Applied Biochemistry and Biotechnology (v.166, #4)

Identification of WA-Type Three-Line Hybrid Rice with Real-Time Polymerase Chain Reaction (PCR) Method by Y. Cheng; B. D. Gao; H. Y. Chen; J. J. Mao; A. X. Cao; J. G. Zhu; S. F. Zhu (819-829).
A real-time fluorescent PCR (RTF-PCR) was developed to detect and quantify wild abortive (WA)-type three-line hybrid rice (Oryza sativa L.). The mitochondrial R2-630 WA gene was reported to be closely related to male sterility in plants, and developed as a molecular maker to identify the cytoplasmic male sterility system of hybrid rice. First, we got the DNA sequence of R2-630 WA gene in 17 rice species with traditional PCR. Then, a pair of specific primers (P3, P4) and TaqMan fluorescence probe (P3-14) were designed based on the R2-630 DNA sequence. The following RTF-PCR was performed on the 17 rice species finally. The results indicate that the probes used here are specific for three-line hybrid rice F1 and male sterile lines. We can even identify a single hybrid seed using the probes, which confirmed that the probes can be applied to the identification and quantification of the WA-type three-line hybrid rice. In addition, the RFT-PCR system can be optimized when the annealing temperature is 60 °C and the Mg2+ concentration is 3.5 mmol/L.
Keywords: Real-time fluorescent PCR; Hybrid rice; Molecular identification; Male sterility; mtDNA; R2-630 WA

Investigation on Ultrasound-Assisted Extraction and Separation of Enduracidin from Streptomyces sp. NJWGY3665 by Yonghong Hu; Wenge Yang; Wenqian Wan; Fei Shen; Ziyu Lei; Dandan Wang (830-838).
Ultrasound-assisted extraction of enduracidin from Streptomyces sp. NJWGY3665 was studied. The effects of various factors on the yield of target components were investigated. The results showed that the extraction by ultrasound-assisted extraction is four times faster than those by conventional solvent extraction. The results also indicated that fast extraction rate was obtained in the first 30 min, and the maximum yield was obtained at the power of 150 W. The effects of other factors such as different solvents, solvent concentration, solvent to solid ratio, and extraction batches on the field were also discussed. The optimum conditions were found at solvent of methanol, time of 30 min, power of 150 W, pH of 7.0, solvent to solid ratio of 50 ml/g, solvent concentration of 70% and extraction batches of four times.
Keywords: Enduracidin; Streptomyces ; Ultrasound-assisted extraction

Process Evaluation of Enzymatic Hydrolysis with Filtrate Recycle for the Production of High Concentration Sugars by Ying Xue; Jannov Rusli; Hou-min Chang; Richard Phillips; Hasan Jameel (839-855).
Process simulation and lab trials were carried out to demonstrate and confirm the efficiency of the concept that recycling hydrolysate at low total solid enzymatic hydrolysis is one of the options to increase the sugar concentration without mixing problems. Higher sugar concentration can reduce the capital cost for fermentation and distillation because of smaller retention volume. Meanwhile, operation cost will also decrease for less operating volume and less energy required for distillation. With the computer simulation, time and efforts can be saved to achieve the steady state of recycling process, which is the scenario for industrial production. This paper, to the best of our knowledge, is the first paper discussing steady-state saccharification with recycling of the filtrate form enzymatic hydrolysis to increase sugar concentration. Recycled enzymes in the filtrate (15–30% of the original enzyme loading) resulted in 5–10% higher carbohydrate conversion compared to the case in which recycled enzymes were denatured. The recycled hydrolysate yielded 10% higher carbohydrate conversion compared to pure sugar simulated hydrolysate at the same enzyme loading, which indicated hydrolysis by-products could boost enzymatic hydrolysis. The high sugar concentration (pure sugar simulated) showed inhibition effect, since about 15% decrease in carbohydrate conversion was observed compared with the case with no sugar added. The overall effect of hydrolysate recycling at WinGEMS simulated steady-state conditions with 5% total solids was increasing the sugar concentration from 35 to 141 g/l, while the carbohydrate conversion was 2% higher for recycling at steady state (87%) compared with no recycling strategy (85%). Ten percent and 15% total solid processes were also evaluated in this study.
Keywords: Steady state; Process simulation; Enzymatic hydrolysis; Stream recycling; Hydrolysis booster

