Applied Biochemistry and Biotechnology (v.162, #3)

Ethanol production from lignocellulose by recombinant yeast with high level expression of heterologous cellulase genes has been a major anticipation. The native secretion signal sequence of the cellulase endoglucanase I (eg1) gene was replaced by Saccharomyces cerevisiae mating factor α prepro-leader sequence (MFα). The transformants containing native secretion signal (Y 1) and MFα secretion signal (Y 2) were characterized with respect to gene expression and growth on cellulose substrate. Increased enzyme activity and cellulose utilization were observed. The enzyme activity of Y 2 was 0.084 U/ml, 61.5% higher than Y 1 (0.052 U/ml). The sufficiency parameter (S value) was raised from 0.6 to 0.84. MFα signal peptide was more efficient than the native signal peptide of eg1 gene, suggesting that signal peptide replacement is an efficient way to enhance the cellulase expression level in yeast, for cellulose-derived ethanol production.
Keywords: Gene expression; Signal peptide; Endoglucanase I; Yeast

Marinobacter sp. (MSI032) isolated from the marine sponge Dendrilla nigra was optimized for the production of extracellular cellulolytic enzyme (CMCase) by submerged fermentation. Initial experiments showed that the culture medium containing 1% maltose as carbon source and 1% peptone and casein as nitrogen source supported maximal enzyme production at 27 °C and at a pH of 9.0. Further optimization carried out showed the maximal enzyme production was supported by the presence of 2% NaCl and 10 mM Zn2+ ions in the production media. The production of enzyme cellulase occurred at 48 h of incubation which proved the importance of this strain for cellulase production in large scale. Further, the enzyme was purified to 12.5-fold with a 37% yield and a specific activity of 2,548.75 U/mg. The purified enzyme displayed maximum activity at mesophilic temperature (27–35 °C) and at a broad pH range with optimal activity at pH 9.0. The purified enzyme was stable even at a higher alkaline pH of 12.0 which is greater than the pH stability that has not been reported in any of the cellulolytic isolates studied so far. Thus, from the present study, it is crucial that, instead of exploring the thermophilic resource that is limited in natural environments, the mesophilic bacteria that occurs commonly in nature can be added up to the database of cellulolytic bacteria. Thus, it is possible that a wide diversity of mesophilic bacteria associated with marine sponges opens up a new doorstep for the degradation of cellulosic waste material for the production of liquid fuels. This is the first report elucidating the prospects of sponge-associated marine bacterium for the production of extracellular alkaline cellulase.
Keywords: Extracellular cellulase; Sponge bacteria; Marinobacter ; Marine cellulase; Alkaline cellulase

Kinetic Evaluation of Aminoethylisothiourea on Mushroom Tyrosinase Activity by Shu-Bai Li; Hua-Li Nie; Hai-Tao Zhang; Yong Xue; Li-Min Zhu (641-653).
This study demonstrates that aminoethylisothiourea (AET), a potent inhibitor of inducible nitric oxide synthase, is an irreversible competitive inhibitor of mushroom tyrosinase by chelation to the active site of tyrosinase when l-3,4-dihydroxyphenylalanine was assayed spectrophotometrically. The spectrophotometric recordings of the inhibition of tyrosinase by AET were characterized by the presence of a lag period prior to the attainment of an inhibited steady-state rate. The lag period corresponded to the time in which AET was reacting with the enzymatically generated o-quinone. Increasing AET concentrations provoked longer lag periods as well as a concomitant decrease in the tyrosinase activity. Both lag period and steady-state rate were dependent on AET, substrate, and tyrosinase concentrations. The inhibition of diphenolase activity of tyrosinase by AET showed positive kinetic cooperativity which arose from the protection of both substrate and o-quinone against inhibition by AET. The UV-visible spectrum of a mixture of tyrosinase and AET exhibited a characteristic shoulder peak ascribed to the chelation of AET to the active site of tyrosinase. Moreover, the presence of copper ions only partially prevented but not reverted mushroom tyrosinase inhibition when CuSO4 was added to the assay medium on tyrosinase activity.
Keywords: Aminoethylisothiourea; Mushroom tyrosinase; Irreversible inhibition; Cooperativity; Copper chelation

