Investigation of Halohydrins Degradation by Whole Cells and Cell-free Extract of Pseudomonas putida DSM 437: A Kinetic Approach

The biodegradation of two halohydrins (1,3-dichloro-2-propanol and 3-chloro-1,2-propanediol) by P. putida DSM 437 was investigated. Intact cells of previously acclimatized P. putida DSM 437 as well as cell-free extracts were used in order to study the degradation kinetics. When whole cells were used, a maximum biodegradation rate of 3-CPD (vmax= 1.28.10–5 mmol mg–1 DCW h–1) was determined, which was more than 4 times higher than that of 1,3-DCP. However, the affinity towards both halohydrins (Km) was practically the same. When using cell-free extract, the apparent vmax and Km values for 1,3-DCP were estimated at 9.61.10–6 mmol mg–1 protein h–1 and 8.00 mM, respectively, while for 3-CPD the corresponding values were 2.42.10–5 mmol mg–1 protein h–1 and 9.07 mM. GC-MS analysis of cell-free extracts samples spiked with 1,3-DCP revealed the presence of 3-CPD and glycerol, intermediates of 1,3-DCP degradation pathway. 3-CPD degradation was strongly inhibited by the presence of epichlorohydrin and to a lesser extent by glycidol, intermediates of dehalogenation pathway. This work is licensed under a Creative Commons Attribution 4.0 International License

Application of Different Processes for the Biodegradation of 1,3-dichloro-2-propanol by the Bacterium Pseudomonas putidaDSM437

1,3-Dichloro-2-propanol (1,3-DCP), is a highly toxic compound used in many industrial processes. Biodegradation of 1,3-DCP, by the bacterial strain Pseudomonas putida DSM 347, was studied applying three different processes. A number of combinations, with respect to glucose and 1,3-DCP concentration were examined during batch process. When the initial concentration of 1,3-DCP was 600 mg L–1 in the presence of 400 mg L–1 glucose, the biodegradation degree and rate were 10.8 % and 0.68 mg L–1 h–1 respectively. 1,3-DCP biodegradation by the resting cells of P. putida DSM 347 was tested at mass concentrations from = 200 to 1 000 mg L–1 using biomass concentration of 5 g dry cell mass L–1. Biodegradation of 1,3-DCP ranged from 84 to 90 %, initial biodegradation rates ranged from r = 2.36 to 10.55 mg L–1 h–1, while dependence of both parameters from the initial concentration of halohydrin was observed. A system of two Continuous Stirred Tank Reactors (CSTRs) in series was developed for the biodegradation of a highly toxic stream of 1,3-DCP (2000 mg L–1). The overall biodegradation degree of the system was 68 %, while biodegradation rates of the first and second bioreactor were r = 2.88 and 5.21 mg L–1 h–1 respectively

Biochemical and catalytic properties of two intracellular β-glucosidases from the fungus Penicillium decumbens active on flavonoid glucosides

In the presence of rutin as sole carbon source, Penicillium decumbens produces two intracellular β-glucosidases named GI and G II, with molecular masses of 56,000 and 460,000Da, respectively. The two proteins have been purified to homogeneity. GI and G II composed of two and four equal sub-units, respectively and displayed optimal activity at pH 7.0 and temperature 65-75°C. Both β-glucosidases were competitively inhibited by glucose and glucono-δ-lactone. GI and GII exhibited broad substrate specificity, since they hydrolyzed a range of (1,3)-, (1,4)- and (1,6)-β-glucosides as well as aryl β-glucosides. Determination of kcat/Km revealed that GII hydrolyzed 3-8 times more efficiently the above-mentioned substrates. The ability of GI and GII to deglycosylate various flavonoid glycosides was also investigated. Both enzymes were active against flavonoids glycosylated at the 7 position but GII hydrolyzed them 5 times more efficiently than G I. Of the flavanols tested, both enzymes were incapable of hydrolyzing quercetrin and kaempferol-3-glucoside. The main difference between GI and GII as far as the hydrolysis of flavanols is concerned, was the ability of GII to hydrolyze the quercetin-3-glucoside. © 2003 Elsevier B.V. All rights reserved

Adsorption of major endoglucanase from Thermoascus aurantiacus on cellulosic substrates

A thermostable endoglucanase (EndoI) was produced by the thermophilic fungus Thermoascus aurantiacus when grown on cellulosic materials under submerged culture (SC) and solid-state fermentation (SSF). In both cultivation techniques a considerable amount of enzyme activity remained adsorbed onto solid particles, and this was taken into consideration when modeling enzyme production. The results were compatible with the assumption that, following its synthesis, an amount of EndoI was bound on substrate and gradually released into the liquid medium. Adsorption of the enzyme on crystalline cellulose was confirmed in vitro by experiments with purified endoglucanase, which was isolated by anion exchange chromatography. The Langmuir isotherm could efficiently describe the adsorption kinetics, and the estimated A max and K ad values compared with those obtained for cellulases bearing a binding domain. EndoI displayed high affinity for crystalline cellulose and low binding capacity, which could be beneficial in textile processing. © 2009 Springer Science+Business Media B.V

Cell bound and extracellular glucose oxidases from Aspergillus niger BTL: Evidence for a secondary glycosylation mechanism

Two glucose oxidase (GOX) isoforms where purified to electrophoretic homogeneity from the mycelium extract (GOXI) and the extracellular medium (GOXII) of Aspergillus niger BTL cultures. Both enzymes were found to be homodimers with nonreduced molecular masses of 148 and 159 kDa and pI values of 3.7 and 3.6 for GOXI and GOXII, respectively. The substrate specificity and the kinetic characteristics of the two GOX forms, as expressed through their apparent K m values on glucose, as well as pH and T activity optima, were almost identical. The only structural difference between the two enzymes was in their degrees of glycosylation, which were determined equal to 14.1 and 20.8% (w/w) of their molecular masses for GOXI and GOXII, respectively. The above difference in the carbohydrate content between the two enzymes seems to influence their pH and thermal stabilities. GOXII proved to be more stable than GOX I at pH values 2.5, 3.0, 8.0, and 9.0. Half-lives of GOXI at pH 3.0 and 8.0 were 8.9 and 17.5 h, respectively, whereas the corresponding values for GOXII were 13.5 and 28.1 h. As far as the thermal stability is concerned, GOXII was also more thermostable than GOXI as judged by the deactivation constants determined at various temperatures. More specifically, the half-lives of GOXI and GOX II, at 45°C, were 12 and 49 h, respectively. These results suggest A. niger BTL probably possesses a secondary glycosylation mechanism that increases the stability of the excreted GOX. © 2007 Springer-Verlag