Applied Biochemistry and Biotechnology (v.169, #8)

Construction of the ie1-Bacmid Expression System and Its Use to Express EGFP and BmAGO2 in BmN Cells by Fang Zhou; Zhen Gao; Zhengbing Lv; Jian Chen; Yeting Hong; Wei Yu; Dan Wang; Caiying Jiang; Xiangfu Wu; Yaozhou Zhang; Zuoming Nie (2237-2247).
The presently available expression tools and vectors (e.g., eukaryotic expression vectors and the adenovirus expression system) for studying the functional genes in Bombyx mori are insufficient. The baculovirus expression system is only used as a protein production tool; therefore, recombinant proteins expressed by B. mori using the baculovirus expression system equipped with a polyhedrin promoter cannot be used for in vivo research applications. In this work, we constructed and screened a eukaryotic expression vector for silkworm cells The EGFP and B. mori Argonaute2 proteins were found to be efficiently expressed using the screened pIEx-1 vector with the FuGENE 6 transfection reagent. Additionally, we constructed a novel nucleopolyhedrovirus ie1-Bacmid expression system for the production of recombinant protein; we then used the system to highly express the EGFP and B. mori Argonaute2 proteins. In this system, the protein of interest can be efficiently expressed 13 h after infection by controlling the B. mori nucleopolyhedrovirus immediate early ie1 promoter. The ie1-Bacmid system provides a powerful “adenovirus-like” expression tool; not only can the tool be used to study baculovirus molecular biology for the silkworm but it is also useful in other research applications as well, such as the study of gene functions involved in cellular physiological processes.
Keywords: BmN; pIEx-1; ie1-Bacmid system; Expression; BmAGO2

Enzymatic Hydrolysis of Spent Coffee Ground by T. Jooste; M. P. García-Aparicio; M. Brienzo; W. H. van Zyl; J. F. Görgens (2248-2262).
Spent coffee ground (SCG) is the main residue generated during the production of instant coffee by thermal water extraction from roasted coffee beans. This waste is composed mainly of polysaccharides such as cellulose and galactomannans that are not solubilised during the extraction process, thus remaining as unextractable, insoluble solids. In this context, the application of an enzyme cocktail (mannanase, endoglucanase, exoglucanase, xylanase and pectinase) with more than one component that acts synergistically with each other is regarded as a promising strategy to solubilise/hydrolyse remaining solids, either to increase the soluble solids yield of instant coffee or for use as raw material in the production of bioethanol and food additives (mannitol). Wild fungi were isolated from both SCG and coffee beans and screened for enzyme production. The enzymes produced from the selected wild fungi and recombinant fungi were then evaluated for enzymatic hydrolysis of SCG, in comparison to commercial enzyme preparations. Out of the enzymes evaluated on SCG, the application of mannanase enzymes gave better yields than when only cellulase or xylanase was utilised for hydrolysis. The recombinant mannanase (Man1) provided the highest increments in soluble solids yield (17 %), even when compared with commercial preparations at the same protein concentration (0.5 mg/g SCG). The combination of Man1 with other enzyme activities revealed an additive effect on the hydrolysis yield, but not synergistic interaction, suggesting that the highest soluble solid yields was mainly due to the hydrolysis action of mannanase.
Keywords: Coffee bean; Spent coffee ground; Cellulose; Mannan; Cellulase; Mannanase; Water extraction

As a kind of folk medicine, edible mushrooms are known to be of medicinal characteristics, for example antitumor activity. However, the mechanism is not clear. In this study, the fluorescence (FL) spectroscopy was used to study the binding reactions of polysaccharide extracted from Auricularia polytricha with quantum dots (QDs). The modified Stern–Volmer quenching constant at different temperatures and corresponding thermodynamic parameters were calculated (∆G < 0, ∆H < 0, and ∆S < 0). The results show that the quenching constant is inversely correlated with temperature. It indicates that the quenching mechanism is the static quenching in nature rather than dynamic quenching. The negative values of free energy (∆G < 0) suggest that the binding process is spontaneous; ∆H < 0 and ∆S < 0 suggest that the binding of polysaccharide to QDs is enthalpy-driven. QDs were found to damage the plasmid DNA according to atomic force microscopy images. DNA damage is the important factor to induce tumor. Many chemical substances can induce tumor, which has been proved by modern medical science. In the presence of polysaccharide from A. polytricha, however, DNA was protected from damage due to polysaccharide winding around QDs, which is the basis for the bioeffect of polysaccharides. This study helps to understand that antitumor activity of edible mushrooms is attributed to protection of DNA from damage in the presence of harmful substances.
Keywords: Auricularia polytricha ; Polysaccharides; QDs; Damage; Protection

