Applied Biochemistry and Biotechnology (v.166, #1)

Conversion of Isoeugenol to Vanillin by Psychrobacter sp. Strain CSW4 by Morahem Ashengroph; Iraj Nahvi; Hamid Zarkesh-Esfahani; Fariborz Momenbeik (1-12).
To screen strains of halotolerant or halophile bacteria which are able to convert isoeugenol to vanillin, 36 different strains of bacteria isolated from the salty environments in Iran were investigated. During growth on isoeugenol, a moderately halotolerant Gram-negative coccobacil showed capability of converting isoeugenol to vanillin. Based on morphological, physiological, and phylogenetic studies, strain CSW4 was classified as a bacterium belonging to the genus Psychrobacter. The bioconversion products were confirmed by thin-layer chromatography, high-performance liquid chromatography, and spectral data obtained from UV/Vis spectroscopy, FTIR, and mass-spectroscopy. Using growing cells, vanillin reached its maximum level of 88.18 mg L−1 after 24 h of reaction time in the presence of 1 g L−1 isoeugenol, resulting in a molar yield of 10.2%. The use of resting cells led to the optimal yield of vanillin (16.4%) which was obtained after 18-h reaction using 1 g L−1 isoeugenol and 3.1 g of dry weight of cells per liter harvested at the end of the exponential growth phase. To improve vanillin yield, the effect of substrate concentration on vanillin production under resting cells conditions was also investigated. Using 10 g L−1 isoeugenol, the maximal vanillin concentration (1.28 g L−1) was achieved after a 48-h reaction, without further optimization. The present study brings the first evidence for biotransformation of isoeugenol to vanillin in the genus Psychrobacter.
Keywords: Bioconversion; Isoeugenol; Vanillin; Psychrobacter sp. strain CSW4

Solvent-Free Production of Bioflavors by Enzymatic Esterification of Citronella (Cymbopogon winterianus) Essential Oil by Natália Paroul; Luana Paula Grzegozeski; Viviane Chiaradia; Helen Treichel; Rogério L. Cansian; J. Vladimir Oliveira; Débora de Oliveira (13-21).
Enzymatic esterification of citronella essential oil towards the production of geranyl and citronellyl esters may present great scientific and technological interest due to the well-known drawbacks of the chemical-catalyzed route. In this context, this work reports the maximization of geranyl and citronellyl esters production by esterification of oleic and propionic acids in a solvent-free system using a commercial immobilized lipase as catalyst. Results of the reactions showed that the strategy adopted for the experimental design proved to be useful in evaluating the effects of the studied variables on the reaction conversion using Novozym 435 as catalyst. The operating conditions that maximized the production of each ester were determined, leading, in a general way, to conversions of about 90% for all systems. New experimental data on enzymatic esterification of crude citronella essential oil for geranyl and citronellyl esters production in solvent-free system are reported in this work.
Keywords: Cymbopogon winterianus ; Essential oil; Esterification; Lipase

In this paper, four nonionic surfactants with different hydrophilic–lipophilic balance (HLB) based on sorbitan monolaurate were synthesized by introducing ethylene oxide gas (n = 20, 40, 60, and 80 ethylene oxide units). The chemical structure of the prepared ethoxylated surfactants was confirmed using Fourier transform-infrared and 1H NMR spectroscopes. The surface tension and thermodynamic properties of the prepared surfactants have been studied. The simultaneous saccharification and fermentation (SSF) process for ethanol production from microwave/alkali pretreated wheat straw has been assayed using nonionic surfactants have different ethylene oxide units. Ethanol yield was 82% and 61% for Kluyveromyces marxianus and Saccharomyces cerevisiae, respectively, with the addition of 2.5 g/l of the prepared nonionic surfactant (HLB = 18.2). Results show that the production of ethanol from microwave/alkali pretreated wheat straw increased with increasing the (HLB) value of the nonionic surfactant.
Keywords: Nonionic surfactants; Simultaneous; Saccharification and fermentation (SSF); Wheat straw; Ethanol; Yeast

