Applied Biochemistry and Biotechnology (v.149, #1)

The metabolism of polycyclic aromatic hydrocarbons (PAHs) was studied in vivo and in vitro in systems consisting of Rigidoporus lignosus and its laccase, in the presence of so-called “mediator” compounds. The static culture of the native fungal strain was able to metabolize anthracene and 2-methylanthracene, but not 9-nitroanthracene. The addition of redox mediators 2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 1-hydroxybenzotriazole (HBT) or violuric acid (VA) led to a significant increase in the degradation of substrates. The oxidation of PAHs was not significant when purified laccase was used without the addition of mediators. The addition of these compounds increased the oxidation of all substrates by approximately 70–80% after 72 h of incubation. The degradation rate was highest for 2-methylanthracene in the presence of VA.
Keywords: Rigidoporus lignosus ; Laccase; Polycyclic aromatic hydrocarbons; Degradation; Redox potential; Mediators

Spectroscopic Characterization of Thiazole Orange-3 DNA Interaction by J. Ghasemi; Sh. Ahmadi; A. I. Ahmad; S. Ghobadi (9-22).
The interaction of a new derivative of thiazole orange (TO-3) with calf thymus DNA (ctDNA) has been investigated by fluorescence and absorption spectroscopy. When TO-3 binds to ctDNA, absorption bands exhibit significant hypochromicity at low base pair/dye ratio (BP/D ratio), and high BP/D show hyperchromicity with red shift. The spectral changes are attributed to the different species formed between TO-3 and ctDNA in the titration course of the dye molecule with DNA. Multivariate curve resolutions–alternating least squares (MCR–ALS) is applied to the absorption measurements recorded to recover the concentration profiles and the pure spectra of the DNA/TO-3 complexes involved in the process. The binding constant and size of the binding site have been determined spectrophotometrically using the McGhee von Hippel equation. MCR–ALS has been used to reveal the precise concentration profiles of all detectable species formed between ctDNA and TO-3 and their pure spectral profiles.
Keywords: ctDNA; TO-3; Fluorescence; Absorption spectra; MCR–ALS

The potential of a laccase mediator system on the delignification of Eucalyptus globulus kraft pulp was investigated under diverse operating conditions. A series of enzymatic treatments were performed to determine how biodelignification is influenced by pulp consistency, oxygen pressure, reaction time, dose of laccase, and dose of mediator. The mediator used was violuric acid. The results revealed that the extent of biodelignification remains practically constant between 1% and 2.5% of pulp consistency then decreases. However, its effect is less pronounced than the mediator or laccase charge. In fact, an increase in the mediator charge from 1% to 4% leads to an increase in the extent of delignification (fixed laccase charge = 20 IU/g and reaction time 120 min) from 28.4% to 52.2%. On the other hand, at a mediator charge of 4%, the increase of laccase charge from 10 to 40 IU/g has a similar impact on delignification. As for oxygen pressure, the extent of delignification increases with oxygen pressure, from 38.9% to 48.6%, when air and pure oxygen at 4 bar gauge are used. From the standpoint of reaction time, delignification is characterized by a very fast phase followed by a much slower one, leading to a plateau.
Keywords: Pulp consistency; Eucalyptus globulus ; Kraft pulp; Laccase; Oxygen pressure; Violuric acid

