Applied Biochemistry and Biotechnology (v.172, #1)

We described a simple and quick miniaturized sequencing gel system for DNA analysis. Two major modifications were made to the previously reported miniaturized DNA sequencing gel system to achieve high-resolution hydroxyl radical cleavage analysis: including formamide in the miniaturized gel and providing uniform heating during electrophoresis. Our method enables one to reduce the cost for chemicals and to significantly reduce electrophoresis time. Furthermore, minimal gel handling simplifies the entire process. We show that the resolution of DNA fragments obtained by hydroxyl radical cleavage for the miniaturized gel is similar to that of a large conventional sequencing gel.
Keywords: Rapid DNA sequencing gel system; DNA analysis; Hydroxyl radical cleavage

Hepatoprotective Effect of β-Chitosan from Gladius of Sepioteuthis lessoniana Against Carbon Tetrachloride-Induced Oxidative Stress in Wistar Rats by Namasivayam Subhapradha; Ramachandran Saravanan; Pasiyappazham Ramasamy; Alagiri Srinivasan; Vairamani Shanmugam; Annaian Shanmugam (9-20).
Chitosan has attracted much attention as a biomedical material, owing to its unique biological activities. In this study, hepatoprotective effect of β-chitosan obtained from the gladius of squid Sepioteuthis lessoniana was studied against carbon tetrachloride (CCl4)-induced oxidative stress and liver injury in rats. The rats that received β-chitosan along with the administration of CCl4 showed significantly decreased plasma and tissue alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and total cholesterol, triglyceride (TG) and free fatty acid (FFA) contents, whereas the treatment with β-chitosan alone markedly increased rat hepatic and circulatory superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) and reduced glutathione (GSH) levels and decreased the malondialdehyde level. Histopathological observations recommended the marked hepatoprotective effect of β-chitosan. The CCl4-induced alterations on circulatory and hepatic antioxidant defence system were normalised by β-chitosan, and it could be concluded that the hepatoprotective effect of chitosan may be due to its antioxidant and antilipidemic property. Therefore, β-chitosan could be considered as antihepatotoxic agent.
Keywords: Sepioteuthis lessoniana ; β-Chitosan; Hepatoprotective; Carbon tetrachloride

Multi-lineage Potential Research of Bone Marrow-Derived Stromal Cells (BMSCs) from Cattle by Taofeng Lu; Yina Huang; Hui Wang; Yuehui Ma; Weijun Guan (21-35).
Bovine bone marrow-derived mesenchymal stem cells (bBMSCs) were isolated from the bone marrow of a 4–6-month-old fetal bovine and then characterized by immunofluorescence and reverse transcriptase polymerase chain reaction. We found that primary bBMSCs could be expanded for 46 passages; the total culture time in vitro was 125 days. The results of surface antigen detection showed that bBMSCs expressed CD29, CD44, and CD73 but did not express endothelial cells and hematopoietic cells-specific marker CD31, CD34, and CD45. The cells from four passages (passages 3, 9, 15, and 25) were successfully induced to differentiate into osteoblasts, adipocytes, hepatic, and islet-like cells. The results indicate the potential for multi-lineage differentiation of bBMSCs that may represent an ideal candidate for cellular transplantation therapy.
Keywords: BMSCs; Multi-lineage differentiation; Hepatic cells; Islet-like cells

Emulsifying, Flocculating, and Physicochemical Properties of Exopolysaccharide Produced by Cyanobacterium Nostoc flagelliforme by Pei-pei Han; Ying Sun; Xiao-ying Wu; Ying-jin Yuan; Yu-jie Dai; Shi-ru Jia (36-49).
The emulsifying, flocculating, and physicochemical properties of purified exopolysaccharide (EPS) of terrestrial cyanobacterium Nostoc flagelliforme cultured in liquid media were investigated. The EPS was defined as heteropolysaccharide composed by 41.2 % glucose, 21.1 % galactose, 21.0 % mannose, 2.5 % fructose, 3.6 % ribose, 1.7 % xylose, 0.6 % arabinose, 3.0 % rhamnose, 0.9 % fucose, and 4.3 % glucuronic acid. The EPS possessed higher intrinsic viscosity than other cyanobacterial strains as reported and displayed pseudoplastic behavior in aqueous solution. The EPS produced more stable emulsions with tested hydrocarbons and oils than xanthan gum, and the emulsification indexes with n-hexadecane, liquid paraffin, and peanut oil were higher than 50 %, indicating the strong emulsion-stabilizing capacity. The EPS showed peak flocculating rates of 93.5 and 86.1 % in kaolin and MgO suspension, respectively, and exhibited a better flocculation performance than Al2(SO4)3 and xanthan gum. These results demonstrated that the EPS of N. flagelliforme was a very promising candidate for numerous industrial applications, as it had higher intrinsic viscosity, good emulsification activity, and excellent flocculation capability.
Keywords: Exopolysaccharide; Nostoc flagelliforme ; Rheology; Emulsification; Flocculation

Genome Shuffling of Aspergillus niger for Improving Transglycosylation Activity by Wei Li; Guiguang Chen; Lingli Gu; Wei Zeng; Zhiqun Liang (50-61).
Isomaltooligosaccharides (IMO), the glucosylsaccharides used as food additives, are made from saccharified starch by enzymes or microbial cells with transglycosylation activity. This study aimed to generate shuffled futants of Aspergillus niger with enhanced transglycosylation activity for industrial IMO production. The starting mutant population was generated by 60Co-γ radiation; mutants with higher transglycosylation activity were selected and subjected to recursive protoplast fusion. The resulting fusants were screened by a novel high-throughput method based on detecting non-fermentable reducing sugar. After three rounds of genome shuffling, the best performing strain GS3-3 was obtained, its transglycosylation activity (14.91 U/mL) was increased by 194.1 % compared to that of original strain C-6181. In fermentor test, transglycosylation activity of GS3-3 was obtained at 16.61 U/mL. The mycelia of GS3-3 were reused ten times to produce IMO syrup from liquefied cassava starch containing about 280 g/L total sugar within 4 days. The conversion of liquefied cassava starch to IMO was at 71.3–72.1 %, which was higher than the best conversion (68 %) ever reported. GS3-3 shows a great potential for industrial IMO production.
Keywords: Transglycosylation activity; Isomaltooligosaccharides; Genome shuffling; Aspergillus niger

In this study, complicated model sulfur compounds in bunker oil and de-asphalted bunker oil were biodesulfurized in a batch process by microbial consortium enriched from oil sludge. Dibenzothiophene (DBT) and benzo[b]naphtho[1,2-d]thiophene (BNT1) were selected as model sulfur compounds. The results show that the mixed culture was able to grow by utilizing DBT and BNT1 as the sole sulfur source, while the cell density was higher using DBT than BNT1 as the sulfur source. GC-MS analysis of their desulfurized metabolites indicates that both DBT and BNT1 could be desulfurized through the sulfur-specific degradation pathway with the selective cleavage of carbon–sulfur bonds. When DBT and BNT1 coexisted, the biodesulfurization efficiency of BNT1 decreased significantly as the DBT concentrations increased (>0.1 mmol/L). BNT1 desulfurization efficiency also decreased along with the increase of 2-hydroxybiphenyl as the end product of DBT desulfurization. For real bunker oil, only 2.8 % of sulfur was removed without de-asphalting after 7 days of biotreatment. After de-asphalting, the biodesulfurization efficiency was significantly improved (26.2–36.5 %), which is mainly attributed to fully mixing of the oil and water due to the decreased viscosity of bunker oil.
Keywords: Biodesulfurization; Benzonaphthothiophene; Heavy oil; Dibenzothiophene; De-asphalting