Simulated Microgravity Affects Growth of Escherichia coli and Recombinant β-d-Glucuronidase Production by Liang Xiang; Feng Qi; DaZhang Dai; Chun Li; YuanDa Jiang (654-661).
Effects of simulated microgravity (SMG) on bacteria have been studied in various aspects. However, few reports are available about production of recombinant protein expressed by bacteria in SMG. In this study growth of E. coli BL21 (DE3) cells transformed with pET-28a (+)-pgus in double-axis clinostat that could model low shear SMG environment and the recombinant β-d-glucuronidase (PGUS) expression have been investigated. Results showed that the cell dry weights in SMG were 16.47%, 38.06%, and 28.79% more than normal gravity (NG) control, and the efficiency of the recombinant PGUS expression in SMG were 18.33%, 19.36%, and 33.42% higher than that in NG at 19 °C, 28 °C, and 37 °C, respectively (P < 0.05).
Keywords: Simulated microgravity (SMG); Normal gravity (NG); Recombinant PGUS; Expression; E. coli

Inhibition of Pro-inflammatory Secreted Phospholipase A2 by Extracts from Cynara cardunculus L. by Maher Kammoun; Imed Koubaa; Yassine Ben Ali; Raoudha Jarraya; Youssef Gargouri; Mohamed Damak; Sofiane Bezzine (662-670).
The aim of the present work was to evaluate the anti-inflammatory properties of Cynara cardunculus L. (Asteraceae) during its growth using various solvents such as n-hexane, dichloromethane, acetone, and methanol for air-dried leaves and stems. The anti-inflammatory activities of crude extracts were evaluated by measuring the inhibition potency of mammalian non-pancreatic phospholipases A2 (hG-IIA). The methanol and acetone extracts of leaves harvested in February exhibit potent inhibition of hG-IIA (IC50 = 50 and 70 µg/ml, respectively). However, the acetone extract of stems harvested in December inhibits the hG-IIA with a lower IC50 around 130 µg/ml. Fractionation on silica gel and hydrophobic gel of the methanol extract of leaves harvested in February increases the inhibitory effect, and the IC50 reached 10 µg/ml.
Keywords: Enzyme; Secreted phospholipases A2; Extraction; Inhibitors; Anti-inflammatory; Cynara cardunculus

Synthesis of Cholesterol-Conjugated Magnetic Nanoparticles for Purification of Human Paraoxonase 1 by Zahoor Qadir Samra; Sadaf Shabir; Zainab Rehmat; Mariam Zaman; Aqsa Nazir; Nadia Dar; Muhammad Amin Athar (671-686).
Human serum paraoxonase 1 (PON1) is known as an antioxidant and is also involved in the detoxification of many compounds. In this study, a novel purification strategy was employed to purify the PON1 by using cholesterol-conjugated magnetic nanoparticles. Magnetic nanoparticles were synthesized and conjugated with cholesterol through diazotized p-aminohippuric acid. In Fourier transform infrared spectrum of cholesterol-p-aminohippuric acid-Fe3O4 nanoparticles, the appearance of peaks at 3,358.3, 1,645 cm−1, and at 2,334.9 cm−1 confirmed the conjugation. The molecular weight of purified PON1 was nearly 45 kDa on sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE), and isoelectric point was 5.3. The specific activity was 438 U mg−1 protein, and the purification fold was 515 with 73% yield. The K m values were 1.3 and 0.74 mM with paraoxon and phenyl acetate, respectively. Western blot of 2D-PAGE confirmed the homogeneity and stability of the enzyme. Mg+2, Mn+2, glycerol, (NH4)2SO4, PEG 6000, Triton X-100, and phenylmethylsulfonyl fluoride did not show any effect on activity. Pb+2, Co+2, Zn2+, ethanol, β-mercaptoethanol, and acetone reduced the activity while Ni2+, Cd2+, Cu2+, iodoacetic acid, SDS, dimethylformamide, DMSO inhibited the activity. In vitro enzyme activity was slightly reduced by acetyl salicylic and acetaminophen and reduced 50% with amino glycosides and ampicillin antibiotics at concentrations of 0.6 and 30 mg ml−1, respectively. This is the first report for the synthesis of cholesterol-conjugated magnetic nanoparticles for simple purification of PON1 enzyme.
Keywords: Paraoxonase 1; Nanoparticles; FTIR; Antibiotics; 2D-PAGE; Western blot

