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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.170, #1)

Reductive Decolourisation of Sulphonated Mono and Diazo Dyes in One- and Two-Stage Anaerobic Systems by Marcos Erick Rodrigues da Silva; Paulo Igor Milen Firmino; André Bezerra dos Santos (pp. 1-14).

This work assessed the application of one- and two-stage mesophilic anaerobic systems to colour removal of sulphonated mono and diazo dyes with ethanol as electron donor. The dyes Congo Red (CR), Reactive Black 5 (RB5) and Reactive Red 2 (RR2) were selected as model compounds and tested separately in seven different periods. The one-stage system (R₁) consisted of a single up-flow anaerobic sludge blanket (UASB) reactor, whereas the two-stage system (R₂) consisted of an acidogenic UASB reactor (R_A), a settler and a methanogenic UASB reactor (R_M). For CR and RB5, no remarkable difference was observed between the colour removal performance of both anaerobic systems R₁ and R₂. The experiments with RR2 revealed that R₂ was more efficient on colour removal than R₁, showing efficiencies almost 2-fold (period VI) and 2.5-fold (period VII) higher than those found by R₁. Additionally, R₂ showed a higher stability, giving a good prospect for application to textile wastewaters. Finally, the acidogenic reactor (R_A) had an important role in the overall decolourisation achieved by R₂ during the experiments with CR and RB5 (>78 %), whereas for RR2, a more recalcitrant dye, R_A was responsible for up to 38 % of the total colour removal.

Keywords: Anaerobic treatment; Sulphonated azo dyes; Reductive decolourisation; Two-stage system; Acidogenic reactor

Apoptosis Induced by Aqueous Extracts of Crocodile Bile in Human Heptacarcinoma SMMC-7721 by Wei Song; Shan-Shan Li; Ping-ping Qiu; Dong-Yan Shen; Ling Tian; Qiu-Yan Zhang; Long-Xing Liao; Qing-Xi Chen (pp. 15-24).

In the present study, effects of aqueous extracts from Crocodylus siamensis bile (AE-CB) on SMMC-7721 cell growth, cell cycle, and apoptosis were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, inverted microscopy, fluorescence microscopy, propidium iodide (PI) single- and fluorescein isothiocyanate (FITC)/PI double-staining flow cytometry, and western blotting. Our data have revealed that AE-CB significantly inhibited the growth of SMMC-7721 cell and arrested cell cycle at G0/G1 phase. SMMC-7721 cells showed typical apoptotic morphological changes after treated with AE-CB for 48 h. Cell death assay indicated that SMMC-7721 cells underwent apoptosis in a dose-dependent manner induced by AE-CB. In addition, AE-CB treatment could downregulate the protein level of Bcl-2 and upregulate the Bax, leading to the increase in the ratio of Bax to Bcl-2 in SMMC-7721 cells. Meanwhile, it was observed that the expression of Survivin and c-Myc decreased, but the expression of P53 increased. All these events were associated with increase of reactive oxygen species. The data indicated that mitochondrial pathway might play an important role in bile extract-induced apoptosis in SMMC-7721 cells. These results provide significant insight into the anticarcinogenic action of bile extract on SMMC-7721 cells.

Keywords: Crocodylus siamensis Bile; Antiproliferation; Apoptosis

Silaffin Peptides as a Novel Signal Enhancer for Gravimetric Biosensors by Dong Hyun Nam; Jeong-O Lee; Byoung-In Sang; Keehoon Won; Yong Hwan Kim (pp. 25-31).

Application of biomimetic silica formation to gravimetric biosensors has been conducted for the first time. As a model system, silaffin peptides fused with green fluorescent protein (GFP) were immobilized on a gold quartz crystal resonator for quartz crystal microbalances using a self-assembled monolayer. When a solution of silicic acid was supplied, silica particles were successfully deposited on the Au surface, resulting in a significant change in resonance frequency (i.e., signal enhancement) with the silaffin–GFP. However, frequency was not altered when bare GFP was used as a control. The novel peptide enhancer is advantageous because it can be readily and quantitatively conjugated with sensing proteins using recombinant DNA technology. As a proof of concept, this study shows that the silaffin domains can be employed as a novel and efficient biomolecular signal enhancer for gravimetric biosensors.

