Applied Biochemistry and Biotechnology (v.174, #8)

exo-Xylanase X from Aeromonas punctata ME-1 was functionally expressed in Escherichia coli with a carboxy terminal His tag (6×) and a molecular mass of 39.42 kDa, which is in agreement with the prediction from its amino acid composition. The recombinant exo-xylanase reached 186 mg l−1 after induction by isopropyl β-d-1-thiogalactopyranoside. Its optimal temperature and pH were 50 °C and 6, respectively. The enzyme showed not only an exo-xylanase activity with K m of 3.90 mg ml−1 and V max of 12.9 U μg−1 for hydrolysis of Remazol Brilliant Blue-xylan but also a considerable exo-glucanase activity (27.9 U mg−1) on P-nitrophenyl β-d-cellobioside. It hydrolyzed xylan predominantly to xylobiose, xylotriose, xylotetraose, and xylose. An enzyme mixture of exo-xylanase and endo-xylanase (50 μg ml−1 each) yielded a larger amount (330 mg l−1) of xylose from beechwood xylan than the controls (270 and 150 mg l−1) using them alone at 100 μg ml−1, indicating a synergistic action between the two xylanases favoring the hydrolysis of beechwood xylan to release more xylose.
Keywords: exo-Xylanase; Expression; Activity; Xylan; Hydrolysis

Tuberculosis is the most deadly infectious disease. In particular, pulmonary tuberculosis, being the predominant one, is highly contagious. In past the 200 years, one billion tuberculosis (TB) deaths had occurred, and it is anticipated that in the next 25 years, more than 40 million people may be killed by TB unless control measures are implemented. There are various causes which increase the susceptibility to Mycobacterium tuberculosis infection; these include weakened immune system which occurs through various diseases and medications like human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS), type II diabetes, end-stage kidney disease, alcoholism and intravenous drug use, certain cancers, cancer treatment such as chemotherapy, malnutrition and very young or advanced age. Some other factors include tobacco use, which increases the risk of getting TB and dying from it. In this manuscript, the authors tried to summarize all the alterations occurring in immune system at cellular and molecular level which occur due to infection, metabolic changes and chemical exposure, which increase susceptibility to mycobacterial infection.
Keywords: Susceptibility; Mycobacterium; Tuberculosis; Macrophages; Monocytes; HIV

Among forty endophytic fungal isolates derived from the mangrove plant Avicennia marina, thirty-seven isolates (92.5 %) shown vary antimycotic activity against clinical Trichophyton, Microsporum, and Epidermophyton isolates. The hyperactive wild antagonistic strains Acremonium sp. MERV1 and Chaetomium sp. MERV7 were subjected to intergeneric protoplast fusion technique, and out of 20 fusants obtained, the fusant MERV6270 showed the highest antimycotic activity with the broadest spectrum against all dermatophytes under study. Solid-state fermentation (SSF) showed its superiority for antimycotic/antiviral metabolite production using cost-effective agroindustrial residues. Low-cost novel fermentation medium containing inexpensive substrate mixture of molokhia stalk, lemon peel, pomegranate peel, peanut peel (2:1:1:1) moistened with potato, and meat processing wastewaters (2:1, at moisture content of 60 %) provided a high antimycotic metabolite yield, 33.25 mg/gds, by the fusant MERV6270. The optimal parameters for antimycotic productivity under SSF were incubation period (4 days), incubation temperature (27.5–30 °C), initial pH (6), initial moisture level (60 %), substrate particle size (1.0 mm), and inoculum size (2 × 106 spores/gds), which elucidated antimycotic activity to 44.19 mg/gds. Interestingly, wild mangrove Acremonium sp. MERV1 and Chaetomium sp. MERV7 strains and their fusant MERV6270 showed significant inhibition of hepatitis C virus with viral knockdown percent of −82.48, −82.44, and −97.37 %, respectively, compared to the control (100 %), which open a new era in combat epidemic viral diseases.
Keywords: Endophytes; Avicennia marina ; Intergeneric protoplast fusion; Antidermatophytes; Anti-HCV; Solid-state fermentation

