ABSTRACTABSTRACTAN ESTERASE FROM A STRAIN OF BACILLUS SUBTILIS RRL BB1 CATALYSES THE KINETIC AN ESTERASE FROM A STRAIN OF BACILLUS SUBTILIS RRL BB1 CATALYSES THE KINETIC RESOLUTION OF SEVERAL RACEMATES INCLUDING DRUG INTERMEDIATES, EXHIBITING RESOLUTION OF SEVERAL RACEMATES INCLUDING DRUG INTERMEDIATES, EXHIBITING HIGH ENANTIOSELECTIVITY (EE~99%) WITH ACYL DERIVATIVES OF UNSUBSTITUTED AND HIGH ENANTIOSELECTIVITY (EE~99%) WITH ACYL DERIVATIVES OF UNSUBSTITUTED AND SUBSTITUTED 1-(PHENYL)ETHANOLS & 1-(6-METHOXY-2-NAPHTHYL)ETHANOLS. PRESENT SUBSTITUTED 1-(PHENYL)ETHANOLS & 1-(6-METHOXY-2-NAPHTHYL)ETHANOLS. PRESENT WORK DESCRIBES: (I) CLONING OF GENE (BEST) ENCODING ENANTIOSELECTIVE WORK DESCRIBES: (I) CLONING OF GENE (BEST) ENCODING ENANTIOSELECTIVE ESTERASE (II) PROVIDES THE NUCLEOTIDE SEQUENCE OF THE BEST ORF, AND (III) THE ESTERASE (II) PROVIDES THE NUCLEOTIDE SEQUENCE OF THE BEST ORF, AND (III) THE AMINO ACID SEQUENCE DEDUCED FROM THE NUCLEOTIDE SEQUENCE OF ORF MATCHED AMINO ACID SEQUENCE DEDUCED FROM THE NUCLEOTIDE SEQUENCE OF ORF MATCHED WITH EXPERIMENTALLY DETERMINED N-TERMINAL AMINO ACID SEQUENCE OF THE WITH EXPERIMENTALLY DETERMINED N-TERMINAL AMINO ACID SEQUENCE OF THE NATIVE ENZYME. RECOMBINANT ENZYME WAS 6 FOLD MORE ACTIVE THAN NATIVE ENZYME. RECOMBINANT ENZYME WAS 6 FOLD MORE ACTIVE THAN WILD STRAIN. WILD STRAIN. (1V)HYPEREXPRESSION OF THE ESTERASE GENE ESTBB1 FROM B. SUBTILIS (RRL BB1) WAS (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 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 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 BLUEBEST. 3D STRUCTURE OF THE BBE MODELED USING THE ATOMIC COORDINATES FROM THE CRYSTAL STRUCTURE OF PNB ESTERASE FROM B. SUBTILIS PROTEIN (PDB ID FROM THE CRYSTAL STRUCTURE OF PNB ESTERASE FROM B. SUBTILIS PROTEIN (PDB ID 1C7J.A) SHOWED A SIMILAR 1C7J.A) SHOWED A SIMILAR // HYDROLASE FOLD COMMON FOR ALL ESTER HYDROLASES HYDROLASE FOLD COMMON FOR ALL ESTER HYDROLASES DESPITE SEQUENCE SIMILARITY OF ONLY 57.5%. THE RESIDUES OF INTEREST IN BBE 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 WERE SELECTED THEN VIRTUALLY MUTATED AND ALLOWED TO RELAX, IN THE ENZYME THROUGH ENERGY MINIMIZATION AND MOLECULAR DYNAMICS. SITE DIRECTED THROUGH ENERGY MINIMIZATION AND MOLECULAR DYNAMICS. SITE DIRECTED MUTATION GENERATED NUMBER OF MUTANTS OF ESTBBE WITH AMINO ACID 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 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 353 353 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 VAL SUBSTITUTIONS DISPLAYED A RELATIVELY GREATER THERMOSTABILITY THAN THE UNMODIFIED ENZYME. `UNMODIFIED ENZYME. `

