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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.164, #6)

Construction, Expression, and Characterization of a Single-Chain Variable Fragment Antibody Against 2,4-Dichlorophenoxyacetic Acid in the Hemolymph of Silkworm Larvae by Seiichi Sakamoto; Benyakan Pongkitwitoon; Seiko Nakamura; Kaori Sasaki-Tabata; Yusuke Tanizaki; Katsumi Maenaka; Hiroyuki Tanaka; Satoshi Morimoto (pp. 715-728).

A single-chain variable fragment antibody against herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D-scFv) has been successfully expressed in the hemolymph of silkworm larvae using a rapid Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Variable heavy- and light-chain domains were cloned directly from the cDNA of the hybridoma cell line 2C4 and assembled together with flexible peptide linker (Gly₄Ser)₃ between two domains. The yield of functional 2,4-D-scFv after purification was 640 μg per 30 ml of hemolymph, which is equivalent to 21.3 mg per liter of hemolymph. The characterization of 2,4-D-scFv using an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that it has wide cross-reactivities against 2,4,5-trichlorophenoxyacetic acid (65.5%), 2,4-dichlorophenol (47.9%), and 2,4-dichlorobenzoic acid (26.0%), making it possible to apply 2,4-D-scFv to icELISA for detecting/determining 2,4-D and its metabolites. Judging from its cost and time requirements and its ease of handling, this BmNPV bacmid DNA expression system is more useful for expressing functional scFv than bacterial systems, which frequently require costly and time-consuming refolding.

Keywords: Bombyx mori nucleopolyhedrovirus; 2,4-dichlorophenoxyacetic acid; Enzyme-linked immunosorbent assay; Silkworm; Single-chain variable fragment antibody

Two-Stage Fractionation of Corn Stover Using Aqueous Ammonia and Hot Water by Chang Geun Yoo; Chi-Woo Lee; Tae Hyun Kim (pp. 729-740).

Hot water and aqueous ammonia fractionation of corn stover were used to separate hemicellulose and lignin and improve enzymatic digestibility of cellulose. A two-stage approach was used: The first stage was designed to recover soluble lignin using aqueous ammonia at low temperature, while the second stage was designed to recover xylan using hot water at high temperature. Specifically, the first stage employed a batch reaction using 15 wt.% ammonia at 60 °C, in a 1:10 solid:liquid ratio for 8 h, while the second stage employed a percolation reaction using hot water, 190–210 °C, at a 20 ml/min flow rate for 10 min. After fractionation, the remaining solids were nearly pure cellulose. The two-stage fractionation process achieved 68% lignin purity with 47% lignin recovery in the first stage, and 78% xylan purity, with 65% xylan recovery in the second stage. Two-stage treatment enhanced the enzymatic hydrolysis of remaining cellulose to 96% with 15 FPU/g of glucan using commercial cellulase enzymes. Enzyme hydrolyses were nearly completed within 12–24 h with the remaining solids fraction.

Keywords: SAA (soaking in aqueous ammonia); Biorefinery; Lignocellulosic biomass; Lignin; Xylan

Mass Spectrometry and X-ray Diffraction Analysis of Two Crystal Types of Dioclea virgata Lectin: An Antinociceptive Protein Candidate to Structure/Function Analysis by Plínio Delatorre; Bruno A. M. Rocha; Rafael C. Simões; Francisco N. Pereira-Júnior; Helton C. Silva; Eduardo Henrique S. Bezerra; Maria Julia B. Bezerra; Emmanuel S. Marinho; Carlos A. A. Gadelha; Tatiane Santi-Gadelha; Daniel L. Farias; Ana Maria S. Assreuy; Gabriela F. O. Marques-Domingos; Celso S. Nagano; Benildo S. Cavada (pp. 741-754).

The lectin from seeds of Dioclea virgata (DvirL) was purified in a single step affinity chromatography, sequenced by tandem mass spectrometry and submitted to crystallization and biological experiments. DvirL has a molecular mass of 25,412 ± 2 Da and the chains β and γ has 12,817 Da ± 2 and 12,612 Da ± 2, respectively. Primary sequence determination was assigned by tandem mass spectrometry and revealed a protein with 237 amino acids and 87% of identify with ConA. The protein crystals were obtained native and complexed with X-Man using vapor-diffusion method at a constant temperature of 293 K. A complete X-ray dataset was collected at 1.8 Å resolution. DvirL crystals were found to be orthorhombic, belonging to the space group I222, with a unit cell parameters a = 647.5 Å, b = 86.6 Å, c = 90.2 Å. Molecular replacement search found a solution with a correlation coefficient of 77.1% and an R _factor of 44.6%. The present study also demonstrated that D. virgata lectin presents edematogenic and antinociceptive activities in rodents electing this protein as a candidate to structure/function analysis.