Microbial hydrolysis of lignocellulosic biomass is becoming increasingly important for the production of renewable biofuels to address global energy concerns. Hemicellulose is the second most abundant lignocellulosic biopolymer consisting of mostly xylan and other polysaccharides. A variety of enzymes is involved in complete hydrolysis of xylan into its constituent sugars for subsequent biofuel fermentation. Two enzymes, endo-β-xylanase and β-xylosidase, are particularly important in hydrolyzing the xylan backbone into xylooligosaccharides and individual xylose units. In this study, we describe the cloning, expression, and characterization of xylanase and β-xylosidase isolated from Bacillus subtilis M015 in Escherichia coli. The genes were identified to encode a 213 amino acid protein for xylanase (glycoside hydrolase (GH) family 11) and a 533 amino acid protein for β-xylosidase (GH family 43). Recombinant enzymes were produced by periplasmic-leaky E. coli JE5505 and therefore secreted into the supernatant during growth. Temperature and pH optima were determined to be 50 °C and 5.5–6 for xylanase and 35 °C and 7.0–7.5 for β-xylosidase using beech wood xylan and p-nitrophenyl-β-D-xylopyranoside as the substrates, respectively. We have also investigated the synergy of two enzymes on xylan hydrolysis and observed 90 % increase in total sugar release (composed of xylose, xylobiose, xylotriose, and xylotetraose) for xylanase/β-xylosidase combination as opposed to xylanase alone.
Keywords: Hemicellulose; Xylan; Xylanase; β-xylosidase; Xylooligosaccharides; Bacillus subtilis ; Secretory expression; Escherichia coli JE5505

Stability and Activity of Porcine Lipase Against Temperature and Chemical Denaturants by P. Krishna Chaitanya; N. Prakash Prabhu (2711-2724).
Lipases are the class of hydrolases with wide industrial applications. The present study analyses the stability of porcine pancreatic lipase (PPL) against urea, guanidine hydrochloride (Gdn), sodium dodecyl sulphate (SDS), and temperature using different spectroscopic techniques. Interestingly, this two-domain protein shows a two-state unfolding transition against urea and Gdn. The free energy of unfolding of PPL calculated from global analysis of the unfolding transitions obtained from different spectroscopic techniques is ~2.2 kcal/mol. In the presence of SDS, PPL shows a cooperative loss of secondary and tertiary structures above 0.2 mM of SDS. At above 2 mM of SDS, PPL forms irreversible, non-native, thermally stable structure. PPL loses its activity even at lower concentrations of urea (3 M), Gdn (0.5 M), and SDS (0.8 mM). Thermal denaturation of PPL shows an irreversible unfolding, and the protein lost its activity even by increasing the temperature to 45 °C. Though PPL in higher concentrations of SDS (>5 mM) shows stable conformation against temperature, its activity is completely lost. The results suggest that the structure and activity of PPL are more sensitive against chemical denaturants and temperature, and forms irreversible, non-native (in SDS) or completely unfolded (in urea, Gdn, and at higher temperature) conformations in different denaturing conditions.
Keywords: Porcine lipase; Activity; Guanidine hydrochloride; Urea; Irreversible unfolding; Thermal denaturation; SDS

A lipopeptide biosurfactant production from a probiotic type strain of Propionibacterium freudenreichii subsp. freudenreichii is being reported here for the first time. This biosurfactant is able to reduce the surface tension of water from 72 to 38 mN/m with an increase of the biosurfactant concentration up to critical micelle concentration value of 1.59 mg/ml. The production of the biosurfactant was found to be much higher in medium containing sunflower oil compared to the glucose-containing medium. The maximum emulsifying activity (E24 = 72 %) was attained with used frying sunflower oil, while kerosene and starch had the lowest emulsifying activity. Biosurfactant production seems to be parallel to cell growth. The produced biosurfactant was relatively thermo-stable and no appreciable changes in biosurfactant activity occurred at temperature ranges of 25–85 °C. The analysis of the extracted biosurfactant by thin layer chromatography, infrared spectroscopy, and 1H and 13CNMR spectroscopy revealed the chemical nature of the biosurfactant as lipopeptide. Produced lipopeptide was evaluated for its antimicrobial and antiadhesive activity and showed significant antimicrobial and antiadhesive action against a wide range of pathogenic bacteria and fungi. A total growth inhibition was observed over Rhodococcus erythropolis, while the best result of antiadhesion was obtained against Pseudomonas aeruginosa.
Keywords: Propionibacterium freudenreichii ; Lipopeptide; Antimicrobial activity; Antiadhesive activity