Keywords: Silaffin peptides; Biomimetic silica formation; R5 peptide; Quartz crystal microbalances; Signal enhancer

Screening, Gene Sequencing and Characterising of Lipase for Methanolysis of Crude Palm Oil by Enny Ratnaningsih; Dewi Handayani; Fatiha Khairunnisa; Ihsanawati; Sari Dewi Kurniasih; Bill Mangindaan; Sinta Rismayani; Cica Kasipah; Zeily Nurachman (pp. 32-43).

Staphylococcus sp. WL1 lipase (LipFWS) was investigated for methanolysis of crude palm oil (CPO) at moderate temperatures. Experiments were conducted in the following order: searching for the suitable bacterium for producing lipase from activated sludge, sequencing lipase gene, identifying lipase activity, then synthesising CPO biodiesel using the enzyme. From bacterial screening, one isolated specimen which consistently showed the highest extracellular lipase activity was identified as Staphylococcus sp. WL1 possessing lipFWS (lipase gene of 2,244 bp). The LipFWS deduced was a protein of 747 amino acid residues containing an α/β hydrolase core domain with predicted triad catalytic residues to be Ser474, His704 and Asp665. Optimal conditions for the LipFWS activity were found to be at 55 °C and pH 7.0 (in phosphate buffer but not in Tris buffer). The lipase had a K _M of 0.75 mM and a V _max of 0.33 mM min⁻¹ on p-nitrophenyl palmitate substrate. The lyophilised crude LipFWS performed as good as the commonly used catalyst potassium hydroxide for methanolysis of CPO. ESI-IT-MS spectra indicated that the CPO was converted into biodiesel, suggesting that free LipFWS is a worthy alternative for CPO biodiesel synthesis.

Keywords: Biodiesel; Lipase; Crude palm oil; Methanolysis; Staphylococcus sp. WL1

Chemical Modification of Saccharomycopsis fibuligera R64 α-Amylase to Improve its Stability Against Thermal, Chelator, and Proteolytic Inactivation by Wangsa Tirta Ismaya; Khomaini Hasan; Idar Kardi; Amalia Zainuri; Rinrin Irma Rahmawaty; Satyawisnu Permanahadi; Baiq Vera El Viera; Gunawan Harinanto; Shabarni Gaffar; Dessy Natalia; Toto Subroto; Soetijoso Soemitro (pp. 44-57).

α-Amylase catalyzes hydrolysis of starch to oligosaccharides, which are further degraded to simple sugars. The enzyme has been widely used in food and textile industries and recently, in generation of renewable energy. An α-amylase from yeast Saccharomycopsis fibuligera R64 (Sfamy) is active at 50 °C and capable of degrading raw starch, making it attractive for the aforementioned applications. To improve its characteristics as well as to provide information for structural study ab initio, the enzyme was chemically modified by acid anhydrides (nonpolar groups), glyoxylic acid (GA) (polar group), dimethyl adipimidate (DMA) (cross-linking), and polyethylene glycol (PEG) (hydrophilization). Introduction of nonpolar groups increased enzyme stability up to 18 times, while modification by a cross-linking agent resulted in protection of the calcium ion, which is essential for enzyme activity and integrity. The hydrophilization with PEG resulted in protection against tryptic digestion. The chemical modification of Sfamy by various modifiers has thereby resulted in improvement of its characteristics and provided systematic information beneficial for structural study of the enzyme. An in silico structural study of the enzyme improved the interpretation of the results.