Improvement of Ethanol Yield from Glycerol via Conversion of Pyruvate to Ethanol in Metabolically Engineered Saccharomyces cerevisiae by Kyung Ok Yu; Ju Jung; Ahmad Bazli Ramzi; Seung Wook Kim; Chulhwan Park; Sung Ok Han (856-865).
The conversion of low-priced glycerol to higher value products has been proposed as a way to improve the economic viability of the biofuels industry. In a previous study, the conversion of glycerol to ethanol in a metabolically engineered strain of Saccharomyces cerevisiae was accomplished by minimizing the synthesis of glycerol, the main by-product in ethanol fermentation processing. To further improve ethanol production, overexpression of the native genes involved in conversion of pyruvate to ethanol in S. cerevisiae was successfully accomplished. The overexpression of an alcohol dehydrogenase (adh1) and a pyruvate decarboxylase (pdc1) caused an increase in growth rate and glycerol consumption under fermentative conditions, which led to a slight increase of the final ethanol yield. The overall expression of the adh1 and pdc1 genes in the modified strains, combined with the lack of the fps1 and gpd2 genes, resulted in a 1.4-fold increase (about 5.4 g/L ethanol produced) in fps1Δgpd2Δ (pGcyaDak, pGupCas) (about 4.0 g/L ethanol produced). In summary, it is possible to improve the ethanol yield by overexpression of the genes involved in the conversion of pyruvate to ethanol in engineered S. cerevisiae using glycerol as substrate.
Keywords: Glycerol; Ethanol production; Saccharomyces cerevisiae ; Alcohol dehydrogenase; Pyruvate decarboxylase

Start-up of Completely Autotrophic Nitrogen Removal Over Nitrite Enhanced by Hydrophilic-Modified Carbon Fiber by You-Peng Chen; Shan Li; Yun-Fang Ning; Na-Na Hu; Hai-Hua Cao; Fang Fang; Jin-Song Guo (866-877).
In order to assess the effects of the surface hydrophilicity of supports on the biofilm formation and evaluate the performance of completely autotrophic nitrogen removal over nitrite (CANON) process in a sequencing batch biofilm reactor (SBBR), unmodified activity carbon fibers (ACFs) and ACFs hydrophilic modified by heat treatment were used as supports. CANON process was initiated in a SBBR from conventional activated sludge. An operation temperature of 32 ± 2 °C, dissolved oxygen (DO) level at 1.5 mg L−1 and free ammonia (FA) concentration with 3.98–15.93 mg L−1 were maintained in the SBBR. Fourier transform infrared (FT-IR) spectra and Boehm’s neutralizing titration exhibited that modified ACFs had more oxygen-containing groups than unmodified ACFs. Larger biofilm growth on the modified surfaces examined by scanning electron microscopy and biofilm’s total dry weight, and the biofilm on the modified surfaces were more active, compared with those on the unmodified surfaces. This study demonstrates the hydrophilic-modified ACFs have better biological affinity than unmodified ACFs. Maximal total nitrogen removal rate of 0.088 k g N m−3 day−1 was achieved for the CANON process on day 80, indicating the CANON process was successfully started up. Apart from supports, the strategies of DO supplying and controlling FA concentration were also keys in starting up the CANON process within a shorter period.
Keywords: CANON; Biofilm; Activity carbon fibers; Hydrophilic