Anti-adipocyte scFv-Fc Antibody Suppresses Subcutaneous Adipose Tissue Development and Affects Lipid Metabolism in Minipigs by M. L. Cheng; S. M. Zhao; W. Z. Li; X. Zhang; C. R. Ge; G. Duan; S. Z. Gao (687-697).
Anti-adipocyte monoclonal antibody has been shown to reduce body fat mass in animals. Here, we investigated the effects of an anti-adipocyte antibody (single-chain variable fragment and crystallizable fragment, scFv-Fc) on pig subcutaneous adipose tissue development and lipid metabolism. The scFv-Fc antibody did not alter feed intake or body weight of treated pigs. It suppressed subcutaneous adipose tissue development by reducing the percentage of larger adipocytes, which led to a reduction in body fat mass and subcutaneous adipose tissue thickness. Body fat mass was reduced by reducing triglyceride biosynthesis and promoting triglyceride lipolysis in adipose tissue. There was an increase in lipoprotein lipase mRNA expression in adipose tissue and activity in blood and an enhanced transportation of circulating high-density lipoprotein, low-density lipoprotein, and free fatty acids. Blood concentrations of triglyceride, total cholesterol, glucose, insulin, and adiponectin and mRNA expression of adiponectin in adipose tissue remained unaffected. These findings suggest that anti-adipocyte scFv-Fc antibody may have an application for reducing body fat mass in obese subjects.
Keywords: scFv-Fc antibody; Adipocyte; Body fat; Obesity; Minipigs

Enterocin LR/6, a purified bacteriocin, exhibited broad inhibitory spectrum both against related as well as some food-borne pathogens such as Listeria monocytogenes, Yersinia enterocolitica, Aeromonas sp., Shigella sp., and Bacillus licheniformis. In this investigation, we have focused on L. monocytogenes as the target organism, as it is not only an important pathogen but can also survive over a wide range of environmental conditions such as refrigeration temperature, low pH, and high-salt concentration. This allows the pathogen to overcome many food preservation and safety barriers and poses a potential risk to human health. The enterocin LR/6 showed a bactericidal action against L. monocytogenes and completely inhibited the growth on agar plates, supplemented with 200 AU/ml of enterocin LR/6. The effectiveness of enterocin LR/6 in completely killing a population of acid-adapted (pH 5.2, 2 h) L. monocytogenes exposed to different temperatures (437 °C), pH (2.5–8.0), and osmotic (up to 30% NaCl) stress is reported here. This paper focuses on the key issue of killing of the acid-adapted L. monocytogenes cells under adverse environmental conditions.
Keywords: Acid adaptation; Enterocin LR/6; Inhibitory spectrum; Listeria monocytogenes

Gene Cloning, Expression, and Characterization of a Family 51 α-l-Arabinofuranosidase from Streptomyces sp. S9 by Pengjun Shi; Ning Li; Peilong Yang; Yaru Wang; Huiying Luo; Yingguo Bai; Bin Yao (707-718).
An α-l-arabinofuranosidase gene, abf51S9, was cloned from Streptomyces sp. S9 and successfully expressed in Escherichia coli BL21 (DE3). The full-length gene consisted of 1,506 bp and encoded 501 amino acids with a calculated mass of 55.2 kDa. The deduced amino acid sequence was highly homologous with the α-l-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. The recombinant protein was purified to electrophoretic homogeneity by Ni-NTA affinity chromatography and subsequently characterized. The optimal pH and temperature for the recombinant enzyme were 6.0 and 60∼65 °C, respectively. The enzyme showed a broad pH range of stability, retaining over 75% of the maximum activity at pH 5.0 to 11.0. The specific activity, K m, and V max with p-nitrophenyl-α-l-arabinofuranoside as substrate were 60.0 U mg−1, 1.45 mM, and 221 μmol min−1 mg−1, respectively. Abf51S9 showed a mild but significant synergistic effect in combination with xylanase on the degradation of oat-spelt xylan and soluble wheat arabinoxylan substrates with a 1.19- and 1.21-fold increase in the amount of reducing sugar released, respectively. These favorable properties make Abf51S9 a good candidate in various industrial applications.
Keywords: α-l-Arabinofuranosidase; Streptomyces sp. S9; Gene cloning and expression; Synergistic action

Isolation and Screening of Microorganisms for R-(+)-Limonene and (−)-β-Pinene Biotransformation by Ieda Rottava; Priscila F. Cortina; Camila E. Grando; André R. S. Colla; Eduarda Martello; Rogério L. Cansian; Geciane Toniazzo; Helen Treichel; Octávio A. C. Antunes; Enrique G. Oestreicher; Débora de Oliveira (719-732).
This work is focused on the biotransformation of R-(+)-limonene and (−)-β-pinene to bioflavor production. To carry out the present study, 405 microorganisms were tested for their ability to bioconvert the substrates. From the isolated microorganisms, 193 were selected in the prescreening using mineral medium for limonene degradation. At the screening step, eight strains were able to convert R-(+)-limonene and 15 to transform (−)-β-pinene, both in α-terpineol. The highest concentration in α-terpineol from R-(+)-limonene was about 3,450 mg/L for Penicillium sp. isolated from eucalyptus steam. From (−)-β-pinene, the highest product concentration of 675.5 mg/L was achieved using an Aspergillus sp. strain isolated from orange tree stem.
Keywords: Screening; Biotransformation; R-(+)-limonene; (−)-β-Pinene; α-Terpineol; Monoterpenes

