Applied Biochemistry and Biotechnology (v.165, #2)

Insights to Sequence Information of Polyphenol Oxidase Enzyme from Different Source Organisms by Neha Malviya; Mugdha Srivastava; Sanjeev Kumar Diwakar; Sarad Kumar Mishra (397-405).
Polyphenol oxidases (PPOs) are widely distributed enzymes among animals, plants, bacteria, and fungi. PPOs often have significant role in many biologically essential functions including pigmentation, sclerotization, primary immune response, and host defense mechanisms. In the present study, forty-seven full-length amino acid sequences of PPO from bacteria, fungi, and plants were collected and subjected to multiple sequence alignment (MSA), domain identification, and phylogenetic tree construction. MSA revealed that six histidine, two phenylalanine, two arginine, and two aspartic acid residues were highly conserved in all the analyzed species, while a single cysteine residue was conserved in all the plant and fungal PPOs. Two major sequence clusters were constructed by phylogenetic analysis. One cluster was of the plant origin, whereas the other one was of the fungal and bacterial origin. Motif GGGMMGDVPTANDPIFWLHHCNVDRLWAVWQ was found in all the species of bacterial and fungus sources. In addition, seven new motifs which were unique for their group were also identified.
Keywords: Polyphenol oxidase; Sequence analysis; Phylogenetic analysis; Conserved regions; Motifs

Black Liquor Decolorization by Selected White-Rot Fungi by Verónica Da Re; Leandro Papinutti (406-415).
Five different strains of white-rot fungi have been tested for their ability to decolorize black liquor on plates and on solid-state fermentation using vermiculite as the solid inert support. Since the high salt concentration inhibited the growth of all fungi, the black liquor was dialyzed against distilled water prior to use. A preliminary step on plates was carried out to qualitatively determine the capacity of the fungal strains for black liquor decolorization. Out of the five fungi studied, Phanerochaete sordida, Pycnoporus sanguineus, and Trametes elegans exhibited the more conspicuous decolorization halos in malt extract medium, while the decolorization by all the strains was not evident when a defined culture medium was used. Cultures on solid-state fermentation using vermiculite as solid support were also tested, the liquid phase was malt extract or glucose-based medium and supplemented with different black liquor concentrations. Decolorization of black liquor was largely affected by the fungal strain, the concentration of black liquor, and the carbon source. The percentage of color removal ranged from 6.14% to 91.86% depending on the fungal strain and culture conditions. Maximal decolorization was observed in malt extract cultures after 60 cultivation days. Interestingly, decolorization in malt extract medium increased with increasing black liquor concentration. The highest decolorization value was achieved by Steccherinum sp. which reduced up to 91.86% the color of the black liquor in malt extract medium; this percentage is equivalent to 5.2 g L−1 of decolorized black liquor, the highest value reported to date. Traditional technologies used for the treatment of black liquor are not always effective and may not to be an environmentally friendly process. Vermiculite–white-rot fungi systems are presented in this work as a promising efficient alternative for the treatment of black liquor.
Keywords: Black liquor; Decolorization; Laccase; MnP; White-rot fungi

Efficient Production of a Thermophilic 2-Deoxyribose-5-Phosphate Aldolase in Glucose-Limited Fed-Batch Cultivations of Escherichia coli by Continuous Lactose Induction Strategy by Xiao-Lin Pei; Qiu-Yan Wang; Cheng-Lu Li; Xiao-Feng Qiu; Kai-Lin Xie; Li-Feng Huang; An-Ming Wang; Zhao-Wu Zeng; Tian Xie (416-425).
The production of a thermophilic 2-deoxyribose-5-phosphate aldolases (DERA) in Escherichia coli BL21 under continuous lactose induction strategy was investigated. The process was combined with the exponential feeding method, controlling the feeding rate to maintain the specific growth rate at 0.15 h−1. The results indicate that the lactose concentration in the feed medium affected directly the expression of the target protein. The use of 50 g/L in the feed medium resulted in the biomass concentration of 39.3 g DCW/L, and an expression level of above 30%, and the maximum final DERA concentration of 16,200 U/L. Furthermore, the acetate concentration remained at a low level in the fed-batch phase, less than 0.5 g/L. In conclusion, combining glucose feeding with lactose induction is a more powerful way to achieve high cell density cultures and to efficiently produce the thermophilic DERA. The results also indicate the potential industrial utility in the scale production of other recombinant proteins.
Keywords: 2-Deoxyribose-5-phosphate aldolase; Lactose induction; Continuous; Glucose limited; Fed-batch cultivation