Keywords: α-Amylase; Saccharomycopsis fibuligera ; Tryptic digestion; Chemical modification; Enzyme engineering; Structure–function relationship

A New Enrichment Method for Isolation of Bacillus thuringiensis from Diverse Sample Types by Ketan D. Patel; Forum C. Bhanshali; Avani V. Chaudhary; Sanjay S. Ingle (pp. 58-66).

New or more efficient methodologies having different principles are needed, as one method could not be suitable for isolation of organisms from samples of diverse types and from various environments. In present investigation, growth kinetics study revealed a higher germination rate, a higher growth rate, and maximum sporulation of Bacillus thuringiensis (Bt) compared to other Bacillus species. Considering these facts, a simple and efficient enrichment method was devised which allowed propagation of spores and vegetative cells of Bt and thereby increased Bt cell population proportionately. The new enrichment method yielded Bt from 44 out of 58 samples. Contrarily, Bt was isolated only from 16 and 18 samples by sodium acetate selection and dry heat pretreatment methods, respectively. Moreover, the percentages of Bt colonies isolated by the enrichment method were higher comparatively. Vegetative whole cell protein profile analysis indicated isolation of diverse population of Bt from various samples. Bt strains isolated by the enrichment method represented novel serovars and possibly new cry2 gene.

Keywords: Bacillus ; Spore; Enrichment; Growth kinetics

Expression and Purification of Biologically Active Human FGF2 Containing the b′a′ Domains of Human PDI in Escherichia coli by Jung-A Song; Bon-Kyung Koo; Seon Ha Chong; Jihye Kwak; Han-Bong Ryu; Minh Tan Nguyen; Thu Trang Thi Vu; Boram Jeong; Seong Who Kim; Han Choe (pp. 67-80).

Among the members of the fibroblast growth factor (FGF) family that affect the growth, differentiation, migration, and survival of many cell types, FGF2 is the most abundant in the central nervous system. Because of its wound healing effects, FGF2 has potential as a therapeutic agent. The protein is also added to the culture media to maintain stem cells. Expression and purification procedures for FGF2 that are highly efficient and low cost have been intensively investigated for the past two decades. Our current study focuses on the purification of FGF2 fused with b′a′ domains of human protein disulfide isomerase to elevate overexpression, solubility, and stability with a simplified experimental procedure using only ion exchange chromatography, as well as on the confirmation of the biological activity of FGF2 on fibroblast Balb/c 3T3 cells and hippocampal neural cells.

Keywords: hFGF2; Escherichia coli ; PDI; Protein purification

Kinetic and Thermodynamic Characterization of Lysine Production Process in Brevibacterium lactofermentum by Sibtain Ahmed; Munazza Afzal; Muhammad Ibrahim Rajoka (pp. 81-90).

Detailed kinetic and thermodynamic parameters for lysine production from Brevibacterium lactofermentum are investigated for the first time in this study. Production of the essential amino acid, l-lysine, by B. lactofermentum was assessed in a flask and a continuously stirred tank fermentor (22 L). Maximum lysine production was achieved after 40 h of growth and at 35 °C. The effect of different nitrogen sources such as NH₄NO₃, (NH₄)₂SO₄, (NH₄)₂HPO₄, corn steep liquor, NaNO₃, and urea showed that corn steep liquor gave a better lysine yield. Lysine production was increased when dissolved oxygen was maintained at 50 % saturation. The use of dissolved oxygen was critical for high productivity. This indicates that dissolved oxygen greatly affects l-lysine productivity. Kinetic and thermodynamic parameters during lysine production from molasses and glucose mixture showed that B. lactofermentum efficiently converted the substrate mixture into cell mass and lysine. Kinetic and thermodynamic parameters were significantly higher compared with other microorganisms which may be due to the high metabolic activity of B. lactofermentum. This study will have a significant impact on future strategies for lysine production at industrial scale.