INTRODUCTIONINTRODUCTIONIDENTIFICATION OF AN MICROBIAL ENZYME IDENTIFICATION OF AN MICROBIAL ENZYME POSSESSING THE DESIRED ENANTIOSELECTIVITY IS POSSESSING THE DESIRED ENANTIOSELECTIVITY IS IMPOSSIBLE. AN APPROACH WHICH ALLOWS THE IMPOSSIBLE. AN APPROACH WHICH ALLOWS THE CREATION OF A HIGHLY ENANTIOSELECTIVE CATALYSTS CREATION OF A HIGHLY ENANTIOSELECTIVE CATALYSTS IS OF GREAT IMPORTANCE. ONE OF THE APPROACHES IS OF GREAT IMPORTANCE. ONE OF THE APPROACHES TO GET DESIRED ENANTIOSELECTIVITY IS BY TO GET DESIRED ENANTIOSELECTIVITY IS BY SUBJECTING THE ENZYME GENES TO SITE-SPECIFIC SUBJECTING THE ENZYME GENES TO SITE-SPECIFIC MUTAGENESIS OR DIRECTED EVOLUTION. FOR THIS MUTAGENESIS OR DIRECTED EVOLUTION. FOR THIS PURPOSE, IDENTIFICATION, CLONING AND HYPER PURPOSE, IDENTIFICATION, CLONING AND HYPER EXPRESSION OF THE GENE ENCODING THE ENZYME IS EXPRESSION OF THE GENE ENCODING THE ENZYME IS THE PREREQUISITE.THE PREREQUISITE. IN THIS PRESENTATION WE IN THIS PRESENTATION WE DESCRIBE THE CLONING, SEQUENCING & EXPRESSION DESCRIBE THE CLONING, SEQUENCING & EXPRESSION OF AN ENANTIOSELECTIVE ESTER HYDROLASE GENE OF AN ENANTIOSELECTIVE ESTER HYDROLASE GENE FROM B. SUBTILIS RRL BB1. PURIFICATION AND SOME FROM B. SUBTILIS RRL BB1. PURIFICATION AND SOME PHYSIOCHEMICAL PROPERTIES OF RECOMBINANT PHYSIOCHEMICAL PROPERTIES OF RECOMBINANT ENZYME ASS WELL AS ENZYME ASS WELL AS THERMOSTABILISATION OF THE THERMOSTABILISATION OF THE ENZYME BY SITE DIRECTED MUTAGENESIS. ENZYME BY SITE DIRECTED MUTAGENESIS. ..

COLLECTION OF ENVIOURNMENTAL SAMPLES COLLECTION OF ENVIOURNMENTAL SAMPLES FOR ISOLATION FOR ISOLATION

OF MICROORGANISMS FROM NW HIMALAYAS OF MICROORGANISMS FROM NW HIMALAYAS FOR NOVEL FOR NOVEL

ENANTIOSELECTIVE ENZYMESENANTIOSELECTIVE ENZYMES

~1500 MICROORGANISMS SCREENED FOR ENANTIO-~1500 MICROORGANISMS SCREENED FOR ENANTIO-SPECIFIC ESTER HYDROLASES. SPECIFIC ESTER HYDROLASES.

10 SHORTLISTED & 4 STUDIED IN DETAIL 10 SHORTLISTED & 4 STUDIED IN DETAIL

((IDENTIFIED BY 16S RIBOTYPING)IDENTIFIED BY 16S RIBOTYPING) (Enzyme activity) (Enzyme activity)

Arthrobacter spsArthrobacter sps, , RRL-1:RRL-1: (1.7U/mg wet cell mass) (1.7U/mg wet cell mass)

Trichosporon spsTrichosporon sps. . RRLY-15 : RRLY-15 : (0.02U/mg wet cell mass)(0.02U/mg wet cell mass)

BacillusBacillus subtilissubtilis RRL-BB1 :RRL-BB1 : (0.06U/(0.06U/ mg wet cell mass)mg wet cell mass)

Bacillus pumilusBacillus pumilus DBRL-191: DBRL-191: (0.003(0.003mg wet cell mass)mg wet cell mass)

BROAD SUBSTRATE SPECIFICITYBROAD SUBSTRATE SPECIFICITY

MODERATE TO HIGH ENANTIOSELECTIVITYMODERATE TO HIGH ENANTIOSELECTIVITY

CLONING OF ESTER HYDROLASE GENESCLONING OF ESTER HYDROLASE GENES..