Optimization of Laccase Production by Trametes versicolor Cultivated on Industrial Waste by Marina Tišma; Polona Žnidaršič-Plazl; Đurđa Vasić-Rački; Bruno Zelić (36-46).
Laccases are very interesting biocatalysts for several industrial applications. Its production by different white-rot fungi can be stimulated by a variety of inducing substrates, and the use of lignocellulosic wastes or industrial by-products is one of the possible approaches to reduce production costs. In this work, various industrial wastes were tested for laccase production by Trametes versicolor MZKI G-99. Solid waste from chemomechanical treatment facility of a paper manufacturing plant showed the highest potential for laccase production. Enzyme production during submerged cultivation of T. versicolor on the chosen industrial waste has been further improved by medium optimization using genetic algorithm. Concentrations of five components in the medium were optimized within 60 shake-flasks experiments, where the highest laccase activity of 2,378 U dm−3 was achieved. Waste from the paper industry containing microparticles of CaCO3 was found to stimulate the formation of freely dispersed mycelium and laccase production during submerged cultivation of T. versicolor. It was proven to be a safe and inexpensive substrate for commercial production of laccase and might be more widely applicable for metabolite production by filamentous fungi.
Keywords: Laccase production; Industrial waste; Medium optimization; Genetic algorithm; Trametes versicolor ; Microparticles

Okadaic acid (OA), a lipophilic toxin, is produced by Dinophysis and Prorocentrum, and causes diarrheic shellfish poisoning to humans. The mechanism of OA action is based on the reversible inhibition of protein phosphatase type 2A (PP2A) by the toxin. Therefore, this inhibition could be used to develop assay for OA detection. In this work, a colorimetric test based on the PP2A inhibition was developed for OA detection. PP2A from GTP and Millipore was immobilized on silica sol-gel, and the detection was performed. A limit of detection of 0.29 and 1.14 μg/L was respectively observed for enzyme from GTP and Millipore. The immobilization technique provided a tool to preserve the enzymatic activity, which is very unstable in solution. The PP2A immobilized sol-gel exhibited a storage stability of near 5 months, when microtiter plate with enzyme-immobilized polymer was kept at −18C°. The combination of the simplicity of the colorimetric method, along with long storage stability achieved by sol-gel immobilization, demonstrated the potentiality of this technique to be used for commercial purpose.
Keywords: Okadaic acid; Protein phosphatase 2A; Sol gel; Storage test; Colorimetric enzymatic-assay

N-acetylgalactosamine Kinase: A Naturally Promiscuous Small Molecule Kinase by Helena Kristiansson; David J. Timson (57-63).
N-acetylgalactosamine kinase is a member of the GHMP family of small molecule kinases which catalyses the ATP-dependent phosphorylation of N-acetylgalactosamine. It is highly similar in structure and sequence to galactokinase. Alteration of galactokinase at a key tyrosine residue (Tyr-379 in the human enzyme) has been shown to dramatically enhance the substrate range of this enzyme. Here, we investigated the substrate specificity of the wild type N-acetylgalactosamine kinase and demonstrated that it can also catalyse the phosphorylation of N-acetylglucosamine and N-acetylmannosamine. In human N-acetylgalactosamine kinase, the equivalent residue to Tyr-379 in galactokinase is Phe-444. Alteration of this residue did not result in dramatic changes to the specificity of the enzyme. The more relaxed substrate specificity of N-acetylgalactosamine kinase, compared to galactokinase, can be explained by the greater flexibility of a glycine rich loop in the active site of the enzyme. These results suggest that N-acetylgalactosamine kinase is a potential biocatalyst for the phosphorylation of N-acetyl sugars. However, it is unlikely that it will be possible to further broaden the substrate range by alteration of Phe-444.
Keywords: GALK2; GHMP kinase; Sugar phosphate; N-acetyl sugar; Biocatalysis; Protein flexibility