Evaluation of Textile Dye Degradation Due to the Combined Action of Enzyme Horseradish Peroxidase and Hydrogen Peroxide by A. R. Pereira; R. S. da Costa; L. Yokoyama; E. M. Alhadeff; L. A. C. Teixeira (2741-2747).
The kinetic parameters of the oxidant action of the combination of enzyme horseradish peroxidase (HRP) with hydrogen peroxide in the degradation of methylene blue dye were investigated. Twenty-one percent of color removal was obtained at pH 5.0 and temperature of 30 °C. Under these conditions, the kinetic parameters K m and V max of enzymatic reactions were determined for hydrogen peroxide in the absence of methylene blue dye (K m = 17.3 mM; V max = 1.97 mM/min) and in the presence of methylene blue dye (K m = 0.27 mM, V max = 0.29 μM/min). By means of analysis of phosphorescence, the presence of reactive oxygen species was detected in the form of singlet oxygen through the redox reaction between HRP and hydrogen peroxide. The existence of this reactive species is directly dependent on the concentration of hydrogen peroxide in the aqueous solution.
Keywords: Singlet oxygen; Horseradish peroxidase; Degradation; Methylene blue

Enzymes Produced by Halotolerant Spore-Forming Gram-Positive Bacterial Strains Isolated From a Resting Habitat (Restinga de Jurubatiba) in Rio de Janeiro, Brazil: Focus on Proteases by Anderson Fragoso d. Santos; Clarissa Almeida Pacheco; Roberta d. Santos Valle; Lucy Seldin; André Luis Souza d. Santos (2748-2761).
The screening for hydrolases-producing, halotolerant, and spore-forming gram-positive bacteria from the root, rhizosphere, and non-rhizosphere soil of Blutaparon portulacoides, a plant found in the Restinga de Jurubatiba located at the northern region of Rio de Janeiro State, Brazil, resulted in the isolation of 22 strains. These strains were identified as Halobacillus blutaparonensis (n = 2), Oceanobacillus picturae (n = 5), and Oceanobacillus iheyensis (n = 15), and all showed the ability to produce different extracellular enzymes. A total of 20 isolates (90.9 %) showed activity for protease, 5 (22.7 %) for phytase, 3 (13.6 %) for cellulase, and 2 (9.1 %) for amylase. Some bacterial strains were capable of producing three (13.6 %) or two (9.1 %) distinct hydrolytic enzymes. However, no bacterial strain with ability to produce esterase and DNase was observed. The isolate designated M9, belonging to the species H. blutaparonensis, was the best producer of protease and also yielded amylase and phytase. This strain was chosen for further studies regarding its protease activity. The M9 strain produced similar amounts of protease when grown either without or with different NaCl concentrations (from 0.5 to 10 %). A simple inspection of the cell-free culture supernatant by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed the presence of three major alkaline proteases of 40, 50, and 70 kDa, which were fully inhibited by phenylmethylsulfonyl fluoride (PMSF) and tosyl-l-phenylalanine chloromethyl ketone (TPCK) (two classical serine protease inhibitors). The secreted proteases were detected in a wide range of temperature (from 4 to 45 °C) and their hydrolytic activities were stimulated by NaCl (up to 10 %). The serine proteases produced by the M9 strain cleaved gelatin, casein, albumin, and hemoglobin, however, in different extensions. Collectively, these results suggest the potential use of the M9 strain in biotechnological and/or industrial processes.
Keywords: Hydrolytic enzymes; Amylase; Cellulase; Phytase; Alkaline protease; Halotolerant bacteria; Industrial applications

A Yeast Isolated from Cashew Apple Juice and Its Ability to Produce First- and Second-Generation Ethanol by E. M. Barros; T. H. S. Rodrigues; A. D. T. Pinheiro; A. L. Angelim; V. M. M. Melo; M. V. P. Rocha; Luciana R. B. Gonçalves (2762-2776).
The aim of this study was to isolate and identify an indigenous yeast from cashew apple juice (CAJ) and then use it in the production of first- and second-generation ethanol, using CAJ and the enzymatic hydrolysate of cashew apple bagasse (MCAB-OH), respectively. The isolated yeast was identified as belonging to the genus Hanseniaspora. Afterward, the effect of the medium initial pH on the production of ethanol from CAJ was evaluated in the range of 3.0 to 5.5, with its maximum ethanol production of 42 g L−1 and Y P/S of 0.44 g g−1 and 96 % efficiency. The effect of temperature (28–38 °C) on ethanol production was evaluated in a synthetic medium, and no difference in ethanol production in the temperature range evaluated (28–36 °C) was observed. At 32 °C, the yield, concentration, efficiency, and productivity of ethanol when using the CAJ medium were higher when compared to the results achieved for the synthetic medium. Regarding second-generation ethanol, the results showed that the yeast produced 24.37 g L−1 of ethanol with an efficiency of 80.23 % and a productivity of 4.87 g L−1 h−1 at 5 h. Therefore, Hanseniaspora sp., isolated from CAJ, is a promising microorganism for the production of first- and second-generation ethanol.
Keywords: Hanseniaspora ; Ethanol; Process integration; Cashew apple juice; Cashew apple bagasse