Electrode-supported hydrogels were conferred with the biospecificity of enzymes during the process of electropolymerization to give rise to a class of bioactive, stimuli-responsive co-joined interpenetrating networks of inherently conductive polymers and highly hydrated hydrogels. Glucose responsive biotransducers were prepared by potentiostatic electropolymerization [750 mV vs. Ag/AgCl (3 M KCl)] of pyrrole at Poly(hydoxyethyl methacrylate)-based hydrogel-coated Pt micro-electrodes (Φ = 100 μm) from aqueous solutions of pyrrole and glucose oxidase (GOx; 0.4 M pyrrole, 1.0 mg/ml GOx) to 1.0 and 10.0 mC/cm2. Polypyrrole was them over-oxidized by cyclic voltammetry (0–1.2 V vs. Ag/AgCl, 40 cycles in PBKCl, pH = 7.0). Biotransducers were stored at 4 °C in PBKCl for up to 18 days. Amperometric dose–response at 0.4 V vs. Ag/AgCl followed by Lineweaver–Burk analysis produced enzyme kinetic parameters as a function of electropolymerization charge density and storage time. Apparent Michaelis constant (K Mapp) increased from 18.6–152.0 mM (1.0 mC/cm2) and from 2.7–6.1 mM (10.0 mC/cm2). Biotransducer sensitivity increased to 21.2 nA/mM after 18 days and to 12.8 pA/mM after 10 days for the 1.0 and 10.0 mC/cm2 membranes, respectively. Maximum current, I max, also increased over time to 2.7 nA (1.0 mC/cm2) and to 170 pA (10.0 mC/cm2). Electropolymerization of polypyrrole is shown to be an effective means for imparting bioactivity to a hydrogel-coated microelectrode. GOx was shown to be stabilized and to increase activity over time within the electroconductive hydrogel.
Keywords: Biotransducer; Polypyrrole; Glucose; Hydrogels; Electroconductive

The Construction of Glucose Biosensor Based on Platinum Nanoclusters—Multiwalled Carbon Nanotubes Nanocomposites by Cheng Yan Wang; Xing Rong Tan; Shi Hong Chen; Fang Xin Hu; Hua An Zhong; Yu Zhang (889-902).
One-step synthesis method was proposed to obtain the nanocomposites of platinum nanoclusters and multiwalled carbon nanotubes (PtNCs–MWNTs), which were used as a novel immobilization matrix for the enzyme to fabricate glucose biosensor. The fabrication process of the biosensor was characterized by cyclic voltammetry, electrochemical impedance spectroscopy, atomic force microscopy and scanning electron microscope. Due to the favorable characteristic of PtNCs–MWNTs nanocomposites, the biosensor exhibited good characteristics, such as wide linear range (3.0 μM–12.1 mM), low detection limit (1.0 μM), high sensitivity (12.8 μA mM−1), rapid response time (within 6 s). The apparent Michaelis–Menten constant ( $$ K_m^{ ext{app}} $$ ) is 2.1 mM. The performance of the resulting biosensor is more prominent than that of most of the reported glucose biosensors. Furthermore, it was demonstrated that this biosensor can be used for the assay of glucose in human serum samples.
Keywords: Biosensor; Platinum nanoclusters; Multiwalled carbon nanotubes; Nanocomposites; Glucose oxidase

Integrated Perspective for Effective Bioremediation by Vasundhara Paliwal; Sampada Puranik; Hemant J. Purohit (903-924).
Identification of factors which can influence the natural attenuation process with available microbial genetic capacities can support the bioremediation which has been viewed as the safest procedure to combat with anthropogenic compounds in ecosystems. With the advent of molecular techniques, assimilatory capacity of an ecosystem can be defined with changing community dynamics, and if required, the essential genetic potential can be met through bioaugmentation. At the same time, intensification of microbial processes with nutrient balancing, expressing and enhancing the degradative capacities, could reduce the time frame of restoration of the ecosystem. The new concept of ecosystems biology has added greatly to conceptualize the networking of the evolving microbiota of the niche that helps in effective application of bioremediation tools to manage pollutants as additional carbon source.
Keywords: Bioremediation; Bioaugmentation; Natural attenuation; Microbial community; Genomics tool; Ecosystem biology

Cross-linking of enzyme aggregates from recombinant Geotrichum sp. lipase based on polyethylenimine (PEI) was applied to hydrolyze fish oil for enrichment of polyunsaturated fatty acids successfully. Through acetone precipitation and cross-linking of physical aggregates using glutaraldehyde in the presence of PEI, firmly cross-linked enzyme aggregates (PEI-CLEAs) were prepared. They could maintain more than 65% of relative hydrolysis degree after incubation in the range of 50–55 °C for 4 h and maintain more than 85% of relative hydrolysis degree after being treated by acetone, tert-butyl alcohol and octane for 4 h. PEI-CLEAs increased hydrolysis degree to 42% from 12% by free lipase. After five batch reactions, PEI-CLEAs still maintained 72% of relative hydrolysis degree. Hydrolysis of fish oil by PEI-CLEAs produced glycerides containing concentrated EPA and DHA in good yield. PEI-CLEAs had advantages over general CLEAs and free lipase in initial reaction rate, hydrolysis degree, thermostability, organic solvent tolerance and reusability.
Keywords: Lipase; Immobilization; Aggregate; Cross-linking; Polyunsaturated fatty acids