Preparation and Characterizations of Polyclonal Antibodies Against STAR Protein QKI7b by Qiuyan Liu; Yu Luo; Xiaotong Li; Li Yuan; Ruian Xu; James Y. Yang (2273-2280).
Quaking (QKI) proteins are important regulators of RNA metabolism and cellular signal transduction. Recent studies have shown that isoforms of QKI proteins, which include QKI5/6/7/7b in human cells, play important roles in the development of neurological diseases and human cancers. In comparison with QKI5/6/7, however, there are little data on QKI7b due to lack of specific antibodies. Here, we reported the preparation and initial characterizations of polyclonal antibodies against human QKI7b. Utilizing a chemically synthesized C-terminal peptide fragment of human QKI7b, we raised two preparations of rabbit antiserum. We found that these antibodies were able to recognize human QKI7b, but not QKI5/6/7. Our immunofluorescence staining showed that in LO2 hepatocytes, QKI7b localizes predominantly in the perinuclear cytoplasm and less abundantly in the nucleus. In clinical samples, we showed that like QKI5/6/7 proteins, QKI7b protein was also significantly downregulated in most human colorectal cancer tissues. These antibodies, therefore, might be useful in future functional studies of QKI7b.
Keywords: QKI7b; Polyclonal antibody; Subcellular localization; Colorectal cancer

The adaptive response of the fungus Blakeslea trispora to the oxidative stress induced by iron ions during carotene production in shake flask culture was investigated. The culture response to oxidative stress was studied by measuring the specific activities of catalase (CAT) and superoxide dismutase (SOD). The addition of 1.0 mM of FeCl3 to the medium was associated with a mild oxidative stress as evidenced by remarkable increase of the specific activities of SOD and CAT. On the other hand, the addition 5.0 mM of FeCl3 caused a strong oxidative stress resulting in a drastic decrease in carotene concentration. The oxidative stress in B. trispora changed the composition of the carotenes and caused a significant increase of γ-carotene ratio. The highest concentration of carotenes (115.0 ± 3.5 mg/g dry biomass) was obtained in the basal medium without the addition of FeCl3 after 8 days of fermentation. In this case, the carotenes consisted of β-carotene (46.3 %), γ-carotene (40.1 %), and lycopene (13.6 %). The addition of 1.0 mM of FeCl3 into the medium did not change the concentration of carotenes. But, the composition of carotenes was changed with a drastic increase of γ-carotene ratio (61.6 %) and a decrease in β-carotene and lycopene ratio (31.2 and 7.2 %, respectively).
Keywords: Carotenes; Blakeslea trispora ; Synthetic medium; Iron ions; Oxidative stress; Shake flask culture

A Natural Isolate Producing Shikimic Acid: Isolation, Identification, and Culture Condition Optimization by Garima Rawat; Priyanka Tripathi; Firdaus Jahan; R. K. Saxena (2290-2302).
Shikimic acid has wide use in pharmaceuticals due to its application in the synthesis of drug Tamiflu used in the treatment of Swine flu. The high cost and limited availability of shikimic acid isolated from plants has impeded the use of this valuable building block of the drug. In this context, fermentation route to produce shikimic acid from renewable resources has become increasingly attractive. The present study was embarked upon isolation of wild-type microorganisms able to produce shikimic acid. Out of the 42 isolates obtained from the soil, isolate GR-21 was selected as the best with initial production of 0.54 g/L shikimic acid and later identified as Citrobacter sp. The process optimization resulted in 14-fold increase in the shikimic acid production, thereby claiming this process to be a sustainable alternative for the production of this important biomolecule. The process was further scaled up to 14 L bioreactor to validate the production of shikimic acid. Further, the product formed is shikimic acid was confirmed by FTIR analysis. The current studies suggest that the selected isolate could be used as a promising agent to fulfill the worldwide demand of shikimic acid.
Keywords: Shikimic acid; Tamiflu; Swine flu; Citrobacter sp.