Keywords: Batch fermentation; Brevibacterium lactofermentum ; Kinetics; Lysine; Thermodynamics

Acetylcholinesterase Immobilization on Polyacrylamide/Functionalized Multi-walled Carbon Nanotube Nanocomposite Nanofibrous Membrane by Navid Amini; Saeedeh Mazinani; Seyed-Omid Ranaei-Siadat; Mohammad Reza Kalaee; Saeed Hormozi; Kaveh Niknam; Nasrin Firouzian (pp. 91-104).

In this work, polyacrylamide/multi-walled carbon nanotubes (MWCNT) solution is electrospun to nanocomposite nanofibrous membranes for acetylcholinesterase enzyme immobilization. A new method for enzyme immobilization is proposed, and the results of analysis show successful covalent bonding of enzymes on electrospun membrane surface besides their non-covalent entrapment. Fourier transform infrared spectroscopy, mechanical and thermal investigations of nanofibrous membrane approve successful cross-linking and enzyme immobilization. The enzyme relative activity and kinetic on both pure and nanocomposite membranes is investigated, and the results show proper performance of designed membrane to even improve the enzyme activity followed by immobilization compared to free enzyme. Scanning electron microscopy images show nanofibrous web of 3D structure with a low shrinkage and hydrogel structure followed by enzyme immobilization and cross-linking. Moreover, the important role of functionalized carbon nanotubes on final nanofibrous membrane functionality as a media for enzyme immobilization is investigated. The results show that MWCNT could act effectively for enzyme immobilization improvement via both physical (enhanced fibers’ morphology and conductivity) and chemical (enzyme entrapment) methods. Figure Mechanism for APTS surface modification of nanofibrous nanoweb for enzyme immobilization

Keywords: Polyacrylamide nanofibers; Carbon nanotube; Acetylcholinesterase enzyme

Anaerobic Treatment of Industrial Biodiesel Wastewater by an ASBR for Methane Production by Renato C. Silva; José A. D. Rodrigues; Suzana M. Ratusznei; Marcelo Zaiat (pp. 105-118).

A mechanically stirred anaerobic sequencing batch reactor (5 L, 30 °C) containing granular biomass was used to treat the effluent of an industrial biodiesel production process with the purpose to produce methane. Process stability and efficiency were analyzed as a function of applied volumetric organic load (AVOL of 1,000 to 3,000 mgCOD/L), reactor feed time, and cycle length (8-h cycles with 10-min or 4-h feeding and 4-h cycles with 10-min or 2-h feeding). Batch operations (B) with 1,000 to 3,000 mgCOD/L involved 10-min feeding/discharge: (1) 1.0-L influent with 4-h cycle and (2) 2.0-L influent with 8-h cycle. Fed-batch operations (FB) with 1,000 to 3,000 mgCOD/L involved 10-min discharge and the following feeding: (1) 1.0-L influent in 2 h with 4-h cycle and (2) 2.0-L influent in 4 h with 8-h cycle. At 1,000 mgCOD/L (AVOL of 18 to 1.29 gCOD/L day), kinetic parameter values were 1.03 and 0.92 h^-1 at conditions B-1000-4 h and FB-1000-8/4 h, respectively. At both conditions, removal efficiency was 88 %, and cycle length could be reduced to 3 h (B-1000-4 h) and 5 h (FB-1000-8/4 h). At 2,000 mgCOD/L (AVOL of 2.38 to 2.52 gCOD/L day), kinetic parameter values were 1.08 and 0.99 h^-1 at conditions B-2000-4/2 h and FB-2000-8/4 h, respectively, and removal efficiencies were 83 and 81 %. Cycle length could be reduced to 3 h (B-2000-4/2 h) and 6 h (FB-2000-8/4 h). At 3,000 mgCOD/L (AVOL of 3.71 to 3.89 gCOD/L day), conditions allowing stable operation were B-3000-4 h, FB-3000-8/4 h, and FB-3000-4/2 h. Stability could not be obtained at condition B-3000-8 h, and the best results were obtained at condition FB-3000-8/4 h. Specific methane production ranged from 41.1 to 93.7 NmLCH₄/gCOD, demonstrating reactor application potential and operation flexibility.