HYPEREXPRESSION & SECRETIONHYPEREXPRESSION & SECRETION

PROTEIN ENGINEERINGPROTEIN ENGINEERING (Stability & desired enantioselectivity)(Stability & desired enantioselectivity)

N

AcO

O PMP

H H

N

AcO

O PMP

H H

O

N

AcO

O PMP

H H

S

(S)OH

OR

N

HO

O PMP

H

OH

R

EE 70-99%EE 70-99%

H3CO

OH

EE 98%EE 98%

NH

COOMe

EE 99%EE 99%

EE 99%EE 99%

EE 99%EE 99%

EE 99%EE 99%EE=98%EE=98%

RRL-1/ RRLBB1RRL-1/ RRLBB1

EE 76%EE 76%

Arylalkyl CarbinolsArylalkyl Carbinols

-lactams-lactams(intermediat(intermediates of es of Phenyl Phenyl isoserine)isoserine)

Indoline 2-carboxylateIndoline 2-carboxylate

2- benzyl –1,3-propanediol2- benzyl –1,3-propanediol

SUBSTRATE PROFILE OF RRL-1 & RRLBB1SUBSTRATE PROFILE OF RRL-1 & RRLBB1

SUBSTRATE PROFILE OF SUBSTRATE PROFILE OF Bacillus subtilisBacillus subtilis

RRLBB-1RRLBB-1

2-Aryl-1- propanol2-Aryl-1- propanol EE~ 67%EE~ 67% Methyl 2-bromopropanoateMethyl 2-bromopropanoate

OH OH

R1

CH3O

OH

Br

COOCH3

OH

COOH

MeO

OCOR

OCOR

MeO

Br

COOCH3

OH

COOH

OCOR

R1MeO

R

R

RRLBB-1

AGAR DIFFUSION ASSAY FOR ESTEROLYTIC AND LIPOLYTIC ACTIVITYAGAR DIFFUSION ASSAY FOR ESTEROLYTIC AND LIPOLYTIC ACTIVITY

0.7% AGAROSE GEL SHOWING INSERTS0.7% AGAROSE GEL SHOWING INSERTS FROM CLONES pBS1 AND pBS21 FROM CLONES pBS1 AND pBS21 LANE 1. HIND 111 / pUC19LANE 1. HIND 111 / pUC19LANE 2. HIND 111 / pBS1 WITH 0.9 KB INSERTLANE 2. HIND 111 / pBS1 WITH 0.9 KB INSERTLANE 3. HIND 111 /pBS21 WITH 4.5 KB INSERTLANE 3. HIND 111 /pBS21 WITH 4.5 KB INSERTLANE 4. DNA MARKERLANE 4. DNA MARKER

PHYSICAL MAP OF POSITIVE PHYSICAL MAP OF POSITIVE CLONE (pBS21) OBTAINED CLONE (pBS21) OBTAINED AFTER DELETION OF 1.7 KB AFTER DELETION OF 1.7 KB WITHIN pBS2.1 WITHIN pBS2.1

11% SDS-PAGE OF BBE FROM 11% SDS-PAGE OF BBE FROM DIFFERENT PURIFICATION STEPS. DIFFERENT PURIFICATION STEPS. LANE1: MW MARKERSLANE1: MW MARKERSLANE2: FRACTION FROM MONO-Q; LANE2: FRACTION FROM MONO-Q; LANE3: FRACTION FROM HIC; LANE3: FRACTION FROM HIC; LANE4: FRACTION FROM SALT LANE4: FRACTION FROM SALT PRECIPITATION PRECIPITATION LANE5: CFELANE5: CFE