Keywords: Lectin; Crystallization; Tandem mass spectrometry; Antinociceptive

Concentration, Partial Characterization, and Immobilization of Lipase Extract from P. brevicompactum by Solid-State Fermentation of Babassu Cake and Castor Bean Cake by Marceli Fernandes Silva; Denise M. G. Freire; Aline Machado de Castro; Marco Di Luccio; Marcio A. Mazutti; J. Vladimir Oliveira; Helen Treichel; Débora de Oliveira (pp. 755-766).

One relevant limitation hindering the industrial application of microbial lipases has been attributed to their production cost, which is determined by the production yield, enzyme stability among other. The objective of this work was to evaluate the concentration and immobilization of lipase extracts from Penicillium brevicompactum obtained by solid-state fermentation of babassu cake and castor bean cake. The precipitation with ammonium sulfate 60% of saturation of crude extract obtained with babassu cake as raw material showed an enhancement in hydrolytic and esterification activities from 31.82 to 227.57 U/g and from 170.92 to 207.40 U/g, respectively. Concentrated lipase extracts showed preference to medium-chain triglycerides and fatty acids. It is shown that the enzyme activity is maintained during storage at low temperatures (4 and −10°C) for up to 30 days. Higher esterification activities were achieved when the lipase extract was immobilized in sodium alginate and activated coal.

Keywords: Lipase; Concentration; Partial characterization; Immobilization; Babassu cake; Castor bean cake

Green Pigment from Bacillus cereus M¹ ₁₆ (MTCC 5521): Production Parameters and Antibacterial Activity by Debopam Banerjee; Sandipan Chatterjee; U. C. Banerjee; Arun K. Guha; Lalitagauri Ray (pp. 767-779).

A bacterial strain, Bacillus cereus M¹ ₁₆ (MTCC 5521), isolated and identified in our laboratory produces a green pigment when grown in nutrient broth at stationary condition. Optimum fermentation parameters for maximum pigment production are pH 7.0, temperature 30°C, time of incubation 72 h and inoculum volume 1% from 20 h grown cell suspension. Magnesium ion enhances pigment production whereas calcium and zinc ions inhibit the process. The pigment is better extracted from the fermented broth with chloroform in comparison with diethyl ether, ethyl acetate, and butanol. The extracted crude pigment consists of three fractions as revealed from thin layer chromatogram on silica gel GF254 using ethyl acetate and hexane (1:1) solvent system. The major fraction C₃ shows antibacterial activity against different gram positive bacteria. The proposed structure of C₃ is 9-methyl-1,4,5,8-tetra-azaphenanthrene obtained by elemental analysis, GC-MS, and NMR spectra studies.

Keywords: Green pigment; Bacillus cereus ; Antibacterial activity; Production; Chemical structure

Oxidative Stress Parameters of L929 Cells Cultured on Plasma-Modified PDLLA Scaffolds by Melike Erol Demirbilek; Murat Demirbilek; Zeynep Karahaliloğlu; Ebru Erdal; Tayfun Vural; Eda Yalçın; Necdet Sağlam; Emir Baki Denkbaş (pp. 780-792).

Oxidative stress may produce high level of reactive oxygen species (ROS) following cell exposure to endogenous and exogenous factors. Recent experiments implicate oxidative stress as playing an essential role in cytotoxicity of many materials. The aim of this study was to measure intracellular malondialdehyde (MDA), advanced oxidation protein product (AOPP) levels, and superoxide dismutase (SOD) activities of L929 fibroblasts cultured on PDLLA, polyethylene glycol (PEG), or ethylenediamine (EDA) grafted PDLLA by plasma polymerization method. Cell proliferation on these scaffolds was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The study showed that MDA, AOPP levels, and SOD activities in L929 fibroblast cells cultured on all scaffolds were significantly different compared to the control group and each other. The highest MDA (0.42 ± 0.76 nmol/mg protein), AOPP (14.99 ± 4.67 nmol/mg protein) levels, and SOD activities (7.49 ± 3.74 U/mg protein) were observed in cells cultured on non-modified scaffolds; meanwhile, the most cell proliferation was obtained in EDA-modified scaffolds (MDA 0.15 ± 0.14 nmol/mg protein, AOPP 13.12 ± 3.86 nmol/mg protein, SOD 4.82 ± 2.64 U/mg protein). According to our finding, EDA- or PEG-modified scaffolds are potentially useful as suitable biomaterials in tissue engineering.