Mining fungal genomes for glucoamylase and α-amylase encoding sequences led to the selection of 23 candidates, two of which (designated TSgam-2 and NFamy-2) were advanced to testing for cooked or raw starch hydrolysis. TSgam-2 is a 66-kDa glucoamylase recombinantly produced in Pichia pastoris and originally derived for Talaromyces stipitatus. When harvested in a 20-L bioreactor at high cell density (OD600 > 200), the secreted TSgam-2 enzyme activity from P. pastoris strain GS115 reached 800 U/mL. In a 6-L working volume of a 10-L fermentation, the TSgam-2 protein yield was estimated to be ∼8 g with a specific activity of 360 U/mg. In contrast, the highest activity of NFamy-2, a 70-kDa α-amylase originally derived from Neosartorya fischeri, and expressed in P. pastoris KM71 only reached 8 U/mL. Both proteins were purified and characterized in terms of pH and temperature optima, kinetic parameters, and thermostability. TSgam-2 was more thermostable than NFamy-2 with a respective half-life (t1/2) of >300 min at 55 °C and >200 min at 40 °C. The kinetic parameters for raw starch adsorption of TSgam-2 and NFamy-2 were also determined. A combination of NFamy-2 and TSgam-2 hydrolyzed cooked potato and triticale starch into glucose with yields, 71–87 %, that are competitive with commercially available α-amylases. In the hydrolysis of raw starch, the best hydrolysis condition was seen with a sequential addition of 40 U of a thermostable Bacillus globigii amylase (BgAmy)/g starch at 80 °C for 16 h, and 40 U TSgam-2/g starch at 45 °C for 24 h. The glucose released was 8.7 g/10 g of triticale starch and 7.9 g/10 g of potato starch, representing 95 and 86 % of starch degradation rate, respectively.
Keywords: Fungal genomes; Amylase; Glucoamylase; Cold starch hydrolysis; Genome mining; Triticale; Potato Starch; Pichia pastoris ; Biocatalysts; Biorefinery

The Challenging Measurement of Protein in Complex Biomass-Derived Samples by Mai Østergaard Haven; Henning Jørgensen (87-101).
Measurement of the protein content in samples from production of lignocellulosic bioethanol is an important tool when studying the adsorption of cellulases. Several methods have been used for this, and after reviewing the literature, we concluded that one of the most promising assays for simple and fast protein measurement on this type of samples was the ninhydrin assay. This method has also been used widely for this purpose, but with two different methods for protein hydrolysis prior to the assay—alkaline or acidic hydrolysis. In samples containing glucose or ethanol, there was significant interference from these compounds when using acid hydrolysis, which was not the case when using the alkaline hydrolysis. We evaluated the interference from glucose, cellulose, xylose, xylan, lignin and ethanol on protein determination of BSA, Accellerase® 1500 and Cellic® CTec2. The experiments demonstrated that the presence of cellulose, lignin and glucose (above 50 g/kg) could significantly affect the results of the assay. Comparison of analyses performed with the ninhydrin assay and with a CN analyser revealed that there was good agreement between these two analytical methods, but care has to be taken when applying the ninhydrin assay. If used correctly, the ninhydrin assay can be used as a fast method to evaluate the adsorption of cellulases to lignin.
Keywords: Ninhydrin assay; Protein hydrolysis; CN analyser; Elemental analyser; Cellulase; Adsorption; Lignocellulosic bioethanol

Human maltase glucoamylase (MGAM) is a potent molecular target for controlling post prandial glucose surplus in type 2 diabetes. Binding of small molecules from Syzygium sp. with α-glucosidase inhibitory potential in MGAM has been investigated in silico. Our results suggest that myricetin was the most potent inhibitor with high binding affinity for both N- and C-terminals of MGAM. Molecular dynamics revealed that myricetin interacts in its stretched conformation through water-mediated interactions with C-terminal of MGAM and by normal hydrogen bonding with the N-terminal. W1369 of the extended 21 amino acid residue helical loop of C-terminal plays a major role in myricetin binding. Owing to its additional sugar sites, overall binding of small molecules favours C-terminal MGAM.
Keywords: Molecular docking; MD dynamics simulation; MGAM; Syzygium; Type 2 diabetes

A novel hybrid epoxy/nano CaCO3 composite matrix for catalase immobilization was prepared by polymerizing epoxy resin in the presence of CaCO3 nanoparticles. The hybrid support was characterized using scanning electron microscopy and Fourier transform infrared spectroscopy. Catalase was successfully immobilized onto epoxy/nano CaCO3 support with a conjugation yield of 0.67 ± 0.01 mg/cm2 and 92.63 ± 0.80 % retention of activity. Optimum pH and optimum temperature of free and immobilized catalases were found to be 7.0 and 35 °C. The value of K m for H2O2 was higher for immobilized enzyme (31.42 mM) than native enzyme (27.73 mM). A decrease in V max value from 1,500 to 421.10 μmol (min mg protein)−1 was observed after immobilization. Thermal and storage stabilities of catalase improved immensely after immobilization. Immobilized enzyme retained three times than the activity of free enzyme when kept at 75 °C for 1 h and the half-life of enzyme increased five times when stored in phosphate buffer (0.01 M, pH 7.0) at 5 °C. The enzyme could be reused 30 times without any significant loss of its initial activity. Desorption of catalase from the hybrid support was minimum at pH 7.0.
Keywords: Enzyme technology; Immobilization; Kinetic parameters; Adsorption; CaCO3 nanoparticles; Epoxy

Effect of Microalgae/Activated Sludge Ratio on Cooperative Treatment of Anaerobic Effluent of Municipal Wastewater by Fatemeh Pourasgharian Roudsari; Mohammad Reza Mehrnia; Akram Asadi; Zohreh Moayedi; Reza Ranjbar (131-140).
In this work, capability of the green microalga (MA), Chlorella vulgaris, in treating synthetic anaerobic effluent of municipal wastewater was investigated. While pure C. vulgaris (100 % MA) provided maximum soluble chemical oxygen demand (sCOD) and N − NH 4 + removal efficiencies of 27 and 72 % respectively, addition of activated sludge (AS) to MA in different mass ratios (91, 80, 66.7, 9 % MA) improved wastewater treatment efficiency. Thus giving maximum sCOD and N − NH 4 + removal efficiencies 85 and 86.3 % (for MA/AS = 10/1), respectively. Utilizing AS without C. vulgaris, for treating the synthetic wastewater resulted in 87 % maximum sCOD and 42 % maximum N − NH 4 + removal efficiencies. Furthermore, algal growth and specific growth rates were measured in the systems with microalga as the dominant cellular population. As a result, faster algal growth was observed in mixed systems. Specific growth rate of C. vulgaris was 0.14 (day−1) in 100 % MA and 0.39 (day−1) in 80 % MA. Finally, data gathered by online measurement of dissolved oxygen indicate that algae-activated sludge mixture improves photosynthetic activity of examined microalga strain during anaerobic effluent treatment.
Keywords: Chlorella vulgaris ; Microalgal-bacterial symbiosis; Mixed culture; Municipal wastewater treatment

