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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.81, #1)
Fungal morphology in submerged cultures and its relation to glucose oxidase excretion by recombinant Aspergillus niger by Hesham El-Enshasy; Karsten Hellmuth; Ursula Rinas (pp. 1-11).
The effect of culture conditions such as medium composition and shear stress on the fungal pellet morphology in shake-flask cultures and its relation to glucose oxidase (GOD) excretion by recombinant Aspergillus niger NRRL 3 (GOD 3–18) was investigated. It was shown that culture conditions resulting in the formation of smaller fungal pellets with an increased mycelial density result in higher yields of exocellular GOD. The pellets obtained in shake-flask cultures showed distinct layers of mycelial density with only the thin outer layer consisting of a dense mycelial network. The performance of the recombinant strain and the process of pellet formation was also analyzed during batch cultivation in a stirred-tank bioreactor. It was shown that the process of pellet formation occurred in two steps: (1) aggregation of free spores to spore clusters with subsequent germination and formation of small aggregates surrounded by a loose hyphal network, and (2) aggregation of the primary aggregates to the final full-size pellets. The fungal pellets formed during bioreactor cultivation were smaller, did not show large differences in mycelial density, and were more efficient with respect to the production of exocellular GOD. The decreasing pellet size also correlated with an increased mycelial density, indicating an improvement of the transport of nutrients to the inner parts of the pellet.
Keywords: Aspergillus niger ; recombinant strain; glucose oxidase; protein excretion; fungal morphology
Influence of ammonium on the performance of a denitrifying culture under heterotrophic conditions by Francisco Cervantes; Oscar Monroy; Jorge Gómez (pp. 13-21).
The effect of ammonium on a denitrifying reactor of the upflow anaerobic sludge blanket type was studied. At a constant nitrate loading rate (2500 mg NO 3 − -N/[L · d]), using acetate as organic electron donor and at a C/NO 3 − -N ratio of 1.23, an increase in the N2 production rate was observed when the ammonium loading rate was increased (25, 250, and 500 mg NH 4 + -N/[L · d]). Dissimilatory nitrate reduction to ammonium (DNRA) was not observed, and the N2 production efficiency was increased from 84 to 100% or higher. Since NH 4 + in the output was lower than in the input, it was suggested that it was used for nitrate reduction. At constant NH 4 + -N/NO 3 − -N and C/NO 3 − -N ratios of 0.2 and 1.63, respectively, the molecular nitrogen production rate was increased at 300 and 500 mg NH 4 + -N/(L · d), whereas at 200 mg NH 4 + -N/(L · d) DNRA took place probably owing to culture conditions of low reductive power. Molecular nitrogen production was not observed under autotrophic conditions, and the addition of acetate to the culture recovered its high nitrogen removal rate. Experimental results and balances indicated that the consumed ammonium was used as an additional reductive source.
Keywords: Nitrogen removal; denitrification; ammonium oxidation; dissimilatory nitrate reduction
Characterization of invertase entrapped into calcium alginate beads by L. M. O. Arruda; M. Vitolo (pp. 23-33).
A solution of 10 g/L of sodium alginate (Satialgine® types used [Sanofi trademark]: SG800® and S1100® with manuronic/guluronic ratio of 0.5 and 1.2, respectively) containing invertase (0.08 g of protein/L) was dropped into 0.1 M CaCl2 solution buffered at pH 4.0, 7.0, or 8.0. The beads were left to harden in CaCl2 solution for 24 h. The high immobilization yield of 60% occurred with SG800 at pH8.0. The activity of soluble and insoluble invertase was measured against pH (2.8–8.0), sucrose concentration (4.5–45 mM), and temperature (30–60°C). Both forms presented an optimum pH of 4.6. However, the soluble invertase was stable at the overall pH interval studied, whereas insoluble invertase lost 30% of its original activity at pH > 5.0. At temperatures above 40°C, the insoluble form was more stable than the soluble one. The kinetic constants and activation energies (E a ) for free invertase were K M =41.2 mM, V max=0.10 mg of TRS/(min · mL), and E a 28 kJ/mol for entrapped invertase they were (K M ) ap =7.2 mM, (V max) ap =0.060 mg of TRS/(min · mL), and (E a )ap=24 kJ/mol.
