Modification of Lipase by Polyethylene Glycol

Lipase was modified using polyethylene glycol activated by p-nitrochloroformate. The hydrolytic activity of the polyethylene glycol-derivatised lipase (PEG-lipase) was relatively low compared with that of the unmodified enzyme in aqueous system. The esterification activity, however, was enhanced following the modification. The rate of esterification of butyric acid was higher than that of oleic acid. Benzene was the best solvent for the esterification reaction.     

Optimisation of rhamnolipids produced byPseudomonas aeruginosa 181 using Response Surface Modeling

This work investigated the optimisation of the fermented culture medium for maximisation of rhamnolipids production produced byPseudomonas aeruginosa 181 using Response Surface Modeling (RSM). A two full factorial central composite experimental design was used in the design of experiments and in the analysis of results. This procedure limited the number of actual experiments performed while allowing for possible interactions between the parameters (pH, stirring rate, casamino acid concentration and incubation period) on the production of biosurfactants. Production carried out at larger volumes of one litre using Bioreactor under RSM-optimised conditions yielded 3.61 g l^?1 of products after purification by acid precipitation.     

Virulotyping of <Emphasis Type="Italic">Salmonella enterica</Emphasis> subsp. <Emphasis Type="Italic">enterica</Emphasis> isolated from indigenous vegetables and poultry meat in Malaysia using multiplex-PCR

The increased occurrence of Salmonella occurrence in local indigenous vegetables and poultry meat can be a potential health hazards. This study is aimed to detect the prevalence of twenty different virulence factors among Salmonella enterica strains isolated from poultry and local indigenous vegetables in Malaysia via an optimized, rapid and specific multiplex PCR assay. The assay encompasses a total of 19 Salmonella pathogenicity islands genes and a quorum sensing gene (sdiA) in three multiplex reaction sets. A total of 114 Salmonella enterica isolates belonging to 38 different serovars were tested. Each isolate in under this study was found to possess up to 70% of the virulence genes tested and exhibited variable pathogenicity gene patterns. Reproducibility of the multiplex PCR assay was found to be 100% and the detection limit of the optimized multiplex PCR was tested with lowest detectable concentration of DNA 0.8 pg μl⁻¹. This study demonstrated various Salmonella pathogenicity island virulence gene patterns even within the same serovar. This sets of multiplex PCR system provide a fast and reliable typing approach based on Salmonella pathogenicity islands, thus enabling an effective monitoring of emerging pathogenic Salmonella strains as an additional tool in Salmonella surveillance studies.     

Secretory expression and characterization of a highly Ca-activated thermostable L2 lipase

Thermostable lipases are important biocatalysts, showing many interesting properties with industrial applications. Previously, a thermophilic Bacillus sp. strain L2 that produces a thermostable lipase was isolated. In this study, the gene encoding for mature thermostable L2 lipase was cloned into a Pichia pastoris expression vector. Under the control of the methanol-inducible alcohol oxidase (AOX) promoter, the recombinant L2 lipase was secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. After optimization the maximum recombinant lipase activity achieved in shake flasks was 125 U/ml. The recombinant 44.5 kDa L2 lipase was purified 1.8-fold using affinity chromatography with 63.2% yield and a specific activity of 458.1 U/mg. Its activity was maximal at 70 °C and pH 8.0. Lipase activity increased 5-fold in the presence of Ca²⁺. L2 lipase showed a preference for medium to long chain triacylglycerols (C₁₀–C₁₆), corn oil, olive oil, soybean oil, and palm oil. Stabilization at high temperature and alkaline pH as well as its broad substrate specificity offer great potential for application in various industries that require high temperature operations.     

S5 Lipase: an organic solvent tolerant enzyme

In this study, an organic solvent tolerant bacterial strain was isolated. This strain was identified as Pseudomonas sp. strain S5, and was shown to degrade BTEX (Benzene, Toluene, Ethyl-Benzene, and Xylene). Strain S5 generates an organic solvent-tolerant lipase in the late logarithmic phase of growth. Maximum lipase production was exhibited when peptone was utilized as the sole nitrogen source. Addition of any of the selected carbon sources to the medium resulted in a significant reduction of enzyme production. Lower lipase generation was noted when an inorganic nitrogen source was used as the sole nitrogen source. This bacterium hydrolyzed all tested triglycerides and the highest levels of production were observed when olive oil was used as a natural triglyceride. Basal medium containing Tween 60 enhanced lipase production to the most significant degree. The absence of magnesium ions (Mg2+) in the basal medium was also shown to stimulate lipase production. Meanwhile, an alkaline earth metal ion, Na+, was found to stimulate the production of S5 lipase.     

Kinetic and mutagenic evidence for the role of histidine residues in the Lycopersicon esculentum 1-aminocyclopropane-1-carboxylic acid oxidase

The ACCO gene from Lycopersicon esculentum (tomato) has been cloned into the expression vector PT7-7. The highly expressed protein was recovered in the form of inclusion bodies. ACCO is inactivated by diethyl pyrocarbonate (DEPC) with a second-order rate constant of 170 M^–1 min^–1. The pH–inactivation rate data imply the involvement of an amino acid residue with a pK value of 6.05. The difference UV spectrum of the the DEPC-inactivated versus native ACCO showed a single peak at 242 nm indicating the modification of histidine residues. The inactivation was reversed by the addition of hydroxylamine to the DEPC-inactivated ACCO. Substrate/cofactor protection studies indicate that both iron and ACC bind near the active site, which contains histidine residues. Four histidines of ACCO were individually mutated to alanine and glycine. H39A is catalytically active, while H177A, H177G, H211A, H211G, H234A, and H234G are basically inactive. The results indicate that histidine residues 177, 211, and 234 may serve as ligands for the active-site iron of ACCO and/or may play some important structural or catalytic role.     

The kinetics of the active and de-energized transport of O-methyl glucose in Ustilago maydis

The kinetics of the uptake and efflux of 3-O-methyl-glucose in sporidia of Ustilago maydis were measured, both in active cells and in cells whose metabolic activity had been inhibited by azide and iodoacetate.The de-energized transport system proved to be carrier mediated with apparent affinity constants 13 ± 2 mM outside (K₀) and 18 ± 2 mM inside (K₁). The apparent maximum rate constants for the same system were 0.66 ± 0.05 mmol/l cell water per min for uptake (V₊ and 0.53 ± 0.04 mmol/l cell water per min for efflux (V_-. For the active system K₀ = 0.08 ± 0.01, K₁ > 40, V₊ = 9.7 ± 0.5 and V₋ 1.1 ± 0.9 (in equivalent units). These results are discussed in the context of the carrier mechanism as proposed by Regen and Morgan (Regen, D.M. and Morgan, H.E. (1964) Biochim. Biophys. Acta 79, 151–166).The antifungal compound carboxin had no effect on de-energized transport but was shown to decrease both K₀ and V₊ in the active system. Phloretin and phlorizin were also found to be without effect on de-energized cells but the former enhanced while the latter inhibited active uptake.     

Uricase immobilized onto nylon tube for use in automated analysis of serum urate

Uricase is immobilized onto glutaraldehyde-activated nylon tube. Immobilized uricase tube is incorporated into a continuous flow automated analyzer for assaying blood urate. The immobilized enzyme shows good storage and operational stability and the assay system compares favorably with an established method.