A mathematical model that describes the heterogeneous reaction–diffusion process involved in penicillin G hydrolysis in a batch reactor with immobilized penicillin G acylase is presented. The reaction system includes the bulk liquid phase containing the dissolved substrate (and products) and the solid biocatalyst phase represented by glyoxyl-agarose spherical porous particles carrying the enzyme. The equations consider reaction and diffusion components that are presented in dimensionless form. This is a complex reaction system in which both products of reaction and the substrate itself are inhibitors. The simulation of a batch reactor performance with immobilized penicillin G acylase is presented and discussed for the internal diffusional restrictions impact on effectiveness and productivity. Increasing internal diffusional restrictions, through increasing catalyst particle size and enzyme loading, causes impaired catalyst efficiency expressed in a reduction of effectiveness factor and specific productivity. High penicillin G initial concentrations decrease the impact of internal diffusional restrictions by increasing the mass transfer towards porous catalyst until product inhibition becomes significant over approximately 50 mM of initial penicillin G, where a drop in conversion rate and a maximum in specific productivity are then obtained. Results highlight the relevance of considering internal diffusional restrictions, reactor performance, and productivity analysis for proper catalyst and reactor design.
Keywords: Heterogeneous biocatalysis; Immobilized enzyme; Penicillin acylase; Penicillin G; Enzyme reactor; Diffusional restrictions

Food waste and municipal wastewater are promising feedstocks for microbial lipid biofuel production, and corresponding production process is to be developed. In this study, different oleaginous yeast strains were tested to grow in hydrolyzed food waste, and growths of Cryptococcus curvatus, Yarrowia lipolytica, and Rhodotorula glutinis in this condition were at same level as in glucose culture as control. These strains were further tested to grow in municipal primary wastewater. C. curvatus and R. glutinis had higher production than Y. lipolytica in media made from primary wastewater, both with and without glucose supplemented. Finally, a process was tested to grow C. curvatus and R. glutinis in media made from food waste and municipal wastewater, and the effluents from these processes were further treated with yeast culture and phototrophic algae culture; 1.1 g/L C. curvatus and 1.5 g/L R. glutinis biomass were further produced in second-step yeast cultures, as well as 1.53 and 0.58 g/L Chlorella sorokiniana biomass in phototrophic cultures. The residual nitrogen concentrations in final effluents were 33 mg/L and 34 mg/L, respectively, and the residual phosphorus concentrations were 1.5 and 0.6 mg/L, respectively. The lipid contents in the produced biomass were from 18.7% to 28.6%.
Keywords: Oleaginous yeast; Algae; Biodiesel; Municipal wastewater; Food waste

An improved single-step protocol has been developed for extracting pure community humic substance-free DNA from alkaline soils and sediments. The method is based on direct cell lysis in the presence of powdered activated charcoal and polyvinylpolypyrrolidone followed by precipitation with polyethyleneglycol and isopropanol. The strategy allows simultaneous isolation and purification of DNA while minimizing the loss of DNA with respect to other available protocols for metagenomic DNA extraction. Moreover, the purity levels are significant, which are difficult to attain with any of the methods reported in the literature for DNA extraction from soils. The DNA thus extracted was free from humic substances and, therefore, could be processed for restriction digestion, PCR amplification as well as for the construction of metagenomic libraries.
Keywords: Humic substances; Metagenomic DNA; Polyvinylpolypyrrolidone; Polyethylene glycol 8000; Powdered activated charcoal

High Levels of Expression of Fibroblast Growth Factor 21 in Transgenic Tobacco (Nicotiana benthamiana) by Hongqi Fu; Shifeng Pang; Ping Xue; Jing Yang; Xiuming Liu; Yanfang Wang; Tingting Li; Haiyan Li; Xiaokun Li (465-475).
Fibroblast growth factor-21 (FGF21) is a hepatic hormone that plays a critical role in metabolism, stimulating fatty acid oxidation in the liver and glucose uptake in adipose tissue. In this study, we produced tobacco plants expressing human recombinant FGF21 (hFGF21) via Agrobacterium-mediated transformation using a potato virus X (PVX)-based vector (pgR107). The vector contained the sequence encoding the human FGF21 gene fused with green florescence protein and a histidine tag. The recombinant plasmid was introduced into leaf cells of Nicotiana benthamiana (a wild Australian tobacco) via Agrobacterium-mediated agroinfiltration. As determined by fluorescence and Western blot of leaf extracts, the hFGF21 gene was correctly translated in tobacco plants. Seven days after agroinfection, the recombinant hFGF21 had accumulated to levels as high as 450 μg g−1 fresh weight in leaves of agroinfected plants. The recombinant hFGF21 was purified from plant tissues by Ni–NTA affinity chromatography, and the purified hFGF21 stimulated glucose uptake in 3T3/L1 cells. This indicated that the recombinant hFGF21 expressed via the PVX viral vector in N. benthamiana was biologically active.
Keywords: Agroinfection; Fibroblast growth factor 21 (FGF21); Fusion protein; Green fluorescent protein; Nicotiana benthamiana ; Potato virus X