Keywords: Invertase; entrapment in calcium alginate
Recent developments in microbial inulinases by Ashok Pandey; Carlos R. Soccol; Ponniah Selvakumar; Vanete T. Soccol; Nadia Krieger; Jose D. Fontana (pp. 35-52).
Microbial inulinases are an important class of industrial enzymes that have gained much attention recently. Inulinases can be produced by a host of microorganisms, including fungi, yeast, and bacteria. Among them, however, Aspergillus sp. (filamentous fungus) and Kluyveromyces sp. (diploid yeast) are apparently the preferred choices for commercial applications. Among various substrates (carbon source) employed for their production, inulin-containing plant materials offer advantages in comparison to pure substrates. Although submerged fermentation has been universally used as the technique of fermentation, attempts are being made to develop solidstate fermentation technology also. Inulinases catalyze the hydrolysis of inulin to d-fructose (fructose syrup), which has gained an important place in human diets today. In addition, inulinases are finding other newer applications. This article reviews more recent developments, especially those made in the past decade, on microbial inulinases—its production using various microorganisms and substrates. It also describes the characteristics of various forms of inulinases produced as well as their applications.
Keywords: Microbial inulinases; production; microorganisms; substrates; properties; applications
Purification and characterization of thermostable d-hydantoinase from Bacillus thermocatenulatus GH-2 by Joo-Ho Park; Geun-Joong Kim; Seung-Goo Lee; Dong-Cheol Lee; Hak-Sung Kim (pp. 53-65).
A thermostable d-hydantoinase was isolated from thermophilic Bacillus thermocatenulatus GH-2 and purified to homogeneity by using immunoaffinity chromatography. The molecular mass of the enzyme was determined to be about 230 kDa, and a value of 56 kDa was obtained as a molecular mass of the subunit on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, implying that oligomeric structure of the enzyme is tetrameric. Isoelectric pH of the enzyme was found to be approx 4.3. The enzyme required Mn2+ for the activity and exhibited its highest activity with phenylhydantoin as a substrate. The optimal pH and temperature for catalytic activity were about 7.5 and 65°C, respectively. The half-life of the enzyme was estimated to be about 45 min at 80°C.
Keywords: Thermostability; d-hydantoinase; Bacillus ; immunoaffinity
Electrophoretic extraction and analysis of DNA from chitosan or poly-l-lysine-coated alginate beads by Douglas Quong; Ronald J. Neufeld (pp. 67-77).
Alginate beads containing entrapped DNA were produced using both external and internal calcium sources, and coated with chitosan or poly-l-lysine membranes. The beads were assayed with DNase nuclease to determine formulation conditions offering the highest level of DNA protection fromnucleic acid hydrolysis, simulating gastrointestinal exposure. A method was developed to extract and assay intracapsular DNA through a modified agarose electrophoresis system. Both external and internally gelled beads were permeable to DNase (Mw=31 kDa), indicated by the absence of DNA after nuclease exposure. At low levels of DNase exposure, coated high guluronic content alginate beads offered a higher level of DNA protection compared with coated beads with low guluronic alginate. No apparent correlation was found with chitosan membrane molecular weight and degree of deacetylation; however, increasing poly-l-lysine molecular weight appeared to increase DNase exclusion from beads. At elevated levels of DNase exposure, DNA hydrolysis was evident within all coated beads with the exception of those coated with the highest molecular weight poly-l-lysine (Mw=197.1 kDa), which provided almost total nuclease protection. Optimal combination then for DNA protection from nucleases is a high guluronic alginate core, coated with high molecular weight poly-l-lysine.
Keywords: DNA; chitosan; poly-l-lysine; alginate; electrophoresis