Keywords: Biocompatibility; Oxidative stress; PDLLA scaffolds; L929 fibroblasts; MDA; SOD; AOPP

Selection of Conditions for Cellulase and Xylanase Extraction from Switchgrass Colonized by Acidothermus cellulolyticus by Farzaneh Rezaei; Lawrence D. Joh; Hiroyuki Kashima; Amitha P. Reddy; Jean S. VanderGheynst (pp. 793-803).

Solid-state fermentation has been widely used for enzyme production. However, secreted enzymes often bind to the solid substrate preventing their detection and recovery. A series of screening studies was performed to examine the role of extraction buffer composition including NaCl, ethylene glycol, sodium acetate buffer, and Tween 80, on xylanase and cellulase recovery from switchgrass. Our results indicated that the selection of an extraction buffer is highly dependent on the nature and source of the enzyme being extracted. While a buffer containing 50 mM sodium acetate at pH 5 was found to have a positive effect on the recovery of commercial fungal-derived cellulase and xylanase amended to switchgrass, the same buffer had a significant negative effect on enzyme extraction from solid fermentation samples colonized by the bacterium Acidothermus cellulolyticus. Xylanase activity was more affected by components in the extraction buffers compared to cellulase. This study demonstrated that extraction followed by diafiltration is important for assessing enzyme recovery from solid fermentation samples. Reduction in activity due to compounds present in the switchgrass extracts is reversible when the compounds are removed via diafiltration.

Keywords: Enzyme extraction; Solid-state fermentation; Cellulase; Xylanase; Acidothermus cellulolyticus

Phosphoproteome Profiling Using a Fluorescent Phosphosensor Dye in Two-Dimensional Polyacrylamide Gel Electrophoresis by Mieko Otani; Taizo Taniguchi; Akiko Sakai; Jouji Seta; Keiichi Kadoyama; Tooru Nakamura-Hirota; Shogo Matsuyama; Keiji Sano; Masaoki Takano (pp. 804-818).

We validated the novel PhosphoQUANTI SolidBlue Complex (PQSC) dye for the sensitive fluorescent detection of phosphorylated proteins in polyacrylamide- and two-dimensional gel electrophoresis (PAGE and 2DE, respectively). PQSC can detect as little as 15.6 ng of ß-casein, a pentaphosphorylated protein, and 61.3 ng of ovalbumin, a diphosphorylated protein. Fluorescence intensity correlates with the number of phosphorylated residues on the protein. To demonstrate the specificity of PQSC for phosphoproteins, enzymatically dephosphorylated lysates of Swiss 3T3 cells were separated in 2DE gels and stained by PQSC. The fluorescence signals in these gels were markedly reduced following dephosphorylation. When the phosphorylated proteins in Swiss 3T3 cell lysates were concentrated using a phosphoprotein enrichment column, the majority of phosphoproteins showed fluorescence signals in the pI 4–5 range. Finally, we performed phosphoproteome analysis to study differences in the protein phosphorylation profiles of proliferating and quiescent Swiss 3T3 cells. Over 135 discernible protein spots were detected, from which a selection of 15 spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS). The PQSC staining procedure for phosphoprotein detection is simple, reversible, and fully compatible with MALDI TOF-MS.

Keywords: Phosphoproteome; Two-dimensional PAGE; MALDI TOF-MS; PhosphoQUANTI solidblue complex; Swiss 3T3 cell

Catalytic Performance of Corn Stover Hydrolysis by a New Isolate Penicillium sp. ECU0913 Producing both Cellulase and Xylanase by Qian-Qian Shi; Jie Sun; Hui-Lei Yu; Chun-Xiu Li; Jie Bao; Jian-He Xu (pp. 819-830).