In Vitro Reduction of Hexavalent Chromium by Cytoplasmic Fractions of Pannonibacter phragmitetus LSSE-09 under Aerobic and Anaerobic Conditions by Lin Xu; Mingfang Luo; Chengying Jiang; Xuetuan Wei; Peng Kong; Xiangfeng Liang; Junmei Zhao; Liangrong Yang; Huizhou Liu (933-941).
Hexavalent chromate reductase was characterized and was found to be localized in the cytoplasmic fraction of a chromium-resistant bacterium Pannonibacter phragmitetus LSSE-09. The Cr(VI) reductase activity of cell-free extract (S12) was significantly improved by external electron donors, such as NADH, glucose, acetate, formate, citrate, pyruvate, and lactate. The reductase activity was optimal at pH 7.0 with NADH as the electron donor. The aerobic and anaerobic Cr(VI)-reduction enhanced by 0.1 mM NADH were respectively 3.5 and 3.4 times as high as that without adding NADH. The Cr(VI) reductase activity was inhibited by Mn2+, Cd2+, Fe3+, and Hg2+, whereas Cu2+ enhanced the chromate reductase activity by 29% aerobically and 33% anaerobically. The aerobic and anaerobic specific Michaelis–Menten constant K m of S12 fraction was estimated to be 64.95 and 47.65 μmol L−1, respectively. The soluble S150 fractions showed similar activity to S12 and could reduce 39.7% and 53.4% of Cr(VI) after 1 h of incubation aerobically and anaerobically while the periplasmic contents showed no obvious reduction activity, suggesting an effective enzymatic mechanism of Cr(VI) reduction in the cytoplasmic fractions of the bacterium. Results suggest that the enzymatic reduction of Cr(VI) could be useful for Cr(VI) detoxification in wastewater.
Keywords: Pannonibacter phragmitetus LSSE-09; Cr(VI) reduction; Chromate reductase; Cytoplasmic fractions

Apoptosis Mechanism of Human Cholangiocarcinoma Cells Induced by Bile Extract from Crocodile by Jin-He Kang; Wen-Qing Zhang; Wei Song; Dong-Yan Shen; Shan-Shan Li; Ling Tian; Yan Shi; Ge Liang; You-Xiong Xiong; Qing-Xi Chen (942-951).
Animal bile is popularly used as a traditional medicine in China, and bile acids are their major bioactive constituents. In the present study, effects of bile extract from crocodile gallbladder on QBC939 cell growth, cell cycle, and apoptosis were investigated by MTT assay, inverted microscopy, fluorescence microscopy, transmission electron microscopy, scanning electron microscopy, PI single- and FITC/PI double-staining flow cytometry, and western blotting. Our data have revealed that bile extract inhibited cells growth significantly, and the cell cycle was arrested in G1 phase. Bile extract induced QBC939 cell apoptosis, which was associated with collapse of the mitochondrial membrane potential and increase of ROS. In bile extract-treated cells, it was observed that the expression of bcl-2 decreased and cytochrome c released to cytosol, but the expression of bax remained unchanged. The data indicated that mitochondrial pathway might play an important role in bile extract-induced apoptosis in QBC939 cells. These results provide significant insight into the anticarcinogenic action of bile extract on cholangiocarcinoma cells.
Keywords: QBC939 cells; Apoptosis; Bcl-2; Cytochrome c ; Crocodile; Bile

A Thermophilic Cellulase Complex from Phialophora sp. G5 Showing High Capacity in Cellulose Hydrolysis by Junqi Zhao; Pengjun Shi; Yingguo Bai; Huoqing Huang; Huiying Luo; Huitu Zhang; Donghao Xu; Yaru Wang; Bin Yao (952-960).
A cellulase-producing mesophilic fungal strain, named G5, was isolated from the acidic wastewater and mud of a tin mine and identified as Phialophora sp. based on the internal transcribed spacer sequence. The volumetric activities and specific activities of cellulase induced by different carbon sources (Avicel, corn cob, wheat bran and corn stover) were compared. The cellulase complex of Phialophora sp. G5 exhibited the optimal activities at 60–65 °C and pH 4.0–5.0, and had good long-term thermostability at 50 °C. Compared with the commercial cellulase (Accellerase 1500, Genencor), the enzyme under study showed 60% and 80% of the capacity to hydrolyze pure cellulose and natural cellulose, respectively. This is the first study to report that a cellulytic enzymes complex from Phialophora genus, and the superior properties of this enzyme complex make strain G5 a potential microbial source to produce cellulase for industrial applications, and the production ability could be improved by mutagenesis.
Keywords: Phialophora sp. G5; Thermophilic; Thermostability; Cellulase complex

