ABSTRACT
AN ESTERASE FROM A STRAIN OF BACILLUS SUBTILIS RRL BB1 CATALYSES THE KINETIC
RESOLUTION OF SEVERAL RACEMATES INCLUDING DRUG INTERMEDIATES, EXHIBITING
HIGH ENANTIOSELECTIVITY (EE~99%) WITH ACYL DERIVATIVES OF UNSUBSTITUTED AND
SUBSTITUTED 1-(PHENYL)ETHANOLS & 1-(6-METHOXY-2-NAPHTHYL)ETHANOLS. PRESENT
WORK DESCRIBES: (I) CLONING OF GENE (BEST) ENCODING ENANTIOSELECTIVE
ESTERASE (II) PROVIDES THE NUCLEOTIDE SEQUENCE OF THE BEST ORF, AND (III) THE
AMINO ACID SEQUENCE DEDUCED FROM THE NUCLEOTIDE SEQUENCE OF ORF MATCHED
WITH EXPERIMENTALLY DETERMINED N-TERMINAL AMINO ACID SEQUENCE OF THE
NATIVE ENZYME. RECOMBINANT ENZYME WAS 6 FOLD MORE ACTIVE THAN WILD STRAIN.
(1V)HYPEREXPRESSION OF THE ESTERASE GENE ESTBB1 FROM B. SUBTILIS (RRL BB1) WAS
DONE BY PLACING THE GENE UNDER THE CONTROL OF T7 PROMOTER IN PET BLUE2
VECTOR. ~80 FOLD INCREASE IN EXPRESSION WAS OBSERVED IN THE CFE OF CLONE PET
BLUEBEST. 3D STRUCTURE OF THE BBE MODELED USING THE ATOMIC COORDINATES
FROM THE CRYSTAL STRUCTURE OF PNB ESTERASE FROM B. SUBTILIS PROTEIN (PDB ID
1C7J.A) SHOWED A SIMILAR / HYDROLASE FOLD COMMON FOR ALL ESTER HYDROLASES
DESPITE SEQUENCE SIMILARITY OF ONLY 57.5%. THE RESIDUES OF INTEREST IN BBE
WERE SELECTED THEN VIRTUALLY MUTATED AND ALLOWED TO RELAX, IN THE ENZYME
THROUGH ENERGY MINIMIZATION AND MOLECULAR DYNAMICS. SITE DIRECTED
MUTATION GENERATED NUMBER OF MUTANTS OF ESTBBE WITH AMINO ACID
SUBSTITUTIONS LIKE ILE 60 VAL, ASN 87 SER, LEU 145 MET, ASN 173 PRO, PHE 266 LEU, MET
353 VAL, PHE 365 TYR. ENZYME FROM MUTANTS WITH ILE 60 VAL, ASN 87SER AND MET 353
VAL SUBSTITUTIONS DISPLAYED A RELATIVELY GREATER THERMOSTABILITY THAN THE
UNMODIFIED ENZYME. `
INTRODUCTION
IDENTIFICATION
OF
AN
MICROBIAL
ENZYME
POSSESSING THE DESIRED ENANTIOSELECTIVITY IS
IMPOSSIBLE. AN APPROACH WHICH ALLOWS THE
CREATION OF A HIGHLY ENANTIOSELECTIVE CATALYSTS
IS OF GREAT IMPORTANCE. ONE OF THE APPROACHES TO
GET DESIRED ENANTIOSELECTIVITY IS BY SUBJECTING
THE ENZYME GENES TO SITE-SPECIFIC MUTAGENESIS
OR DIRECTED EVOLUTION. FOR THIS PURPOSE,
IDENTIFICATION, CLONING AND HYPER EXPRESSION OF
THE GENE ENCODING THE ENZYME IS THE
PREREQUISITE. IN THIS PRESENTATION WE DESCRIBE
THE CLONING, SEQUENCING & EXPRESSION OF AN
ENANTIOSELECTIVE ESTER HYDROLASE GENE FROM B.
SUBTILIS RRL BB1. PURIFICATION AND SOME
PHYSIOCHEMICAL PROPERTIES OF RECOMBINANT
ENZYME ASS WELL AS THERMOSTABILISATION OF THE
ENZYME BY SITE DIRECTED MUTAGENESIS.
COLLECTION OF ENVIOURNMENTAL SAMPLES
FOR ISOLATION
OF MICROORGANISMS FROM NW HIMALAYAS FOR
NOVEL
ENANTIOSELECTIVE ENZYMES
~1500 MICROORGANISMS SCREENED FOR ENANTIOSPECIFIC ESTER HYDROLASES.
10 SHORTLISTED & 4 STUDIED IN DETAIL
(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
(0.06U/ mg wet cell mass)
RRL-BB1 :
Bacillus pumilus DBRL-191:
(0.003mg wet cell mass)
BROAD SUBSTRATE SPECIFICITY
MODERATE TO HIGH ENANTIOSELECTIVITY
CLONING OF ESTER HYDROLASE GENES.