Sox9 Is Upstream of MicroRNA-140 in Cartilage by Yukio Nakamura; Xinjun He; Hiroyuki Kato; Shigeyuki Wakitani; Tatsuya Kobayashi; Sumiko Watanabe; Atsumi Iida; Hideaki Tahara; Matthew L. Warman; Ramida Watanapokasin; John H. Postlethwait (64-71).
MicroRNA-140 (miR-140) is specifically expressed in developing cartilage tissues. We have previously reported that miR-140 plays an important role during palatal cartilage development by modulating platelet-derived growth factor receptor alpha (pdgfra) in zebrafish. However, the regulatory mechanism of miR-140 in cartilage is still unknown. Using developing zebrafish, sox9a mutant (sox9a−/−) and sox9b mutant (sox9b−/−) zebrafish and SOX9 small interfering RNA in human chondrocytes, T/C-28 cells, we found that miR-140 is regulated by the cartilage master transcription regulator Sox9 in zebrafish and mammalian cells.
Keywords: MicroRNA-140 (miR-140); Sox9 ; Cartilage; Zebrafish; T/C-28; siRNA; In situ hybridization; RT-PCR

Cloning and Characterization of Two β-Glucosidase/Xylosidase Enzymes from Yak Rumen Metagenome by Lei Bao; Qiang Huang; Lei Chang; Qingwen Sun; Jungang Zhou; Hong Lu (72-86).
Two β-glucosidase/xylosidase genes, Rubg3A and Rubg3B, were cloned from yak rumen uncultured microorganisms by metagenome method and function-based screening. Recombinant RuBG3A and RuBG3B purified from Escherichia coli were characterized for enzymatic properties, and they exhibited activity against 4-nitrophenyl-β-d-glucopyranoside and 4-nitrophenyl-β-d-xylopyranoside, suggesting bifunctional β-glucosidase/xylosidase activity. Chromatography analysis showed that they could effectively hydrolyze cellooligosaccharide substrates, indicating the facilitation in saccharification of cellulose. RuBG3A and RuBG3B can also increase the reducing sugar released in xylan hydrolysis to 218% and 169%, respectively, through synergism with xylanase, suggesting their application in hemicellulose saccharification. Molecular modeling and substrate docking showed that there should be one active center responsible for the bifunctional activity in each enzyme, since the active site pocket is substantially wide to allow the entry of both β-glucosidic or β-xylosidic substrates, which elucidated the structure–function relationship in substrate specificities. Therefore, the enzymatic properties, the participation in hydrolysis of cellooligosaccharides, and the synergism with xylanase make RuBG3A and RuBG3B very interesting candidates for saccharification of both cellulose and hemicellulose.
Keywords: β-Glucosidase; β-Xylosidase; Uncultured yak rumen microorganism; Molecular modeling; Substrate docking

Design and Evaluation of an Optimal Controller for Simultaneous Saccharification and Fermentation Process by Ganti S. Murthy; David B. Johnston; Kent D. Rausch; M. E. Tumbleson; Vijay Singh (87-111).
Ethanol from corn is produced using dry grind corn process in which simultaneous saccharification and fermentation (SSF) is one of the most critical unit operations. In this work an optimal controller based on a previously validated SSF model was developed by formulating the SSF process as a Bolza problem and using gradient descent methods. Validation experiments were performed to evaluate the performance of optimal controller under different process disturbances that are likely to occur in practice. Use of optimal control algorithm for the SSF process resulted in lower peak glucose concentration, similar ethanol yields (13.38±0.36% v/v and 13.50±0.15% v/v for optimally controlled and baseline experiments, respectively). Optimal controller improved final ethanol concentrations as compared to process without optimal controller under conditions of temperature (13.35±1.28 and 12.52±1.19% v/v for optimal and no optimal control, respectively) and pH disturbances (12.65±0.74 and 11.86±0.49% v/v for optimal and no optimal control, respectively). Cost savings due to lower enzyme usage and reduced cooling requirement were estimated to be up to $1 million for a 151 million L/yr (40 million gal/yr) dry grind plant.
Keywords: Dry grind corn ethanol; Saccharomyces cerevisiae ; Cybernetic model; SSF process; Optimal controller; Gradient descent; Process disturbances