Production of d-tagatose, a Functional Sweetener, Utilizing Alginate Immobilized Lactobacillus fermentum CGMCC2921 Cells by Zheng Xu; Sha Li; Fenggen Fu; Guixiang Li; Xiaohai Feng; Hong Xu; Pingkai Ouyang (961-973).
d-tagatose is a ketohexose that can be used as a novel functional sweetener in foods, beverages, and dietary supplements. This study was aimed at developing a high-yielding d-tagatose production process using alginate immobilized Lactobacillus fermentum CGMCC2921 cells. For the isomerization from d-galactose into d-tagatose, the immobilized cells showed optimum temperature and pH at 65 °C and 6.5, respectively. The alginate beads exhibited a good stability after glutaraldehyde treatment and retained 90% of the enzyme activity after eight cycles (192 h at 65 °C) of batch conversion. The addition of borate with a molar ratio of 1.0 to d-galactose led to a significant enhancement in the d-tagatose yield. Using commercial β-galactosidase and immobilized L. fermentum cells, d-tagatose was successfully obtained from lactose after a two-step biotransformation. The relatively high conversion rate and productivity from d-galactose to d-tagatose of 60% and 11.1 g l−1 h−1 were achieved in a packed-bed bioreactor. Moreover, lactobacilli have been approved as generally recognized as safe organisms, which makes this L. fermentum strain an attracting substitute for recombinant Escherichia coli cells among d-tagatose production progresses.
Keywords: d-Tagatose; Lactobacillus fermentum ; Immobilized cells; Packed-bed bioreactor; l-arabinose isomerase

The recovery of an inhibiting product from a bioreactor soon after its formation is an important issue in industrial bioprocess development. In the present study, the potential of the anion exchanger-based in situ product recovery (ISPR) technique for the biocatalytic production of propionic acid was discussed. The focus of the current work was the selection of a suitable configuration of metabolically active cells for application in propionic acid production. Accumulation of propionic acid in fermentation broth caused feedback inhibition of the growth and biotransformation activity of Propionibacterium freudenreichii CICC 10019. Relevant product inhibition kinetics was discussed, and the results showed that keeping the aqueous propionic acid concentration below 10.02 g L−1 was an essential prerequisite for ISPR process. A batch study, in which three ISPR configuration mode designs were compared, was conducted. The comparison indicated that employing an external direct mode had significant advantages over other modes in terms of increased productivity and product yield, with a corresponding decrease in the number of downstream processing steps, as well as in substrate consumption. The fed-batch culture using an external direct mode for the continuous accumulation of propionic acid resulted in a cumulative propionic acid concentration of 62.5 g L−1, with a corresponding product yield of 0.78 g propionic acid/g glucose.
Keywords: Propionibacterium freudenreichii ; Propionic acid; ISPR; External direct mode; Fed-batch fermentation

A Fungus Capable of Degrading Microcystin-LR in the Algal Culture of Microcystis aeruginosa PCC7806 by Yong Jia; Jingjing Du; Fuqiang Song; Guiying Zhao; Xingjun Tian (987-996).
Microcystins (MCs) are a family of natural toxins produced by cyanobacteria (blue-green algae). Microbial degradation is considered an efficient method for eliminating cyanobacteria and MCs in environmental conditions. This study examines the ability of Trichaptum abietinum 1302BG, a white rot fungus, to degrade microcystin-LR in the harmful algal culture of Microcystis aeruginosa PCC7806. Results showed that microcystin-LR could not be detected by high-performance liquid chromatography after 12 h in algal culture incubated with the fungus. There were also high activities of catalase and peroxidase in algal culture incubated with the fungus. However, similar to the control, they decreased to normal levels after 72 h. Meanwhile, the micronucleus test in the toxicity studies revealed that the degraded algal culture had low toxicity.
Keywords: Cyanobacteria; Trichaptum abietinum 1302BG; Microcystin-LR; Micronucleus