HYPEREXPRESSION & SECRETION
PROTEIN ENGINEERING (Stability & desired enantioselectivity)
OH
Arylalkyl Carbinols
OH
H
AcO
H
R
H3CO
N
EE 98%
EE 70-99%
O
PMP
-lactams
(intermediat
es of
Phenyl
isoserine)
EE 99%
COOMe
AcO
N
H
H
O
RRL-1/ RRLBB1
EE 99%
Indoline 2-carboxylate
N
O
OR
(S)
H
PMP
EE 99%
OH
HO
H
H
S
N
EE 76%
2- benzyl –1,3-propanediol
AcO
H
N
O
O
PMP
PMP
EE=98%
EE 99%
SUBSTRATE PROFILE OF RRL-1 & RRLBB1
SUBSTRATE PROFILE OF
Bacillus subtilis
RRLBB-1
OH
OH
R1
OCOR
R
MeO
OCOR
R1
MeO
R
OCOR
RRLBB-1
OH
COOH
MeO
OH
Br
OH
COOCH3
COOH
Br
CH3O
COOCH3
2-Aryl-1- propanol
EE~ 67%
Methyl 2-bromopropanoate
AGAR DIFFUSION ASSAY FOR ESTEROLYTIC AND LIPOLYTIC ACTIVITY
1 6
1. 4
1 4
1. 2
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
Wet cell mass (g/l)
Enzyme activity
(U/mg wet cell mass)
1. 6
2 1
Gr owt h a nd e nz y m e a c t i v i t y of A r t hr oba c t e r spe c i e s
t r i but yr i n
t r i acet i n
cel l mass
PHYSICAL MAP OF POSITIVE
0.7% AGAROSE GEL SHOWING INSERTS
CLONE (pBS21) OBTAINED
FROM CLONES pBS1 AND pBS21
AFTER DELETION OF 1.7 KB
LANE 1. HIND 111 / pUC19
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
180
116
84
BBE
58
42
36
22
18
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
120
% Relative activity
100
80
60
40
20
0
C2
C4
C6
C8
Triacylglycerols
C12
C16
C18 olive oil
C4
C6
C8
p-Nitrophenyl esters
SUBSTRATE SPECIFICITY OF ABL
C16
NUCLEOTIDE SEQUENCE OF 1446 BP
INSERT CARRYING BEST GENE IN pUC19
1
2
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
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)
pGEMT
Vector
4.6kb
1.45 kb
insert
PROTEIN ENGINEERING OF BACILLUS SUBTILIS
ESTERASE BY RATIONAL DESIGN AND DIRECTED
EVOLUTION (ERROR PRONE PCR)
CONCLUSION
•
BACILLUS SUBTILIS RRL BB1 PRODUCES ESTERASE (BEST) WHICH CATALYSES RESOLUTION OF ACYL
DERIVATIVES OF UNSUBSTITUTED AND SUBSTITUTED 1-(PHENYL) ETHANOLS AND ETHYL 3-HYDROXY-3PHENYLPROPANOATES WITH EE 96-99% AND ACE-INHIBITOR AND ANTI-DEPRESSANT DRUG (PAROXETINE)
INTERMEDIATES WITH MODERATE SELECTIVITY (EE 75-80%).
•
SCREENING OF THE GENOMIC LIBRARY FOR THE CLONE ENCODING BEST LED TO THE IDENTIFICATION OF
POSITIVE CLONE E. COLI/pBS2.
•
SEQUENCING IDENTIFIED best GENE ENCODING ESTERASE BEING EXPRESSED UNDER ITS NATIVE
PROMOTER.
•
LIGATION OF AMPLIFIED ORF`S (1.4 KB) INTO PET BLUE2 RESULTED IN CONSTRUCTS PETBLUEBEST
CORRESPONDING TO ORF OF B. SUBTILIS CARBOXYL ESTERASE. THE CONSTRUCT WAS USED TO
TRANSFORMATION OF E. COLI BL21(DE3)PLACI HOST CELLS WITH PETBLUEBEST RESULTED IN THE
ACCUMULATION OF ENZYME INTRACELLULARLY WITH ~80 FOLD OVEREXPRESSION .
•
HOMOLOGY MODEL OF BBE DEVELOPED BY USING THE 3D CRYSTAL STRUCTURE OF PNB ESTERASE FROM
B. SUBTILIS PROTEIN (PDB ID 1C7J.A) AS TEMPLATE SHOWED A SIMILAR / HYDROLASE FOLD COMMON
FOR ALL ESTER HYDROLASES DESPITE SEQUENCE SIMILARITY OF ONLY 57.5%.
•
3D-STRUCTURE OF THE TWO SUPERIMPOSED MODELS OF TWO ESTERASES APPEARED SIMILAR WITH A
RELATIVE MEAN STANDARD DIFFERENCE OF 1.2 0A FOR THE 481 ALIGNED AMINO ACID RESIDUES WITH NO
HYDROPHOBIC LID WHICH IS THE CHARACTERISTICS OF A TRUE ESTERASE.
•
CONSIDERING THE SIMILARITIES OF THE TWO ENZYMES AT STRUCTURAL LEVEL, STRATEGY FOR THE
MUTATION OF BBE BY SITE DIRECTED MUTAGENESIS WAS DESIGNED USING SUBSTITUTION OF AMINO
ACIDS AT POSITIONS NUMBERED 60, 87, 145, 173, 266, 353 AND 365. NINE SPECIFIC ESTERASE VARIENTS WERE
PRODUCED MODIFIED BY ONE OR MORE AMINO ACID SUBSTITUTIONS ILE 60 VAL, ASN 87 SER, LEU 145 MET,
ASN 173 PRO, PHE 266 LEU, MET 353 VAL AND PHE 365 TYR WHICH SHOWED VARIABLE DEGREE OF THERMAL
STABILITY IN AQUEOUS MEDIA OVER NATURALLY OCCURRING ESTERASE BBE.