An efficient, rapid, and highly reproducible regeneration protocol was successfully developed for Phyllanthus fraternus from the field-derived mature nodal segments. The explants induced multiple shoots on cytokinin containing medium. The highest frequency (99 %) and maximum number of shoots (19.75) were induced on Murashige and Skoog’s (MS) medium supplemented with 2.22 μM 6-benzylaminopurine after 3–4 weeks of culture initiation. The elongated shoots were rooted on MS medium supplemented with indol-3-butyric acid (IBA) or α-naphthalene acetic acid. Pulse treatment of microshoots promoted significant increase in the percentage of rooting and number of root regeneration per shoot. The highest rooting (100 %) and maximum number of roots (8.75) per shoot was obtained when shoots were dipped in IBA solution (0.98 mM) for 5 min and further subcultured on MS basal medium. Plantlets were successfully acclimatized and established in soil. Regenerated plants were grown normally in the field without showing any morphological variations. This cost-effective protocol will help the mass multiplication of P. fraternus for commercial propagation and high biomass production of this valuable medicinal plant.
Keywords: Bhui-amla; Phyllanthus fraternus ; Nodal segments; Regeneration; Tissue culture

Evidence for a Molten Globule State in Cicer α-Galactosidase Induced by pH, Temperature, and Guanidine Hydrochloride by Neelesh Singh; Reetesh Kumar; M. V. Jagannadham; Arvind M. Kayastha (2315-2325).
Physiologically as well as industrially, α-galactosidases are very important enzymes, but very little is known about the stability and folding aspect of enzyme. In the present study, we have investigated the temperature, pH, and guanidine hydrochloride (GuHCl) induced unfolding of Cicer α-galactosidase using circular dichroism and fluorescence spectroscopy. Strong negative ellipticities at 208, 215, and 222 nm indicate the presence of both α and β structures in Cicer α-galactosidase and showed that its secondary structure belongs to α + β class of proteins with 31 % α-helicity. For Cicer α-galactosidase the emission maximum was found to be 345 nm which suggests that tryptophan residues are less exposed to solvent. However, at pH 2.0, protein showed blue-shift. This state of protein lacked activity but it retained significant secondary structure. Enhanced binding of ANS at pH 2.0 indicated significant unfolding and exposure of hydrophobic regions. The unfolded state of Cicer α-galactosidase showed a red-shift of 15 nm with a concomitant decrease in the fluorescence intensity. The enzyme maintained its native structure and full activity up to 40 °C; however, above this temperature, denaturation was observed.
Keywords: α-Galactosidase; Fluorescence; Circular dichroism; Molten globule; Unfolding; Guanidine hydrochloride

Cloning, Expression, and Characterization of β-mannanase from Bacillus subtilis MAFIC-S11 in Pichia pastoris by Junnan Lv; Yiqun Chen; Honglei Pei; Wenhan Yang; Zhimin Li; Bing Dong; Yunhe Cao (2326-2340).
The β-mannanase gene (1,029 nucleotide) from Bacillus subtilis MAFIC-S11, encoding a polypeptide of 342 amino acids, was cloned and expressed in Pichia pastoris. To increase its expression, the β-mannanase gene was optimized for codon usage (mannS) and fused downstream to a sequence-encoding modified α-factor signal peptide. The expression level was improved by 2-fold. This recombinant enzyme (mannS) showed its highest activity of 24,600 U/mL after 144-h fermentation. The optimal temperature and pH of mannS were 50 °C and 6.0, respectively, and its specific activity was 3,706 U/mg. The kinetic parameters V max and K m were determined as 20,000 U/mg and 8 mg/mL, respectively, representing the highest ever expression level of β-mannanase reported in P. pastoris. In addition, the enzyme exhibited much higher binding activity to chitin, chitosan, Avicel, and mannan. The superior catalytic properties of mannS suggested great potential as an effective additive in animal feed industry.
Keywords: Bacillus subtilis ; β-mannanase; Codon bias; Pichia pastoris ; Expression