This was the continuation of our previous study along the same line with more focus on technical details because the data are usually divided into two datasets, one for model development and the other for model validation during the development of predictive model. The widely used validation method is the delete-1 jackknife validation. However, no systematical studies were conducted to determine whether the jackknife validation with different deletions works better because the number of validations with different deletions increases in a factorial fashion. Therefore it is only small dataset that can be used for such an exhausted study. Cellulase is an enzyme playing an important role in modern industry, and many parameters related to cellulase in enzymatic reactions were poorly documented. With increased interests in cellulases in bio-fuel industry, the prediction of parameters in enzymatic reactions is listed on agenda. In this study, two aims were defined (a) which amino acid property works better to predict the temperature optimum and (b) with which deletion the jackknife validation works. The results showed that the amino acid distribution probability works better in predicting the optimum temperature of catalytic reaction by cellulase, and the delete-4, more precisely one-fifth deletion, jackknife validation works better.
Keywords: Cellulase; Enzyme; Jackknife validation; Prediction; Temperature optimum

The possibility of controlling Fusarium wilt—caused by Fusarium oxysporum sp. cubensec (race 4)—was investigated by genetic engineering of banana plants for constitutive expression of rice thaumatin-like protein (tlp) gene. Transgene was introduced to cauliflower-like bodies’ cluster, induced from meristemic parts of male inflorescences, using particle bombardment with plasmid carrying a rice tlp gene driving by the CaMV 35S promoter. Hygromycin B was used as the selection reagent. The presence and integration of rice tlp gene in genomic DNA confirmed by PCR and Southern blot analyses. RT-PCR revealed the expression of transgene in leaf and root tissues in transformants. Bioassay of transgenic banana plants challenged with Fusarium wilt pathogen showed that expression of TLP enhanced resistance to F. oxysporum sp. cubensec (race 4) compared to control plants.
Keywords: Banana; Fusarium wilt; Male inflorescence; Particle bombardment; PR-5; Thaumatin-like protein

A type III polyketide biosynthetic gene cluster has been discovered in the industrially important strain Streptomyces toxytricini NRRL 15443, including four genes stp450-1, stts, stp450-2, and stmo. The stts gene encodes a putative type III polyketide synthase that is homologous to RppA, a 1,3,6,8-tetrahydroxynaphthalene (THN) synthase from Streptomyces griseus. The deduced protein product of stmo resembles the cupin-containing monooxygenase MomA from Streptomyces antibioticus that oxidizes THN into flaviolin. Two cytochrome P450s (CYPs), StP450-1 and StP450-2, are present in the gene cluster. StTS was overexpressed in Escherichia coli BL21(DE3) and identified as a THN synthase. The synthesized THN can be easily oxidized into flaviolin by air. Both CYPs were reconstituted in E. coli BL21(DE3) and can oxidize flaviolin to form oligomers. The k cat/K m values for StP450-1 and StP450-2 were 0.28 and 0.71 min−1 mM−1, respectively. UV irradiation test showed that expression of StTS in E. coli BL21(DE3) significantly protects the cells from UV radiation, and coexpression of StTS and StP450-1 provides even stronger protection.
Keywords: Type III polyketide synthase; Cytochrome P450 monooxygenases; Streptomyces toxytricini ; UV protection

Soybean hull, generated from soybean processing, is a lignocellulosic material with limited industrial applications and little market value. This research is exploring a new application of soybean hull to be converted to fungal lipids for biodiesel production through solid-state fermentation. Mortierella isabellina was selected as the oil producer because of its high lipid content at low C/N ratio. Several cultivation factors were investigated, including moisture content, inoculums size, fungal spore age, and nutrient supplements, in an attempt to enhance the lipid production of the solid-state fermentation process. The results showed that lipid production with the increase of the moisture content and the spore age, while decreased as the size of inoculums increased. Nutrients addition (KH2PO4 1.2 mg and MgSO4 0.6 mg/g soybean hull) improved the lipid production. The total final lipid reached 47.9 mg lipid from 1 g soybean hull after the conversion, 3.3-fold higher than initial lipid reserve in the soybean hull. The fatty acid profile analysis indicated that fatty acid content consisted of 30.0% of total lipid, and 80.4% of total fatty acid was C16 and C18. Therefore, lipid production from soybean hull is a possible option to enable soybean hull as a new resource for biodiesel production and to enhance the overall oil production from soybeans.
Keywords: Biodiesel; Solid-state fermentation; Mortierella isabellina ; Soybean hull