Trehalose Has a Protective Effect on Human Brain-Type Creatine Kinase During Thermal Denaturation by Jiang-Liu Yang; Hang Mu; Zhi-Rong Lü; Shang-Jun Yin; Yue-Xiu Si; Sheng-Mei Zhou; Fang Zhang; Wei-Jiang Hu; Fan-Guo Meng; Hai-Meng Zhou; Zi-Ping Zhang; Guo-Ying Qian (476-484).
We investigated the effects of trehalose on thermal inactivation and aggregation of human brain-type creatine kinase (hBBCK) in this study. In the presence of 1.0 M trehalose, the midpoint temperature of thermal inactivation (T m) of hBBCK increased by 4.6 °C, and the activation energy (E a) for thermal inactivation increased from 29.7 to 41.1 kJ mol−1. Intrinsic fluorescence spectra also showed an increase in the apparent transition temperature (T 1/2) of hBBCK from 43.0 °C to 46.5 °C, 47.7 °C, and 49.9 °C in 0, 0.6, 0.8, and 1.2 M trehalose, respectively. In addition, trehalose significantly blocked the aggregation of hBBCK during thermal denaturation. Our results indicate that trehalose has potential applications as a thermal stabilizer and may aid in the folding of other enzymes in addition to hBBCK.
Keywords: Creatine kinase; Trehalose; Thermal denaturation; Folding; Kinetics

By using three physical techniques (atomic force microscopy (AFM), laser tweezers, and Raman spectroscopy), many excellent works in single-cell/molecule research have been accomplished. In this review, we present a brief introduction to the principles of these three techniques, and their capabilities toward single-cell/molecule research are highlighted. Afterward, the advances in single-cell/molecule research that have been facilitated by these three techniques are described. Following this, their complementary assets for single-cell/molecule research are analyzed, and the necessity of integrating the functions of these three techniques into one instrument is proposed.
Keywords: Single cells; Single molecules; Atomic force microscopy; Laser tweezers; Raman spectroscopy

Conserved Domains, Conserved Residues, and Surface Cavities of C-reactive Protein (CRP) by Suggula Varun Kumar; Roshini Kulampurathu Ravunny; Chiranjib Chakraborty (497-505).
C-reactive protein (CRP) is a highly conserved plasma protein belonging to pentraxins, a superfamily which has significant proinflammatory role. Therefore, CRP can be a good target for drug discovery to prevent disease pathogenesis, especially cardioprotection in acute myocardial infarction and neuroprotection in stroke. Hence, the knowledge of the structure of CRP is very important. In this paper, we have demonstrated three-dimensional structure, conserved domains, and surface structure of human CRP with the help of bioinformatics analysis. We have also depicted that evolutionary relationship of CRP exists among the different species. Simultaneously, WebLogo has been generated to know the more conserved part of this medically important protein.
Keywords: Conserved domains; Conserved residues; Surface cavities; C-reactive protein (CRP)

Oxidative Lime Pretreatment of Alamo Switchgrass by Matthew Falls; Mark T. Holtzapple (506-522).
Previous studies have shown that oxidative lime pretreatment is an effective delignification method that improves the enzymatic digestibility of many biomass feedstocks. The purpose of this work is to determine the recommended oxidative lime pretreatment conditions (reaction temperature, time, pressure, and lime loading) for Alamo switchgrass (Panicum virgatum). Enzymatic hydrolysis of glucan and xylan was used to determine the performance of the 52 studied pretreatment conditions. The recommended condition (110°C, 6.89 bar O2, 240 min, 0.248 g Ca(OH)2/g biomass) achieved glucan and xylan overall yields (grams of sugar hydrolyzed/100 g sugar in raw biomass, 15 filter paper units (FPU)/g raw glucan) of 85.9 and 52.2, respectively. In addition, some glucan oligomers (2.6 g glucan recovered/100 g glucan in raw biomass) and significant levels of xylan oligomers (26.0 g xylan recovered/100 g xylan in raw biomass) were recovered from the pretreatment liquor. Combining a decrystallization technique (ball milling) with oxidative lime pretreatment further improved the overall glucan yield to 90.0 (7 FPU/g raw glucan).
Keywords: Switchgrass; Pretreatment; Lime; Enzymatic digestion

The toxicity of the recombinant protein towards the expression host remains a significant deterrent for bioprocess development. In this study, the expression of human granulocyte macrophage-colony stimulating factor (hGM-CSF), which is known to be toxic to its host, was enhanced many folds using a combination of genetic and bioprocess strategies in Escherichia coli. The N terminus attachment of endoxylanase and asparaginase signal sequences from Bacillus subtilis and E. coli, respectively, in combination with and without His-tag, considerably improved expression levels. Induction and media optimization studies in shake flask cultures resulted in a maximal hGM-CSF concentration of 365 mg/L in the form of inclusion bodies (IBs) with a specific product yield (Y P/X) of 120 mg/g dry cell weight in case of the asparaginase signal. Culturing the cells in nutrient rich Terrific broth maintained the specific product yields (Y P/X) while a 6.6-fold higher volumetric concentration of both product and biomass was obtained. The purification and refolding steps were optimized resulting in a 95% pure protein with a fairly high refolding yield of 45%. The biological activity of the refolded protein was confirmed by a cell proliferation assay on hGM-CSF dependent human erythroleukemia TF-1 cells. This study demonstrated that this indeed is a viable route for the efficient production of hGM-CSF.
Keywords: hGM-CSF; Asparaginase; Endoxylanase; Signal sequence; Recombinant protein; E. coli

