Characterization of biotechnologically relevant extracellular lipase produced by Aspergillus terreus NCFT 4269.10

Abstract Enzyme production by Aspergillus terreus NCFT 4269.10 was studied under liquid static surface and solid-state fermentation using mustard oil cake as a substrate. The maximum lipase biosynthesis was observed after incubation at 30 °C for 96 h. Among the domestic oils tested, the maximum lipase biosynthesis was achieved using palm oil. The crude lipase was purified 2.56-fold to electrophoretic homogeneity, with a yield of 8.44%, and the protein had a molecular weight of 46.3 kDa as determined by SDS-PAGE. Enzyme characterization confirmed that the purified lipase was most active at pH 6.0, temperature of 50 °C, and substrate concentration of 1.5%. The enzyme was thermostable at 60 °C for 1 h, and the optimum enzyme–substrate reaction time was 30 min. Sodium dodecyl sulfate and commercial detergents did not significantly affect lipase activity during 30-min incubation at 30 °C. Among the metal ions tested, the maximum lipase activity was attained in the presence of Zn2+, followed by Mg2+ and Fe2+. Lipase activity was not significantly affected in the presence of ethylenediaminetetraacetic acid, sodium lauryl sulfate and Triton X-100. Phenylmethylsulfonyl fluoride (1 mM) and the reducing, β-mercaptoethanol significantly inhibited lipase activity. The remarkable stability in the presence of detergents, additives, inhibitors and metal ions makes this lipase unique and a potential candidate for significant biotechnological exploitation.

Cloning and characterization of newly isolated lipase from Enterobacter sp. Bn12

A mesophilic Enterobacter sp. Bn12 producing an alkaline thermostable lipase was isolated from soil in Tehran, Iran. The lipase gene (ELBn12) was identified from a genomic library. Sequence analysis of the DNA fragment revealed an open reading frame of 879 bp encoding a lipase with a molecular mass of 31.3 kDa. The deduced amino acid sequence showed 96% identity with a lipase of Enterobacter sp. Ag1 and the identity of their DNA sequences was 88.9%. ELBn12 belongs to the lipase subfamily I.1 and its catalytic triad consists of Ser82, Asp237 and His259. The lipase was expressed in Escherichia coli (BL21) pLysS and partially purified by anion exchange chromatography. The maximum activity of ELBn12 was obtained at temperature of 60 °C and pH 8.0 towards tricaprylin (C8) and its specific activity was around 2900 U/mg. ELBn12 was stable within a broad pH range from 6.0 to 11.0. The enzyme showed high stability in both polar and nonpolar organic solvents at 50% (v/v). The lipase activity was enhanced in the presence of 10 mM of Ca2+, Mg2+ and K+, while heavy metals (Fe3+ and Zn2+) had strong inhibitory effect. ELBn12 showed high activity in the presence of 1% (w/v) nonionic surfactants, however ionic surfactants inhibited the lipolytic activity. ELBn12 characteristics show that it has a potential to be used in various industrial processes.

Screening and isolation of halophilic bacteria producing industrially important enzymes

Halophiles are excellent sources of enzymes that are not only salt stable but also can withstand and carry out reactions efficiently under extreme conditions. The aim of the study was to isolate and study the diversity among halophilic bacteria producing enzymes of industrial value. Screening of halophiles from various saline habitats of India led to isolation of 108 halophilic bacteria producing industrially important hydrolases (amylases, lipases and proteases). Characterization of 21 potential isolates by morphological, biochemical and 16S rRNA gene analysis found them related to Marinobacter, Virgibacillus, Halobacillus, Geomicrobium, Chromohalobacter, Oceanobacillus, Bacillus, Halomonas and Staphylococcus genera. They belonged to moderately halophilic group of bacteria exhibiting salt requirement in the range of 3-20%. There is significant diversity among halophiles from saline habitats of India. Preliminary characterization of crude hydrolases established them to be active and stable under more than one extreme condition of high salt, pH, temperature and presence of organic solvents. It is concluded that these halophilic isolates are not only diverse in phylogeny but also in their enzyme characteristics. Their enzymes may be potentially useful for catalysis under harsh operational conditions encountered in industrial processes. The solvent stability among halophilic enzymes seems a generic novel feature making them potentially useful in non-aqueous enzymology.