Hexavalent Molybdenum Reduction to Molybdenum Blue by S. Marcescens Strain Dr. Y6 by M. Y. Shukor; S. H. M. Habib; M. F. A. Rahman; H. Jirangon; M. P. A. Abdullah; N. A. Shamaan; M. A. Syed (33-43).
A molybdate-reducing bacterium has been locally isolated. The bacterium reduces molybdate or Mo6+ to molybdenum blue (molybdate oxidation states of between 5+ and 6+). Different carbon sources such as acetate, formate, glycerol, citric acid, lactose, fructose, glucose, mannitol, tartarate, maltose, sucrose, and starch were used at an initial concentration of 0.2% (w/v) in low phosphate media to study their effect on the molybdate reduction efficiency of bacterium. All of the carbon sources supported cellular growth, but only sucrose, maltose, glucose, and glycerol (in decreasing order) supported molybdate reduction after 24 h of incubation. Optimum concentration of sucrose for molybdate reduction is 1.0% (w/v) after 24 h of static incubation. Ammonium sulfate, ammonium chloride, valine, OH-proline, glutamic acid, and alanine (in the order of decreasing efficiency) supported molybdate reduction with ammonium sulfate giving the highest amount of molybdenum blue after 24 h of incubation at 0.3% (w/v). The optimum molybdate concentration that supports molybdate reduction is between 15 and 25 mM. Molybdate reduction is optimum at 35 °C. Phosphate at concentrations higher than 5 mM strongly inhibits molybdate reduction. The molybdenum blue produced from cellular reduction exhibits a unique absorption spectrum with a maximum peak at 865 nm and a shoulder at 700 nm. The isolate was tentatively identified as Serratia marcescens Strain Dr.Y6 based on carbon utilization profiles using Biolog GN plates and partial 16s rDNA molecular phylogeny.
Keywords: S. marcescens ; Molybdate-reduction; Molybdenum blue

The growth and the enzymatic production of two microbial fungal associations were studied: Aspergillus niger and Fusarium moniliforme and Trametes versicolor and Aspergillus niger. The synergistic interrelations between the species of the first mixed culture increased the biosynthesis of α-amylase and pectinase. T. versicolor and A. niger proved to be compatible partners in the overproduction of the enzyme laccase, whose synthesis surpassed 8.4 times the enzymatic level in the monoculture, with both of the mixed microbial populations cocultivation facilitating the amplified synthesis of enzymes rather than their growth acceleration. A further proof of the presence of synergism established by the cultures was the enzyme volumetric productivities in both of the mixed microbial cultures, which increased parallel to the rise in the combined biomass synthesis. The competent selection of compatible partners can adjust the desired enzymatic levels and compositions in mixed fungal systems aimed at a number of specified designations. Thus, a very high level of laccase production (97,600 IU/g dry weight) was achieved. The chosen fungal strains produce a variety of different enzymes, but first microbial association produces mainly amylase and pectinase, necessary for their growth, and second association produces mainly laccase and pectinase.
Keywords: Solid substrate fermentation; Laccase; Pectinase; Mixed fungal culture

Purification of β-1,3-1,4-glucanase from the cell-free culture fluid of Bacillus subtilis GN156 by affinity chromatography of epoxy-activated sepharose 6B and ultrafiltration technique resulted in homogeneous J1 and partially purified pJ2 enzymes. The molecular weight and pI of J1 were 25 kDa and 3.5, respectively, while those for J2 were 90 kDa and 3.6, respectively. Both β-1,3-1,4-glucanase J1 and pJ2 had optimum pH values of 6–6.5 and an optimum temperature of 60°C. Both enzymes were not inhibited by Li2+ but were inhibited significantly by Ca2+, Cu2+, Mn2+ and Zn2+. However, J1 was slightly inhibited by Fe2+, while pJ2 was inhibited by Mg2+ as well. They were highly specific to only barley β-glucan. K m and V max values of J1 were 1.53 mg/ml and 8,511 μU/ml.min, respectively, while those for pJ2 were 4.36 mg/ml and 7,397 μU/ml.min, respectively. Degradation of barley β-1, 3-1,4-glucan resulted in four different oligosaccharides with 1,3 linkages triose, tetrose, pentose and a high molecular weight (HMW) with 1,3 linkage estimated from their mobilities. The quantitative degradation by the crude enzyme after of incubation yielded in descending order: triose, pentose and tetrose, while that of J1 in descending order yielded: pentose, triose and tetrose. The pJ2 showed low activity yielding a degradation pattern in descending order: pentose, triose, tetraose and a HMW polysaccharide.
Keywords: β-1; 3-1; 4-Glucanase; Bacillus subtilis ; Degradation patterns; Lichenase