Differential Effects of Cold Exposure on Gene Expression Profiles in White Versus Brown Adipose Tissue by Masahiro Watanabe; Takenori Yamamoto; Atsushi Yamamoto; Eriko Obana; Kanami Niiyama; Takuya Hada; Toshihiko Ooie; Masatoshi Kataoka; Tomoshige Hori; Hitoshi Houchi; Yasuo Shinohara (538-547).
The thermogenic function of brown adipose tissue (BAT) is known to be markedly elevated when animals are exposed to the cold, and intensive studies have been carried out to understand the molecular basis enabling effective thermogenesis in cold-exposed animals. In this study, we used microarray analysis to examine the effects of cold exposure of animals on their gene expression profiles in white adipose tissue (WAT), which seems to function as a counterpart tissue of BAT. The results indicate that the effects of cold exposure on the gene expression profiles of WAT were much more moderate than the effects on those of BAT. Possible reasons for the different responses of BAT and WAT to cold exposure are discussed.
Keywords: White adipose tissue; Brown adipose tissue; Cold exposure; Gene expression; Microarray analysis

Detection of Liposome Lysis Utilizing an Enzyme–Substrate System by Daniel J. Wichelecki; Trisha M. McNew; Aysegul Aygun; Kathryn Torrey; Larry D. Stephenson (548-558).
A novel optical reporter system was developed to verify encapsulation and subsequent release of a foreign molecule in liposomes. The protocol utilizes a single enzyme and substrate. We encapsulate o-nitrophenyl-β,d-galactopyranoside (ONPG) and measure its release by detecting the levels of o-nitrophenol created when the encapsulated ONPG is released and hydrolyzed by β-galactosidase. Using this method, liposome formation and subsequent lysis with Triton X-100 were verified. This new protocol eliminates the complications of multiple reaction enzyme detection methods, along with the chance for false negatives and unreliable data seen when using fluorescent particles as reporters.
Keywords: β-Galactosidase; Encapsulation; Liposome; Lysis; ONP; ONPG

Improved porcine interferon-α (pIFN-α) production by recombinant Pichia pastoris was achieved by culture conditions optimization in a 5-l bioreactor. The results indicated that the pIFN-α concentration, specific methanol consumption rate, specific activities of alcohol oxidase, formaldehyde dehydrogenase, and formate dehydrogenase could be significantly enhanced by decreasing induction temperature. The highest pIFN-α concentration (1.35 g l−1) was obtained by simultaneously controlling methanol concentration at 5 g l−1 and induction temperature at 20 °C, which was about 1.6-fold higher than the maximum obtained with previous optimal methanol concentration level (about 10 g l−1) when inducing at 30 °C. The potential mechanisms behind low temperature and low methanol concentration effect on pIFN-α production may be ascribed to higher cell metabolic activity, more carbon flux towards pIFN-α production, and less intracellular/extracellular protease release.
Keywords: Pichia pastoris ; Fed-batch cultivation; Induction; Metabolic flux; Porcine interferon-α

Effects of Sucrose and Trehalose on Stability, Kinetic Properties, and Thermal Aggregation of Firefly Luciferase by Sanaz Rasouli; Saman Hosseinkhani; Parichehreh Yaghmaei; Azadeh Ebrahim-Habibi (572-582).
In this study, we used sugars as stabilizing additives to improve the thermostability and to inhibit aggregation of firefly luciferase. The combination of sucrose and trehalose has a strong stabilizing effect on firefly luciferase activity and prevents its thermoinactivation. These additives can also increase optimum temperature. It has been shown that the presence of both sucrose and trehalose can inhibit thermal aggregation of firefly luciferase and decrease bioluminescence decay rate. In order to understand the molecular mechanism of thermostabilization, we investigated the effects of sucrose and trehalose combination on the secondary structure of luciferase by Fourier transform infrared spectroscopy. Minor changes in content of secondary structure of firefly luciferase are observed upon treatment with additives.
Keywords: Aggregation; Bioluminescence; Firefly luciferase; Thermostability