Highly Thermostable and pH-Stable Cellulases from Aspergillus niger NS-2: Properties and Application for Cellulose Hydrolysis by Namita Bansal; Chetna Janveja; Rupinder Tewari; Raman Soni; Sanjeev Kumar Soni (141-156).
Optimization of cultural conditions for enhanced cellulase production by Aspergillus niger NS-2 were studied under solid-state fermentation. Significant increase in yields (CMCase 463.9 ± 20.1 U/g, FPase 101.1 ± 3.5 U/g and β-glucosidase 99 ± 4.0 U/g) were obtained under optimized conditions. Effect of different nutritional parameters was studied to induce the maximum production of cellulase complex. Scale-up studies for enzyme production process were carried out. Characterization studies showed that enzymes produced by A. niger NS-2 were highly temperature- and pH stable. At 50 °C, the half life for CMCase, FPase, β-glucosidase were approximately 240 h. Cellulases from A. niger NS-2 were stable at 35 °C for 24 h over a broader pH range of 3.0–9.0. We examined the feasibility of using steam pretreatment to increase the saccharification yields from various lignocellulosic residues for sugar release which can potentially be used in bioethanol production. Saccharification of pretreated dry potato peels, carrot peels, composite waste mixture, orange peels, onion peels, banana peels, pineapple peels by crude enzyme extract from A. niger NS-2, resulted in very high cellulose conversion efficiencies of 92–98 %.
Keywords: Lignocellulosics; Aspergillus niger ; Optimization; Pretreatment; Enzymatic hydrolysis; Bioconversion

Remarkable Improvement of Methylglyoxal Synthase Thermostability by His–His Interaction by Malihe Mohammadi; Mona Atabakhshi Kashi; Shekufeh Zareian; Manoochehr Mirshahi; Khosro Khajeh (157-167).
Lately it has been proposed that interaction between two positively charged side chains can stabilize the folded state of proteins. To further explore this point, we studied the effect of histidine–histidine interactions on thermostability of methylglyoxal synthase from Thermus sp. GH5 (TMGS). The crystal structure of TMGS revealed that His23, Arg22, and Phe19 are in close distance and form a surface loop. Here, two modified enzymes were produced by site-directed mutagenesis (SDM); one of them, one histidine (TMGS-HHO), and another two histidines (TMGS-HHHO) were inserted between Arg22 and His23 (HO). In comparison with the wild type, TMGS-HHO thermostability increased remarkably, whereas TMGS-HHHO was very unstable. To explore the role of His23 in the observed phenomenon, the original His23 in TMGS-HHHO was replaced with Ala (TMGS-HHA). Our data showed that the half-life of TMGS-HHA decreased in relation to the wild type. However, its half-life increased in comparison with TMGS-HHHO. These results demonstrated that histidine–histidine interactions at position 23 in TMGS-HHO probably have the main role in TMGS thermostability.
Keywords: Histidine–histidine interaction; Thermostability; Methylglyoxal synthase; TMGS; Allosteric enzyme

Production of Rhamsan Gum Using a Two-Stage pH Control Strategy by Sphingomonas sp. CGMCC 6833 by Xu Xiao Ying; Zhu Ping; Li Sha; Chen Xiao Ye; Yao Zhong; Xu Hong (168-175).
In this paper, the production of rhamsan gum from Sphingomonas sp. CGMCC 6833 at different pH values was investigated. Based on kinetic analysis, a two-stage strategy for pH control was proposed. During the first 10 h, pH was controlled at 7.5 to maintain high specific cell growth rate and specific glucose consumption rate. After 10 h, pH decreased naturally to 7.0; this value was retained to maintain high specific rhamsan gum formation rate. Using this method, the maximum concentration and productivity of rhamsan gum reached 18.56 ± 1.68 g/L and 0.290 ± 0.026 g/L/h, which are 12.83 and 12.84 % higher than the optimum results obtained at natural pH, respectively.
Keywords: Sphingomonas sp. CGMCC 6833; Rhamsan gum; Two-stage strategy; pH control; Batch fermentation

Characterization of a Layered Methylene Blue/Vanadium Oxide Nanocomposite and its Application in a Reagentless H2O2 Biosensor by Xiaobo Zhang; Dan Li; Fujun Yin; Junyan Gong; Xujie Yang; Zhiwei Tong; Xingyou Xu (176-187).
Layered nanocomposite of methylene blue (MB)-intercalated vanadium oxide was obtained through a simple hydrothermal synthesis method using MB, V2O5, and NaI as starting materials. The intercalation reaction was proven to be successful using X-ray diffraction pattern. The MB-V2O5 nanocomposite was characterized using a scanning electron micrograph, infrared spectra, thermogravimetric analysis, UV spectra, and electrochemical measurements. The intercalated MB cations showed a fine diffusion-controlled electrochemical redox process and facilitated the immobilized horseradish peroxidase’s (HRP) good catalytic reduction upon H2O2. The as-prepared MB-V2O5/HRP biosensor showed a linear response to H2O2 over a range from 2.0 × 10−6 to 9.5 × 10−5 M with a detection limit of 9.7 × 10−7 M (S/N ratio = 3).
Keywords: Methylene blue; Intercalation; Vanadium oxide; Horseradish peroxidase; Hydrogen peroxide biosensor

In Silico Comparison of Low- and High-Risk Human Papillomavirus Proteins by Mahsa Alemi; Hassan Mohabatkar; Mandana Behbahani (188-195).
Human papillomavirus (HPV) is an important pathogen which is classified into two, high- and low-risk groups. The proteins of high-risk and low-risk HPV types have different functions. Therefore, there is a need to develop a computational method for predicting these two groups. In the present study, the physiochemical properties of all early (E1, E2, E4, E5, E6, and E7) and late (L1 and L2) proteins in high- and low-risk HPV types have been studied. The concept of receiver operating characteristic analysis and support vector machines methods has been used for comparison of high- and low-risk HPV types. The results demonstrate that amino acid composition, physiochemical, and secondary structure of E2 protein are significantly different between these two groups. The results demonstrate that in silico properties can create useful information to predict high-risk and low-risk HPV types.
Keywords: Human papillomavirus; In silico classification; High risk; Low risk; ROC; SVM

The purpose of the research was to study the purification and partial characterization of antifungal alkaline chitinase from a newly isolated Citrobacter freundii haritD11. The enzyme was purified in a three-step procedure involving ammonium sulfate precipitation, dialysis, and Sephadex G-100 gel filtration chromatography. The enzyme was shown to have a relative high molecular weight of 64 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and was purified 7.3-fold with a yield of 18.8 %. It was most active at 35 °C, pH 8.0, with colloid chitin as substrate and was very stable at alkaline pH contradicting the characteristic that most of the bacterial chitinases are active at acidic pH. Further, the purified chitinase exhibited remarkable antifungal activity against pathogenic fungi Aspergillus flavus MTCC 2798 and Aspergillus niger MTCC 9652 showing diametric inhibition zones of 27 mm and 21 mm, respectively.
Keywords: Alkaline chitinase; Citrobacter freundii haritD11; Purification; Characterization; Antifungal activity

Identification of Site-Specific Degradation in Bacterially Expressed Human Fibroblast Growth Factor 4 and Generation of an Aminoterminally Truncated, Stable Form by Saiko Sugawara; Toshihiko Ito; Shiori Sato; Yuki Sato; Kano Kasuga; Ikuo Kojima; Masayuki Kobayashi (206-215).
Fibroblast growth factor 4 (FGF4) is considered as a crucial gene for tumorigenesis in humans and the development of mammalian embryos. The secreted, mature form of human FGF4 is thought to be comprised of 175 amino acid residues (proline32 to leucine206, Pro32-Leu206). Here, we found that bacterially expressed, 6× histidine (His)-tagged human FGF4 (Pro32-Leu206) protein, referred to as HishFGF4, was unstable such as in phosphate-buffered saline. In these conditions, site-specific cleavage, including between Ser54 and Leu55, in HishFGF4 was identified. In order to generate stable human FGF4 derivatives, a 6× His-tagged human FGF4 (Leu55-Leu206), termed HishFGF4L, was expressed in Escherichia coli. HishFGF4L could be purified from the supernatant of cell lysates by heparin column chromatography. In phosphate-buffered saline, HishFGF4L was considered as sufficiently stable. HishFGF4L exerted significant mitogenic activities in mouse embryonic fibroblast Balb/c 3T3 cells. In the presence of PD173074, an FGF receptor inhibitor, the growth-stimulating activity of HishFGF4L disappeared. Taken together, we suggest that HishFGF4L is capable of promoting cell growth via an authentic FGF signaling pathway. Our study provides a simple method for the production of a bioactive human FGF4 derivative in E. coli.
Keywords: E. coli ; FGF4; Human; Recombinant; Stable; Truncate