Effects of ELF Magnetic Field in Combination with Iron(III) Chloride (FeCl3) on Cellular Growth and Surface Morphology of Escherichia coli (E. coli) by Meric A. Esmekaya; S. Ipek Acar; Fadime Kıran; Ayşe G. Canseven; Ozlem Osmanagaoglu; Nesrin Seyhan (2341-2349).
This study investigated the effects of extremely low frequency (ELF) magnetic field with/without iron(III) chloride (FeCl3) on bacterial growth and morphology. The ELF exposures were carried out using a pair of Helmholtz coil-based ELF exposure system which was designed to generate 50 Hz sinusoidal magnetic field. The field was approximately uniform throughout the axis of the coil pair. The samples which were treated or non-treated with different concentrations FeCl3 were exposed to 50 Hz, 2 millitesla (mT) magnetic field for 24 h. ELF effect on viability was assessed in terms of viable colony counts (in colony-forming unit per milliliter) with the standard plate count technique. Scanning electron microscopy was used to investigate the magnetic field effect on surface morphology of Escherichia coli. No significant results were seen in terms of cell viability between ELF and sham-exposed bacterial strains. Similarly, FeCl3 treatment did not change cell viability of E. coli samples. However, we observed some morphological changes on E. coli cell surfaces. Pore formations and membrane destruction were seen on the surface of 24 h ELF field-exposed cells. We concluded that ELF magnetic field exposure at 2 mT does not affect cell viability; however, it may affect bacterial surface morphology.
Keywords: Extremely low frequency; Magnetic field; E. coli ; Cell viability; Morphology; Scanning electron microscopy

Surface Display of Malolactic Enzyme from Oenococcus oeni on Saccharomyces cerevisiae by Xiuyan Zhang; Xiaoyan Hou; Fang Liang; Fusheng Chen; Xiaohong Wang (2350-2361).
In order to display malolactic enzyme (MLE) on the cell surface of Saccharomyces cerevisiae, a yeast cell surface display plasmid pADH1-AGG was constructed by fusing the α-factor signal encoding sequence (267 bp) and the C-terminal half of α-agglutinin encoding sequence (1,645 bp) into the plasmid pADH1. The pADH1-AGG could successfully express and anchor the enhanced green fluorescent protein (EGFP) onto the yeast cell surface when the EGFP was used to verify its function. Then the pADH1-MLE was constructed by inserting the MLE encoding sequence (1,600 bp) into the pADH1-AGG and introduced into S. cerevisiae cells. The positive strain carrying pADH1-MLE was confirmed by use of the 6× His monoclonal antibody and fluorescein isothiocyanate-conjugated goat anti-mouse IgG. All results indicated that the MLE was displayed successfully on the cell surface of positive transformant. The MLE activity of genetically engineered yeast strain could turn 21.11 % l-malate into lactic acid after 12 h reaction with l-malate. The constructed yeast strain might be used to conduct malolactic fermentation (MLF) in wine to solve the important issues of sluggish MLF, microbial spoilage, and adverse metabolic substances produced by the lactic acid bacteria.
Keywords: Malolactic enzyme; Yeast cell surface display; Malolactic fermentation; Saccharomyces cerevisiae ; l-malate lactic acid; Enhanced green fluorescent protein

As an efficient means of strain improvement, adaptive evolution is a technique with great potential. Long-term cultivation of Saccharomyces cerevisiae was performed in a polydimethylsiloxane membrane bioreactor system which was constructed by coupling the fermentation with pervaporation. A parent strain was subjected to three rounds of fermentation–screening–transfer procedure lasting 1,500 h in a continuous and closed circulating fermentation (CCCF) system, and its 600-generation descendant S33 was screened. In shaking flask culture test, the selected strain S33 from the third round showed great superiority over the parent strain in the residual broth medium, with the ethanol yield and specific ethanol productivity increasing by 34.5 and 34.7 %, respectively. In the long-term CCCF test, the fermentation performance of the descendant strain in the third round was higher than that of its parent strain in the second round. These results show the potential of this novel adaptive evolution approach in optimization of yeast strains.
Keywords: PDMS membrane bioreactor; Saccharomyces cerevisiae ; Closed circulating fermentation; Adaptive evolution