A rapid and novel microwave-mediated protocol was established for extracellular synthesis of metallic silver (Ag) and zinc oxide (ZnO) nanoparticles using the extracts of macro-algae Gracilaria edulis (GE) and also examined its anticancer activity against human prostate cancer cell lines (PC3). The formation of silver nanoparticles (GEAgNPs) and zinc oxide nanoparticles (GEZnONPs) in the reaction mixture was determined by ultraviolet-visible spectroscopy. The synthesized Ag and ZnO nanoparticles were characterized by X-ray diffraction, Fourier transform infra-red spectroscopy, energy dispersive X-ray, and field emission scanning electron microscopy. The silver and zinc oxide nanoparticles were spherical and rod-shaped, respectively. Cell viability assays were carried out to determine the cytotoxic effects of AgNPs and ZnONPs against PC3 and normal African monkey kidney (VERO) cell line. The inhibitory concentration values were found to be 39.60, 28.55, 53.99 μg/mL and 68.49, 88.05, 71.98 μg/mL against PC3 cells and Vero cells for AgNPs, ZnONPs, and aqueous G. edulis extracts, respectively, at 48 h incubation period. As evidenced by acridine orange/ethidium bromide staining, the percentage of the apoptotic bodies was found to be 62 and 70 % for AgNPs and ZnONPs, respectively. The present results strongly suggest that the synthesized ZnONPs showed an effective anticancer activity against PC3 cell lines than AgNPs.
Keywords: Anticancer activity; Gracilaria edulis ; Nanoparticle synthesis; Macro-algae; Microwave; PC3 cell line

Blackgram (Vigna mungo (L.) Hepper), an important grain legume crop, is sensitive to many fungal pathogens including Corynespora cassiicola, the causal agent of corynespora leaf spot disease. In the present study, plasmid pGJ42 harboring neomycin phosphotransferase (nptII) a selectable marker gene, the barley antifungal genes chitinase (AAA56786) and ribosome-inactivating protein (RIP; AAA32951) were used for the transformation, to develop fungal resistance for the first time in blackgram. The presence and integration of transgene into the blackgram genome was confirmed by PCR and Southern analysis with an overall transformation frequency of 10.2 %. Kanamycin selection and PCR analysis of T0 progeny revealed the inheritance of transgene in Mendelian fashion (3:1). Transgenic plants (T1), evaluated for fungal resistance by in vitro antifungal assay, arrested the growth of C. cassiicola up to 25–40 % over the wild-type plants. In fungal bio-assay screening, the transgenic plants (T1) sprayed with C. cassiicola spores showed a delay in onset of disease along with their lesser extent in terms of average number of diseased leaves and reduced number and size of lesions. The percent disease protection among different transformed lines varies in the range of 27–47 % compare to control (untransformed) plants. These results demonstrate potentiality of chitinase and RIP from a heterologous source in developing fungal disease protection in blackgram and can be helpful in increasing the production of blackgram.
Keywords: Agrobacterium tumefaciens ; Blackgram; Chitinase; Fungal resistance; Ribosome-inactivating protein; Transgenic plants

Cellulolytic Enzymes Production by Utilizing Agricultural Wastes Under Solid State Fermentation and its Application for Biohydrogen Production by Ganesh D. Saratale; Siddheshwar D. Kshirsagar; Vilas T. Sampange; Rijuta G. Saratale; Sang-Eun Oh; Sanjay P. Govindwar; Min-Kyu Oh (2801-2817).
Phanerochaete chrysosporium was evaluated for cellulase and hemicellulase production using various agricultural wastes under solid state fermentation. Optimization of various environmental factors, type of substrate, and medium composition was systematically investigated to maximize the production of enzyme complex. Using grass powder as a carbon substrate, maximum activities of endoglucanase (188.66 U/gds), exoglucanase (24.22 U/gds), cellobiase (244.60 U/gds), filter paperase (FPU) (30.22 U/gds), glucoamylase (505.0 U/gds), and xylanase (427.0 U/gds) were produced under optimized conditions. The produced crude enzyme complex was employed for hydrolysis of untreated and mild acid pretreated rice husk. The maximum amount of reducing sugar released from enzyme treated rice husk was 485 mg/g of the substrate. Finally, the hydrolysates of rice husk were used for hydrogen production by Clostridium beijerinckii. The maximum cumulative H2 production and H2 yield were 237.97 mL and 2.93 mmoL H2/g of reducing sugar, (or 2.63 mmoL H2/g of cellulose), respectively. Biohydrogen production performance obtained from this work is better than most of the reported results from relevant studies. The present study revealed the cost-effective process combining cellulolytic enzymes production under solid state fermentation (SSF) and the conversion of agro-industrial residues into renewable energy resources.
Keywords: Phanerochaete chrysosporium ; Solid state fermentation; Cellulase; Rice husk; Biohydrogen; Dark fermentation

