS-PAGE OF BBE FROM
DIFFERENT PURIFICATION STEPS.
LANE1: MW MARKERS
LANE2: FRACTION FROM MONO-Q;
LANE3: FRACTION FROM HIC;
LANE4: FRACTION FROM SALT
PRECIPITATION
LANE5: CFE
C18 olive oil
C4
C6
C8
C16
p-Nitrophenyl esters
SUBSTRATE SPECIFICITY OF ABL
NUCLEOTIDE SEQUENCE OF 1446 BP
INSERT CARRYING BEST GENE IN pUC19
3
1000
8000
7000
6000
5000
4000
3000
2500
2000
1500
1000
750
500
250
0.7% AGAROSE GEL
ELECTROPHORESIS
OF PCR AMPLIFIED
PRODUCT OF
LANE 1, BB1 T-DNA;
LANE 2, estBB1;
LANE 3, 1Kb LADDER.
PHYSICAL MAP OF pET
BLUEBEST
E. COLI HOST CELLS CARRYING PET
BLUEBEST (D) & ESTBBE (L) PATCHED
ON TRIBUTYRIN AGAR PLATE, SHOWING
ZONES OF CLEARANCE
OVER EXPRESSION OF RRLBB1
ESTERASE GENE PET BLUE2
(A)
(PROTEIN STAINING)
(B)
(ACTIVITY STAINING)
TEMP & pH OPTIMA AND TEMP & pH STABILITY OF BBE
PATENTS & PUBLICATIONS
•Johri, S., Verma, V., Parshad, R., Koul, S., Taneja, S.C. and Qazi, G.N. (2001) Purification and
characterisation of an ester hydrolase from a strain of Arthrobacter species: Its application in
asymmetrisation of 2-benzyl-1,3–propanediol acylates. BMC. 9: 269-273.
•Maqbool, Q. A., Johri, S., Verma, L., Riyaz-ul-Hassan, S., Verma, V., Koul, S., Taneja, S.C., Parshad,
R. and Qazi, G. N. (2002) Purification and characterization of a novel enantioselective hydrolase from
Bacillus subtilis. Biotechnology & Applied Biochemistry 36(3): 227-234.
pGEMT
Vector
4.6kb
HOMOLOGY MODEL OF ESTERASE
FROM B. SUBTILIS RRL-BB1 POSITION
OF SER 190, GLU 304 & HIS 395
RESIDUES THE N- AND C-TERMI
DOMAINS INDICATED
SUPERIMPOSITION OF MODEL OF
ESTBB1 WITH 3-D STRUCTURE OF
PNB ESTERASE ESTBB1 (RED)
TEMPLATE SEQUENCE (GREEN)
PNB ESTERASE (PDB ID 1C7J.A)
•Vakhlu, J., Johri, S., Verma, V., Koul, S., Parshad, R., Taneja, S.C. and Qazi, G.N. (2005) Purification
and properties of enantioselective ester hydrolase from a strain of Trichosporon species (DSMZ 11829).
Enzyme and Microbial Technology, 37 (3): 330-339.
1.45 kb
insert
PROTEIN ENGINEERING OF BACILLUS
SUBTILIS ESTERASE BY RATIONAL DESIGN
AND DIRECTED EVOLUTION (ERROR PRONE
PCR)
•Rasool, S., Johri, S., Riyaz-ul-Hassan, S., Maqbool, Q., Verma, V., Koul, S., Taneja, S.C. and Qazi, G.
N. (2005) Molecular cloning of enantioselective ester hydrolase from Bacillus pumilus DBRL-191.
FEMS Microbiol. Letters, 249(1): 113-120.
•Kaiser, P., Raina, C., Parshad, R., Sarojini Johri, S., Verma, V., Andrabi, K.I. and Qazi, G.N. (2006) A
novel esterase from Bacillus subtilis (RRL 1789): Purification and characterization of the enzyme.
Protein Expression and Purification, 45: 262-268.
•Molecular cloning of carboxylesterase gene and biochemical characterization of encoded protein from
Bacillus subtilis (RRL BB1) Qurrat-ul-Ain Maqbool, Sarojini Johri, Shafaq Rasool, Syed Riyaz-ulHassan,Vijeshwar Verma , Amit Nargotra, Surrinder Koul, Ghulam N. Qazi. (2006) J. Biotechnology.
125, 1-10