Candida Biofilm Disrupting Ability of Di-rhamnolipid (RL-2) Produced from Pseudomonas aeruginosa DSVP20 by Nivedita Singh; Suma C. Pemmaraju; Parul A. Pruthi; Swaranjit S. Cameotra; Vikas Pruthi (2374-2391).
Biosurfactant produced from Pseudomonas aeruginosa DSVP20 was evaluated for its potential to disrupt Candida albicans biofilm formed on polystyrene (PS) surfaces in this investigation. P. aeruginosa DSVP20 exhibited optimum production of biosurfactant (5.8 g L−1) after 96 h of growth with an ability to reduce surface tension of the aqueous solution from 72 to 28 mN m−1. Analysis of purified biosurfactant with FT-IR, 1H and 13C NMR and MALDI-TOF MS revealed it to be di-rhamnolipid (RL-2) in nature. Biofilm disrupting ability of RL-2 (0.16 mg mL−1) on Candida cells when checked using XTT reduction assay revealed that about 50 % of the cells remain adhered to 96-well plate after 2 h of treatment, while up to 90 % reduction in pre-formed C. albicans biofilm on PS surface was observed with RL-2 (5.0 mg mL−1) in a dose-dependent manner. Microscopic analyses (SEM and CLSM) further confirm the influence of RL-2 on disruption of Candida biofilm extracellular matrix on PS surface which can be exploited as a potential alternative to the available conventional therapies.
Keywords: Di-rhamnolipid; Candida albicans ; Biofilm; Surface tension; CLSM

Application of Pigeon Pea (Cajanus cajan) Stalks as Raw Material for Xylooligosaccharides Production by A. K. Samanta; Natasha Jayapal; A. P. Kolte; S. Senani; Manpal Sridhar; Sukriti Mishra; C. S. Prasad; K. P. Suresh (2392-2404).
Pigeon pea (Cajanus cajan) is a perennial plant widely cultivated in tropical and subtropical regions of many countries. The present studies aimed to produce xylooligosaccharides (XOS) from pigeon pea stalks in order to do value addition. The chemical analysis of stalks revealed 18.33 ± 1.40 % hemicelluloses in addition to cellulose, protein, and lignin. Sodium hydroxide coupled with steam application enabled almost 96 % recovery of original xylan, present in the pigeon pea stalks. Enzymatic hydrolysis of xylan led to production of XOS namely, xylobiose and xylotriose. Response surface model indicated a maximum yield of xylobiose (0.502 mg/ml) under the hydrolysis conditions of pH 4.91, temperature at 48.11 °C, enzyme dose at 11.01 U, and incubation time at 15.65 h. The ideal conditions for higher xylotriose yield (0.204 mg/ml) were pH 5.44, temperature at 39.29 °C, enzyme dose at 3.23 U, and incubation time at 15.26 h. The present investigation was successful in assessing the prospect of using pigeon pea stalks as a raw material for xylan extraction vis-à-vis XOS production.
Keywords: Pigeon pea (Cajanus cajan) stalk; Xylan; Xylooligosaccharides; Enzymatic process