An extracellular lipase secreted by Pseudomonas aeruginosa CS-2 was purified to homogeneity about 25.5-fold with an overall yield of 45.5%. The molecular mass of the lipase was estimated to be 33.9 kDa by SDS-PAGE and 36 kDa by gel filtration. The optimum temperature and pH were 50 °C and 8.0. The lipase was found to be stable at pH 4–10 and below 50 °C. Its hydrolytic activity was highest against p-nitrophenyl palmitate (p-NPP) among p-nitrophenyl esters of fatty acids with various chain lengths. The lipase was activated in the presence of Ca2+, while it was inactivated by other metal ions more or less. EDTA significantly reduced the lipase activity, indicating the lipase was a metalloenzyme. Gum Arabic and polyvinyl alcohol 124 enhanced lipase activity but Tween-20, Tween-80, and hexadecyltrimethyl ammonium bromide strongly inhibited the lipase. It exhibited stability in some organic solvents. The lipase was activated in the presence of acetonitrile. Conversely, it was drastically inactivated by methanol and ethanol.
Keywords: Organic solvent-tolerant lipase; Pseudomonas aeruginosa CS-2

The levo-lactonase gene of Fusarium proliferatum ECU2002 (EC3.1.1.25) was cloned and expressed in Escherichia coli JM109 (DE3) for biocatalytic resolution of industrially important chiral lactones, including DL-pantoyl lactone which was a key precursor to calcium d-pantothenate. By increasing the biomass concentration and lowering the inducer (isopropyl-β-d-thiogalactoside) concentration and induction temperature, the lactonase production was significantly enhanced up to 20 kU/L, which was 20 times higher than that of wild-type strain F. proliferatum ECU2002. The recombinant Fusarium lactonase was purified using immobilized metal affinity chromatography, and its SDS-PAGE revealed a molecular mass of 50 kDa for the recombinant protein, suggesting that the enzyme was a simplex protein. Furthermore, biocatalytic properties of the recombinant lactonase were investigated, including kinetic parameters, additive’s effect, and substrate specificity. The results reported in this paper provide a feasible method to make the whole cells of E. coli JM109 (DE3) expressing lactonase gene to be a highly efficient and easy-to-make biocatalyst for asymmetric synthesis of chiral compounds.
Keywords: Levo-lactonase; DL-pantoyl lactone; Recombinant protein; Immobilized metal affinity chromatography; Biocatalytic property; Substrate specificity

Preparation of a Crosslinked Bioimprinted Lipase for Enrichment of Polyunsaturated Fatty Acids from Fish Processing Waste by Jinyong Yan; Lifan Li; Qianli Tang; Manzhou Jiang; Shenzhou Jiang (757-765).
Geotrichum sp. lipase modified with a combined method composed of crosslinking and bioimprinting was employed to selectively hydrolyze waste fish oil for enrichment of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in glycerides. Crosslinked polymerization by monomer (polyethylene glycol 400 dimethyl acrylate), crosslinker (trimethylolpropane trimethylacrylate), and photoinitiator (benzoin methyl ether) coupled to bioimprinting using palmitic acid as imprint molecule, resulted in much more effective enzyme preparation used in aqueous hydrolysis reaction. Since the crosslinked polymerization modification maintained bioimprinted property and gave good dispersion of enzyme in reaction mixture, the crosslinked bioimprinted enzyme exhibited higher hydrolysis temperature, enhanced specific activity, shorter hydrolysis time, and better operational stability compared to free lipase. Crude fish oil was treated at 45 °C with this crosslinked bioimprinted lipase for 8 h, and 46% hydrolysis degree resulted in the production of glycerides containing 41% of EPA and DHA (EPA+DHA), achieving 85.7% recovery of initial EPA and DHA. The results suggested that bioimprinted enzymes did not lose their induced property in aqueous environment when prepared according to the described crosslinking–bioimprinting method. It could also be seen that the crosslinked bioimprinted lipase was effective in producing glycerides that contained a higher concentration of polyunsaturated fatty acid with better yield.
Keywords: Lipase; Crosslinking; Bioimprinting; Hydrolysis; Polyunsaturated fatty acid