Keywords: ASBR; Industrial biodiesel effluent; Organic load; Feeding time; Methane; Anaerobic treatment

Thermostable and Alkalistable Endoxylanase of the Extremely Thermophilic Bacterium Geobacillus thermodenitrificans TSAA1: Cloning, Expression, Characteristics and Its Applicability in Generating Xylooligosaccharides and Fermentable Sugars by Digvijay Verma; Ashima Anand; T. Satyanarayana (pp. 119-130).

Xylanase encoding gene (1,224 bp) from Geobacillus thermodenitrificans was cloned in pET28a (+) vector and successfully expressed in Escherichia coli BL21 (DE3). The deduced amino acid sequence analysis revealed homology with that of glycosyl hydrolase (GH) 10 family with a high molecular mass (50 kDa). The purified recombinant xylanase is optimally active at pH 9.0 and 70 °C with T _1/2 of 10 min at 80 °C, and retains greater than 85 % activity after exposure to 70 °C for 180 min. The enzyme liberates xylose as well as xylooligosaccharides from birchwood xylan and agro-residues, and therefore, this is an endoxylanase. The xylan hydrolytic products (xylooligosaccharides, xylose, and xylobiose) find application as prebiotics and in the production of bioethanol. The xylanase being thermostable and alkalistable, it has released chromophores and phenolics from the residual lignin of pulps, suggesting its utility in mitigating chlorine requirement in pulp bleaching.

Keywords: Geobacillus thermodenitrificans ; Thermo-alkalistable xylanase; Pulp pre-bleaching; Xylooligosaccharides; Prebiotics

Decreased Activity of Ca⁺⁺-ATPase and Na⁺/K⁺-ATPase during Aging in Humans by Pawan Kumar Maurya; Siya Prakash (pp. 131-137).

Aging is a biological process characterized by a progressive functional impairment which is associated with increased susceptibility to a variety of diseases. The main purpose of this study is to understand the gender-based relationship between human aging and activities of two erythrocyte membranes bound enzymes, Ca⁺⁺-ATPase and Na⁺/K⁺-ATPase. Ca⁺⁺-ATPase and Na⁺/K⁺-ATPase activities were determined as per the previous reports. Statistical differences were analyzed with Student’s t test. Our results show a significant (p < 0.0001) decrease in the Ca⁺⁺-ATPase and Na⁺/K⁺-ATPase activities in males and females as a function of age. We also correlate the activities of ATPases with total antioxidant capacity of the plasma in term of ferric reducing ability of plasma values. The Ca⁺⁺-ATPase and Na⁺/K⁺-ATPase activities positively correlated with ferric reducing ability of plasma value. No significant differences in the ATPase activity between males and females were observed. Decreased activity of Ca⁺⁺-ATPase and Na⁺/K⁺-ATPase during human aging may be due to increased free radical generation which leads to oxidative stress and alter the erythrocyte membrane transport function and other activities. Our results emphasize the need to establish age-dependent reference values for membrane bound enzymes in studies involving its role in different disease conditions.

Keywords: Ca⁺⁺-ATPase; Na⁺/K⁺-ATPase; Aging; Oxidative stress; Erythrocyte

Gastrodin Production from p-2-Hydroxybenzyl Alcohol Through Biotransformation by Cultured Cells of Aspergillus foetidus and Penicillium cyclopium by Linlin Fan; Yachen Dong; Tengyang Xu; Haifeng Zhang; Qihe Chen (pp. 138-148).

