1. 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. `

2. 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..

3. COLLECTION OF ENVIOURNMENTAL SAMPLES FOR ISOLATION OF MICROORGANISMS FROM NW HIMALAYAS FOR NOVEL ENANTIOSELECTIVE ENZYMES

4. ~1500 MICROORGANISMS SCREENED FOR ENANTIO- SPECIFIC 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 RRL-BB1 : (0.06U/ mg wet cell mass)  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)

5. EE 70-99% EE 98% EE 99% EE=98% RRL-1/ RRLBB1 EE 76% Arylalkyl Carbinols  -lactams (intermediat es of Phenyl isoserine) Indoline 2-carboxylate 2- benzyl –1,3-propanediol SUBSTRATE PROFILE OF RRL-1 & RRLBB1

6. SUBSTRATE PROFILE OF Bacillus subtilis RRLBB-1 2-Aryl-1- propanol EE~ 67% EE~ 67% Methyl 2-bromopropanoate

7. AGAR DIFFUSION ASSAY FOR ESTEROLYTIC AND LIPOLYTIC ACTIVITY

9. 0.7% AGAROSE GEL SHOWING INSERTS FROM CLONES pBS1 AND pBS21 FROM CLONES pBS1 AND pBS21 LANE 1. HIND 111 / pUC19 LANE 2. HIND 111 / pBS1 WITH 0.9 KB INSERT LANE 3. HIND 111 /pBS21 WITH 4.5 KB INSERT LANE 4. DNA MARKER PHYSICAL MAP OF POSITIVE CLONE (pBS21) OBTAINED AFTER DELETION OF 1.7 KB WITHIN pBS2.1

10. 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 180116845842362218 BBE

11. Triacylglycerols p-Nitrophenyl esters Triacylglycerols p-Nitrophenyl esters SUBSTRATE SPECIFICITY OF ABL SUBSTRATE SPECIFICITY OF ABL % Relative activity

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

13. 0.7% AGAROSE GEL ELECTROPHORESIS OF PCR AMPLIFIED PRODUCT OF LANE 1, BB1 T-DNA; LANE 2, estBB1; LANE 3, 1Kb LADDER. 10008000700060005000400030002500200015001000750500250 1 2 3 1 2 3 PHYSICAL MAP OF pET BLUEBEST

14. OVER EXPRESSION OF RRLBB1 ESTERASE GENE PET BLUE2 ESTERASE GENE PET BLUE2 (A)(PROTEIN STAINING) (B)(ACTIVITY STAINING) E. COLI HOST CELLS CARRYING PET BLUEBEST (D) & ESTBBE (L) PATCHED ON TRIBUTYRIN AGAR PLATE, SHOWING ZONES OF CLEARANCE

15. TEMP & pH OPTIMA AND TEMP & pH STABILITY OF BBE

16. 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)

17. PROTEIN ENGINEERING OF BACILLUS SUBTILIS ESTERASE BY RATIONAL DESIGN AND DIRECTED EVOLUTION (ERROR PRONE PCR) 1.45 kb insert pGEMT Vector 4.6kb

18. 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-3- PHENYLPROPANOATES WITH EE 96-99% AND ACE-INHIBITOR AND ANTI-DEPRESSANT DRUG (PAROXETINE) INTERMEDIATES WITH MODERATE SELECTIVITY (EE 75-80%).BACILLUS SUBTILIS RRL BB1 PRODUCES ESTERASE (BEST) WHICH CATALYSES RESOLUTION OF ACYL 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) 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.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.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.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%.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 0 A FOR THE 481 ALIGNED AMINO ACID RESIDUES WITH NO HYDROPHOBIC LID WHICH IS THE CHARACTERISTICS OF A TRUE ESTERASE.3D-STRUCTURE OF THE TWO SUPERIMPOSED MODELS OF TWO ESTERASES APPEARED SIMILAR WITH A RELATIVE MEAN STANDARD DIFFERENCE OF 1.2 0 A 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.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.