Reactive oxygen species (ROS) production is the first level of response by a host during stress. Even though the ROS are toxic to cell, when present in a limited amount, they act as a signalling molecule for the expression of defence-related genes and later are scavenged by either enzymatic or non-enzymatic mechanisms of the host. The different anti-oxidative enzymes like glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APO), peroxidase (POD) and polyphenol oxidase (PPO) were estimated, and their activities were compared between infected and healthy leaves of the tolerant and susceptible cultivars of tea. The infected leaves of the susceptible cultivars registered higher amount of enzyme activity when compared with the tolerant cultivars. The study reveals that the more anti-oxidative enzymes, the more susceptible the cultivar will be.
Keywords: Anti-oxidative enzymes; Disease resistance; Grey blight; Pestalotiopsis sp.

In this study, different cultivation systems such as roller bottles (RB), 5-L stirred-tank bioreactor (STR), and disposable bioreactors were used to cultivate hybridoma for lab-scale production of Salmonella Enteritidis O-antigen-specific monoclonal antibody (MAb). Hybridoma cell line was cultivated in either serum-containing or serum-free medium (SFM) culture conditions. In STR, MAb production scaled up to 4 L, and production capabilities of the cells were also evaluated in different featured production systems. Moreover, the growth parameters of the cells in all production systems such as glucose consumption, lactate and ammonia production, and also MAb productivities were determined. Collected supernatants from the reactors were concentrated by a cross-flow filtration system. In conclusion, cells were not adapted to SFM in RB and STR. Therefore, less MAb titer in both STR and RB systems with SFM was observed compared to the cultures containing fetal bovine serum-supplemented medium. A higher MAb titer was gained in the membrane-aerated system compared to those in STR and RB. Although the highest MAb titer was obtained in the static membrane bioreactor system, the highest productivity was obtained in STR operated in semicontinuous mode with overlay aeration.
Keywords: Hybridoma; Monoclonal antibody; Salmonella Enteritidis; Bioreactors; Cross-flow filtration

Suramin is a Novel Activator of PP5 and Biphasically Modulates S100-Activated PP5 Activity by Fuminori Yamaguchi; Sho Yamamura; Seiko Shimamoto; Hiroshi Tokumitsu; Masaaki Tokuda; Ryoji Kobayashi (237-247).
Suramin is an activator of ryanodine receptors and competitively binds to the calmodulin-binding site. In addition, S100A1 and calmodulin compete for the same binding site on ryanodine receptors. We therefore studied the effects of suramin on protein phosphatase 5 (PP5) and S100-activated PP5. In the absence of S100 proteins, suramin bound to the tetratricopeptide repeat (TPR) domain of PP5 and activated the enzyme in a dose-dependent manner. In the presence of S100A2/Ca2+, lower concentrations of suramin dose-dependently inhibited PP5 activity as an S100 antagonist, whereas higher concentrations of suramin reactivated PP5. Although the C-terminal fragment of heat shock protein 90 (HspC90) also weakly activated PP5, the binding site of suramin and HspC90 may be different, and addition of suramin showed no clear effect on the phosphatase activity of PP5. Similar biphasic effects of suramin were observed with S100A1-, S100B- or S100P-activated PP5. However, the inhibitory effects of lower concentrations of suramin on S100A6-activated PP5 are weak and high concentrations of suramin further activated PP5. SPR and the cross-linking study showed inhibition of the interaction between S100 protein and PP5 by suramin. Our results revealed that suramin is a novel PP5 activator and modulates S100-activated PP5 activity by competitively binding to the TPR domain.
Keywords: Suramin; S100 protein; Protein phosphatase 5; Hsp90; Tetratricopeptide repeat

Two new water-soluble pip and hpip analogs, 1 and 2 pip = 2-phenylimidazo[4,5-f][1, 10]phenanthroline; hpip = 2-(2-hydroxyphenyl)imidazo[4,5-f][1, 10]phenanthroline, have been synthesized and fully characterized by CHN analysis, MALDI-TOF MS, 1H-NMR, IR (ATR), and UV–Vis methods. The DNA-binding behaviors of both compounds have been studied by viscosity measurements, spectroscopic methods, and gel electrophoresis studies, and potential for antitumor activity was evaluated by measuring their ability to inhibit DNA transcription. The results indicate that both compounds show some strong binding to DNA in a mixture of electrostatic and intercalative mode resulting in the intrinsic binding constants Kb of (4.0 ± 0.5) × 105 M−1 and (7.5 ± 0.5) × 105 M−1 for 1 and 2, respectively. These strong binding affinities for DNA are comparable for that seen for many transition metal-based intercalators. Comparatively, observed difference in the DNA-binding affinities of two complexes can be reasonably explained by the presence of an intra-molecular hydrogen-bonding between the ortho-phenolic group and the nitrogen atom of the imidazole ring. The extended co-planarity of 2 due to the intramolecular hydrogen bonding may lead to an enhancement of DNA binding affinity of 2. In addition, 2 can promote cleavage of pBR322 DNA upon irradiation, it inhibits DNA transcription and it is more cytotoxic at lower concentrations in comparison to 1, as revealed by the spectroscopic measurements.
Keywords: Polypyridyl Ligands; DNA-Binding; Photocleavage; Antitumor Activity; Transcription Inhibition Assay; Cytotoxicity

Characterization of Biocatalysts Prepared with Thermomyces lanuginosus Lipase and Different Silica Precursors, Dried using Aerogel and Xerogel Techniques by Carlos Eduardo Barão; Leandro Daniel de Paris; João Henrique Dantas; Matheus Mendonça Pereira; Lucio Cardozo Filho; Heizir Ferreira de Castro; Gisella Maria Zanin; Flavio Faria de Moraes; Cleide Mara Faria Soares (263-274).
The use of lipases in industrial processes can result in products with high levels of purity and at the same time reduce pollutant generation and improve both selectivity and yields. In this work, lipase from Thermomyces lanuginosus was immobilized using two different techniques. The first involves the hydrolysis/polycondensation of a silica precursor (tetramethoxysilane (TMOS)) at neutral pH and ambient temperature, and the second one uses tetraethoxysilane (TEOS) as the silica precursor, involving the hydrolysis and polycondensation of the alkoxide in appropriate solvents. After immobilization, the enzymatic preparations were dried using the aerogel and xerogel techniques and then characterized in terms of their hydrolytic activities using a titrimetric method with olive oil and by the formation of 2-phenylethyl acetate in a transesterification reaction. The morphological properties of the materials were characterized using scanning electron microscopy, measurements of the surface area and pore size and volume, thermogravimetric analysis, and exploratory differential calorimetry. The results of the work indicate that the use of different silica precursors (TEOS or TMOS) and different drying techniques (aerogel or xerogel) can significantly affect the properties of the resulting biocatalyst. Drying with supercritical CO2 provided higher enzymatic activities and pore sizes and was therefore preferable to drying, using the xerogel technique. Thermogravimetric analysis and differential scanning calorimetry analyses revealed differences in behavior between the two biocatalyst preparations due to the compounds present.
Keywords: Lipase; Immobilization; Sol–gel; Xerogel; Aerogel; Characterization

l-Ribose is an important precursor for antiviral agents, and thus its high-level production is urgently demanded. For this aim, immobilized recombinant Escherichia coli cells expressing the l-arabinose isomerase and variant mannose-6-phosphate isomerase genes from Geobacillus thermodenitrificans were developed. The immobilized cells produced 99 g/l l-ribose from 300 g/l l-arabinose in 3 h at pH 7.5 and 60 °C in the presence of 1 mM Co2+, with a conversion yield of 33 % (w/w) and a productivity of 33 g/l/h. The immobilized cells in the packed-bed bioreactor at a dilution rate of 0.2 h−1 produced an average of 100 g/l l-ribose with a conversion yield of 33 % and a productivity of 5.0 g/l/h for the first 12 days, and the operational half-life in the bioreactor was 28 days. Our study is first verification for l-ribose production by long-term operation and feasible for cost-effective commercialization. The immobilized cells in the present study also showed the highest conversion yield among processes from l-arabinose as the substrate.
Keywords: l-Arabinose isomerase; Mannose-6-phosphate isomerase; Geobacillus thermodenitrificans ; Immobilized cells; Packed-bed bioreactor; l-Ribose production