The objective of this work was to take advantage of the resting cells of suitable fungus as an in vitro model to prepare gastrodin from p-2-hydroxybenzyl alcohol (HBA), which mainly exists in the metabolites of the plant Gastrodia elata Blume. The one-step biotransformation of HBA into gastrodin was examined with the filamentous fungi cells of Aspergillus foetidus and Penicillium cyclopium AS 3.4513 in this study. The fundamental conditions of biotransformation were screened and compared for both fungi. P. cyclopium AS 3.4513 had better gastrodin-producing capability than A. foetidus through one-step bioconversion. The highest yield of gastrodin was 36 mg/L for A. foetidus ZU-G1 and 65 mg/L for P. cyclopium AS 3.4513 under the respective development condition during 6 days of biotransformation. The comparative results show that P. cyclopium AS 3.4513 reveals great potential to form gastrodin using HBA as the precursor. The products catalyzed by the resting cells of P. cyclopium AS 3.4513 were identified through NMR and ESI-MS. Current results can be applied not only to the chemical synthesis processes that may involve the hydroxylation reaction but also to the industrial production. The selected fungus is the potential biocatalyst for HBA glucosylation.

Keywords: Gastrodin; HBA; Aspergillus foetidus ; Penicillium cyclopium AS 3.4513; Biotransformation evaluation

Performance and Microbial Diversity of Aerated Trickling Biofilter Used for Treating Cheese Industry Wastewater by Sumathi K. M. Saminathan; Rosa Galvez-Cloutier; Najat Kamal (pp. 149-163).

Wastewater discharged from cheese industries is often characterized by high values of organic pollutants, solids, and nutrients. An aerated trickling biofilter using peat and perlite as filter media was employed in a pilot-scale level in order to evaluate the performance of biofilter for removal of pollutants from cheese industry wastewater. The biofilter was operated for a period of 33 days under laboratory conditions, and several parameters were monitored. The results showed a significant improvement in the quality of treated effluent. The maximum removal efficiencies of chemical oxygen demand and biological oxygen demand were 99.2 and 99.9 %, respectively. Significant reduction in total suspended solids (>96 %) was also achieved. A stable ammoniacal-nitrogen (NH₄-N) removal was accompanied by biofilter. On an average, NH₄-N and total nitrogen decreased by 98.7 and 72 %, respectively, with a significant portion of NH₄-N being converted to nitrate-nitrogen (NO₃-N). Also, a molecular approach based on 16S rDNA was employed to analyze the bacterial community composition present in the biofilter. A comparative sequence analysis of excised denaturing gradient gel electrophoresis bands revealed the presence of diverse groups of bacteria belonging to α- and β-Proteobacteria and Bacteroidetes phylum. We conclude from the results that the use of trickling biofilter is highly effective and a potential treatment method for polishing cheese industry wastewater before being discharged into the local environment.

Keywords: Cheese industry; Wastewater; Trickling biofilter; Purification efficiency; Microbial community structure; DGGE analysis

From Natural Products to Drugs for Epimutation Computer-Aided Drug Design by Naveed A. Chikan; V. Bhavaniprasad; K. Anbarasu; Nadeem Shabir; Trupti N. Patel (pp. 164-175).

The epimutational event, i.e., ectopic methylation in tumor suppressor genes, can lead to gene silencing, thus promoting prognosis of cancer. The progression of DNA methylation is a cycle of demethylation, de novo methylation, and maintenance methylation. The enzyme responsible for maintenance of methylation status is DNA methyltransferase 1 (DNMT1), the continuous activity of which is required to maintain the pattern of epimutation; thus, its inhibition is a promising strategy for the treatment of cancer. To the best of our knowledge, this study is the first to focus on the recently developed crystal structure of the catalytic site of DNMT1. Here in this study, we have used the crystal structure for the development of non-nucleoside DNMT1 inhibitors using virtual screening (VS), absorption, distribution, metabolism, elimination/toxicology analysis, and molecular docking studies. In this study, VS was carried out on 48,531 natural products to create a subset of lead-like natural products. Three of them were found to form hydrogen bonds with the catalytic site of the DNMT1 (Cys 1226). Thus, this study adumbrates potential lead compounds for treatment of epimutation.