Kinetics of Alloxan-Induced Inhibition on δ-Aminolevulinate Dehydratase Activity in Mouse Liver Homogenates by Verônica B. Brito; Vanderlei Folmer; Inês Isabel R. Guerra; João Batista T. Rocha (1047-1056).
This study evaluated the effects of alloxan on the kinetics properties of the δ-aminolevulinate dehydratase (δ-ALA-D) using mouse liver homogenates. δ-ALA-D is an important sulfhydryl enzyme that catalyses the second step in heme biosynthesis and is commonly diminished in experimental and human diabetes. Despite the known effects of alloxan in models of experimental diabetes, there are no data in the literature demonstrating the effects of alloxan on the kinetics properties of the δ-ALA-D. The results showed that alloxan (1.25–20 μM) caused a concentration-dependent inhibition of hepatic δ-ALA-D activity. The inhibition constant (K i ) for alloxan-induced inhibition on δ-ALA-D was 3.64 μM. The alloxan (5 μM) caused a decrease in V max (65.8%) and in K m (53.1%), which is suggestive of an uncompetitive inhibition of enzyme. In addition, dithiothreitol (700 and 1,000 μM) completely prevented the δ-ALA-D activity inhibition induced by 10 and 20 μM alloxan. Similar protection was obtained in the presence of 2,000 μM glutathione. Therefore, this work showed that the inhibition of hepatic δ-ALA-D activity can be obtained in vitro at low micromolar levels of alloxan, and can also be prevented by reducing agents. Moreover, these results may help to understand the abnormalities in heme pathway found in models of experimental diabetes in vivo.
Keywords: δ-ALA-D; Alloxan; Diabetes; Kinetics; Mice

Sequential Anaerobic/Aerobic Treatment of Dye-Containing Wastewaters: Colour and COD Removals, and Ecotoxicity Tests by Marcos Erick Rodrigues da Silva; Paulo Igor Milen Firmino; Márcia Rodrigues de Sousa; André Bezerra dos Santos (1057-1069).
Colour and COD removals of the azo dyes Congo Red (CR) and Reactive Black 5 (RB5) were individually evaluated in a sequential anaerobic/aerobic treatment system. Additionally, dye toxicity was assessed by using acute ecotoxicity tests with Daphnia magna as the indicator-organism. The anaerobic reactor was operated at approximately 27 °C and with hydraulic retention times of 12 and 24 h. The aerobic reactor was operated in batch mode with a total cycle of 24 h. During anaerobic step, high colour removals were obtained, 96.3% for CR (400 mg/L) and 75% for RB5 (200 mg/L). During the aerobic phase, COD effluent was considerably reduced, with an average removal efficiency of 52% for CR and 85% for RB5, which resulted in an overall COD removal of 88% for both dyes. Ecotoxicity tests with CR revealed that the anaerobic effluent presented a higher toxicity compared with the influent, and an aerobic post-treatment was not efficient in reducing toxicity. However, the results with RB5 showed that both anaerobic and aerobic steps could decrease dye toxicity, especially the aerobic phase, which removed completely the toxicity in D. magna. Therefore, the anaerobic/aerobic treatment is not always effective in detoxifying dye-containing wastewaters, sometimes even increasing dye toxicity.
Keywords: Anaerobic/aerobic treatment; Azo dyes; Reductive decolourisation; Toxicity; Daphnia magna