Cloning, Sequencing, and Identification Using Proteomic Tools of a Protease from Bromelia hieronymi Mez by Mariela Anahí Bruno; Sebastián Alejandro Trejo; Francesc Xavier Avilés; Néstor Oscar Caffini; Laura Maria Isabel López (583-593).
Fruits of Bromelia hieronymi, a tropical South American plant, possess a high content of peptidases with potential biotechnological uses. Total RNA was extracted from unripe fruits and peptidase cDNA was obtained by 3′RACE-PCR. The consensus sequence of the cysteine peptidase cDNA contained 875 bp, the 690 first ones codifying for a hypothetical polypeptide chain of the mature peptidase, named Bh-CP1 (molecular mass 24.773 kDa, pI 8.6, extinction molar coefficient 58,705 M−1 cm−1). Bh-CP1 sequence shows a high percentage of identity with those of other cysteine plant proteases. The presence of highly preserved residues is observed, like those forming the catalytic site (Gln19, Cys25, His159, and Asn175, papain numbering), as well as other six Cys residues, involved in the formation of disulfide bounds. Molecular modeling results suggest the enzyme belongs to the α + β class of proteins, with two disulfide bridges (Cys23–Cys63 and Cys57–Cys96) in the α domain, while the β domain is stabilized by another disulfide bridge (Cys153–Cys203). Additionally, peptide mass fingerprints (PMFs) of the three peptidases previously isolated from B. hieronymi fruits (namely hieronymain I, II, and III) were performed and compared with the theoretical fingerprint of PMF of Bh-CP1, showing a partial matching between the in silico-translated protein and hieronymain II.
Keywords: Bromelia hieronymi ; Bromeliaceae; Cloning; Sequencing; Cysteine endopeptidase; Hieronymain II; Proteomic tools

Cellulases and Hemicellulases from Endophytic Acremonium Species and Its Application on Sugarcane Bagasse Hydrolysis by Maíra Nicolau de Almeida; Valéria Monteze Guimarães; Kenneth M. Bischoff; Daniel Luciano Falkoski; Olinto Liparini Pereira; Dayelle S. P. O. Gonçalves; Sebastião Tavares de Rezende (594-610).
The aim of this work was to have cellulase activity and hemicellulase activity screenings of endophyte Acremonium species (Acremonium zeae EA0802 and Acremonium sp. EA0810). Both fungi were cultivated in submerged culture (SC) containing l-arabinose, d-xylose, oat spelt xylan, sugarcane bagasse, or corn straw as carbon source. In solid-state fermentation, it was tested as carbon source sugarcane bagasse or corn straw. The highest FPase, endoglucanase, and xylanase activities were produced by Acremonium sp. EA0810 cultivated in SC containing sugarcane bagasse as a carbon source. The highest β-glucosidase activity was produced by Acremonium sp. EA0810 cultivated in SC using d-xylose as carbon source. A. zeae EA0802 has highest α-arabinofuranosidase and α-galactosidase activities in SC using xylan as a carbon source. FPase, endoglucanase, β-glucosidase, and xylanase from Acremonium sp. EA0810 has optimum pH and temperatures of 6.0, 55 °C; 5.0, 70 °C; 4.5, 60 °C; and 6.5, 50 °C, respectively. α-Arabinofuranosidase and α-galactosidase from A. zeae EA0802 has optimum pH and temperatures of 5.0, 60 °C and 4.5, 45 °C, respectively. It was analyzed the application of Acremonium sp. EA0810 to hydrolyze sugarcane bagasse, and it was achieved 63% of conversion into reducing sugar and 42% of conversion into glucose.
Keywords: Cellulase; Hemicellulase; Acremonium ; Endophyte; Ethanol; Agroindustrial residue

Expression and Production of Therapeutic Recombinant Human Platelet-Derived Growth Factor-BB in Pleurotus eryngii by Jun-Hui Choi; Seung Kim; Kumar Sapkota; Se-Eun Park; Sung-Jun Kim (611-623).
Recombinant human platelet-derived growth factor-BB (rhPDGF-BB) is widely used in many therapeutic applications. Until now, there has been no report on rhPDGF-BB expressed in fungi. In this study, we tested whether Pleurotus eryngii could support the expression of human therapeutic rhPDGF-BB protein. A binary vector pCAMBIA1304 containing the hPDGF-BB gene was constructed and introduced into P. eryngii via Agrobacterium tumefaciens-mediated transformation. The transformation of hPDGF-BB gene was confirmed by Southern blot and PCR, whereas the expression was confirmed by Western blot analysis. The recombinant hPDGF-BB reached a maximum expression level of 1.98% of total soluble protein in transgenic mycelia and was in dimeric form. A bioassay revealed that hPDGF-BB expressed in P. eryngii increased proliferation of NIH-3T3 cells similarly to standard material. These results suggest that P. eryngii can be a robust system for the production of human therapeutic proteins including the hPDGF-BB.
Keywords: Agrobacterium tumefaciens ; Human platelet-derived growth factor-BB; Pleurotus eryngii ; Gene transfer; Gene expression