A fungal strain, marked as ECU0913, producing high activities of both cellulase and xylanase was newly isolated from soil sample collected near decaying straw and identified as Penicillium sp. based on internal transcribed spacer sequence homology. The cultivation of this fungus produced both cellulase (2.40 FPU/ml) and xylanase (241 IU/ml) on a stepwisely optimized medium at 30 °C for 144 h. The cellulase and xylanase from Penicillium sp. ECU0913 was stable at an ambient temperature with half-lives of 28 and 12 days, respectively. Addition of 3 M sorbitol greatly improved the thermostability of the two enzymes, with half-lives increased by 2.3 and 188-folds, respectively. Catalytic performance of the Penicillium cellulase and xylanase was evaluated by the hydrolysis of corn stover pretreated by steam explosion. With an enzyme dosage of 50 FPU/g dry substrate, the conversions of cellulose and hemicellulose reached 77.2% and 47.5%, respectively, without adding any accessory enzyme.

Keywords: Penicillium sp.; Cellulase; Xylanase; Enzymatic hydrolysis; Corn stover

Production of a Monoclonal Antibody by Simultaneous Immunization of Staphylococcal Enterotoxin A and B by Bin Liang; Yongxia Zhang; Aiping Liu; Youxiang Zhou; Fusheng Chen; Xiaohong Wang (pp. 831-840).

In this paper, a method of simultaneous immunizing BALB/c mice with staphylococcal enterotoxin (SE) A and B (SEA and SEB) to prepare a monoclonal antibody (3F2) for detecting both of SEA and SEB was developed. The results showed that antibody 3F2 had high titers against both SEA and SEB by enzyme-linked immunosorbent assay (ELISA). The sensitivities of 3F2 to SEA and SEB detected by ELISA were 133.2 and 82.5 ng/mL, respectively, and the detection limits for the two enterotoxins were about 1 ng/mL. The antibody 3F2 had high specificities and affinities to both SEA and SEB, and had no cross-reaction with SEC₁, bovine serum albumin, and ovalbumin. SEs-free skimmed milk samples were spiked with different concentrations of SEA, SEB, or both of them, respectively. Average recoveries of SEA and SEB from the spiked samples were all nearly between 82% and 104%. The result suggested that one cell fusion with simultaneous immunization by multiple antigen to prepare monoclonal antibody against them was possible, simple, and economic. The monoclonal antibody could be used in simultaneous detecting multifarious SEs.

Keywords: Simultaneous immunization; Staphylococcal enterotoxin; Monoclonal antibody; Indirect competitive ELISA

Fucoidans from Brown Seaweeds Sargassum hornery, Eclonia cava, Costaria costata: Structural Characteristics and Anticancer Activity by Svetlana Ermakova; Roza Sokolova; Sang-Min Kim; Byung-Hun Um; Vladimir Isakov; Tatyana Zvyagintseva (pp. 841-850).

Fucoidans were isolated by water extraction and ion-exchange chromatography from brown algae Eclonia cava, Sargassum hornery, and Costaria costata collected near of Korean coasts. The structures of fucoidans were investigated. Fucoidan from E. cava was mixture of sulfated rhamnogalactofucan and galactofucan. Fucoidan from C. costata was a sulfated galactofucan. Fucoidan isolated from S. hornery was separated into three fractions: a homofucan sulfate, a homofucan but without sulfate groups, and a sulfated rhamnofucan. The results clearly showed that fucoidans play an inhibitory role in colony formation in human melanoma and colon cancer cells and may be effective antitumor agents.

Keywords: Fucoidans; Structure; Anticancer activity

Citrate Stimulates Oligosaccharide Synthesis in Metabolically Engineered Agrobacterium sp. by Anne M. Ruffing; Rachel Ruizhen Chen (pp. 851-866).