Pentavalent arsenate reductase activity was localized and characterized in vitro in the cytosolic fraction of a newly isolated bacterial strain from arsenic-contaminated sites. The bacterium was gram negative, rod-shaped, nonmotile, non-spore-forming, and noncapsulated, and the strain was identified as Pseudomonas sp. DRBS1 following biochemical and molecular approaches. The strain Pseudomonas sp. DRBS1 exhibited enzymatic machinery for reduction of arsenate(V) to arsenite(III). The suspended culture of the bacterium reduced more than 97% of As(V) (40–100 mM) to As(III) in 48 h. The growth rate and total cellular yield decreased in the presence of higher concentration of arsenate. The suspended culture repeatedly reduced 10 mM As(V) within 5 h up to five consecutive inputs. The cell-free extracts reduced 86% of 100 µM As(V) in 40 min. The specific activity of arsenate reductase enzyme in the presence of 100 µM arsenate is 6.68 µmol/min per milligram protein. The arsenate reductase activity is maximum at 30 °C and at pH 5.2. The arsenate reductase activity increased in the presence of electron donors like citrate, glucose, and galactose and metal ions like Cd+2, Cu+2, Ca+2, and Fe+2. Selenate as an electron donor also supports the growth of strain DRBS1 and significantly increased the arsenate reduction.
Keywords: Arsenate reductase; Pseudomonas sp.; Cell-free extracts; Selenate; Arsenite

Among all endophytic keratinolytic fungal isolates recovered from marine soft coral Dendronephthya hemprichii, Penicillium spp. Morsy1 was selected as the hyperactive keratinolytic strain under solid substrate fermentation of different agriculture and poultry wastes. The optimization of extraction process, physicochemical parameters affecting the keratinase production in solid-state fermentation, and the purified keratinase parameters were studied. Maximum keratinase activity (1,600 U g−1, initial dry substrate) was recovered from moldy bran with 0.1% Tween 80. The optimized production conditions were rice straw as carbon source, pH of medium 6, growth temperature 26 °C, initial moisture content of 80% (v/w), inoculum size of 105 spores ml−1, and an average particle size of the substrate 0.6 mm (3,560 U g−1, initial dry substrate after 5 days of fermentation). Two types of keratinase (Ahm1 and Ahm2) were purified from the culture supernatant through ammonium sulfate precipitation, DEAE-Sepharose, and gel filtration chromatography. Enzyme molecular weights were 19 kDa (Ahm1) and 40 kDa (Ahm2). The kinetic parameters of purified keratinases were optimized for the hydrolysis of azokeratin by Ahm1 (pH 7.0–8.0, stable in pH range of 6.0 to 8.0 at 50 °C) and Ahm2 enzymes (pH 10.0–11.0, stable in pH range of 6.0 to 11.0 at 60–65 °C). Whereas inhibitors of serine (phenylmethylsulfonyl fluoride) and cysteine (iodoacetamide) proteases had minor effects on both Ahm1 and Ahm2 activity, both keratinases were strongly inhibited by chelating agents EDTA and EGTA. These findings suggest that serine and cysteine residues are not involved in the catalytic mechanisms, and they are metalloproteases.
Keywords: Endophytic Penicillium spp.; Keratinase; Solid-state fermentation; Process parameters; Purification

The effect of different doses of visible (Vis), ultraviolet-А (UVA), and mixed light (UVA + Vis) upon coenzyme Q10 (CoQ10) and β-carotene synthesis and biomass yield by the Sporobolomyces salmonicolor AL1, Cryptococcus albidus AS55, Cryptococcus laurentii AS56, and C. laurentii AS58 strains isolated from Antarctic samples was investigated. The β-carotene concentration in the red strain biomass increased by 52% under irradiation with 11 J/cm2 Vis, and the CoQ10 concentration rose by 37% in relation to the control quantity obtained through dark cultivation. Under irradiation with 6 J/cm2 UVA, the S. salmonicolor AL1 strain synthesized 15% more β-carotene; C. albidus AS55, 22%; C. laurentii AS56, 44%; and C. laurentii AS58, 35% in relation to the control quantity. Irradiation with a low UVА + Vis dose significantly stimulated β-carotene biosynthesis by the strains of the Cryptococcus genus (87%, 138%, and 100%), whereas S. salmonicolor AL1 increased the β-carotene content to a smaller degree (55%). Higher doses of all three irradiation types inhibited β-carotene accumulation. Vis suppressed CoQ10 biosynthesis in the Cryptococcus strains, whereas UVА and UVА + Vis inhibited it in all four strains. The S. salmonicolor AL1 strain pre-treated with 0.02 J/cm2 UVA synthesized twice as much CoQ10 and β-carotene when cultivated in the presence of Vis light in an 11-J/cm2 dose.
Keywords: S. salmonicolor AL1 ; C. albidus AS55 ; C. laurentii AS56 ; C. laurentii AS58 ; β-carotene; Coenzyme Q10 ; UV; Vis

