1 / 1

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

pGEMT Vector 4.6kb. 1.45 kb insert. Arylalkyl Carbinols. EE 98%. EE 70-99%. EE 99%. -lactams (intermediates of Phenyl isoserine). RRL-1/ RRLBB1. EE 99%. Indoline 2-carboxylate. EE 99%. EE 76%. 2- benzyl –1,3-propanediol. EE 99%. EE=98%.

irish
Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. pGEMT Vector 4.6kb 1.45 kb insert Arylalkyl Carbinols EE 98% EE 70-99% EE 99% -lactams (intermediates of Phenyl isoserine) RRL-1/ RRLBB1 EE 99% Indoline 2-carboxylate EE 99% EE 76% 2- benzyl –1,3-propanediol EE 99% EE=98% PHYSICAL MAP OF POSITIVE CLONE (pBS21) OBTAINED AFTER DELETION OF 1.7 KB WITHIN pBS2.1 180 116 84 58 42 36 22 18 0.7% AGAROSE GEL SHOWING INSERTS 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 BBE 1 2 3 % Relative activity 1000 8000 7000 6000 5000 4000 3000 2500 2000 1500 1000 750 500 250 Triacylglycerols p-Nitrophenyl esters 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 SUBSTRATE SPECIFICITY OF ABL 0.7% AGAROSE GEL ELECTROPHORESIS OF PCR AMPLIFIED PRODUCT OF LANE 1, BB1 T-DNA; LANE 2, estBB1; LANE 3, 1Kb LADDER. 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 E. COLI HOST CELLS CARRYING PET BLUEBEST (D) & ESTBBE (L) PATCHED ON TRIBUTYRIN AGAR PLATE, SHOWING ZONES OF CLEARANCE SUPERIMPOSITION OF MODEL OF ESTBB1 WITH 3-D STRUCTURE OF PNB ESTERASE ESTBB1 (RED) TEMPLATE SEQUENCE (GREEN) PNB ESTERASE (PDB ID 1C7J.A) NUCLEOTIDE SEQUENCE OF 1446 BP INSERT CARRYING BEST GENE IN pUC19 PHYSICAL MAP OF pET BLUEBEST OVER EXPRESSION OF RRLBB1 ESTERASE GENE PET BLUE2 (A) (PROTEIN STAINING) (B) (ACTIVITY STAINING) ENANTIOSELECTIVE MICROBIAL ENZYMES Indian Institute of Integrative Medicine Canal Road, Jammu Tawi - 180,001, India Tel.: +91-191-2546368; Fax No. 91-191-2543829 • ~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) • BacillussubtilisRRL-BB1 :(0.06U/mg wet cell mass) • Bacillus pumilusDBRL-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) COLLECTION OF ENVIOURNMENTAL SAMPLES FOR ISOLATION OF MICROORGANISMS FROM NW HIMALAYAS FOR NOVEL ENANTIOSELECTIVE ENZYMES SUBSTRATE PROFILE OF RRL-1 & RRLBB1 AGAR DIFFUSION ASSAY FOR ESTEROLYTIC AND LIPOLYTIC ACTIVITY 1 2 3 4 kD 1 2 3 4 5 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. • 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)

More Related