Isolation of New Polyketide Synthase Gene Fragments and a Partial Gene Cluster from East China Sea and Function Analysis of a New Acyltransfrase by Yu-Liang Jiao; Liang-Hua Wang; Xiao-Yi Dong; Yu-Feng Chen; Ying Zong; Yun Gao; Na Ren; Ai-Yun Guo; Xin-Qun Zhang; Bing-Hua Jiao (67-78).
Using the consensus-degenerate hybrid oligonucleotide primer polymerase chain reaction method, 26 new ketoacyl synthase (KS) fragments were isolated from a marine sediment sample in the East China Sea (ECS) and analyzed by construction of a phylogenetic tree. With a digoxigenin-labeled KS gene fragment used as a probe, a partial polyketide synthase (PKS) gene cluster was isolated and identified by hybridization screening of a marine sediment sample metagenome fosmid library constructed for this study. A new acyltransferase (AT) gene was cloned from the PKS gene cluster and heterogeneously expressed as a protein fused to maltose-binding protein (MBP). Ultraviolet spectrophotometry was used to study the binding of the MBP–AT fusion protein and single AT domain to substrates using MBP and bovine serum albumin as control proteins. Binding constants (Ka, per micromolar) were calculated and used to analyze the substrate specificity of the acyltransferase. We concluded that there are many unrevealed new PKS gene clusters in marine sediments in the ECS. The acyltransferase is presumably an acetyltransferase from a new PKS gene cluster.
Keywords: Acetyltransferase; Acyltransferase; Ketoacyl synthase; Polyketide synthase; Substrate specificity

Microorganisms producing lipase were isolated from soil and sewage samples and screened for enantioselective resolution of (R,S)-methyl mandelate to (R)-mandelic acid. A strain designated as GXU56 was obtained and identified as Burkholderia sp. Preparing immobilized GXU56 lipase by simple adsorption on octyl sepharose CL-4B, the optimum temperature was shifted from 40 °C (free lipase) to 50 °C (immobilized lipase), and the optimum pH was shifted from 8.0 (free lipase) to 7.2 (immobilized lipase). The immobilized enzyme displayed excellent stability in the pH range of 5.0–8.0, at the temperatures below 50 °C and in organic solvents compared with free enzyme. Enantioselectivity ratio for (R)-mandelic acid (E) was dramatically improved from 29.2 to more than 300 by applying immobilized lipase in the resolution of (R,S)-methyl mandelate. After five cycles of use of immobilized lipase, conversion and enantiomeric excess of (R)-mandelic acid were 34.5% and 98.5%, respectively, with enantioselectivity ratio for (R)-mandelic acid (E) of 230. Thus, octyl-sepharose-immobilized GXU56 lipase can be used as a bio-resolution reagent for producing (R)-mandelic acid.
Keywords: Burkholderia sp. GXU56; Immobilization; Lipase; (R)-Mandelic acid

A Simple Substrate Feeding Strategy using a pH Control Trigger in Fed-Batch Fermentation by Tiong-Ee Ting; Gregory J. Thoma; Robert R. Beitle Jr.; Ralph K. Davis; Rugkiat Perkins; Khursheed Karim; Hui-Min Liu (89-98).
A simple automated glucose feeding strategy based on pH control was developed to produce high-cell-density fed-batch fermentation. In this strategy, the pH control scheme utilized an acidified concentrated glucose solution to lower the pH. The frequency of glucose addition to the fermentor is determined by the culture’s growth kinetics. To demonstrate the effectiveness of the coupled pH and glucose control strategy in biomass and/or secondary metabolite production, several fed-batch fermentations of indigenous Escherichia coli and recombinant E. coli were carried out. Both strains produced biomass with optical density of greater than 40 at 600 nm. We also tested the glucose control strategy using two types of pH controller: a less sophisticated portable pH controller and a more sophisticated online proportional-integral-derivative (PID) controller. Our control strategy was successfully applied with both controllers, although better control was observed using the PID controller. We have successfully demonstrated that a glucose feeding strategy based on a simple pH control scheme to indirectly control the glucose concentration can be easily achieved and adapted to conventional bioreactors in the absence of online glucose measurement and control.
Keywords: E. coli ; Fed-batch; Glucose regulation; pH control