The importance of thermostable and alkaline lipases is growing rapidly, since these enzymes are relatively stable as well as are capable of carrying out diversified reactions. The inability of the plant and animal lipases to meet current world demands has led to an increased interest in microbial lipases. Microorganisms represent an excellent source of enzymes owing to their broad biochemical diversity and their susceptibility of genetic manipulation. Lipases have a large variety of applications mainly in the detergent, cosmetic, drug, leather, paper, food industry and in several bioremediation processes. So owing to their vast and varied applications newer microbes are to be screened for production of lipases of desirable properties. Cloning and sequences analysis of the lipase gene will help to understand the structure and activity relationship of the enzyme, which will enable researchers to tailor new lipases for biotechnological applications.

          Screening of lipase producing microorganism from soil, Optimization of process variables, Physico-chemical characterization and purification of the enzyme

          Lipase producing microorganism, identified by MTCC (IMTECH, Chandigarh) as Streptococcus sp. (N1) was isolated from soil using enrichment technique. To the best of our knowledge, to date there is no report available on lipase from genus Streptococcus, and more than hundred species and subspecies of Streptococcus are listed on Taxonomy browser at NCBI site (Taxonomy ID: 1301). The isolate Streptococcus sp. (N1) has been given Taxonomic ID: 167506 at the Taxonomy browser at NCBI site. A two parameter complete experimental design for optimization of carbon and nitrogen sources was applied. Other requirements such as soyameal, potatoflour, trace elements and parameters such as pH, temperature, inoculum age etc. were optimized to give a maximum production of 3.54 x 103 lipolytic units per ml of fermentation broth in 48 h.

         The biochemical properties namely pH optima and stability, temperature optima and thermal stability, organic solvent stability, activation and inactivation by different metal ions, detergents and inhibitors were studied. The enzyme activity was maximum between pH 8.0 and 8.4 and was stable between a pH range of 5.0 – 10.5 upto 96 h. 37oC was the optimum temperature for N1 lipase. The majority of tested metal ions had no inhibitory effect on N1 lipase activity except Hg2+. The activity of the lipase was influenced by DEPC, whereas PMSF had little effect. Enzyme stability was affected by PMSF. No effect of EDTA was seen on enyme activity and stability. Streptococcus sp. N1 lipase retained 100% activity on incubation with Protenaise K for 90 min. No effect on stability was observed on incubation with organic solvents. An electrophorically homogenous lipase having specific activity of 295.3 X 103 U/mg and 361 purification fold has been obtained. The mol. Wt. of purified lipase was deduced to be 44.7 kD.

Probe experiments on biotransformations

          Lipase are today enzymes of choice for organic chemists, pharmacists, biophysicists and biochemists. Lipase from Streptococcus sp. N1 has been successfully used for different biotransformation reactions using medicinally important CDRI compounds such as Loganin, Acetyl protected D-ribose and Spermicidal compound.

Molecular cloning, sequencing and analysis of Lipase gene

          A 3489 bp from mesophilic soil isolate Streptococcus sp. (N1) having structural lipase gene have been cloned in vector pBst II KS at EcoRI site (MCS) using genomic library approach. Southern hybridization confirmed the origin of insert from the host. Lipase producing recombinant clone C1N1 conferred a stable lipolytic phenotype. Full length sequencing was performed adopting the strategy of subcloning, end terminal sequencing, designing of primers for over lap and orientation. The 3489 bp fragment is having four significant ORFs. ORF-1 having a size of 1032 bp is giving signatures with alpha/beta hydrolase fold [104-325] and Esterase/lipase/thioesterase family active site [78-168]. ORF-1 is the structural lipase gene and encodes a protein of 343 a.a. with theoretical mass of 38.6 kD, and pI of 9.31. The instability index was computed to be 34.73, which classifies the protein to be stable. Streptococcus sp. N1 lipase contain highly conserved pentapeptide (G-X1-S-X2-G) containing the active site serine (G-H-S-I-G) [151-155]. This has been identified as interfacial lipid recognition site in several lipase sequences from prokaryotic and eukaryotic organisms. It also contain another consensus seuqnece (His-Gly) [84-85] in the N-terminal part of the enzyme. The searches made on databanks showed it to be novel sequence. The predicted amino acid sequence are giving significant homology with cold adapted lipases of antarctic psychrophiles Moraxella sp. TA144 (lip 3) and Psychrobacter immobilis B-10 (lip 1). ORF-2 of 642 bp is giving signatures with GDSL lipolytic enzyme [34-116] and ORF-3 of 729 bp with ABC transporter family [45-232]. The nucleotide sequence has been assigned Genbank Accession No. AF 395190.