Mercury(II) Biosorption Using Lessonia sp. Kelp by Mariana Reategui; Holger Maldonado; Martha Ly; Eric Guibal (805-822).
Lessonia nigrescens and Lessonia trabeculata kelps have been tested for the sorption of mercury from aqueous solutions. A pretreatment (using CaCl2) allowed stabilizing the biomass that was very efficient for removing Hg(II) at pH 6–7. Sorption isotherms were described by the Langmuir equation with sorption capacities close to 240–270 mg Hg g−1 at pH 6. The temperature had a negligible effect on the distribution of the metal at equilibrium. The presence of chloride anions had a more marked limiting impact than sulfate and nitrate anions. The uptake kinetics were modeled using the pseudo-second-order equation that fitted better experimental data than the pseudo-first-order equation. The particle size hardly influenced sorption isotherms and uptake kinetics, indicating that sorption occurs in the whole mass of the biosorbent and that intraparticle mass transfer resistance was not the limiting rate. Varying the sorbent dosage and the initial metal concentration influenced the equilibrium, but the kinetic parameters were not drastically modified. Metal can be eluted with hydrochloric acid, citric acid, or acidic KI solutions.
Keywords: Mercury(II); Lessonia ; Kelp; Isotherms; Kinetics; Pseudo-second-order equation; Desorption

Optimized Culture Conditions for the Efficient Production of Porcine Adenylate Kinase in Recombinant Escherichia coli by Toru Matsui; Takashi Togari; Satoru Misawa; Tomoyuki Namihira; Naoya Shinzato; Hitoshi Matsuda; Seigo Sato (823-829).
Temperature shift cultivations with amino acid supplementation were optimized to produce porcine adenylate kinase (ADK) in recombinant Escherichia coli harboring a pUC-based recombinant plasmid under the control of the trp promoter. With regard to temperature control, the culture condition was initially maintained at 35 °C for cellular growth, but ADK expression was suppressed until the late logarithmic growth phase; subsequently, a temperature shift was applied (from 35 °C to 42 °C), which resulted in maximal ADK production. In addition, supplementation of amino acids, especially valine and leucine, during the temperature shift stimulated ADK expression from 3.5% to 9.2% and 8.6% of the total protein, respectively. After optimization, 1 g ADK per liter was produced within 16 h of cultivation with a dry cell weight of 21.8 g/l. In this system, there was no loss of the recombinant plasmid during cultivation without selective pressure.
Keywords: High cell density cultivation; Escherichia coli ; Porcine adenylate kinase; Temperature shift cultivation

Production and Stability of Protease from Candida buinensis by Daniela de Araújo Viana; Carolina de Albuquerque Lima; Rejane Pereira Neves; Cristina Souza Mota; Keila Aparecida Moreira; José Luiz de Lima-Filho; Maria Taciana Holanda Cavalcanti; Attilio Converti; Ana Lúcia Figueiredo Porto (830-842).
Cow raw milk from dairy cooperatives was examined for its microbial composition. Among the isolates identified, 17.6% were yeasts. The most frequent genus was Candida, although members belonging to the genera Brettanomyces, Dekkera, and Geotricum were also identified. Although qualitative and quantitative tests for extracellular proteolytic activity were positive for all the species isolated, Candida buinensis showed the highest response (23.5 U/mg); therefore, it was selected for subsequent investigation. The results of fermentations carried out at variable temperature, pH, and soybean flour concentration, according to a 23 full factorial design, demonstrated that this yeast ensured the highest production of extracellular proteases (573 U/mL) when cultivated at 35 °C, pH 6.5, and using soybean flour concentrations in the range 0.1–0.5% (w/v). The cell-free supernatants showed the highest activity at 25 °C and pH 7.0, and satisfactory stability in the ranges 25–30 °C and pH 7–9. The first-order rate constants of protease inactivation in the cell-free supernatants were calculated at different temperatures from semi-log plots of the residual activity versus time and then used in Arrhenius and Eyring plots to estimate the main thermodynamic parameters of thermoinactivation (E* = 40.0 kJ/mol; ΔH* = 37.3 kJ/mol; ΔS* = −197.5 J/mol K; ΔG* = 101 kJ/mol).
Keywords: Candida buinensis ; Protease production; Soybean flour medium; Raw milk

