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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.127, #3)
Purification and characterization of Bacillus cereus protease suitable for detergent industry by Monika Prakash; Rathindra Mohan Banik; Claudia Koch-Brandt (pp. 143-155).
An extracellular alkaline protease from an alkalophilic bacterium, Bacillus cereus, was produced in a large amount by the method of extractive fermentation. The protease is thermostable, pH tolerant, and compatible with commercial laundry detergerts. The protease purified and characterized in this study was found to be saperior to endogenous protease already present in commercial laundry detergents. The enzyme was purified to homogeneity by ammonium sulfate precipitation, concentration by ultrafiltration, anionexchange chromatography, and gel filtration. The purified enzyme had a specific activity of 3256.05 U/mg and was found to be amonomeric protein with a molecular mass of 28 and 31 kDa, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and nondenaturing PAGE, respectively. Its maximum protease activity against casein was found to be at pH 10.5 and 50°C. Proteolytic activity of the enzyme was detected by casein and gelatin zymography, which gave a very clear protease activity zone on gel that corresponded to the band obtained on SDS-PAGE and nondenaturing PAGE with a molecular mass of nearly 31 kDa. The purified enzyme was analyzed through matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and identified as a subtilisin class of protease. Specific serine protease inhibitors, suggesting the presence of serine residues at the active site, inhibited the enzme significantly.
Keywords: Alkaline protease; Bacillus cereus ; detergent; purification; characterization
Preparation of trehalase inhibitor validoxylamine A by biocatalyzed hydrolysis of validemycin a with honeybee (Apis cerana fabr.) β-glucosidase by Ya-Ping Xue; Yu-Guo Zheng; Yin-Chu Shen (pp. 157-171).
Validoxylamine A is structurally similar to trehalose and acts a potent competivive inhibitor of trehalase. It has recently been receiving increased attention as a potential material for the development of new insecticides or drugs. In this study, β-glucosidase extracted from honeybees (Apis cerana Fabr.) was used as a catalyst to produce validoxylamine A through enzymatic hydrolysis of validamycin A. β-Glucosidase was separated and purified from honeybees, and its characteristics were examined. The results showed that β-glucosidase was stable across a range of temperatures from 30 to 40°C and across a relatively wide range of pH values from 5.0 to 7.5. Investigation of the biocatalyzed hydrolysis process from validamycin A to validoxylamine A with β-glucosidase revealed that both the substrate (validamycin A) and the product (validoxylamine A) inhibited β-glucosidase activity. The inhibition constant of the substrate K value was 5.01 mM, and that of the product K ip value was 1.32 mM. This product inhibition was competitive.
Keywords: Enzyme inhibitor; validamycin A; β-glucosidase; hydrolysis; validoxylamine A; biocatalysis
Reactive oxygen species, cell growth, and taxol production of Taxus cuspidata cells immobilized on polyurethane foam by De-Ming Yin; Jin-Chuan Wu; Ying-Jin Yuan (pp. 173-185).
Dynamic changes in reactive oxygen species (ROS) of Taxus cuspidata cells immobilized on polyurethane foam were investigated and the relation between ROS content and taxol production was discussed. Immobilization shortened the lag period of cell growth and moderately increased H2O2 and O2 −• contents inside the microenvironment within the first 15 d. After 20 d, excessive production of H2O2 and O2 −• was observed accompanied by marked increases in membrane lipid peroxidation and cell membrane permeability. The taxol content of immobilized cells was fourfold that of suspended cells at d 35. The addition of exogenous H2O2 barely affected malondialdehyde content and cell membrane permeability but led to an obvious accumulation of taxol. It is inferred that the intracellular and extracellular H2O2 inside the microenvironment might be one factor promoting taxol biosynthesis under the immobilization stress.
Keywords: Immobilization; polyurethane foam; reactive oxygen species; taxol; Taxus cuspidata
Uncoupled metabolism stimulated by chemical uncoupler and oxic-settling-anaerobic combined process to reduce excess sludge production by Fen Xia Ye; Ying Li (pp. 187-199).
The effects of three uncoupled metabolic systems (conventional activated sludge process with the addition of 3,3′,4′,5-tetrachlorosalicylanilide [TCS], oxic-settling-anaerobic [OSA] process modified by insertion of a sludge-holding tank in the sludge return line, and TCS and OSA combined process) on reducing excess sludge production were studied. Compared with the control conventional activated sludge process, the most effective system was the combined process, which could reduce excess sludge production by 46.90%. The 180-d operation results confirmed that TCS is an effective chemical uncoupler in reducing the sludge yield but that it had an adverse effect on substrate removal capability, effluent nitrogen concentration, and sludge settleability. The OSA process decreased excess sludge production by only 26% but had less adverse effect on effluent quality and could improve sludge settleability. The effluent total phosphorous concentration of the three systems was slightly lower than of the control unit. Microbial populations were monitored by both microscopic and molecular biologic analysis method (polymerase chain reaction [PCR]-denaturing gradient gel electrophoresis [DGGE]). The presence of TCS caused metazoans to disappear and decreased the number and activity of protozoa. PCR amplification of 16S rRNA and sequent DGGE analysis found a shift in the diversity of the predominant species. The results imply that OSA combined with the chemical uncoupler process may effectively reduce excess sludge yield and not affect process performance significantly.
Keywords: Activated sludge process; excess sludge reduction; oxicsettling-an aerobic process; 3,3′,4′,5-tetrachlorosalicylanilide; uncoupler
A new thermostable peroxidase from garlic Allium sativum by Saida Medjeldi Marzouki; Farid Limam; M. Issam Smaali; Roland Ulber; M. Néjib Marzouki (pp. 201-214).
Analysis of peroxidase activity by native polyacrylamide gel electrophoresis (PAGE) from a garlic bulb (Allium sativum L) extract showed two major activities (designated POX1 and POX2). The POX2 isoenzyme was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and cation-exchange chromatography. The purified enzyme was found to be monomeric with a molecular mass of 36.5 kDa, as determined by sodium dodecyl sulfate-PAGE. The optimum temperature ranged from 25 to 40°C and optimum pH was about 5.0. The apparent K m values for guaiacol and H2O2 were 9.5 and 2 mM, respectively. POX2 appeared highly stable since 50% of its activity was conserved at 50°C for 5h. Moreover POX2 was stable over a pH range of 3.5–11.0. Immobilization of POX2 was achieved by covalent binding of the enzyme to an epoxy-Sepharose matrix. The immobilized enzyme showed great stability toward heat and storage when compared with soluble enzyme. These properties permit the use of this enzyme as a biosensor to detect H2O2 in some food components such as milk or its derivatives.
Keywords: Plant peroxidases; garlic; protein purification; immobilization; kinetic characterization