1. Indian Institute of Integrative Medicine Canal Road, Jammu Tawi - 180,001, India Tel.: +91-191-2546368; Fax No. 91-191-2543829 NUCLEOTIDE SEQUENCE OF 1446 BP INSERT CARRYING BEST GENE IN pUC19 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 OVER EXPRESSION OF RRLBB1 ESTERASE GENE PET BLUE2 ESTERASE GENE PET BLUE2 (A)(PROTEIN STAINING) (B)(ACTIVITY STAINING) 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) E. COLI HOST CELLS CARRYING PET BLUEBEST (D) & ESTBBE (L) PATCHED ON TRIBUTYRIN AGAR PLATE, SHOWING ZONES OF CLEARANCE 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 kD 1 2 3 4 5 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 ~1500 MICROORGANISMS SCREENED FOR ENANTIO-SPECIFIC ESTER HYDROLASES. 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) ENANTIOSELECTIVE MICROBIAL ENZYMES 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 1 2 3 4 AGAR DIFFUSION ASSAY FOR ESTEROLYTIC AND LIPOLYTIC ACTIVITY COLLECTION OF ENVIOURNMENTAL SAMPLES FOR ISOLATION OF MICROORGANISMS FROM NW HIMALAYAS FOR NOVEL ENANTIOSELECTIVE ENZYMES SUBSTRATE PROFILE OF RRL-1 & RRLBB1 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. 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. 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): 113-120.249(1 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-ul- Hassan,Vijeshwar Verma, Amit Nargotra, Surrinder Koul, Ghulam N. Qazi. (2006) J. Biotechnology. 125, 1-10 PROTEIN ENGINEERING OF BACILLUS SUBTILIS ESTERASE BY RATIONAL DESIGN AND DIRECTED EVOLUTION (ERROR PRONE PCR) 1.45 kb insert pGEMT Vector 4.6kb Triacylglycerols p-Nitrophenyl esters Triacylglycerols p-Nitrophenyl esters SUBSTRATE SPECIFICITY OF ABL SUBSTRATE SPECIFICITY OF ABL % Relative activity