Effect of Physicochemical Parameters on Enzymatic Biodecaffeination During Tea Fermentation by V. R. Sarath Babu; M. S. Thakur; Sanjukta Patra (112-126).
We report for the first time the development of a biodecaffeination process for tea synchronised with tea fermentation process using enzymes isolated from Pseudomonas alcaligenes. Cell-free extract was used for biodecaffeination of tea during fermentation of tea and 80% of the caffeine in the tea dhool was degraded within 90 min of incubation. Several factors that tend to effect the biodecaffeination during this stage, like moisture, aeration, intermittent enzyme addition and mixing, were optimized, and inhibitory interactions of proteins with polyphenols, caffeine–polyphenol interactions, which directly influence the biodecaffeination process were prevented by the use of glycine (5% w/w) in the dhool. Tea decaffeinated through the enzymatic route retained the original flavor and aroma, and there was an increase in the total polyphenol content of the tea.
Keywords: Biodecaffeination; Caffeine demethylase; Multi-enzyme system; Dhool; Glycine

Optimization of Culture Conditions for 1,3-Propanediol Production from Glycerol Using a Mutant Strain of Klebsiella pneumoniae by Baek-Rock Oh; Jeong-Woo Seo; Sun-Yeon Heo; Won-Kyung Hong; Lian Hua Luo; Seonghun Kim; Don-Hee Park; Chul Ho Kim (127-137).
In the present work, mutant strains of Klebsiella pneumoniae with deletions of the als gene encoding acetolactate synthase involved in synthesis of 2,3-butanediol, the ldhA gene encoding lactate dehydrogenase required for lactate synthesis, or both genes, were prepared. Production of 1,3-propanediol (1,3-PD) from glycerol was enhanced in the ldhA mutant strain (ΔldhA), but lower in Δals or Δals ΔldhA mutant strains compared to the parent strain, concomitant with a reduction in the glycerol consumption rate, indicating that deletion of ldhA alone was useful to improve 1,3-PD production. Fed-batch fermentation analysis revealed that, in the ΔldhA mutant strain, 1,3-PD production was higher at low pH than at neutral pH; the reverse was true for the parent strain. Further optimization of culture conditions, by variation of aeration and glycerol feed rates, dramatically improved the production of 1,3-PD by the mutant strain. The maximum level attained was 102.7 g l−1 of 1,3-PD from glycerol.
Keywords: Klebsiella pneumoniae ; Glycerol; 1,3-Propanediol; Gene deletion; Optimized culture conditions

NADH is a coenzyme which plays a central role in cellular growth and metabolism. It is an intracellular fluorophore which fluoresces at 460 nm when cells are irradiated by 340 nm wavelength of light. The application of NADH+H+ fluorescence measurement for characterization of biomass and its metabolic activity during batch fermentation of 1,3-propanediol (1,3-PD) using Clostridium diolis was investigated in this study. A linear correlation between net fluorescence and biomass concentration was observed during both the initial and final phases of 1,3-PD fermentation. This could be used as an on-line indicator of biomass concentration inside the bioreactor thereby eliminating the need for sampling and off-line analysis for establishing biomass concentration during these phases. Also a sharp decline in the NADH+H+ fluorescence value was obtained towards the end of fermentation which could be a significant on-line, in situ signal of substrate depletion in the bioreactor and therefore possible fresh nutrient feed for enhanced production of 1,3-PD by repetitive and/or various fed-batch cultivation(s). This is the first report on the use of NADH + H+ fluorescence measurement technique for 1,3-PD fermentation.
Keywords: 1,3-Propanediol; Clostridium diolis ; NADH+H+ ; Fluorescence; Metabolic state; Batch cultivation