Tobacco Arabinogalactan Protein NtEPc Can Promote Banana (Musa AAA) Somatic Embryogenesis by H. Shu; L. Xu; Z. Li; J. Li; Z. Jin; S. Chang (2818-2826).
Banana is an important tropical fruit worldwide. Parthenocarpy and female sterility made it impossible to improve banana varieties through common hybridization. Genetic transformation for banana improvement is imperative. But the low rate that banana embryogenic callus was induced made the transformation cannot be performed in many laboratories. Finding ways to promote banana somatic embryogenesis is critical for banana genetic transformation. After tobacco arabinogalactan protein gene NtEPc was transformed into Escherichia coli (DE3), the recombinant protein was purified and filter-sterilized. A series of the sterilized protein was added into tissue culture medium. It was found that the number of banana immature male flowers developing embryogenic calli increased significantly in the presence of NtEPc protein compared with the effect of the control medium. Among the treatments, explants cultured on medium containing 10 mg/l of NtEPc protein had the highest chance to develop embryogenic calli. The percentage of lines that developed embryogenic calli on this medium was about 12.5 %. These demonstrated that NtEPc protein can be used to promote banana embryogenesis. This is the first paper that reported that foreign arabinogalactan protein (AGP) could be used to improve banana somatic embryogenesis.
Keywords: Arabinogalactan protein; NtEPc; Banana (Musa AAA); Somatic embryogenesis

Nutrient Removal Using Algal-Bacterial Mixed Culture by Vaishali Ashok; Amritanshu Shriwastav; Purnendu Bose (2827-2838).
Simultaneous nitrate (N), phosphate (P), and COD removal was investigated in photobioreactors containing both algae and bacteria. The reactors were operated in the semi-batch mode with a hydraulic retention time of 2 days. Reactors were operated in two phases, (1) with 33 % biomass recycle and (2) with no biomass recycle. In both phases, more than 90 % of N and P and 80 % of COD present in synthetic wastewaters with initial N and P concentrations of up to 110 and 25 mg/L, respectively, and initial COD of 45 mg/L could be removed. Biomass growth in reactors did not increase with the increase in initial N and P concentration in either phase. However, biomass growth was slightly more in reactors operated with no biomass recycle. In both phases, N and P uptake was greater in reactors with greater initial N and P concentrations. Also in all cases, N and P uptake in the reactors was far in excess of the stoichiometric requirements for the observed biomass growth. This “luxury uptake” of nitrogen and phosphorus by biomass was responsible for excellent nitrogen and phosphorus removal as observed. However, based on the results of this study, no advantage of biomass recycling could be demonstrated.
Keywords: Algal-bacterial consortia; Chlorella vulgaris ; Chlamydomonas reinhardtii ; Nitrates; Phosphates removal; Semi-batch reactors

Molecular Profiling of Systemic Acquired Resistance (SAR)-Responsive Transcripts in Sugarcane Challenged with Colletotrichum falcatum by Nagarathinam Selvaraj; Ashwin Ramadass; Ramesh Sundar Amalraj; Malathi Palaniyandi; Viswanathan Rasappa (2839-2850).
Red rot disease of sugarcane caused by Colletotrichum falcatum is one of the serious constraints affecting the productivity of the crop. The strategy of employing systemic acquired resistance (SAR) against red rot yielded consistently good results at field level. However, elucidation of genes involved in the induction of SAR continues to be a challenging area of research for a critical understanding of red rot disease resistance in sugarcane. Here, temporal expression of 22 putative defense-related genes were analyzed by semiquantitative reverse transcription-PCR (RT-PCR) in red rot-susceptible cultivar (CoC 671) with response to priming using various SAR inducers, viz. benzothiadiazole (BTH), salicylic acid (SA), and C. falcatum elicitor. Among the 22 genes studied, 12 transcripts were found to be differentially expressed, of which seven transcripts represent phenylpropanoid pathway and five transcripts represent resistant gene analogues (RGAs). Differentially regulated phenylpropanoid pathway genes such as cinnamic acid 4-hydroxylase, 4-coumarate:coenzyme A ligase, chalcone synthase, and chalcone reductase were reported to play a major role in the regulation of phytoalexin synthesis, whereas R genes such as NBS-LRR genes and basal layer antifungal peptide (BAF) genes were upregulated upon SAR induction in response to pathogen challenge. These upregulated genes presumably play a potential role in SAR induction and might contribute to defense against C. falcatum.
Keywords: Sugarcane; Systemic acquired resistance; BTH; C. falcatum elicitor; Defense genes