Characterization of pathogenic Aeromonas veronii bv. veronii associated with ulcerative syndrome from Chinese longsnout catfish (Leiocassis longirostris Günther)

273 bacterial strains were isolated from 20 Chinese longsnout catfish samples. The biochemical characteristics of all strains conformed to the species description of Aeromonas veronii bv. veronii on the basis of Vitek GNI+ card. Furthermore, 16S rDNA, gyrB and rpoD sequences of the representative strain PY50 were sequenced and showed high similarity with A. veronii bv. veronii in Genbank. Antibiotic-resistance of the representative strain PY50 was assessed by the Kirby-Bauer disk diffusion method, and the results showed it was susceptible and moderately susceptible to 13 and 4 of the 21 antimicrobial agents tested. Extracellular products of strain PY50 contained gelatinase, lecithinase, elastase, most of lipase and lipopolysaccharide. Virulence of strain PY50 and extracellular products to Chinese longsnout catfish were also tested, and LD50 were about 3.47~10(4) CFU per fish and 11.22 ƒÊg per fish in intraperitoneal injection respectively. This is the first report that A. veronii bv. veronii was the pathogenic agent of ulcerative syndrome in Chinese longsnout catfish.

Comparative study on production of a-Amylase from Bacillus licheniformis strains

Alpha amylase (α-1, 4-glucan-glucanhydrolase, EC 3.2.1.1), an extracellular enzyme, degrades α, 1-4 glucosidic linkages of starch and related substrates in an endo-fashion producing oligosaccharides including maltose, glucose and alpha limit dextrin (7). The present study deals with the production and comparative study of production of α-amylase from two strains of Bacillus licheniformis, MTCC 2617 and 2618, by using four different substrates, starch, rice, wheat and ragi powder as carbon source by submerged fermentation. The effect of varying pH and incubation temperature, activator, inhibitor, and substrate concentration was investigated on the activity of α-amylase produced by MTCC strain 2618. The results shows that the production of the α-amylase by the B.licheniformis strain MTCC 2618, using four different substrates were found to be maximum (Starch 3.64 IU/ml/minutes, Rice powder 2.93 IU/ml/minutes, Wheat powder 2.67 IU/ml/minutes, Ragi powder 2.36 IU/ml/minutes) on comparing the enzyme production of two strains. It was also observed that the maximum production was found on the 3rd day (i.e. 72 hr) and characterization of crude enzyme revealed that optimum activity was at pH 7 and 37ºC.

Xylanase production by Aspergillus awamori under solid state fermentation conditions on tomato pomace

In this work, tomato pomace, a waste abundantly available in the Mediterranean and other temperate climates agro-food industries, has been used as raw material for the production of some hydrolytic enzymes, including xylanase, exo-polygalacturonase (exo-PG), cellulase (CMCase) and ¥á-amylase. The principal step of the process is the solid state fermentation (SSF) of this residue by Aspergillus awamori. In several laboratory experiments, maximum xylanase and exo-PG activities were measured during the first days of culture, reaching values around 100 and 80 IU/gds (international units of enzyme activity per gram of dried solid), respectively. For CMCase and ¥á-amylase production remained almost constant along fermentation, with average values of 19 and 21.5 IU/gds, respectively. Experiments carried out in a plate-type bioreactor at lab scale showed a clear positive effect of aeration on xylanase and CMCase, while the opposite was observed for exo-PG and ¥á-amylase. In general, xylanase was the enzyme produced in higher levels, thus the optimum conditions for the determination of the enzyme activity was characterized. The xylanase activity shows an optimum pH of 5 and an optimum temperature of 50 ¨¬C. The enzyme is activated by Mg2+, but strongly inhibited by Hg2+ and Cu2+. The enzymatic activity remains quite high if the extract is preserved in a range of pH from 3 to 10 and a temperature between 30 ¨¬C to 40 ¨¬C.