Lipids Containing Polyunsaturated Fatty Acids Synthesized by Zygomycetes Grown on Glycerol by Stamatia Bellou; Anna Moustogianni; Anna Makri; George Aggelis (146-158).
Several strains of Zygomycetes cultivated on glycerol produced mycelia rich in lipids containing higher amounts of neutral lipids (NL) than glycolipids plus sphingolipids and phospholipids (P), while biosynthesis of P in Mortierella ramanniana, Mucor sp., and Cunninghamella echinulata occurred though NL accumulation process was in progress. Polyunsaturated fatty acids (PUFA) concentration gradually decreased in all lipid fractions of M. ramanniana during growth. In contrast, in C. echinulata concentration of both linoleic and γ-linolenic acids increased with time, especially in P. Taking for granted that the main function of PUFA is associated to their participation in mycelial membranes, we could suppose that biosynthesis of these fatty acids is associated to mycelial growth. However, this is accurate only for some Zygomycetes, e.g., M. ramanniana. On the contrary, PUFA biosynthesis in C. echinulata persists after growth cessation, suggesting that in this species biosynthetic ability is not a strictly growth-associated process. Phosphatidyl-inositol and phosphatidyl-choline were the major P classes in C. echinulata and M. ramanniana, respectively. In M. ramanniana, a decrease of PUFA concentration was noticed even when mycelia were incubated in low temperature (conditions that normally favor PUFA biosynthesis), indicating that PUFA biosynthesis in this fungus is associated to primary metabolism.
Keywords: Zygomycetes; Neutral lipids; Polar lipids; Fatty acids distribution; γ-linolenic acid; Bioreactor; Temperature

Decolorization of Remazol Brilliant Blue R by a Purified Laccase of Polyporus brumalis by Hyewon Kim; Sungsuk Lee; Sunhwa Ryu; Hyoung T. Choi (159-164).
A white rot basidiomycete Polyporus brumalis has been reported to induce two laccase genes under degradation conditions of dibutylphthalate. When this fungus was grown in a minimal medium, one laccase enzyme was detected by the native polyacrylamide gel electrophoresis. A laccase was purified through ammonium sulfate precipitation and ion exchange chromatography, and the estimated molecular weight was 70 kDa. The optimum pH and temperature of the purified laccase was pH 4.0 and 20 °C, respectively. The K m value of the enzyme was 685.0 μM, and the V max was 0.147 ODmin−1 unit−1 for o-tolidine. Purified laccase showed effective decolorization of a dye, Remazol Brilliant Blue R (RBBR), without any laccase mediator. However, this effect was reduced by a laccase inhibitor, kojic acid, which confirmed that the laccase was directly involved in the decolorization of RBBR.
Keywords: Dye decolorization; Enzyme purification; Laccase; Polyporus brumalis ; RBBR

Photoprotective Effects of a Formulation Containing Tannase-Converted Green Tea Extract Against UVB-Induced Oxidative Stress in Hairless Mice by Yang-Hee Hong; Eun Young Jung; Kwang-Soon Shin; Tae Young Kim; Kwang-Won Yu; Un Jae Chang; Hyung Joo Suh (165-175).
Ultraviolet B (UVB) irradiation may induce the acceleration of skin aging. The purpose of this study was to develop an effective formulation containing tannase-converted green tea extract (FTGE) to inhibit UVB-induced oxidative damage. Significant (p < 0.05) prevention of the reduced form of glutathione (GSH) depletion was observed in mice treated with FTGE. The hydrogen peroxide levels of mice treated with FTGE were similar to those of UVB non-irradiated mice. No significant difference was observed between No UVB control and FTGE mice. Also, mice treated with FTGE had significant (p < 0.05) decreases in thiobarbituric acid-reactive substance levels by lipid peroxidation compared with No UVB control mice. Our data suggest that this formulation may be effective in protecting skin from UVB photodamage.
Keywords: Green tea; Tannase; Formulation; Oxidative stress; Photoprotection