180180116116

8484

58584242

3636

2222

1818

BBE

Triacylglycerols Triacylglycerols p-Nitrophenyl estersp-Nitrophenyl esters

SUBSTRATE SPECIFICITY OF ABLSUBSTRATE SPECIFICITY OF ABL

% R

ela

tive

acti

vity

% R

ela

tive

acti

vity

NUCLEOTIDE SEQUENCE OF 1446 BP NUCLEOTIDE SEQUENCE OF 1446 BP INSERT CARRYING BEST GENE IN pUC19 INSERT CARRYING BEST GENE IN pUC19

0.7% AGAROSE GEL 0.7% AGAROSE GEL ELECTROPHORESISELECTROPHORESISOF PCR AMPLIFIED PRODUCT OF OF PCR AMPLIFIED PRODUCT OF LANE 1, BB1 T-DNA;LANE 1, BB1 T-DNA;LANE 2, estBB1;LANE 2, estBB1;LANE 3, 1Kb LADDER.LANE 3, 1Kb LADDER.

10001000800080007000700060006000500050004000400030003000250025002000200015001500

10001000

750750

500500

250250

1 2 31 2 3

PHYSICAL MAP OF pET PHYSICAL MAP OF pET BLUEBESTBLUEBEST

OVER EXPRESSION OF OVER EXPRESSION OF RRLBB1RRLBB1 ESTERASE GENE PET BLUE2 ESTERASE GENE PET BLUE2 (A)(A) (PROTEIN STAINING) (PROTEIN STAINING) (B)(B) (ACTIVITY STAINING)(ACTIVITY STAINING)

E. COLI HOST CELLS CARRYING PET E. COLI HOST CELLS CARRYING PET BLUEBEST (D) & ESTBBE (L) PATCHED BLUEBEST (D) & ESTBBE (L) PATCHED ON TRIBUTYRIN AGAR PLATE, SHOWING ON TRIBUTYRIN AGAR PLATE, SHOWING ZONES OF CLEARANCE ZONES OF CLEARANCE

TEMP & pH OPTIMA AND TEMP & pH STABILITY OF BBETEMP & pH OPTIMA AND TEMP & pH STABILITY OF BBE

HOMOLOGY MODEL OF ESTERASE FROM B. HOMOLOGY MODEL OF ESTERASE FROM B. SUBTILIS RRL-BB1 POSITION OF SER 190, GLU SUBTILIS RRL-BB1 POSITION OF SER 190, GLU 304 & HIS 395 RESIDUES THE N- AND C-TERMI 304 & HIS 395 RESIDUES THE N- AND C-TERMI DOMAINS INDICATED DOMAINS INDICATED

SUPERIMPOSITION OF MODEL OF SUPERIMPOSITION OF MODEL OF ESTBB1 WITH 3-D STRUCTURE OF ESTBB1 WITH 3-D STRUCTURE OF PNB ESTERASE ESTBB1 (RED) PNB ESTERASE ESTBB1 (RED) TEMPLATE SEQUENCE (GREEN) TEMPLATE SEQUENCE (GREEN) PNB ESTERASE (PDB ID 1C7J.A)PNB ESTERASE (PDB ID 1C7J.A)

PROTEIN ENGINEERING OF PROTEIN ENGINEERING OF BACILLUS SUBTILIS BACILLUS SUBTILIS ESTERASE BY RATIONAL DESIGN AND DIRECTED ESTERASE BY RATIONAL DESIGN AND DIRECTED EVOLUTION (ERROR PRONE PCR)EVOLUTION (ERROR PRONE PCR)

1.45 kb1.45 kb insertinsert

pGEMTVector 4.6kb

CONCLUSIONCONCLUSION

• BACILLUS SUBTILISBACILLUS SUBTILIS RRL BB1 PRODUCES ESTERASE (BEST) WHICH CATALYSES RESOLUTION OF ACYL RRL BB1 PRODUCES ESTERASE (BEST) WHICH CATALYSES RESOLUTION OF ACYL DERIVATIVES OF UNSUBSTITUTED AND SUBSTITUTED 1-(PHENYL) ETHANOLS AND ETHYL 3-HYDROXY-3-DERIVATIVES OF UNSUBSTITUTED AND SUBSTITUTED 1-(PHENYL) ETHANOLS AND ETHYL 3-HYDROXY-3-PHENYLPROPANOATES WITH EE 96-99% AND ACE-INHIBITOR AND ANTI-DEPRESSANT DRUG (PAROXETINE) PHENYLPROPANOATES WITH EE 96-99% AND ACE-INHIBITOR AND ANTI-DEPRESSANT DRUG (PAROXETINE) INTERMEDIATES WITH MODERATE SELECTIVITY (EE 75-80%). INTERMEDIATES WITH MODERATE SELECTIVITY (EE 75-80%).