Agrobacterium sp. ATCC 31749 was previously shown to be an advantageous host for oligosaccharide production. Unexpectedly, the addition of citrate to the oligosaccharide synthesis reaction resulted in up to a sixfold improvement in the production N-aceytl-lactosamine, a disaccharide. The possible mechanisms for this citrate-induced stimulation of oligosaccharide production were investigated, including the consumption of citrate as a carbon and energy source, enhanced metal ion solubility from citrate chelation, and the ability of citrate to act as a buffer. The main mechanisms for the effect of citrate on oligosaccharide production were determined to be carbon and energy provision from citrate consumption and pH maintenance. ATCC 31749 was shown to co-metabolize citrate along with sucrose, a preferred carbon source, indicating the lack of a catabolite repression system in this Agrobacterium. Metabolic flux analysis suggested an increase in flux through TCA cycle for the citrate-containing reaction, which may provide additional energy supply to support enhanced oligosaccharide production. The citrate stimulation of oligosaccharide synthesis was shown to be unique to the Agrobacterium strain, as a similarly engineered Escherichia coli strain did not show significant improvement in oligosaccharide production with citrate addition. This work provides insight into the metabolism of Agrobacterium sp. ATCC 31749 and highlights important factors in whole-cell oligosaccharide synthesis.

Keywords: Oligosaccharide synthesis; Agrobacterium ; ATCC 31749; Whole-cell synthesis; Metabolic flux analysis; Citrate; Co-metabolism of citrate; Catabolite repression

Biosynthesis and Characterization of Poly (3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) Terpolymer with Various Monomer Compositions by Cupriavidus sp. USMAA2-4 by Hema Ramachandran; Nurhezreen Md Iqbal; Coswald Stephen Sipaut; Amirul Al-Ashraf Abdullah (pp. 867-877).

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)] terpolymer was produced using Cupriavidus sp. USMAA2-4 via one-step cultivation process through combination of various carbon sources such as 1,4-butanediol or γ-butyrolactone with either 1-pentanol, valeric acid, or 1-propanol. Oleic acid was added to increase the biomass production. The composition of 3HV and 4HB monomers were greatly affected by the concentration of 1,4-butanediol and 1-pentanol. Terpolymers with 3HV and 4HB molar fractions ranging from 2 to 41 mol.% and 5 to 31 mol.%, respectively, were produced by varying the concentration of carbon precursors. The thermal and mechanical properties of the terpolymers containing different proportions of the constituent monomers were characterized using gel permeation chromatography (GPC), DSC, and tensile machine. GPC analysis showed that the molecular weights (M _w) of the terpolymer produced were within the range of 346 to 1,710 kDa. The monomer compositions of 3HV and 4HB were also found to have great influences on the thermal and mechanical properties of the terpolymer P(3HB-co-3HV-co-4HB) produced.

Keywords: Biosynthesis; Terpolymer; One-step cultivation; 3-Hydroxyvalerate; 4-Hydroxybutyrate

Converting Carbohydrates Extracted from Marine Algae into Ethanol Using Various Ethanolic Escherichia coli Strains by Soojin Lee; Younghoon Oh; Donghyun Kim; Doyeon Kwon; Choulgyun Lee; Jinwon Lee (pp. 878-888).

Marine algae, which make up about 80% of the world’s living organisms, contain many energy sources, such as sugars and lipids. Therefore, the possibility of utilizing structural carbohydrates from marine algae for bioethanol production has been studied. In order to obtain monosaccharides, Undaria pinnatifida, Chlorella vulgaris, and Chlamydomonas reinhardtii were used for the saccharification experiments. The pretreatment was carried out by dilute acid hydrolysis and enzymatic treatment. To find the optimal conditions, experiments were performed at several temperatures, acid concentrations, pH conditions and durations. To test bioethanol production, several ethanolic E. coli W3110 strains, which were developed previously, were used. The maximum yield of bioethanol, 0.4 g ethanol/g biomass, was achieved with pretreated C. vulgaris and E. coli SJL2526, derived from wild-type E. coli W3110 and which includes the adhB, pdc, galP, and glk genes.

Keywords: Marine algae; Bioethanol; Pretreatment; Ethanolic E. coli ; Dilute acid hydrolysis; Enzymatic treatment

Isolation and Partial Characterization of Halotolerant Lactic Acid Bacteria from Two Mexican Cheeses by Fredy Morales; Jesús I. Morales; César H. Hernández; Humberto Hernández-Sánchez (pp. 889-905).