Either the natural biodegradation process or the industrial hydrolytic process requires synergistic interactions between various cellulases. However, it is sometimes impeded by low hydrolytic rate of existing cellulases and the lack of accessory enzymes. Herein, the ability of a commercial cellulase (Spezyme CP, from Genencor) to degrade steam explosion-pretreated corn stover was significantly improved. Firstly, a fungal cellulase producer, Aspergillus fumigatus ECU0811, was isolated from hundreds of soil samples. A 96-deep-well microscale-based platform was developed here to reduce the labor-intensive screening work and proved to be consistent with macroscale screening work. After optimization of fermentation, 3% corn cob could induce A. fumigatus ECU0811 to yield the highest cellulase production. Based on the high activities of β-glucosidase and xylanase by A. fumigatus ECU0811, 0.91 and 125 U/mg protein, respectively, an enzyme cocktail was composed with a fixed dosage of Spezyme CP (CPCel) at 14.2 filter paper units (FPU)/g glucan and varied dosages of A. fumigatus cellulase (AFCel). Consequently, the glucan-to-glucose conversion of corn stover was increased from 25.6% in the presence of CPCel at a dosage of 14.2 FPU/g glucan to 99.5% in the presence of the enzyme cocktail (14.2 FPU CPCel plus 1.21 FPU AFCel per gram of glucan). On the other side, it reduced the total protein amount of CPCel by as much as tenfold, which extremely improved the hydrolytic rate of Spezyme CP and reduced its dosage.
Keywords: Aspergillus fumigatus ; β-glucosidase; Microplate; Enzyme cocktail; Corn stover

Characterization and Enhanced Production of Prodigiosin from the Spoiled Coconut by R. Siva; K. Subha; D. Bhakta; A. R. Ghosh; S. Babu (187-196).
Many bacterial secondary products are bioactive substances that play an important role in biotechnology and pharmacology (e.g., as antibiotics or antitumor agents). Over the past few years interest in prodigiosin has been increased due to its promising anti-cancer activity. Prodigiosin is also of potential clinical interest because it is reported to have anti-fungal, anti-bacterial, anti-protozoal/anti-malarial, and immunosuppressive activity. Thus there is a need to develop a high-throughput and cost-effective bioprocess for the production of prodigiosin. In the present study, Serratia rubidaea was isolated from colored portion of a spoiled coconut and further it was authenticated by MTCC, India. The various parameters like temperature, pH, salt concentration, and precursors were optimized for the production of prodigiosin. We now report that the pigment production was higher in our isolated strain than S. marcescens. It was observed that prodigiosin binds with plastic, paper, and fibers and thus in near future, it can also be used as a natural dye.
Keywords: Serratia rubidaea ; Serratia marcescens ; Prodigiosin; Natural dye

Agar is a complex mixture of gel-forming polysaccharides. Gelling agents are very often used to provide proper support for plants grown in semisolid culture media. And agar is the most frequently used gelling agent in plant tissue culture media. Galactomannans, another group of gel-forming polysaccharides, consists of a (1 → 4)-linked β-d-mannopyranosyl backbone partially substituted at O-6 with d-galactopyranosyl side groups. In this work, we demonstrate that a statistically significant 2.7-fold increase on the multiplication rate (MR) for in vitro-grown Marubakaido (Malus prunifolia) shoots was associated with a 12.5% replacement of agar in the semi-solid culture media for a galactomannan obtained from seeds of Schizolobium paraybae. This increase on MR was due mainly to a 1.9-fold increase in the number of main branches and an 8.6-fold increase in the number of primary lateral branches. Gas liquid chromatography and thin layer chromatography analyzes demonstrated that the galactomannan-driven enhancement of the in vitro multiplication rate of the Marubakaido apple rootstock was not related to the galactomannan degradation. To the best of our knowledge, this is the first report on the successful use of partial replacement of high quality agar by a galactomannan from S. paraybae in a micropropagation system for a tree species.
Keywords: Agar replacement; Galactomannan; HPSEC–MALLS; Gas–liquid chromatograph (GLC); Schizolobium paraybae