• SCREENING OF THE GENOMIC LIBRARY FOR THE CLONE ENCODING BEST LED TO THE IDENTIFICATION SCREENING OF THE GENOMIC LIBRARY FOR THE CLONE ENCODING BEST LED TO THE IDENTIFICATION OF POSITIVE CLONE OF POSITIVE CLONE E. COLI/E. COLI/pBS2.pBS2.

• SEQUENCING IDENTIFIED SEQUENCING IDENTIFIED best best GENE ENCODING ESTERASE BEING EXPRESSED UNDER ITS NATIVE GENE ENCODING ESTERASE BEING EXPRESSED UNDER ITS NATIVE

PROMOTER. PROMOTER.

• LIGATION OF AMPLIFIED ORF`S (1.4 KB) INTO PET BLUE2 RESULTED IN CONSTRUCTS PETBLUEBEST LIGATION OF AMPLIFIED ORF`S (1.4 KB) INTO PET BLUE2 RESULTED IN CONSTRUCTS PETBLUEBEST CORRESPONDING TO ORF OF CORRESPONDING TO ORF OF B. SUBTILIS B. SUBTILIS CARBOXYL ESTERASE. THE CONSTRUCT WAS USED TO CARBOXYL ESTERASE. THE CONSTRUCT WAS USED TO TRANSFORMATION OF TRANSFORMATION OF E. COLI E. COLI BL21(DE3)PLACI HOST CELLS WITH PETBLUEBEST RESULTED IN THE BL21(DE3)PLACI HOST CELLS WITH PETBLUEBEST RESULTED IN THE ACCUMULATION OF ENZYME INTRACELLULARLY WITH ~80 FOLD OVEREXPRESSION .ACCUMULATION OF ENZYME INTRACELLULARLY WITH ~80 FOLD OVEREXPRESSION .

• HOMOLOGY MODEL OF BBE DEVELOPED BY USING THE 3D CRYSTAL STRUCTURE OF PNB ESTERASE FROM HOMOLOGY MODEL OF BBE DEVELOPED BY USING THE 3D CRYSTAL STRUCTURE OF PNB ESTERASE FROM B. SUBTILISB. SUBTILIS PROTEIN (PDB ID 1C7J.A) AS TEMPLATE SHOWED A SIMILAR PROTEIN (PDB ID 1C7J.A) AS TEMPLATE SHOWED A SIMILAR // HYDROLASE FOLD COMMON HYDROLASE FOLD COMMON FOR ALL ESTER HYDROLASES DESPITE SEQUENCE SIMILARITY OF ONLY 57.5%.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 3D-STRUCTURE OF THE TWO SUPERIMPOSED MODELS OF TWO ESTERASES APPEARED SIMILAR WITH A RELATIVE MEAN STANDARD DIFFERENCE OF 1.2 RELATIVE MEAN STANDARD DIFFERENCE OF 1.2 00A FOR THE 481 ALIGNED AMINO ACID RESIDUES WITH NO A FOR THE 481 ALIGNED AMINO ACID RESIDUES WITH NO HYDROPHOBIC LID WHICH IS THE CHARACTERISTICS OF A TRUE ESTERASE.HYDROPHOBIC LID WHICH IS THE CHARACTERISTICS OF A TRUE ESTERASE.

• CONSIDERING THE SIMILARITIES OF THE TWO ENZYMES AT STRUCTURAL LEVEL, STRATEGY FOR THE 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 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 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 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 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. THERMAL STABILITY IN AQUEOUS MEDIA OVER NATURALLY OCCURRING ESTERASE BBE.