The present study aimed to synthesize Zinc oxide (ZnO) nanoparticles and to analyze the dose-dependent effect on the oxidative stress and antioxidant enzyme activities in 3T3-L1 adipocytes. ZnO is widely used in the several cosmetic lotions and biomedical products. There are several studies that have reported the ZnO nanoparticle-mediated cytotoxicity on various tissues. However, there are no studies carried out on dose-dependent effect of ZnO nanoparticles in the adipose tissue. ZnO nanoparticle was synthesized by chemical pyrolysis method and characterized by the SEM. A cytotoxicity assay was carried out to determine 3T3-L1 cell viability. 3T3-L1 cell morphology was significantly altered, and most of the cells are dead at higher concentration of ZnO nanoparticles. ZnO nanoparticles increased reactive oxygen species (ROS), lipid peroxidation (MDA), and reduced glutathione (GSH) in 3T3-L1 adipocytes. In addition, antioxidant enzyme activity and its mRNA expression were also upregulated in 3T3-L1 adipocytes. In conclusion, the present study showed that ZnO nanoparticles significantly altered oxidative stress and antioxidant enzyme activity in 3T3-L1 adipocytes on a dose-dependent manner.
Keywords: 3T3-L1 cell; Antioxidant; ZnO nanoparticles; Cytotoxicity; Enzymes; mRNA

Purification and Characterization of Recombinant Cel7A from Maize Seed by Nathan C. Hood; Kendall R. Hood; Susan L. Woodard; Shivakumar P. Devaiah; Tina Jeoh; Lisa Wilken; Zivko Nikolov; Erin Egelkrout; John A. Howard; Elizabeth E. Hood (2864-2874).
The corn grain biofactory was used to produce Cel7A, an exo-cellulase (cellobiohydrolase I) from Hypocrea jecorina. The enzymatic activity on small molecule substrates was equivalent to its fungal counterpart. The corn grain-derived enzyme is glycosylated and 6 kDa smaller than the native fungal protein, likely due to more sugars added in the glycosylation of the fungal enzyme. Our data suggest that corn seed-derived cellobiohydrolase (CBH) I performs as well as or better than its fungal counterpart in releasing sugars from complex substrates such as pre-treated corn stover or wood. This recombinant protein product can enter and expand current reagent enzyme markets as well as create new markets in textile or pulp processing. The purified protein is now available commercially.
Keywords: Cel7A; Recombinant protein; Cellobiohydrolase I; Protein purification; Cellulase; Biomass conversion

Behaviors of Biomass and Kinetic Parameter for Nitrifying Species in A2O Process at Different Sludge Retention Time by Tzu-Yi Pai; Huang-Mu Lo; Terng-Jou Wan; Shun-Cheng Wang; Pei-Yu Yang; Yu-Ting Huang (2875-2885).
The effect of sludge retention time (SRT) on biomass, kinetic parameters, and stoichiometric parameters of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in anaerobic/anoxic/oxic (A2O) process were explored in this study. The results showed that the growth rate constants were 1.52, 1.22, and 0.85 day−1, respectively, for AOB, those were 1.59, 1.19, and 0.87 day−1, respectively, for NOB when SRT was 20, 10, and 5 days. The lysis rate constants of AOB and NOB were 0.14 and 0.09 day−1, respectively. The yield coefficients were 0.23 and 0.22, respectively, for AOB and NOB. They did not change with SRT obviously. The biomass of AOB was 50.94, 26.35, and 14.68 mg L−1, respectively, and the biomass of NOB was 116.77, 60.00, and 44.25 mg L−1, respectively, at SRT of 20, 10, and 5 days. When SRT diminished from 20 to 5 days, the biomass of AOB and NOB diminished by 36.26 and 75.52 mg L−1, respectively. The removal efficiency of NH4 +–N diminished by 68.9 %. The removal efficiency of total nitrogen diminished by 42.9 %.
Keywords: Sludge retention time (SRT); Oxygen uptake rate (OUR); Ammonia-oxidizing bacteria (AOB); Nitrite-oxidizing bacteria (NOB); Growth rate constant; Lysis rate constant; Yield coefficient