Preliminary Studies for Cephamycin C Purification Technique by Álvaro de Baptista Neto; Maritza Catalina Condori Bustamante; Jaine Honorata Hortolan Luiz de Oliveira; Ana Cláudia Granato; Carolina Bellão; Alberto Colli Badino Junior; Marlei Barboza; Carlos Osamu Hokka (208-221).
A study was made for purification of cephamycin C from fermentation of Streptomyces clavuligerus. Initially, the culture broth was clarified by microfiltration and ultrafiltration, after which the resulting permeates were subjected to nonspecific adsorption and ion-exchange chromatography on resin columns. The antibiotic activity was measured by the biological method at each stage by assaying its activity against the Escherichia coli ESS, super sensitive to β-lactam antibiotic. The purification processes were assessed in relation to the variables affecting each step. The purification efficiency by nonspecific adsorption was monitored by UV spectrophotometry, while the ion-exchange adsorption fractions were assessed by NMR spectroscopy. Some of the fractions obtained during purification were also analyzed by mass spectrometry (LC/MS and LC/MS/MS) to identify the cephamycin C molecule. These preliminary results proved the process feasibility.
Keywords: Cephamycin C; Purification; Adsorption; Bioassay; NMR

Molecular Analysis of OsLEA4 and Its Contributions to Improve E. coli Viability by Tingzhang Hu; Hua Zeng; Shuai He; Yingmei Wu; Guixue Wang; Xiaoyun Huang (222-233).
OsLEA4, a late embryogenesis abundant (LEA) protein gene from rice (Oryza sativa L.), contains a 312-bp open reading frame encoding a putative polypeptide of 103 amino acids with a calculated molecular mass of 11.19 kDa and a theoretical pI of 10.04. OsLEA4 polypeptide is rich in Ala (22%), Lys (15%), Glu (9%), His (8%), Thr (8%), and Arg (7%) and lacking in Trp, Cys, Asn, and Phe residues. OsLEA4 protein contains a Pfam:LEA_1 domain architecture at positions 1–73 with three α-helical domains and without β-sheet domain. In silico predictions showed that OsLEA4 protein was strongly hydrophilic with the grand average of hydropathy value of −0.816 and instability index of 27.31. The hydrophilic regions were found in the conserved motif of OsLEA4. OsLEA4 gene was introduced into Escherichia coli, and a fusion protein (∼29.4 kDa) was expressed after isopropylthio-β-d-galactoside inducting by sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. OsLEA4 protein enhanced the tolerance of E. coli recombinant to high salinity, heat, freezing, and UV radiation, which suggested that OsLEA4 protein may play a protective role under stressed conditions. This is the first successful use of E. coli as a prokaryotic system for LEA production from rice.
Keywords: Abiotic stresses; Late embryogenesis abundant proteins; Oryza sativa L.; Protein expression