In this study, an enzymatic procedure for the determination of glycine (Gly) was developed by using a column containing immobilized glutamate dehydrogenase (GDH) on glyoxal agarose beads. Ammonia is produced from the enzymatic reactions between Gly and GDH with NAD+ in phosphate buffer medium. The indophenol blue method was used for ammonia detection based on the spectrophotometric measurements of blue-colored product absorbing at 640 nm. The calibration graph is linear in the range of 0.1–10 mM of Gly concentrations. The effect of pH, temperature, and time interval was studied to find column stability, and also the interference effects of other amino acids was investigated. The interaction between GDH and glyoxal agarose beads was analyzed by Fourier transform infrared (FTIR) spectroscopy. The morphology of the immobilized and non-immobilized agarose beads were characterized by atomic force microscopy (AFM).
Keywords: Glycine; Glutamate dehydrogenase; Glyoxal agarose; Indophenol blue; FTIR

Evaluation of Genetic Homogeneity in Tissue Culture Regenerates of Jatropha curcas L. using Flow Cytometer and DNA-based Molecular Markers by Mangal S. Rathore; P. Yadav; Shaik G. Mastan; Ch. R. Prakash; A. Singh; Pradeep K. Agarwal (298-310).
The present investigation aimed to evaluate the reliability of in vitro propagation methods for elite genotypes of Jatropha curcas L., that maintain genetic integrity of tissue culture (TC) regenerates among two regeneration systems developed through direct shoot bud regeneration using nodal/apical shoot segments (protocol-A) and in vitro-derived leaves (protocol-B) as explants. Random amplified polymorphic DNA (RAPD), intersimple sequence repeat (ISSR), simple sequence repeat (SSR) molecular markers, and flow cytometery (FCM) were employed to evaluate genetic homogeneity in TC-regenerates at different passages of subcultures. RAPD markers showed genetic homogeneity in fifth-generation TC-regenerates of both protocols. ISSR markers showed genetic stability of leaf regenerates (protocol-B) at 10th generation. FCM analysis of TC-regenerates at 10th generation in protocol-B and at 20th generation in both protocols, showed stability of ploidy level. SSR assessment of TC-regenerates at 20th generation in both protocols confirmed genetic homogeneity. The results confirmed the genetic stability of the TC-regenerates and demonstrated the reliability of the regeneration systems developed so far using explants of two different origins, for large-scale multiplication of elite genotypes of Jatropha.
Keywords: Biodiesel; Genetic homogeneity; Jatropha; Microsatellite; Ploidy; Wasteland

Extract from Eugenia punicifolia is an Antioxidant and Inhibits Enzymes Related to Metabolic Syndrome by Denise Morais Lopes Galeno; Rosany Piccolotto Carvalho; Ana Paula de Araújo Boleti; Arleilson Sousa Lima; Patricia Danielle Oliveira de Almeida; Carolina Carvalho Pacheco; Tatiane Pereira de Souza; Emerson Silva Lima (311-324).
The present study aimed to investigate in vitro biological activities of extract of Eugenia punicifolia leaves (EEP), emphasizing the inhibitory activity of enzymes related to metabolic syndrome and its antioxidant effects. The antioxidant activity was analyzed by free radicals scavengers in vitro assays: DPPH·, ABTS·+, O2 ·−, and NO· and a cell-based assay. EEP were tested in inhibitory colorimetric assays using α-amylase, α-glucosidase, xanthine oxidase, and pancreatic lipase enzymes. The EEP exhibited activity in ABTS·+, DPPH·, and O2 ·− scavenger (IC50 = 10.5 ± 1.2, 28.84 ± 0.54, and 38.12 ± 2.6 μg/mL), respectively. EEP did not show cytotoxic effects, and it showed antioxidant activity in cells in a concentration-dependent manner. EEP exhibited inhibition of α-amylase, α-glucosidase, and xanthine oxidase activities in vitro assays (IC50 = 122.8 ± 6.3; 2.9 ± 0.1; 23.5 ± 2.6), respectively; however, EEP did not inhibit the lipase activity. The findings supported that extract of E. punicifolia leaves is a natural antioxidant and inhibitor of enzymes, such as α-amylase, α-glucosidase, and xanthine oxidase, which can result in a reduction in the carbohydrate absorption rate and decrease of risks factors of cardiovascular disease, thereby providing a novel dietary opportunity for the prevention of metabolic syndrome.
Keywords: Eugenia punicifolia ; Antioxidant; Digestive enzymes; Metabolic syndrome; In vitro assay

Isolation and Functional Characterization of a Novel Seed-Specific Promoter Region from Peanut by Sowmini Sunkara; Pooja Bhatnagar-Mathur; Kiran Kumar Sharma (325-339).
The importance of using tissue-specific promoters in the genetic transformation of plants has been emphasized increasingly. Here, we report the isolation of a novel seed-specific promoter region from peanut and its validation in Arabidopsis and tobacco seeds. The reported promoter region referred to as groundnut seed promoter (GSP) confers seed-specific expression in heterologous systems, which include putative promoter regions of the peanut (Arachis hypogaea L.) gene 8A4R19G1. This region was isolated, sequenced, and characterized using gel shift assays. Tobacco transgenics obtained using binary vectors carrying uidA reporter gene driven by GSP and/or cauliflower mosaic virus 35S promoters were confirmed through polymerase chain reaction (PCR), RT-PCR, and computational analysis of motifs which revealed the presence of TATA, CAAT boxes, and ATG signals. This seed-specific promoter region successfully targeted the reporter uidA gene to seed tissues in both Arabidopsis and tobacco model systems, where its expression was confirmed by histochemical analysis of the transgenic seeds. This promoter region is routinely being used in the genetic engineering studies in legumes aimed at targeting novel transgenes to the seeds, especially those involved in micronutrient enhancement, fungal resistance, and molecular pharming.
Keywords: Floral dip; Arabidopsis ; Tobacco; Gel shift assay; Legumes; Promoter; Seed protein; GUS