Keywords: Cancer epigenetics; Epimutation; DNMT1; Virtual screening; Molecular docking; Non-nucleoside inhibitor

A Comparative Study of Anti-Candida Activity and Phenolic Contents of the Calluses from Lythrum salicaria L. in Different Treatments by Azadeh Manayi; Soodabeh Saeidnia; Mohammad Ali Faramarzi; Nasrin Samadi; Samineh Jafari; Mahdi Vazirian; Ardeshir Ghaderi; Tahmineh Mirnezami; Abbas Hadjiakhoondi; Mohammad Reza Shams Ardekani; Mahnaz Khanavi (pp. 176-184).

In the study, anti-Candida activity and phenol contents of Lythrum salicaria L. calli and wild species have been evaluated. The seeds of L. salicaria (Lythraceae), collected from Lahidjan City in the north of Iran, were cultured in Murashige and Skoog medium (MSM) with a supplement, gibberellin, to germinate. Callus inductions were performed from segments of seedling on MSM containing different concentrations of plant growth regulators, 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP). The activity of calluses extracts, wild plant, gallic acid, and 3,3′,4′-tri-O-methylellagic acid-4-O-β-d-glucopyranoside (TMEG) as the main phenolic compounds against Candida albicans was assessed using cup plate diffusion method. The total phenols contents of calli and wild plant extracts were analyzed using Folin–Ciocalteu reagent. The callus formation in MSM supplemented with various concentrations of 2,4-D and BAP were 0–100 %. Anti-Candida activity of callus extract which obtained from MSM supplemented with 2,4-D and BAP (1 mg dm⁻³) was similar to the wild plant extract. Minimum inhibitory concentration values of gallic acid and TMEG were obtained as 0.312 and 2.5 mg cm⁻³, respectively. Gallic acid equivalent values in all treatments were from 0 to 288 μg GAE mg⁻¹. Phenolic contents of plant aerial parts (331 ± 3.7 μg GAE mg⁻¹) and the callus, which developed in MSM including 1 mg dm⁻³ of both 2,4-D and BAP, showed the same phenolic value and exhibited anti-Candida extract activity.

Keywords: BAP; 2,4-D; Lythraceae; Fungicidal activity; 3,3′,4′-tri-O-methylellagic acid-4-O-β-d-glucopyranoside

Comparison of Glucose and Glycerol as Carbon Sources for ε-Poly-l-Lysine Production by Streptomyces sp. M-Z18 by Xu-Sheng Chen; Zhong-Gui Mao (pp. 185-197).

Glucose is widely used in the production of ε-poly-l-lysine (ε-PL); however, glycerol is an emerging carbon source for ε-PL production in recent years. Glycerol is superior to glucose for ε-PL production according to batch and fed-batch fermentations by Streptomyces sp. M-Z18 in this study. To elaborate this difference, physiological metabolism of Streptomyces sp. M-Z18 on glycerol has compared with glucose during batch fermentation. The activities of key enzymes showed that aspartate kinase (ASPK) and ε-PL synthetase (Pls) in glycerol medium was higher than those in glucose, and especially the activity of Pls could enhance by 2.3- and 3.6-fold at 24 and 36 h, respectively. Moreover, metabolism flux analysis demonstrated that a 25 % higher fluxes derived from glycerol are directed into ε-PL synthesis than glucose. As a result, the mechanism of glycerol better than glucose is determined: the activities of ASPK and Pls in glycerol higher than those in glucose, and result in carbon fluxes directed into ε-PL synthesis increased. This study offers a reference to substitute glycerol for conventional glucose as carbon source for ε-PL production.

Keywords: ε-Poly-l-lysine; Glucose; Glycerol; Batch and fed-batch fermentation; Metabolism flux analysis; Streptomyces

Purification of Acetylcholinesterase by 9-Amino-1,2,3,4-tetrahydroacridine from Human Erythrocytes by Habibe Budak Kaya; Bilge Özcan; Melda Şişecioğlu; Hasan Ozdemir (pp. 198-209).