Extraction of Soluble Fiber from Distillers' Grains by Hunter R. Flodman; Elizabeth J. Boyer; Arthy Muthukumarappan; Hossein Noureddini (1070-1081).
The feasibility of using coproducts from dry grind corn ethanol production as a substrate for the production of soluble fiber was examined. Acid- and base-catalyzed hydrolysis experiments were performed using sulfuric acid and sodium hydroxide to partially hydrolyze hemicellulose content of whole stillage, a precursor to distillers' grains, to soluble fiber. The influences of temperature, reaction time, and hydrolyzing agent concentration on the formation of soluble fiber were studied. Soluble fiber was recovered by precipitation in a 95% ethanol solution. Results indicate that appreciable quantities of soluble fiber may be extracted using either acid- or base-catalyzed reactions. The highest yield of soluble fibers was 13.2 g per 100 g-db of treated whole stillage using one weight percent sodium hydroxide at 80ºC for 1 h. HPLC analysis was used to quantify the amount of monomeric sugars which were formed during the hydrolysis procedures.
Keywords: Soluble fiber; Whole stillage; Hemicellulose; Hydrolysis; Distillers’ grains; Ethanol

A novel laccase producing Basidiomycete Peniophora sp. (NFCCI-2131) was isolated from pulp and paper mill effluent. The optimal temperature and initial pH for laccase production by the isolate in submerged culture were found to be 30 and 4.6° C, respectively. Maltose (20 g l−1) and tryptone (1.0 g l−1) were the most suitable carbon and nitrogen sources for laccase production. Cu2+ (1.0 mM) and veratryl alcohol induced maximum laccase production giving 6.6 and 6.07 U/ml laccase activity, respectively. Under optimised culture conditions, 7.6 U/ml activity was obtained, which was 2.4 times higher than that was achieved in basal medium. An evaluation of the delignification efficiency of the crude enzyme in the presence of redox mediators [2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and (1-hydroxybenzotriazole)] revealed structural changes in lignin and existence of many active centres for both chemical and biological degradation of lignin following enzymatic treatment.
Keywords: Peniophora sp.; Laccase; Lignin; Delignification; FTIR

Isolation of Thermo-stable and Solvent-Tolerant Bacillus sp. Lipase for the Production of Biodiesel by Ramachandran Sivaramakrishnan; Karuppan Muthukumar (1095-1111).
This study presents the production of biodiesel from algae oil by transesterification using thermophilic microorganism. The microorganism used in this study was isolated from the soil sample obtained near the furnace. The organism was identified as Bacillus sp., and the lipase obtained was purified by ammonium sulfate precipitation and ion exchange chromatography leading to 8.6-fold purification and 13% recovery. Molecular weight of the enzyme was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it was found to be 45 kDa. The effect of pH, temperature, and solvent addition on lipase activity was investigated. The enzyme showed maximum activity at 55 °C and at pH 7 and was also found to be highly active in the presence of organic solvents such as hexane and t-butanol. The isolated lipase was successfully used for the production of biodiesel. The transesterification activity of the isolated lipase showed 76% of fatty acid methyl esters yield in 40 h, which indicated that this enzyme can be used as a potential biocatalyst for the biodiesel production.
Keywords: Lipase; Thermo-stable; Solvent-tolerant; Biodiesel

Cloning, Differential Expression, and Association Analysis with Fat Traits of Porcine IDH3γ Gene by Zhuqing Ren; Yuanzhu Xiong; Changyan Deng; Siwen Jiang (1112-1120).
Mitochondrial NAD+-dependent isocitrate dehydrogenase (IDH3) catalyzes the allosterically regulated rate-limiting step of the tricarboxylic acid cycle activated. In pigs, very little is known about this gene. Here, we cloned 1,346 bp full-length cDNA and 8,778 bp genomic sequence of porcine γ subunit of IDH3 (IDH3γ). IDH3γ contains 12 exons separated by 11 introns. Real-time PCR revealed that IDH3γ mRNA were upregulated in backfat of Large White compared with Meishan and F1 hybrids, and most abundant in small intestine via tissue distribution profile. A microsatellite (“GT” repeats) in second intron was found. The selected pigs were genotyped at this microsatellite. The IDH3γ genotypes showed a significant effect on backfat thickness at thorax–waist (P < 0.05), backfat thickness at sixth to seventh thorax (P < 0.01), and average backfat thickness (P < 0.05). This site seemed to be significantly dominant in action (P < 0.05 for backfat thickness at sixth to seventh thorax, backfat thickness at thorax–waist, and average backfat thickness), and allele B was associated with increase of thickness values of these traits. This locus is possibly considered as a marker for adipose deposition traits.
Keywords: IDH3γ; Differential expression; Microsatellite; Pig