Immunomodulatory and Therapeutic Potential of a Mycelial Lectin from Aspergillus nidulans by Ram Sarup Singh; Ranjeeta Bhari; Vikas Rana; Ashok Kumar Tiwary (624-638).
Lectins bind to surface receptors on target cells, and activate a cascade of events, eventually leading to altered immune status of host. The immunomodulatory potential of purified lectin from Aspergillus nidulans was evaluated in Swiss albino mice treated intraperitoneally with seven different doses of purified lectin. Lectin prevented BSA-induced Arthus reaction and systemic anaphylaxis. The enhanced functional ability of macrophages was evident from respiratory burst activity and nitric oxide production in splenocyte cultures. Interferon-gamma and interleukin-6 levels were significantly up-regulated in treated groups. Maximum stimulatory effect was observed at the dose of 1.5 mg/kg body weight. Therapeutic potential of A. nidulans lectin was assessed against trinitrobenzene sulfonic acid-induced ulcerative colitis in male Wistar rats. Rats pre-treated with 80 mg/kg body weight of purified lectin intraperitoneally prior to colitis induction showed lesser disease severity and recovery within 7 days, while rats post-treated with the same dose showed recovery in 11 days. The results demonstrate immunomodulatory effects of A. nidulans lectin in Swiss albino mice, resulting in improved immune status of the animals and unfold its curative effect against ulcerative colitis in rat model. This is the first report on immunomodulatory and therapeutic potential of a lectin from microfungi.
Keywords: Aspergillus nidulans ; Lectin; Immune stimulation; Colitis; Therapeutic potential

Fungal isolates (Aspergillus wentii 1, A. wentii 2, Penicillium citrinum, Penicillium granulatum) were selected to study their in vitro antioxidant potential by various assay procedures. Czapek–Dox’s medium was selected for the growth of fungi as it supported the best antioxidant activity based on their EC50 values, P. citrinum was the best followed by P. granulatum, A. wentii 1, and A. wentii 2. The chromatographic analyses showed several compounds possessing antioxidant activity in the fungal extracts. Two such compounds were partially purified from P. citrinum which demonstrated potent antioxidant activity, equally effective or better than some of the standard antioxidants.
Keywords: Antioxidant activity; Dot blot assay; Fungi; HPLC; Aspergillus wentii ; Penicillium citrinum ; Penicillium granulatum

Molecular Cloning and Transgenic Expression of a Synthetic Human Erythropoietin Gene in Tobacco by Fernanda Sperb; Isabel C. R. Werlang; Marcia Margis-Pinheiro; Luiz A. Basso; Diógenes S. Santos; Giancarlo Pasquali (652-665).
Erythropoietin (EPO) is a hormone belonging to a group of hematopoietic growth factors that control the proliferation and differentiation of bone marrow cells. It induces the production of erythrocytes, thereby increasing the amount of circulating hemoglobin and oxygen. Previous attempts to transgenically express human EPO in plants failed to succeed because the plants exhibited abnormal morphology and infertility. In the present work, we describe the generation of fertile transgenic tobacco plants able to express a synthetic version of human EPO. A 582-bp fragment of the human EPO gene was synthesized using a PCR-based method and ligated into pCR-Blunt. After sequencing, the human EPO fragment was transferred to pWUbi.tm1 and the expression cassette was then transferred to the binary vector pWBVec4a. After Agrobacterium-mediated transformation of Nicotiana tabacum SR1 plants, integration of the transgene into T0 and T1 plant genomes was confirmed by PCR. The human EPO gene was found to be expressed in tobacco leaves at the mRNA and protein levels. Self-crossing allowed us to obtain T1 plants exhibiting Mendelian segregation of the transgene. None of the plants presented any kind of malformation or deformity.
Keywords: Agrobacterium ; Recombinant protein; Erythropoietin; Heterologous expression; Molecular breeding; Transgenic tobacco; Ubiquitin promoter

Escherichia coli is one of the most commonly used host strains for recombinant protein production. More and more research works on the production of recombinant protein indicate that extracellular production throughout a culture medium is more convenient and attractive compared to intracellular production. In present work, inducing temperature and isopropyl β-d-1-thiogalactopyranoside (IPTG) concentration were investigated to decrease the formation of inclusion body and increase the amount of soluble recombinant cutinase initially. Enzyme activity in the culture medium reached to 118.9 U/ml at 64 h of culture, and no inclusion body was detected in cytoplasm under the inducement condition of 0.2 mM IPTG and 30°C. In addition, it was found that a large amount of cutinase had been accumulated in periplasm since 16-h cultivation under the same inducement condition. Therefore, glycine and surfactant sodium taurodeoxycholate (TDOC) were further used to promote the leakage of recombinant cutinase from periplasm. Supplied with 100 mM glycine and 1 mM TDOC, the amount of cutinase in periplasm decreased remarkably, and the activity in the culture medium reached to 146.2 and 149.2 U/ml after 54 h of culturing, respectively.
Keywords: Thermobifida fusca ; Cutinase; Escherichia coli ; Extracellular production