An efficient, cyclic, two-step protocol for clonal in vitro regeneration system of an antiallergenic plant, Cassia alata, has been successfully developed. Nodal explants from a 5-year-old tree were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations (1.0, 2.5, 5.0, 7.5, and 10.0 μM) of thidiazuron (TDZ). TDZ (5.0 μM) was found to be optimal for the formation of maximum shoot induction. Shoot proliferation and elongation increased when the regenerated shoots were subcultured on hormone-free MS medium after 4 weeks of exposure to TDZ. Nodal explants from in vitro regenerated microshoots to developed shoots, thus making the process recurrent. In 6 months duration, owing to the recurring nature of the protocol, large number of shoots could be produced from a single nodal explant from an adult tree. Shoots rooted best on MS supplemented medium with 0.5 μM IBA. Regenerated plantlets were acclimatized and successfully transplanted to the garden soil, where they grew well without any morphological and genetic variations. To confirm the uniformity, the genetic fidelity of in vitro raised C. alata clones was also assessed by using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. The present regeneration process not only favored the clonal multiplication but also expressed the regeneration capability of in vitro regenerated microshoots and can be subjugated for catering enough raw materials to various pharma industries by continuous cyclic shoot production.
Keywords: Acclimatization; Cyclic regeneration; In vitro rooting; Genetic fidelity

The total phenolic and flavonoid content and percentage of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of callus and in vivo plant parts of Heliotropium indicum Linn. were estimated. Murashige and Skoog (MS) basal medium supplemented with α-naphthaleneacetic acid (NAA) 2.0 mg/l with benzyladenine (BA) 0.5 mg/l showed the highest amount of callus biomass (1.87 g/tube). The morphology of callus was significantly different according to the plant growth regulators and their concentrations used in the medium. The highest amount of total phenolic (21.70 mg gallic acid equivalent per gram (GAE/g)) and flavonoid (4.90 mg quercetin equivalent per gram (QE/g)) content and the maximum percentage (77.78 %) of radical scavenging activity were estimated in the extract of inflorescence. The synergistic effect of NAA (2.0 mg/l) and BA (0.5 mg/l) enhances the synthesis of total phenolic (9.20 mg GAE/g) and flavonoid (1.25 mg QE/g) content in the callus tissue. The callus produced by the same concentration shows 45.24 % of free radical scavenging activity. While comparing the various concentrations of NAA with 2,4-dichlorophenoxyacetic acid (2,4-D) for the production of callus biomass, total phenolic and flavonoid content and free radical scavenging activity, all the concentrations of NAA were found to be superior than those of 2,4-D.
Keywords: Callus biomass; Flavonoids; Free radical scavenging activity; Relative growth rate of callus; Total phenolic

Influence of Calcium, Magnesium, and Iron Ions on Aerobic Granulation by Beata Kończak; Jagna Karcz; Korneliusz Miksch (2910-2918).
In this study, we investigated the effect of different multivalent cations on granule formation. Previous experiments showed that formation of matrix EPS and their structure depend of the presence of divalent cations. This study indicates that trivalent cations are also playing an important role. However, the more compact granules were formed in the presence of all cations. The authors tried also to determine changes in proteomic profile of slime and tightly bound EPS. These results showed that matrix EPS is composed of a variety of large and complex proteins, but there are also small proteins, like for example, lectins. These small proteins have a role in the interaction between cells and exopolysaccharides and in granules formation.
Keywords: Aerobic granule; Multivalent cations; Formation; Stability

High Sulfate Reduction Efficiency in a UASB Using an Alternative Source of Sulfidogenic Sludge Derived from Hydrothermal Vent Sediments by Selene Montserrat García-Solares; Alberto Ordaz; Oscar Monroy-Hermosillo; Janet Jan-Roblero; Claudia Guerrero-Barajas (2919-2940).
Sulfidogenesis in reactors is mostly achieved through adaptation of predominantly methanogenic granular sludge to sulfidogenesis. In this work, an upflow anaerobic sludge blanket (UASB) reactor operated under sulfate-reducing conditions was inoculated with hydrothermal vent sediments to carry out sulfate reduction using volatile fatty acids (VFAs) as substrate and chemical oxygen demand (COD)/SO4 −2 ratios between 0.49 and 0.64. After a short period of adaptation, a robust non-granular sludge was capable of achieving high sulfate reduction efficiencies while avoiding competence with methanogens and toxicity to the microorganisms due to high sulfide concentration. The highest sulfide concentration (2,552 mg/L) was obtained with acetate/butyrate, and sulfate reduction efficiencies were up to 98 %. A mixture of acetate/butyrate, which produced a higher yielding of HS, was preferred over acetate/propionate/butyrate since the consumption of COD was minimized during the process. Sludge was analyzed, and some of the microorganisms identified in the sludge belong to the genera Desulfobacterium, Marinobacter, and Clostridium. The tolerance of the sludge to sulfide may be attributed to the syntrophy among these microorganisms, some of which have been reported to tolerate high concentrations of sulfide. To the best of our knowledge, this is the first report on the analysis of the direct utilization of hydrothermal vent sediments as an alternate source of sludge for sulfate reduction under high sulfide concentrations.
Keywords: Sulfidogenic UASB; Sulfidogenic sludge; Sulfate reduction; Sulfide tolerance; Hydrothermal vent sediments; Clostridium difficile ; Desulfobacterium