Effect of Subchronic Hypobaric Hypoxia on Oxidative Stress in Rat Heart by Mrinalini Singh; Pauline Thomas; Dhananjay Shukla; Rajkumar Tulsawani; Saurabh Saxena; Anju Bansal (2405-2419).
We examined the effect of subchronic hypobaric hypoxia in rat heart. Adult male Sprague–Dawley rats were exposed at 25,000 ft for different time periods (2 and 5 days). Susceptibility of their hearts to oxidative stress as well as modulation in gene expression was evaluated. The results showed a crosstalk between reactive oxygen species (ROS) and nitric oxide (NO), initial response was accompanied by increase in ROS generation and development of oxidative stress as confirmed by increased lipid peroxidation, protein oxidation and accumulation of 2, 4-dinitrophenyl hydrazine and 4-hydroxy-2-nonenal adducts. At the same time, glutathione activity decreased; however, antioxidant enzymatic activities of superoxide dismutases, glutathione-S-transferase, and glutathione peroxidase rose in response to 5-days hypoxia. Interestingly, NO level increased till 5 days, however ROS decreased after 5 days; this observation suggests that ROS/NO balance plays an important role in cardioprotection. This observation is further supported by upregulation of antioxidant genes hemeoxygenase (HO-1) and metallothionein (MT). In addition, hypoxia also induces gradual upregulation of hypoxia-inducible transcription factor (HIF-1α), which in turn induces the expression of adaptive genes erythropoiesis, vascular endothelial growth factor, glucose transporter-1, nitric oxide synthase. Collectively, our data suggests a reciprocal regulation of ROS and NO and this effect is mediated by the increase in antioxidant proteins HO-1 and MT. Along with this HIF-1-mediated induction of various cardioprotective genes also plays an important role in acclimatization.
Keywords: Hypobaric hypoxia; Cardioprotection; Oxidative stress; Antioxidants; Metallothionein

The Oxidation of Phenylhydrazine by Tyrosinase by Yi-Ming Sung; Srivardhan Reddy Gayam; Shu-Pao Wu (2420-2429).
Tyrosinase was found to catalyze the oxidation of phenylhydrazine to phenol in a reaction that did not resemble those typically performed by tyrosinase. The kinetics of this reaction was investigated by measuring the initial velocity of the formation of phenol (25 °C). The values of k cat and K M for the oxidation of phenylhydrazine were obtained as 11.0 s−1 and 0.30 mM, respectively. The generation of superoxides during the oxidation of phenylhydrazine by tyrosinase was monitored by nitroblue tetrazolium (NBT) assay. In the phenylhydrazine-tyrosinase reaction, 1 mol O2 was required for the production of 1 mol phenol and 1/6 mol superoxide. The decomposition of superoxide by superoxide dismutase enhanced the rate constant of the oxidation of phenylhydrazine. Phenol formed in the oxidation of phenylhydrazine by tyrosinase was further oxidized by tyrosinase to an o-quinone, after the oxidation of phenylhydrazine by tyrosinase was almost completed.
Keywords: Tyrosinase; Phenylhydrazine; Phenol; Superoxide

Maximum Production of Fermentable Sugars from Barley Straw Using Optimized Soaking in Aqueous Ammonia (SAA) Pretreatment by Chang Geun Yoo; Nhuan P. Nghiem; Kevin B. Hicks; Tae Hyun Kim (2430-2441).
Soaking in aqueous ammonia (SAA) pretreatment was investigated to improve enzymatic digestibility and consequently to increase total fermentable sugar production from barley straw. Various effects of pretreatment process parameters, such as reaction temperature, reaction time, solid:liquid ratio, and ammonia concentration, were evaluated, and the optimum conditions for two of the most important factors, reaction temperature and time were determined using response surface methodology. Optimized reaction conditions were 77.6 °C treatment temperature, 12.1 h. treatment time, 15 wt.% ammonia concentration, and 1:8 solid-to-liquid ratio, which gave a sugar recovery yield of 71.5 % (percent of theoretical sugar recovered from the untreated barley straw) with enzyme loading of 15 FPU/g-glucan. In the optimization of the SAA pretreatment process, ammonia concentration, reaction temperature, and reaction time were determined to be the most significant factors correlated to subsequent enzyme digestibility. Based on tested conditions exhibiting high sugar recovery yields of >60 %, it appeared that reaction temperature affected total fermentable sugar production more significantly than reaction time.
Keywords: Fermentable sugars; Sugar recovery yield; Lignocellulosic biomass; Ammonia pretreatment