The acetylcholinesterase enzyme was purified from human erythrocyte membranes using a simple and effective method in a single step. Tacrine (9-amino-1,2,3,4-tetrahydroacridine) is a well-known drug for the treatment of Alzheimer's disease, which inhibits cholinesterase. We have developed a tacrine ligand affinity resin that is easy to synthesize, inexpensive and selective for acetylcholinesterase. The affinity resin was synthesized by coupling tacrine as the ligand and l-tyrosine as the spacer arm to CNBr-activated Sepharose 4B. Acetylcholinesterase was purified with a yield of 23.5 %, a specific activity of 9.22 EU/mg proteins and 658-fold purification using the affinity resin in a single step. During purification, the enzyme activity was measured using acetylthiocholine iodide as a substrate and 5,5′-dithiobis-(2-nitrobenzoicacid) as the chromogenic agent. The molecular weight of the enzyme was determined as about 70 kDa monomer upon disulphide reduction by sodium dodecyl sulphate polyacrylamide gel electrophoresis. K _m, V _max, optimum pH and optimum temperature for acetylcholinesterase were found by means of graphics for acetylthiocholine iodide as the substrate. The optimum pH and optimum temperature of the acetylcholinesterase were determined to be 7.4 and 25–35 °C. The Michaelis–Menten constant (K _m) for the hydrolysis of acetylthiocholine iodide was found to be 0.25 mM, and the V _max was 0.090 μmol/mL/min. Maximum binding was achieved at 2 °C with pH 7.4 and an ionic strength of approximately 0.1 M. The capacity for the optimum condition was 0.07 mg protein/g gel for acetylcholinesterase.

Keywords: Acetylcholinesterase; Enzyme purification; Tacrine; Affinity chromatography

Analysis of Mutation Effects on PIDDosome Core Complex by Tae-Ho Jang; En Kyung Seo; Hyun Ho Park (pp. 210-218).

PIDDosome is a recently-identified caspase-2-activating molecular complex formed by genotoxic stress that leads to caspase-2-dependent apoptosis. PIDD, RAIDD, and caspase-2 are three protein components of PIDDosome. The core portion of PIDDosome is formed by the unique screw rotation of seven RAIDD DD and five PIDD DD. In the current study, we found that two mutations generated during structural-based mutagenesis studies, Q169E and R170A on RAIDD DD, were dominant negative. Because the discovery of dominant-negative mutants might implicate the disease and therapeutic intervention, newly identified dominant-negative mutants could lead to new potential applications for treatment of human diseases caused by excessive or reduced apoptosis.

Keywords: Apoptosis; Caspase-2; RAIDD; PIDD; PIDDosome; Dominant-negative mutant

A Novel Ribonuclease with HIV-1 Reverse Transcriptase Inhibitory Activity from the Edible Mushroom Hygrophorus russula by Mengjuan Zhu; Lijing Xu; Xiao Chen; Zengqiang Ma; Hexiang Wang; T. B. Ng (pp. 219-230).

A 28-kDa ribonuclease, with an optimum pH of 4.0 and an optimum temperature at 58 °C, was isolated from fruiting bodies of the edible mushroom Hygrophorus russula. It was purified by ion exchange chromatography on carboxymethyl-cellulose, dithyaminoethyl-cellulose, quaternary amine-sepharose and sulphopropyl-sepharose, followed by fast protein liquid chromatography gel filtration on Superdex 75. The N-terminal amino acid sequence was ASAGG which showed homology to those of other fungal RNases to some degree. It exerted the highest RNase activity on poly C and poly U. The Michaelis constant (K _m) value of the RNase on yeast tRNA was 3.6 μM, and the maximal velocity (V _max) was 0.6 μM. The RNase activity was suppressed by some ions including Fe²⁺ and Zn²⁺ ions. The RNase inhibited the activity of HIV-1 reverse transcriptase with an IC₅₀ of 4.64 μM.

Keywords: Ribonuclease; Purification; HIV-1 reverse transcriptase; Edible mushroom; Hygrophorus russula

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.170, #1)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.170, #1)