Indian Institute of Integrative Medicine Indian Institute of Integrative Medicine Canal Road, Jammu Tawi - 180,001, IndiaCanal Road, Jammu Tawi - 180,001, India

Tel.: +91-191-2546368; Fax No. 91-191-2543829Tel.: +91-191-2546368; Fax No. 91-191-2543829

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 OF PCR AMPLIFIED PRODUCT OF 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)

HOMOLOGY MODEL OF ESTERASE HOMOLOGY MODEL OF ESTERASE FROM B. SUBTILIS RRL-BB1FROM B. SUBTILIS RRL-BB1 POSITION POSITION OF SER 190, GLU 304 & HIS 395 OF SER 190, GLU 304 & HIS 395 RESIDUES THE N- AND C-TERMI 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)

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

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

kD 1 2 3 4 5

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

~1500 MICROORGANISMS SCREENED FOR ENANTIO-SPECIFIC~1500 MICROORGANISMS SCREENED FOR ENANTIO-SPECIFIC

ESTER HYDROLASES. 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 ENGINEERING PROTEIN ENGINEERING (Stability & desired enantioselectivity)(Stability & desired enantioselectivity)

ENANTIOSELECTIVE MICROBIAL ENZYMESENANTIOSELECTIVE MICROBIAL ENZYMES

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

1 2 3 4 1 2 3 4

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

COLLECTION OF ENVIOURNMENTAL SAMPLES FOR ISOLATION COLLECTION OF ENVIOURNMENTAL SAMPLES FOR ISOLATION OF MICROORGANISMS FROM NW HIMALAYAS FOR NOVEL OF MICROORGANISMS FROM NW HIMALAYAS FOR NOVEL

ENANTIOSELECTIVE ENZYMESENANTIOSELECTIVE ENZYMES

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

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

PATENTS & PUBLICATIONSPATENTS & 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.

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

1.45 kb1.45 kb insertinsert

pGEMTVector 4.6kb

Triacylglycerols Triacylglycerols p-Nitrophenyl estersp-Nitrophenyl esters

SUBSTRATE SPECIFICITY OF ABLSUBSTRATE SPECIFICITY OF ABL

% R

ela

tive

acti

vity

% R

ela

tive

acti

vity