Succinic Acid Production from Corn Cob Hydrolysates by Genetically Engineered Corynebacterium glutamicum by Chen Wang; Hengli Zhang; Heng Cai; Zhihui Zhou; Yilu Chen; Yali Chen; Pingkai Ouyang (340-350).
Corynebacterium glutamicum wild type lacks the ability to utilize the xylose fractions of lignocellulosic hydrolysates. In the present work, we constructed a xylose metabolic pathway in C. glutamicum by heterologous expression of the xylA and xylB genes coming from Escherichia coli. Dilute-acid hydrolysates of corn cobs containing xylose and glucose were used as a substrate for succinic acid production by recombinant C. glutamicum NC-2. The results indicated that the available activated charcoal pretreatment in dilute-acid hydrolysates of corn cobs could be able to overcome the inhibitory effect in succinic acid production. Succinic acid was shown to be efficiently produced from corn cob hydrolysates (55 g l−1 xylose and 4 g l−1 glucose) under oxygen deprivation with addition of sodium carbonate. Succinic acid concentration reached 40.8 g l−1 with a yield of 0.69 g g−1 total sugars within 48 h. It was the first report of succinic acid production from corn cob hydrolysates by metabolically engineered C. glutamicum. This study suggested that dilute-acid hydrolysates of corn cobs may be an alternative substrate for the efficient production of succinic acid by C. glutamicum.
Keywords: Corynebacterium glutamicum ; Succinic acid; Corn cob hydrolysates; Xylose utilization; Biomass

Purification and Characterization of a Urethanase from Penicillium variabile by Nan-di Zhou; Xiao-lei Gu; Xiao-hong Zha; Ya-ping Tian (351-360).
Urethanase produced by Penicillium variabile was purified through ultrasonication, concentration by polyethylene glycol 20,000, and Superdex G-200 gel filtration chromatography. The molecular weight of urethanase was determined to be around 96 kDa by gel filtration. The purified enzyme showed a single band in SDS-PAGE with the molecular weight of ~13.7 kDa, which suggests that the enzyme has a multimeric structure composed of the same subunits. Peptide map fingerprinting analysis was then carried out by MALDI/TOF-TOF MS. Within the known sequences in NCBI, glucosamine-6-phosphate deaminase and 6-phosphogluconate dehydrogenase get high score as compared with urethanase. Sequence analysis informs that N-terminal sequence of urethanase is GTNTADNDAA. The Minchaelis constant (K m) and maximum reaction rate (V m) of urethanase are 27.2 mmol/L and 156.25 μmol/L min, respectively.
Keywords: Urethanase; Penicillium variabile ; Purification; Sequence analysis; Characterization

Kinetic Modeling of Ethanol Production by Scheffersomyces stipitis from Xylose by Daniele Farias; Rafael R. de Andrade; Francisco Maugeri-Filho (361-379).
This work focuses on the kinetics of ethanol production by Scheffersomyces stipitis on xylose with the development of a mathematical model considering the effect of substrate and product concentrations on growth rate. Experiments were carried out in batch and continuous modes, with substrate concentration varying from 7.2 to 145 g L−1. Inhibitory effects on cell growth, substrate uptake, and ethanol production rates were found to be considerable. Kinetic parameters were obtained through linear and non-linear regression methods. Experiments in continuous mode were performed at different dilution rates to evaluate the inhibitory effect of ethanol. A mixed mathematical model which combined Andrews and Levenspiel's models, combining substrate and product inhibition, was used. A quasi-Newton routine was applied to obtain a more accurate fitting of kinetic parameters. The parameters such as cell to product factor (Y P/X) and limiting cell yield (Y X) were shown to be dependent on substrate concentration. The kinetic model fitted satisfactorily the experimental data.
Keywords: Ethanol fermentation; Scheffersomyces stipitis ; Kinetic parameter estimation; Mathematical modeling

The thermotolerant yeast Pichia etchellsii produces multiple cell bound β-glucosidases that can be used for synthesis of important alkyl- and aryl-glucosides. Present work focuses on enhancement of β-glucosidase I (BGLI) production in Pichia pastoris. In the first step, one-factor-at-a-time experimentation was used to investigate the effect of aeration, antifoam addition, casamino acid addition, medium pH, methanol concentration, and mixed feed components on BGLI production. Among these, initial medium pH, methanol concentration, and mixed feed in the induction phase were found to affect BGLI production. A 3.3-fold improvement in β-glucosidase expression was obtained at pH 7.5 as compared to pH 6.0 on induction with 1 % methanol. Addition of sorbitol, a non-repressing substrate, led to further enhancement in β-glucosidase production by 1.4-fold at pH 7.5. These factors were optimized with response surface methodology using Box–Behnken design. Empirical model obtained was used to define the optimum “operating space” for fermentation which was a pH of 7.5, methanol concentration of 1.29 %, and sorbitol concentration of 1.28 %. Interaction of pH and sorbitol had maximum effect leading to the production of 4,400 IU/L. The conditions were validated in a 3-L bioreactor with accumulation of 88 g/L biomass and 2,560 IU/L β-glucosidase activity.
Keywords: β-glucosidase I; Pichia pastoris ; Response surface methodology; Box–Behnken Design; Pichia etchellsii

The L-Cell Isolation from Heterogonous Population of Intestinal Cell Line Using Antibiotic Selection Method by Mina Rasouli; Sakineh Abbasi; Kazhal Sarsaifi; Homayoun Hani; Zalinah Ahmad; Abdul Rahman Omar (394-404).
Enteroendocrine cells are the largest population of hormone-producing cells in the body and play important roles in many aspects of body functions. The enteroendocrine cell population is divided into different subpopulations that secrete different hormones and peptides. Characterization of each subpopulation is particularly useful for analyzing the cellular mechanisms responsible for specific cell types. Therefore, the necessity of a pure cell line for a specific study purpose was the important motivation for the separation of cell lines for each subpopulation of enteroendocrine cells. The present research introduces a method for the isolation of L-cells, one of the important subpopulations of enteroendocrine cells. The antibiotic selection method was conducted in order to isolate the L-cells from a heterogonous population of intestinal cell line. In this method, a neomycin resistance gene (as selected marker) was expressed under the control of a specific promoter of L-cells. After transfection of manipulated plasmid, only the cells which determine the specific promoter and express neomycin resistance protein would be able to survive under Geneticin antibiotic treatment condition. In order to confirm that the isolated cells were L-cells, reverse transcriptase polymerase chain reaction (PCR) and quantitative PCR assays were performed. Based on the results, the isolated cells were pure L-cells that could be able to express specific mRNA of L-cells efficiently. This technique provides a unique method for the isolation and purification of any cell line. The purified isolated L-cells by this method can be used for future studies and for analyzing cellular mechanisms that involve L-cells' functions.
Keywords: Enteroendocrine cells; L-cells; Antibiotic selection method; Neomycin resistance gene; Pure cell line

Nutrient Removal and Biomass Production in an Outdoor Pilot-Scale Phototrophic Biofilm Reactor for Effluent Polishing by N. C. Boelee; M. Janssen; H. Temmink; R. Shrestha; C. J. N. Buisman; R. H. Wijffels (405-422).
An innovative pilot-scale phototrophic biofilm reactor was evaluated over a 5-month period to determine its capacity to remove nitrogen and phosphorus from Dutch municipal wastewater effluents. The areal biomass production rate ranged between 2.7 and 4.5 g dry weight/m2/day. The areal nitrogen and phosphorus removal rates averaged 0.13 g N/m2/day and 0.023 g P/m2/day, which are low compared to removal rates achieved in laboratory biofilm reactors. Nutrient removal increased during the day, decreased with decreasing light intensity and no removal occurred during the night. Additional carbon dioxide supply was not requisite as the wastewater was comprised of enough inorganic carbon to sustain microalgal growth. The study was not conclusive for the limiting factor that caused the low nutrient removal rate, possibly the process was limited by light and temperature, in combination with pH increases above pH 9 during the daytime. This pilot-scale study demonstrated that the proposed phototrophic biofilm reactor is not a viable post-treatment of municipal wastewater effluents under Dutch climate conditions. However, the reactor performance may be improved when controlling the pH and the temperatures in the morning. With these adaptations, a phototrophic biofilm reactor could be feasible at lower latitudes with higher irradiance levels.
Keywords: Microalgae; Biofilm; Wastewater treatment; Nitrogen removal; Phosphorus removal