Discrimination of A1555G and C1494T Point Mutations in the Mitochondrial 12S rRNA Gene by On/Off Switch by Zi-Fen Guo; Wu-Shuang Guo; Li Xiao; Guo-Qiang Gao; Fen Lan; Xue-Guan Lu; Kai Li; Duan-Fang Liao (234-242).
The objective of this study was to apply the “on/off” switch consisting of 3′ phosphorothioate-modified allele specific primers and exo+ polymerase in single base discrimination of A1555G and C1494T mutations in the highly conserved sites of the mitochondrial 12S rRNA. The two point mutations are the hotspot mutations associated with either aminoglycoside antibiotics induced deafness or inherited nonsyndromic hearing loss. The PCR products of mitochondrial DNA (mtDNA) 12S rRNA gene were inserted into the pMD19-T vector for transformation into Escherichia coli JM109 competent cells for preparing wild-type pMD19-T/mt vector. Inverse PCR was carried out for mtDNA 12S rRNA gene C1494T and A1555G mutagenesis and DpnI endonuclease degradating methylated pMD19-T/mt vector existing in the inverse PCR products was carried out to construct the mutation-type pMD19-T/mtM vector. These constructed vectors were confirmed by DNA sequencing. Allelic specific primers targeting wild-type and mutation-type templates were designed with 3′ terminal phosphorothioate modification. Two-directional primer extension was performed using Pfu polymerases. Amplified by exo+ polymerase, allelic specific primers perfectly matching wild-type allele were extended while no products were produced from primers targeting point-mutated deafness-related allele. Similarly, allelic specific primers perfectly matching point-mutated deafness-related mutation-type allele were extended and no products were yielded from primers targeting wild-type allele. No specific product was observed in the primer extension reaction mediated by on/off switch in screening the mtDNA 12S rRNA gene harboring either C1494T or A1555G mutation in 40 healthy volunteers tested. These data suggest that the “off switch” mediated by exo+ polymerase is highly reliable in the diagnosis of monogenic diseases and the novel “on/off” switch has enormous applications in systematic and extended screening of the12S rRNA gene A1555G and C1494T mutations. The established assay can be widely used not only for hearing loss patients but also for normal subjects before the use of aminoglycoside antibiotics.
Keywords: Mitochondrial 12S rRNA; Exo+ polymerase; Phosphorothioate modification; Aminoglycoside antibiotics

In Silico Characterization of Alkaline Proteases from Different Species of Aspergillus by Vivek Kumar Morya; Sangeeta Yadav; Eun-Ki Kim; Dinesh Yadav (243-257).
A total of 49 protein sequences of alkaline proteases retrieved from GenBank representing different species of Aspergillus have been characterized for various physiochemical properties, homology search, multiple sequence alignment, motif, and super family search and phylogenetic tree construction. The sequence level homology was obtained among different groups of alkaline protease enzymes, viz alkaline serine protease, oryzin, calpain-like protease, serine protease, subtilisin-like alkaline proteases. Multiple sequence alignment of alkaline protease protein sequence of different Aspergillus species revealed a stretch of conserved region for amino acid residues from 69 to 110 and 130–204. The phylogenetic tree constructed indicated several Aspergillus species-specific clusters for alkaline proteases namely Aspergillus fumigatus, Aspergillus niger, Aspergillus oryzae, Aspergillus clavatus. The distributions of ten commonly observed motifs were analyzed among these proteases. Motif 1 with a signature amino acid sequence of 50 amino acids, i.e., ASFSNYGKVVDIFAPGQDILSCWIGSTTATNTISGTSMATPHIVGLSCYL, was uniformly observed in proteases protein sequences indicating its involvement with the structure and enzymatic function. Motif analysis of acidic proteases of Aspergillus and bacterial alkaline proteases has revealed different signature amino acid sequences. The superfamily search for these proteases revealed the presence of subtilases, serine-carboxyl proteinase, calpain large subunit, and thermolysin-like superfamilies with 45 representing the subtilases superfamily.
Keywords: Alkaline serine protease; Aspergillus ; Homology; Multiple sequence alignment; Phylogenetic tree; Motif; Superfamily

Erratum to: Preliminary Studies for Cephamycin C Purification Technique by Álvaro de Baptista Neto; Maritza Catalina Condori Bustamante; Jaine Honorata Hortolan Luiz de Oliveira; Ana Cláudia Granato; Carolina Bellão; Alberto Colli Badino; Marlei Barboza; Carlos Osamu Hokka (258-258).