Immobilization of Pectinesterase in Genipin-Crosslinked Chitosan Membrane for Low Methoxyl Pectin Production by Adriana Marisol Rangel-Rodríguez; Solans Conxita; Vílchez Susana; Sergio Gabriel Flores-Gallardo; Juan Carlos Contreras-Esquivel; Liliana Licea-Jiménez (2941-2950).
In this study, an environmentally friendly method for preparation of chitosan crosslinked membranes with different genipin concentrations (0, 125, 250, and 500 mg/L) was developed. Genipin-crosslinked chitosan membranes were used for the immobilization of fungal pectinesterase (PE). PE was efficiently immobilized in chitosan membranes and used for modification of high-methoxylated pectins into low-methoxylated pectins. The charge density (ζ-potential), infrared spectroscopy (FTIR), and ion-exchange chromatography (IEC) revealed the modification of pectin for immobilized PE in genipin-crosslinked chitosan membrane. The ζ-potential results indicated a decrease of −5.2 mV in the pectin control solution, whereas using the PE immobilized in chitosan and genipin-chitosan membranes, the obtained values were −30.45 mV and −36.38 mV, respectively. Genipin-crosslinked chitosan membrane jointly with the immobilized PE represents an eco-friendly support to prepare tailor-made low-methoxylated lime pectin.
Keywords: Pectinmethylesterase; Genipin; Chitosan; Membrane; Immobilization

Spore Production of Clonostachys rosea in a New Solid-state Fermentation Reactor by Yuanyuan Zhang; Junhong Liu; Yuanming Zhou; Yinlin Ge (2951-2959).
A new solid-state fermentation (SSF) reactor was developed for the spore production of biocontrol agent Clonostachys rosea. The greatest spore production in the reactor, 3.36 × 1010 spores g DM−1 occurred with mixings, which was about 10 times greater than that in traditional tray reactor. The reactor provides about two times sporulation surface area for spore formation. Moisture content of the medium was adjusted to meet the spore production by changing the surface porosity. Two mixings were carried out during cultivation to make the medium loose, which resulted in a mass of new sporulation surface. The fermentation period shortened from 14–15 to 10–11 days. It is suggested that the new reactor has great potential in the mass production of spores of C. rosea and other fungal biocontrol agents.
Keywords: Clonostachys rosea ; Spore; Solid-state fermentation; Reactor; Mixing

The Efficient Production of 3β,7α,15α-Trihydroxy-5-Androsten-17-One from Dehydroepiandrosterone by Gibberella intermedia by Hui Li; Zhenzhen Fu; Xiaomei Zhang; Heng Li; Jinsong Shi; Zhenghong Xu (2960-2971).
The biotransformation of dehydroepiandrosterone (DHEA) by Gibberella intermedia was investigated. The formation of the main products was monitored by high-performance liquid chromatography (HPLC). Products were purified and identified as 3β,7α-dihydroxy-5-androsten-17-one (7α-OH-DHEA) and 3β,7α,15α-trihydroxy-5-androsten-17-one (7α,15α-diOH-DHEA) using mass spectroscopy (MS) and nuclear magnetic resonance (NMR) analyses. 7α,15α-DiOH-DHEA is a key intermediate for the synthesis of pharmacologically active steroids. A biotransformation process was optimized to obtain a high concentration of 7α,15α-diOH-DHEA. The results showed the optimal biotransformation process under the following conditions: a culture medium containing 15 g/L glucose, 16 g/L yeast extract, 12 g/L corn steep liquor, and 0.15 g/L ferrous sulfate; an initial pH of 6.5; culture temperature of 30 °C; inoculum size of 4 %; 40 mL volume of the culture medium in 250 mL flasks; the addition of 1 % Tween 80 as co-solvent; and the transformation period of 78 h. After optimization, the molar yield of 7α,15α-diOH-DHEA from 5 g/L substrate DHEA was 77.4 %, which was increased by 115 % than the yield obtained in the original bioconversion process.
Keywords: Biotransformation; Dihydroxylation; DHEA; 7α,15α-diOH-DHEA; Gibberella intermedia