Impact of High Pyruvate Concentration on Kinetics of Rabbit Muscle Lactate Dehydrogenase by Matthew Warren Eggert; Mark E. Byrne; Robert P. Chambers (676-686).
In order to evaluate the effectiveness of l-lactate dehydrogenase (LDH) from rabbit muscle as a regenerative catalyst of the biologically important cofactor nicotinamide adenine dinucleotide (NAD), the kinetics over broad concentrations were studied to develop a suitable kinetic rate expression. Despite robust literature describing the intricate complexations, the mammalian rabbit muscle LDH lacks a quantitative kinetic rate expression accounting for simultaneous inhibition parameters, specifically at high pyruvate concentrations. Product inhibition by l-lactate was observed to reduce activity at concentrations greater than 25 mM, while expected substrate inhibition by pyruvate was significant above 4.3 mM concentration. The combined effect of ternary and binary complexes of pyruvate and the coenzymes led to experimental rates as little as a third of expected activity. The convenience of the statistical software package JMP allowed for effective determination of experimental kinetic constants and simplification to a suitable rate expression: $$ v = frac{{{V_{max}}left( {AB} ight)}}{{{K_{ia}}{K_b} + {K_b}A + {K_a}B + AB + frac{P}{{{K_{I - Lac}}}} + frac{{{B^2}A}}{{{K_{I - Pyr}}}} + frac{{{B^2}Q}}{{{K_{I - Pyr - NAD}}}}}} $$ where the last three terms represent the inhibition complex terms for lactate, pyruvate, and pyruvate–NAD, respectively. The corresponding values of K I–Lac, K I–Pyr, and K I–Pyr–NAD for rabbit muscle LDH are 487.33 mM−1 and 29.91 mM and 97.47 mM at 22 °C and pH 7.8.
Keywords: l-Lactate dehydrogenase; Enzyme kinetics; Pyruvate substrate inhibition; Rabbit muscle; Statistical software kinetic modeling

Microbial Biosensors for Organophosphate Pesticides by Ashok Mulchandani; Rajesh (687-699).
Organophosphates, amongst the most toxic substance known, are used widely in agriculture around the world. Their extensive use, however, has resulted in their occurrence in the water and food supply threatening humans and animals. Therefore, there is a need for determination of these neurotoxic compounds sensitively, selectively, and rapidly in the field. The present work is a brief review on the recent advancements in amperometric, potentiometric, and optical biosensors using genetically engineered microorganisms expressing organophosphate hydrolyzing enzyme intracellularly or anchored on the cell surface for the detection of organophosphate pesticides. The benefits and limitations associated with such microbial biosensors are delineated.
Keywords: Biosensor; Microbial; Organophosphate; Nerve agents; Pesticides

Co-cultured Production of Lignin-Modifying Enzymes with White-Rot Fungi by Chen Qi-he; Sven Krügener; Thomas Hirth; Steffen Rupp; Susanne Zibek (700-718).
Co-cultivation was a potential strategy in lignocellulolytic biodegradation with producing high activity enzymes due to their synergistic action. The objective of this study was to investigate the rarely understood effects of co-culturing of two white-rot fungi on lignin-modifying enzymes (LMEs) production. Six species, Bjerkandera adusta, Phlebia radiata, Pleurotus ostreatus, Dichomitus squalens, Hypoxylon fragiforme and Pleurotus eryngii, were cultured in pairs to study the production of LMEs. The paired hyphal interaction observed showed that P. eryngii is not suitable for co-growth. The use of agar plates containing dye RBBR showed elevated decolourisation at the confrontation zone between mycelia. Laccase was significantly stimulated only in the co-culture of P. radiata with D. squalens under submerged cultivation; the highest value was measured after 4 days of incubation (120 U mg−1). The improved productions of MnP and LiP were simultaneously observed at the co-culture of P. ostreatus and P. radiata (MnP = 800 nkat L−1 after 4 days of incubation; LiP = 60 nkat L−1 after 7 days of incubation), though it was not a good producer of laccase. P. ostreatus appeared to possess specific potential to be used in co-cultured production of LMEs. The phenotype of LMEs production was not only dependent on the species used but also regulated by different nutritions available in the culture medium. The present data will provide evidence for illustrating the regulatory roles of C/N on LMEs production under the co-cultures’ circumstances.
Keywords: LMEs; White-rot fungi; Co-cultivation; Interspecies interaction; Nutrients regulation