Gene Cloning, Expression, and Characterization of a Cyclic Nucleotide Phosphodiesterase from Arthrobacter sp. CGMCC 3584 by Zhifang Zheng; Mengzhu Zhu; Ying He; Nan Li; Ting Guo; Yong Chen; Jinglan Wu; Hanjie Ying; Jingjing Xie (2442-2456).
Based on thermal asymmetric interlaced polymerase chain reaction, the arpde gene encoding a cyclic nucleotide-specific phosphodiesterase was cloned from Arthrobacter sp. CGMCC 3584 for the first time. The 930-bp region encoded a 309-amino-acid protein with a molecular weight of 33.6 kDa. The recombinant ArPDE was able to hydrolyze 3′,5′-cAMP, 3′,5′-cGMP, and 2′,3′-cAMP. The K m values of ArPDE for 3′,5′-cAMP and 3′,5′-cGMP were 6.82 and 12.82 mM, respectively. ArPDE was thermostable and displayed optimal activity at 45 °C and pH 7.5. The enzyme did not require any metal cofactors, although its activity was stimulated by 2 mM Co2+ and inhibited by Zn2+. Nucleotides, reducing agents, and sulfhydryl reagents had different inhibitory effects on the activity of ArPDE. NaF, the actual compound used to improve the industrial yield of cAMP, exhibited 62 % inhibitions at concentrations of 10 mM.
Keywords: Arthrobacter ; cAMP; Characterization; ArPDE; Expression; NaF

Anaerobic Digestion of Cassava Wastewater Pre-treated by Fungi by Paula Loureiro Paulo; Thais Adriana Colman-Novaes; Laynara Dayene Soares Obregão; Marc Árpád Boncz (2457-2466).
Cassava wastewater (cww) contains high concentrations of easily acidifying compounds, requiring a buffered system to allow a stable operation during anaerobic digestion (AD). The possibility to include a preliminary one-step fungi treatment aimed at raising the pH and buffering the cww prior to AD was studied. Preliminary tests were performed with a naturally grown fungal mixed culture, under aerated (AE), non-aerated (NAE) and initially oxygen-deprived (IOD) conditions. The cww was pre-treated by the NAE condition, until reaching a soluble chemical oxygen demand (COD) of 10 g L−1 and pH 6.4 (batch A) and pH 5.7 (batch B). The fungal mixed culture showed ability to biodegrade the cww with initial pH of 4.4 and 14,500 mg COD L-1, raising the pH over 8.5, with only 13 % of COD remaining within 27 days for both AE and NAE condition. The fungal pre-treated-cww (FPTcww) was subjected to anaerobic digestion under different buffered (CaCO3 and NaHCO3) and non-buffered conditions. The FPTcww with initial pH at 6.4 provided stability during the anaerobic biodegradability tests, showing the possibility of system operation without buffer addition, with final pH around 7. The application of a fungal pre-treatment can be a promising strategy to permit the anaerobic digestion of carbohydrate-rich wastewaters.
Keywords: Buffer; High strength; Rapid acidification; Starch; UASB

In the present study, we report the optimisation of batch conditions for improved α-1,4-glucan-glucanohydrolase (GGH) secretion by a nitrous acid (NA)-treated Bacillus alcalophilus. The wild (isolate GCB-18) and NA-derivative (mutant GCBNA-4) were grown in a medium containing 10 g/L nutrient broth, 10 g/L starch, 5 g/L lactose, 2 g/L ammonium sulphate, 2 g/L CaCl2 and phosphate buffer (pH 7.6). Sodium dodecyl sulphate (SDS) was used as an enzyme inducer while batch fermentations were carried out at 40 °C. The mutant produced GGH in 40 h which was 15-fold higher than the wild in presence of SDS. Thermodynamic studies revealed that the mutant culture exhibited the capability for improved enzyme activity over a broad range of temperature (35–70 °C). The enzyme was purified by cation-exchange column chromatography with ∼80 % recovery. The performance of fuzzy-logic system control was found to be highly promising for the improved substrate conversion rate. The correlation (1.045E + 0025) among variables demonstrated the model terms as highly significant indicating commercial utility of the culture used (P < 0.05).
Keywords: Bacillus alcalophilus ; Enzyme inducer; 2-factorial design; Glucanhydrolase secretion; Sodium dodecyl sulphate; Thermal inactivation