Cloning of a Heat-Stable Chitin Deacetylase Gene from Aspergillus nidulans and its Functional Expression in Escherichia coli by Yun Wang; Jin-Zhu Song; Qian Yang; Zhi-Hua Liu; Xiao-Mei Huang; Yan Chen (843-854).
A gene encoding chitin deacetylase was cloned by polymerase chain reaction from Aspergillus nidulans. Sequencing result showed 40% homology to the corresponding gene from Colletotrichum lindemuthianum. The complete gene contains an open reading frame of 747 nucleotides encoding a sequence of 249 amino acid residues. The chitin deacetylase gene was subcloned into a pET28a expression vector and expressed in Escherichia coli BL21 and then purified by metal affinity chromatography using a His-bind column. The purified chitin deacetylase demonstrated an activity of 0.77 U ml−1 for the glycol chitin substrates, and its specific activity was 4.17 U mg−1 for it. The optimal temperature and pH of the purified enzyme were 50 °C and 8.0, respectively. When glycol chitin was used as the substrate, K m was 4.92 mg ml−1, and K cat showed 6.25 s−1, thus the ratio of K cat and K m was 1.27 ml s−1 mg−1. The activity of chitin deacetylase was affected by a range of metal ions and ethylenediaminetetraacetic acid.
Keywords: Aspergillus nidulans ; Chitin deacetylase; Gene cloning; Expression

Genome-Wide Expression Changes in Saccharomyces cerevisiae in Response to High-LET Ionizing Radiation by Satomi Mizukami-Murata; Hitoshi Iwahashi; Shinzou Kimura; Kumie Nojima; Yoshinori Sakurai; Takeshi Saitou; Noriko Fujii; Yoshinori Murata; Shinzi Suga; Kazuhide Kitagawa; Kenichi Tanaka; Satoru Endo; Masaharu Hoshi (855-870).
To understand the yeast response to high-linear energy transfer (LET) ionizing radiation (IR), we investigated global gene expression in yeast irradiated by three types of high-LET IR (fast neutrons, heavy ions, and thermal neutrons) and gamma rays using DNA microarray analysis. Stationary cells were irradiated by each IR and recultured in yeast–peptone–dextrose medium to allow repair for 40 min. RNA was then isolated from three independent samples of irradiated yeast. Genes involved in the Mec1p kinase pathway, which functions in DNA damage response, were induced by all forms of high-LET IR and by gamma rays. Some genes related to oxidative stress and the cell wall were induced by all forms of high-LET IRs. Gene expression patterns as a function of each type of high-LET IR were examined statistically by one-way analysis of variance. This analysis demonstrated the existence of irradiation-specific responses. For example, genes involved in ribosomal DNA synthesis were specifically induced by fast neutron irradiation, while the ubiquitin–proteasome system and heat shock response were specifically induced by thermal neutron irradiation. The study characterizes high-LET IR-induced gene expression and provides a molecular understanding of subsequent adaptation in yeast.
Keywords: High-LET IRs; Fast neutron; Heavy ion; Thermal neutron; Gene expression; ANOVA analysis; Mec1p kinase pathway; Ubiquitin–proteasome system

Antibacterial Activity of Alpinia galanga (L) Willd Crude Extracts by Kiranmayee Rao; Bhuvaneswari Ch; Lakshmi M. Narasu; Archana Giri (871-884).
Methanol, acetone and diethyl ether extracts of Alpinia galanga have been evaluated against pathogens viz. Bacillus subtilis MTCC 2391, Enterobacter aerogene, Enterobacter cloacae, Enterococcus faecalis, Escherichia coli MTCC 1563, Klebsiella pneumoniae, Pseudomonas aeruginosa MTCC 6642, Salmonella typhimurium, Staphylococcus aureus and Streptococcus epidermis using Agar well diffusion method. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of all the extracts were determined using the macrodilution method. Methanol extracts have shown excellent activity towards all the pathogens with MIC and MBC values ranging from 0.04–1.28 mg/ml and 0.08–2.56 mg/ml, respectively. The GC–MS analysis of methanol extracts have yielded compounds like 5-hydroxymethyl furfural (59.9%), benzyl alcohol (57.6%), 1,8 cineole (15.65%), methylcinnamate (9.4%), 3-phenyl-2-butanone (8.5%) and 1,2 benzenedicarboxylic acid (8.9%), which could be responsible for its broad spectrum activity. So, A. galanga can be quite resourceful for the development of new generation drugs.
Keywords: Alpinia galanga ; Antimicrobial activity; MIC; MBC; GC–MS analysis; Pathogens; Plant extracts