Biosolubilization of Rock Phosphate by Three Stress-Tolerant Fungal Strains by Chunqiao Xiao; Ruan Chi; Xiaohong Li; Min Xia; Zhongwu Xia (719-727).
Three stress-tolerant phosphate-solubilizing fungal strains identified as Aspergillus niger, Aspergillus japonicus, and Penicillium simplicissimum were isolated from wheat rhizospheric soil. The strains demonstrated different capabilities of phosphate solubilization in National Botanical Research Institute’s phosphate medium containing rock phosphate (RP) as sole phosphorus (P) source, and the solubilization of RP by P. simplicissimum was the most effective among these strains, followed by A. niger and A. japonicus. All the strains exhibited high levels of stress tolerance like 10∼45°C temperature, 4∼11 pH, 0∼3.5% NaCl, and 0∼35% PEG 10000. The strains also differed in their abilities to survive and release soluble P from RP under different stresses. A. niger showed significantly higher tolerance to temperature and pH over the other two strains. Higher amount of spores and content of soluble P in the medium were observed in the presence of 3.5% NaCl with P. simplicissimum, followed by A. niger and A. japonicus. P. simplicissimum could not solubilize RP in the presence of 35% PEG 10000, which exhibited the lowest tolerance to desiccation stress among the three strains.
Keywords: Biosolubilization; Rock phosphate (RP); Phosphate-solubilizing fungi; Stress; Phosphorus (P)

Heterologous Expression Characteristics of Trichoderma viride Endoglucanase V in the Silkworm, Bombyx mori L. by Xing-hua Li; Mei-xian Wang; Peng Zhang; Jia-biao Hu; Chun-guang Sun; Xin-ju Liu; Fang Zhou; Yan-shan Niu; Firdose Ahmad Malik; Roy Bhaskar; Hua-jun Yang; Yun-gen Miao (728-736).
Efficient degradation of cellulose needs a synergistic reaction of the cellulolytic enzymes, which include exoglucanases, endoglucanases, and β-1,4-glucosidase. In this study, we used an improved Bac-to-Bac/BmNPV baculovirus expression system, which lacks the virus-encoded chitinase cathepsin (v-cath) genes of Bombyx mori nucleopolyhedrovirus (BmNPV), to express the endoglucanase V (EG V) gene from Trichoderma viride in silkworm BmN cells and silkworm larvae, and analyzed the characteristics of the recombinant enzyme in silkworm larvae. The result showed that an around 36-kDa protein was visualized in BmN cells at 48 h after the second-generation recombinant mBacmid/BmNPV/EG V baculovirus infection. The crude enzyme extract from the recombinant baculoviruses-infected silkworms exhibited a significant maximum activity at the environmental condition of pH 5.0 and a temperature of 50 °C, and increased 39.86% and 37.76% compared with that from blank mBacmid/BmNPV baculovirus-infected silkworms and normal silkworms, respectively. It was stable at pH range from 5.0 to 10.0 and at temperature range from 40 to 60 °C. The availability of large quantities of EG V that the silkworm provides might greatly facilitate the future research and the potential application in industries.
Keywords: Trichoderma viride ; Cellulase; Bombyx mori

Bacteriocin Release Protein-Mediated Secretory Expression of Recombinant Chalcone Synthase in Escherichia coli by Iffah Izzati Zakaria; Raja Noor Zaliha Raja Abdul Rahman; Abu Bakar Salleh; Mahiran Basri (737-747).
Flavonoids are secondary metabolites synthesized by plants shown to exhibit health benefits such as anti-inflammatory, antioxidant, and anti-tumor effects. Thus, due to the importance of this compound, several enzymes involved in the flavonoid pathway have been cloned and characterized in Escherichia coli. However, the formation of inclusion bodies has become a major disadvantage of this approach. As an alternative, chalcone synthase from Physcomitrella patens was secreted into the medium using a bacteriocin release protein expression vector. Secretion of P. patens chalcone synthase into the culture media was achieved by co-expression with a psW1 plasmid encoding bacteriocin release protein in E. coli Tuner (DE3) plysS. The optimized conditions, which include the incubation of cells for 20 h with 40 ng/ml mitomycin C at OD600 induction time of 0.5 was found to be the best condition for chalcone synthase secretion.
Keywords: Bacteriocin release protein; Chalcone synthase; Extracellular expression

Treatment of Bleached Wool with Trans-Glutaminases to Enhance Tensile Strength, Whiteness, and Alkali Resistance by Majid Montazer; Fatemeh Lessan; Elmira Pajootan; Fatemeh Dadashian (748-759).
Trans-glutaminases is known as a cross-linking enzyme for proteins. Wool is a proteinous fiber conventionally is treated through several processes to obtain the desirable characteristics. Bleaching is also one of the most important processes usually carried out by using an oxidizing agent in a conventional method. The tensile strength of wool yarns was reduced as a consequence of oxidative bleaching. Here, with the help of microbial trans-glutaminases (m-TGases), a novel bleaching process was disclosed in a way to obtain a bleached wool yarn with no significant reduction in the tensile strength. The results confirmed that the bleached wool yarns with H2O2 could be modified by m-TGases post-treatment. The m-TGases treatment on the bleached wool yarns improved the tensile strength and whiteness along with the higher alkali resistance.
Keywords: Microbial trans-glutaminases; Bleaching; Wool; Tensile strength; Whiteness; Alkali solubility