The effect of cultivation condition of two locally isolated ascomycetes strains namely Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2 were compared in submerged and solid state fermentation. Physical evaluation on water absorption index, solubility index and chemical properties of lignin, hemicellulose and cellulose content as well as the cellulose structure on crystallinity and amorphous region of treated oil palm empty fruit bunch (OPEFB) (resulted in partial removal of lignin), sago pith residues (SPR) and oil palm decanter cake towards cellulases production were determined. Submerged fermentation shows significant cellulases production for both strains in all types of substrates. Crystallinity of cellulose and its chemical composition mainly holocellulose components was found to significantly affect the total cellulase synthesis in submerged fermentation as the higher crystallinity index, and holocellulose composition will increase cellulase production. Treated OPEFB apparently induced the total cellulases from T. asperellum UPM1 and A. fumigatus UPM2 with 0.66 U/mg FPase, 53.79 U/mg CMCase, 0.92 U/mg β-glucosidase and 0.67 U/mg FPase, 47.56 U/mg and 0.14 U/mg β-glucosidase, respectively. Physical properties of water absorption and solubility for OPEFB and SPR also had shown significant correlation on the cellulases production.
Keywords: Cellulases; Submerged fermentation; Solid state fermentation; Enzymatic hydrolysis; Water absorption and solubility; Crystallinity

Expression and Characterization of Recombinant GH11 Xylanase from Thermotolerant Streptomyces sp. SWU10 by Wasana Sukhumsirichart; Warin Deesukon; Takuya Kawakami; Shotaro Matsumoto; Weeranuch Seesom; Tatsuji Sakamoto (436-446).
Xylans are major hemicellulose components of plant cell wall which can be hydrolyzed by xylanolytic enzymes. Three forms of endo-β-1,4-xylanases (XynSW1, XynSW2A, and XynSW2B) produced by thermotolerant Streptomyces sp. SWU10 have been reported. In the present study, we described the expression and characterization of the fourth xylanase enzyme from this bacteria, termed XynSW3. The gene containing 726 bp was cloned and expressed in Escherichia coli. The recombinant enzyme (rXynSW3) was purified from cell-free extract to homogeneity using Ni-affinity column chromatography. The apparent molecular mass of rXynSW3 was 48 kDa. Amino acid sequence analysis revealed that it belonged to a xylanase of glycoside hydrolase family 11. The optimum pH and temperature for enzyme activity were 5.5–6.5 and 50 °C, respectively. The enzyme was stable up to 40 °C and in wide pH ranges (pH 0.6–10.3). Xylan without arabinosyl side chain is the most preferable substrate for the enzyme. By using birch wood xylan as substrate, rXynSW3 produced several oligosaccharides in the initial stage of hydrolysis, and their levels increased with time, demonstrating that the enzyme is an endo-acting enzyme. The major products were xylobiose, triose, and tetraose. The rXynSW3 can be applied in several industries such as food, textile, and biofuel industries, and waste treatment.
Keywords: Expression; Characterization; Xylanase; Glycoside hydrolase family 11; Streptomyces sp.

Using microalgae to capture CO2 from flue gas is an ideal way to reduce CO2 emission, but this is challenged by the high cost of carbon capture and transportation. To address this problem, a bicarbonate-based integrated carbon capture and algae production system (BICCAPS) has been proposed, in which bicarbonate is used for algae culture, and the regenerated carbonate from this process can be used to capture more CO2. High-concentration bicarbonate is obligate for the BICCAPS. Thus, different strains of microalgae and cyanobacteria were tested in this study for their capability to grow in high-concentration NaHCO3. The highest NaHCO3 concentrations they are tolerant to were determined as 0.30 M for Synechocystis sp. PCC6803, 0.60 M for Cyanothece sp., 0.10 M for Chlorella sorokiniana, 0.60 M for Dunaliella salina, and 0.30 M for Dunaliella viridis and Dunaliella primolecta. In further study, biomass production from culture of D. primolecta in an Erlenmeyer flask with either 0.30 M NaHCO3 or 2 % CO2 bubbling was compared, and no significant difference was detected. This indicates BICCAPS can reach the same biomass productivity as regular CO2 bubbling culture, and it is promising for future application.
Keywords: Bicarbonate; CO2 ; Dunaliella ; Chlorella ; Synechocystis ; Cyanothece

Endoglucanase and Total Cellulase from Newly Isolated Rhizopus oryzae and Trichoderma reesei: Production, Characterization, and Thermal Stability by Larine Kupski; Fernanda Arnhold Pagnussatt; Jaqueline Garda Buffon; Eliana Badiale Furlong (458-468).
A multienzymatic complex production was evaluated, as well as endoglucanase and total cellulase characterization, during solid-state fermentation of rice industry wastes with Rhizopus oryzae CCT 7560 (newly isolated microorganism) and Trichoderma reesei QM 9414 (control). R. oryzae produced enzymes with higher activity at 15 h of fermentation (5.1 and 2.3 U g−1 to endoglucanase and total cellulase), while T. reesei produced them at 55 h (15.3 and 2.8 U g−1 to endoglucanase and total cellulase). The optimum temperature for total cellulase and endoglucanase was 60 °C. For Trichoderma and Rhizopus, the optimum pH was 5.0 and 6.0 for total cellulase and 6.0 and 5.0 for endoglucanase, respectively. The enzymes produced by Rhizopus presented higher stability at the temperature range evaluated (25–100 °C); the endoglucanase K M value was 20 times lower than the one found for Trichoderma. The characterization of the cellulolytic enzymes from the fungal species native of rice husk revealed that they can be more efficient than the genetically modified enzymes when rice husk and rice bran are used as substrates.
Keywords: Solid-state fermentation; Cellulase; Biochemical characterization; Rice bran; Rice husk

Water Miscible Mono Alcohols’ Effect on the Proteolytic Performance of Bacillus clausii Serine Alkaline Protease by Yonca (Avcı) Duman; Dilek Kazan; Aziz Akın Denizci; Altan Erarslan (469-486).
In this study, our investigations showed that the increasing concentrations of all examined mono alcohols caused a decrease in the V m, k cat and k cat/K m values of Bacillus clausii GMBE 42 serine alkaline protease for casein hydrolysis. However, the K m value of the enzyme remained almost the same, which was an indicator of non-competitive inhibition. Whereas inhibition by methanol was partial non-competitive, inhibition by the rest of the alcohols tested was simple non-competitive. The inhibition constants (K I) were in the range of 1.32–3.10 M, and the order of the inhibitory effect was 1-propanol>2-propanol>methanol>ethanol. The ΔG and ΔG E − T values of the enzyme increased at increasing concentrations of all alcohols examined, but the ΔG ES value of the enzyme remained almost the same. The constant K m and ΔG ES values in the presence and absence of mono alcohols indicated the existence of different binding sites for mono alcohols and casein on enzyme the molecule. The k cat of the enzyme decreased linearly by increasing log P and decreasing dielectric constant (D) values, but the ΔG and ΔG E − T values of the enzyme increased by increasing log P and decreasing D values of the reaction medium containing mono alcohols.
Keywords: Bacillus clausii ; Alkaline protease; Mono alcohols; Proteolytic performance