Effect of Fill Time on the Performance of Pilot-scale ASBR and AnSBBR Applied to Sanitary Wastewater Treatment by Luciano Farias de Novaes; Lucas Oliveira Borges; José Alberto Domingues Rodrigues; Suzana Maria Ratusznei; Marcelo Zaiat; Eugenio Foresti (885-899).
Many lab-scale studies have been carried out regarding the effect of feed strategy on the performance of anaerobic sequencing batch reactors (ASBR); however, more detailed pilot-scale studies should be performed to assess the real applicability of this type of operation. Therefore, the objective of this work was to assess the effect of feed strategy or fill time in a 1-m3 mechanically stirred pilot-scale sequencing batch reactor, treating 0.65 m3 sanitary wastewater in 8-h cycles at ambient temperature. Two reactor configurations were used: one containing granular biomass (denominated ASBR) and the other immobilized biomass on polyurethane foam as inert support (denominated anaerobic sequencing batch biofilm reactor (AnSBBR)). The reactors were operated under five distinct feed strategies, namely: typical batch and fed-batch for 25%, 50%, 75%, and 100% of the cycle length. Stirring frequency in the ASBR was 40 rpm with two flat-blade turbine impellers and 80 rpm in the AnSBBR with two helix impellers. The results showed that both the ASBR and AnSBBR when operated under typical batch, fed-batch for 50% and 75% of the cycle length, presented improved organic matter removal efficiencies, without significant differences in performance, thus showing important operational flexibility. In addition, the reactors presented operation stability under all conditions.
Keywords: ASBR; AnSBBR; Fill time; Mechanical stirring; Anaerobic reactor; Sanitary wastewater

Purification, Molecular Cloning, and Biochemical Characterization of Subtilisin JB1 from a Newly Isolated Bacillus subtilis JB1 by Ji Hea Sung; Sang Jung Ahn; Na Young Kim; Soo-Kyoung Jeong; Joong Kyun Kim; Joon Ki Chung; Hyung Ho Lee (900-911).
An extracellular gelatinolytic enzyme obtained from the newly isolated Bacillus subtilis JB1, a thermophilic microorganism relevant to the aerobic biodegradation process of fish-meal production, was purified via ammonium sulfate precipitation, Sephadex G-200 Gel filtration chromatography, and one-dimensional gel electrophoresis separation and subsequently identified via peptide mass fingerprinting and chemically assisted fragmentation matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The subtilisin JB1 gene was sequenced and its recombinant protein prosubtilisin JB1 was expressed in Escherichia coli, and the purified prosubtilisin JB1 (62 kDa) protein was digested with gelatin, bovine serum albumin, azocasein, fibrinogen, and the fluorogenic peptide substrate Ala-Ala-Phe-7-amido-4-methylcoumarin hydrochloride, whereas the serine protease inhibitors phenylmethylsulfonyl fluoride and chymostatin completely inhibited its enzyme activity at an optimal pH of 7.5. Thus, our results show that subtilisin JB1 may serve as a potential source material for use in industrial applications of proteolytic enzymes and microorganisms for fishery waste degradation and fish by-product processing.
Keywords: Skim milk agar plate; MALDI-TOF-MS; Peptide-mass fingerprinting (PMF); Chemically assisted fragmentation (CAF); Subtilisin; Bacillus subtilis JB1

Antifungal and Antiproliferative Activities of Lectin from the Rhizomes of Curcuma amarissima Roscoe by Norhameemee Kheeree; Polkit Sangvanich; Songchan Puthong; Aphichart Karnchanatat (912-925).
A lectin was purified from the rhizomes of Curcuma amarissima Roscoe by aqueous extraction, fractionation with 80% saturated ammonium sulfate, and a combination of affinity and gel chromatography on ConA Sepharose and Superdex G-75, respectively. The molecular mass of the purified lectin was 32.4 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lectin showed no significant specificity in its ability to hemagglutinate erythrocytes from human blood groups (A, B, AB, and O), but for other animals, it only agglutinated rabbit and rat, and not mouse, guinea pig, goose, and sheep erythrocytes. The lectin was stable at temperatures below 40°C, but the hemagglutinating activity halved when it was heated to 45–85°C and was completely lost at 95°C. The hemagglutinating activity was more stable at 80°C than at 70°C and was rapidly inactivated at 90°C. It showed a maximum hemagglutination activity within the pH range of 8.0–11.0. The deduced amino acid sequence of an internal tryptic peptide sequence of this purified lectin showed sequence similarity (homology) to other members of the leucoagglutinating phytohemagglutinin precursor family, whilst the complete lectin inhibited the in vitro growth of three plant pathogenic fungi, Fusarium oxysporum, Exserohilum turicicum, and Colectrotrichum cassiicola, at a concentration of 17.5 to 35 µg, and showed in vitro cytotoxicity against the BT474 breast cancer cell line with an IC50 of approximately 21.2 μg.
Keywords: Lectin; ConA Sepharose; Curcuma amarissima ; Antifungal; Antiproliferative

Erratum to: Properties of a Cationic Peroxidase from Citrus jambhiri cv. Adalia by Saleh A. Mohamed; Mohamed O. El-Badry; Ehab A. Drees; Afaf S. Fahmy (926-926).