Optimization of physical parameters for lipase production from Arthrobacter sp. BGCC#490.

The physical parameters for the production of thermostable, alkaline lipase from Arthrobacter sp. BGCC# 490 were optimized using response surface methodology (RSM), employing face centered central composite design (FCCCD). The design was employed by selecting pH, temperature and incubation period as the model factors and to achieve maximum yield, interaction of these factors was studied by RSM. A second-order quadratic model and response surface method showed that the optimum conditions for lipase production (pH 10.0, temperature 40oC and incubation period 48 h) resulted in 1.6-fold increase in lipase production (13.75 EUml-1), as compared to the initial level (8.6 EUml-1) after 48 h of incubation, whereas its value predicted by the quadratic model was 12.8 EUml-1. Lipase showed stability in the pH range 8-10 and temperature range 40-60oC, with maximum activity at pH 9.0 and temperature 50oC. Lipase activity was enhanced in the presence of K+, Ca2+ and Mg2+ ions, but inhibited by Hg2+ ions. The enzyme exhibited high activity in the presence of acetone, isopropanol and ethanol, but was unaffected by methanol. These properties suggest that the lipase may find potential applications in the detergent industry. The present work also demonstrated the feasibility of using experimental design tools to optimize physical parameters for lipase production by an indigenous Arthrobacter sp.

Evaluation of antifouling activity of eight commercially available organic chemicals against the early foulers marine bacteria and Ulva spores.

Environmental impacts caused by tin and copper based commercial antifouling (AF) paints were proved to be detrimental to aquatic ecosystems. Therefore, a search of environmental friendly AF compounds to be used in marine paint to protect the surface of maritime developmental structures from the unwanted biofouling is a burning issue of the present time. Commercially available eight organic chemicals--allyl isothiocyanate, beta-myrecene, cis-3-hexenyl acetate, citral, ethyl heptanoate, eugenol, methyl caproate, and octyl alcohol were evaluated forAF activities using both laboratory and field assays. The test chemicals were found to repel the target motile marine bacteria--Alteromonas marina, Bacillus atrophaeus, Roseobactergallaeciensis and Shewanella oneidensis and motile spores of the green alga, Ulva pertusa. The bacterial and Ulva spore repulsion activities of the test chemicals were measured by chemotaxis and agar diffusion methods respectively interestingly these test chemicals were less toxic to the test fouling species. The toxicity of the test chemicals was measured by using antibiotic assay disks against the bacteria and motility test against Ulva spores. Moreover, in field assay, all test chemicals showed a perfect performance ofAF activity showing no fouling during the experimental period of one year Such results and commercial as well as technical feasibility of the test chemicals firmly showed the possibility of using as alternatives of the existing toxic AF agents.

Rapid Detection of Duplication/Deletion of the PMP22 Gene in Patients with Charcot-Marie-Tooth Disease Type 1A and Hereditary Neuropathy with Liability to Pressure Palsy by Real-time Quantitative PCR using SYBR Green I Dye

Mutations and altered gene dosage of the peripheral myelin protein (PMP22) gene in chromosome 17p11.2-12 are the main causes for hereditary neuropathies, accounting for approximately 70% of all cases. Patients with duplication of the PMP22 develop Charcot-Marie-Tooth disease type 1A (CMT1A) and deletion of one PMP22 allele leads to hereditary neuropathy with liability to pressure palsy (HNPP). Twenty patients with CMT1A, 17 patients with HNPP, and 18 normal family members and 28 normal controls were studied by real-time quantitative PCR using SYBR Green I on the ABI 7700 Sequence Detection System. The copy number of the PMP22 gene was determined by the comparative threshold cycle method and the albumin was used as a reference gene. The PMP22 duplication ratio ranged from 1.45 to 2.06 and the PMP22 deletion ratio ranged from 0.42 to 0.64. The PMP22 ratio in normal controls, including normal family members, ranged from 0.85 to 1.26. No overlap was found between patients with CMT1A or patients with HNPP and normal controls. This method is fast, highly sensitive, specific, and reproducible in detecting PMP22 duplication and deletion in CMT1A and HNPP patients, respectively.

Chemical modification studies of Rhizomucor miehei protease: evidence for the role of basic amino acids in enzyme catalysis.

The effect of chemical modification on milk clotting and proteolytic activities of aspartyl protease obtained from Rhizomucor miehei NRRL 3500 was examined in the absence and the presence of its specific inhibitor pepstatin A. The effect on the ratio of milk clotting activity (MC) to proteolytic activity (PA), an index of the quality of milk clotting proteases was also determined. Modification of the enzyme with trinitrobenzenesulfonic acid, diethylpyrocarbonate and phenylglyoxal produced an increase in the ratio of MC/PA, while modification with 2- hydroxy-5-nitrobenzyl bromide did not affect the ratio. Modification with N-acetylimidazole resulted in a marginal increase in MC/PA ratio. Protection using pepstatin A during modification with phenylglyoxal, N-acetylimidazole and 2-hydroxy-5-nitrobenzyl bromide, protected both MC and PA. In the case of modification by diethylpyrocarbonate, pepstatin A protected only MC. Pepstatin A did not protect both the activities on the modification of the enzyme by trinitrobenzene sulfonic acid. These observations indicate the presence of arginine, tyrosine and tryptophan at the catalytic site of the enzyme, for eliciting MC and PA of the enzyme. In general, modification of the positively charged residues increases the MC/PA ratio of the enzyme. In addition the modified lysine residues responsible for the inactivation of the enzyme were not involved in the active site of the enzyme. Thus the lysine residues might have a secondary role in enzyme catalysis. Further, histidine at the catalytic site was found to be exclusively involved in milk clotting activity. The enzyme with modified histidine residues were more susceptible to autocatalysis, indicating that histidine residues protect the enzyme against autolysis.