Leptospira interrogans, a Gram-negative pathogen, could cause infections in a wide variety of mammalian hosts, but due to their fastidious cultivation requirements and the lack of genetic systems, the pathogenic factor is still not clear. Isocitrate dehydrogenase (IDH) is a key enzyme in the tricarboxylation (TCA) cycle, which could have an important impact on the growth and pathogenesis of the bacteria. In the present study, we first report the cloning, heterologous expression, and detailed characterization of the IDH gene from L. interrogans serovar Lai strain 56601(LiIDH). The molecular weight of LiIDH was determined to be 87 kDa by filtration chromatography, suggesting LiIDH is a typical homodimer. The optimum activity of LiIDH was found at 60 °C, and its optimum pH was 7.0 (Mn2+) and 8.0 (Mg2+). Heat inactivation studies showed that heat treatment for 20 min at 50 °C caused a 50 % loss of enzyme activity. LiIDH was completely divalent cation dependent as other typical dimeric IDHs and Mg2+ was its best activator. The recombinant LiIDH specificities (k cat/K m values for NADP+ and NAD+) in the presence of Mg2+ and Mn2+ were 6,269-fold and 1,000-fold greater for NADP+ than NAD+, respectively. This current work is expected to shed light on the functions of metabolic enzymes in L. interrogans and provide useful information for LiIDH to be considered as a possible candidate for serological diagnostics and detection of L. interrogans infection.
Keywords: Leptospira interrogans ; Isocitrate dehydrogenase; Kinetics; Coenzyme specificity; Diagnosic candidate

Canine Parvovirus Type 2a (CPV-2a)-Induced Apoptosis in MDCK Involves Both Extrinsic and Intrinsic Pathways by Juwar Doley; Lakshya Veer Singh; G. Ravi Kumar; Aditya Prasad Sahoo; Lovleen Saxena; Uttara Chaturvedi; Shikha Saxena; Rajiv Kumar; Prafull Kumar Singh; R. S. Rajmani; Lakshman Santra; S. K. Palia; S. Tiwari; D. R. Harish; Arvind Kumar; G. S. Desai; Smita Gupta; Shishir K. Gupta; A. K. Tiwari (497-508).
The canine parvovirus type 2 (CPV-2) causes an acute disease in dogs. It has been found to induce cell cycle arrest and DNA damage leading to cellular lysis. In this paper, we evaluated the apoptotic potential of the “new CPV-2a” in MDCK cells and elucidated the mechanism of the induction of apoptosis. The exposure of MDCK cells to the virus was found to trigger apoptotic response. Apoptosis was confirmed by phosphatidylserine translocation, DNA fragmentation assays, and cell cycle analysis. Activation of caspases-3, -8, -9, and -12 and decrease in mitochondrial potential in CPV-2a-infected MDCK cells suggested that the CPV-2a-induced apoptosis is caspase dependent involving extrinsic, intrinsic, and endoplasmic reticulum pathways. Increase in p53 and Bax/Bcl2 ratio was also observed in CPV-2a-infected cells.
Keywords: Apoptosis; Caspase; CPV-2; MDCK cells; Apoptotic pathways

Candida rugosa lipase was immobilized with a sol–gel encapsulation procedure in the presence and absence of a calix[n]arene carboxylic acid derivative grafted onto magnetic nanoparticles or in the presence of the calix[n]arene carboxylic acid derivative with Fe3O4 magnetic nanoparticles as an additive. Through the enantioselective hydrolysis of racemic naproxen methyl ester and the hydrolysis of p-nitrophenylpalmitate, the relative enzyme activity was evaluated and tested. These results show that the encapsulated lipase without supports has lower conversion and enantioselectivity compared to the Calix[n]COOH-based encapsulated lipase. It has also been observed that the Calix[4]COOH-based encapsulated lipase has excellent enantioselectivity (enantiomeric ratio (E) > 400) as compared to encapsulated-free lipase enantioselectivity (E = 137), and it also has an enantiomeric excess value of ~98 % for S-naproxen.
Keywords: Lipase; Calix[n]arene; Enantioselectivity; Magnetic nanoparticles; Sol–gel encapsulation

Cold-Active Xylanase Produced by Fungi Associated with Antarctic Marine Sponges by Abdiel Del-Cid; Pamela Ubilla; María-Cristina Ravanal; Exequiel Medina; Inmaculada Vaca; Gloria Levicán; Jaime Eyzaguirre; Renato Chávez (524-532).
Despite their potential biotechnological applications, cold-active xylanolytic enzymes have been poorly studied. In this work, 38 fungi isolated from marine sponges collected in King George Island, Antarctica, were screened as new sources of cold-active xylanases. All of them showed xylanase activity at 15 and 23 °C in semiquantitative plate assays. One of these isolates, Cladosporium sp., showed the highest activity and was characterized in detail. Cladosporium sp. showed higher xylanolytic activity when grown on beechwood or birchwood xylan and wheat bran, but wheat straw and oat bran were not so good inducers of this activity. The optimal pH for xylanase activity was 6.0, although pH stability was slightly wider (pH 5–7). On the other hand, Cladosporium sp. showed high xylanase activity at low temperatures and very low thermal stability. Interestingly, thermal stability was even lower after culture media were removed and replaced by buffer, suggesting that low molecular component(s) of the culture media could be important in the stabilization of cold-active xylanase activity. To the best of our knowledge, this study is the first report on extracellular xylanase production by fungi associated with Antarctic marine sponges.
Keywords: Cold-active xylanases; Antarctic marine sponges; Fungi; Cladosporium sp.; Thermostability

The potentiality of Pongamia pinnata L. as a sustainable source of feedstock for the biodiesel industry is dependent on an extensive knowledge of the genome structure of the plant. Flow cytometry, with propidium iodide (PI) as the DNA stain, was used to estimate the nuclear DNA content of P. pinnata, with respect to Zea mays ‘CE-777’ as standard. The internal and pseudo-internal standardization was followed on account of the inhibitory effect of secondary compounds on PI intercalation. The antioxidants (PVP-40 and β-mercaptoethanol) were added to the nuclear isolation buffer for the reduction of inhibitory effect of P. pinnata cytosol. Nuclear DNA content estimation was done for P. pinnata leaves from different altitudes (37–117 m height from sea level) of Assam. Flow cytometry analysis indicated that the nuclear DNA content of P. pinnata is 2.66 pg with predicted 1C value of 1,300 Mb using Z. mays as standard. Coefficient of variation in flow cytometric analysis was within the limit of 5 % indicating that the results were reliable. Somatic chromosome numbers were counted from root–tip cells and was found to be 2n = 22 corresponding to the diploid level (x = 11). A decreasing trend in the nuclear DNA content was observed for the species of different altitudes.
Keywords: Flow cytometry; Internal standardization; Nuclear DNA content; PI intercalation; Pseudo-internal standardization; Pongamia pinnata

Siderophore Production by Bacillus megaterium: Effect of Growth Phase and Cultural Conditions by Sofia Santos; Isabel F. F. Neto; Manuela D. Machado; Helena M. V. M. Soares; Eduardo V. Soares (549-560).
Siderophore production by Bacillus megaterium was detected, in an iron-deficient culture medium, during the exponential growth phase, prior to the sporulation, in the presence of glucose; these results suggested that the onset of siderophore production did not require glucose depletion and was not related with the sporulation. The siderophore production by B. megaterium was affected by the carbon source used. The growth on glycerol promoted the very high siderophore production (1,182 μmol g−1 dry weight biomass); the opposite effect was observed in the presence of mannose (251 μmol g−1 dry weight biomass). The growth in the presence of fructose, galactose, glucose, lactose, maltose or sucrose, originated similar concentrations of siderophore (546–842 μmol g−1 dry weight biomass). Aeration had a positive effect on the production of siderophore. Incubation of B. megaterium under static conditions delayed and reduced the growth and the production of siderophore, compared with the incubation in stirred conditions.
Keywords: Bacillus megaterium ; Carbon source; Environmental-friendly chelating agents; Iron chelator; Siderophore production