Isolated strains of halotolerant or halophilic lactic acid bacteria (HALAB) from Cotija and doble crema cheeses were identified and partially characterized by phenotypic and genotypic methods, and their technological abilities were studied in order to test their potential use as dairy starter components. Humidity, a_w, pH, and salt concentration of cheeses were determined. Genotypic diversity was evaluated by randomly amplified polymorphic DNA-polymerase chain reaction. Molecular identification and phylogenetic reconstructions based on 16S rRNA gene sequences were performed. Additional technological abilities such as salt tolerance, acidifying, and proteolytic and lipolytic activities were also investigated. The differences among strains reflected the biodiversity of HALAB in both types of cheeses. Lactobacillus acidipiscis, Tetragenococcus halophilus, Weissella thailandensis, and Lactobacillus pentosus from Cotija cheese, and L. acidipiscis, Enterococcus faecium, Lactobacillus plantarum, Lactobacillus farciminis, and Lactobacillus rhamnosus from doble crema cheese were identified based on 16S rRNA. Quantitative and qualitative assessments showed strains of T. halophilus and L. plantarum to be proteolytic, along with E. faecium, L. farciminis, and L. pentosus to a lesser extent. Lipolytic activity could be demonstrated in strains of E. faecium, L. pentosus, L. plantarum, and T. halophilus. Strains belonging to the species L. pentosus, L. plantarum, and E. faecium were able to acidify the milk media. This study evidences the presence of HALAB that may play a role in the ripening of cheeses.

Keywords: Cotija cheese; Lactic acid bacteria; Halotolerant; Isolation; Identification

Determination of Methyl Parathion in Water and Its Removal on Zirconia Using Optical Enzyme Assay by Kanchanmala Deshpande; Rupesh K. Mishra; Sunil Bhand (pp. 906-917).

A simple, miniaturized microplate chemiluminescence assay for determination of methyl parathion (MP) was developed in 384-microwell plates. Zirconia (ZrO₂) was added in microwell for adsorption of acetylcholinesterase (AChE). The developed assay is based on inhibition of AChE by MP. A good dynamic range 0.008–1,000 ng/mL was obtained for MP with limit of detection 0.008 ng/mL. Intrabatch and interbatch reproducibility for miniaturized assay was obtained with % RSD up to 3.07 and 5.66, respectively. In 384 well plate formats, 70 samples were simultaneously analyzed within 20 min with assay volume of 41.5 μL. The application of developed assay was extended for MP remediation. Column containing ZrO₂ was utilized for remediation where MP was selectively adsorbed. Under optimized condition, adsorption of MP on ZrO₂ was found to be 98–99% with 2-h contact time in real water samples. Adsorption of MP on ZrO₂ column followed by quantification using developed bioassay provides a novel approach to monitor remediation. The applicability of assay was successfully extended for determination of MP in water samples after removal through ZrO₂.

Keywords: Optical enzymatic assay; Methyl parathion (MP); Acetylcholinesterase (AChE); Zirconia (ZrO₂); Drinking water; Remediation; Inhibition

Pretreatment of Corn Stover Silage with Fe(NO₃)₃ for Fermentable Sugar Production by Youshan Sun; Xuebin Lu; Rui Zhang; Xinying Wang; Shuting Zhang (pp. 918-928).

Corn stover silage is an attractive raw material for the production of biofuels and chemicals due to its high content of carbohydrates and easy degradability. The effects of Fe(NO₃)₃ pretreatment conditions on sugar yields were investigated for corn stover silage. In addition, a combined severity factor was used to evaluate the effect of pretreatment conditions on the concentration of total sugars and inhibitors. Optimum pretreatment condition was obtained at 150 °C for 10 min with 0.05 M Fe(NO₃)₃, at which the yields of soluble xylose and glucose in liquid achieved 91.80% of initial xylose, 96.74% of initial arabinose and 19.09% of initial glucose, respectively, meanwhile, 91.84% of initial xylose, 98.24% of initial arabinose, and 19.91% of initial glucose were removed. In addition, a severity analysis showed that the maximum sugar concentration of 33.48 g/l was achieved at combined severity parameter value of 0.62, while the inhibitor concentration was only 0.03 g/l. Fe(NO₃)₃ is an effective catalyst to enhance hemicellulose hydrolysis in corn stover silage, the yields of monomeric xylose in the liquid fraction reached as high as 91.06% of initial xylose and 96.22% of initial arabinose, respectively.

Keywords: Fe(NO₃)₃ ; Corn stover silage; Saccharification; Pretreatment; Inhibitor

Ethanol Production from Cashew Apple Bagasse: Improvement of Enzymatic Hydrolysis by Microwave-Assisted Alkali Pretreatment by Tigressa Helena Soares Rodrigues; Maria Valderez Ponte Rocha; Gorete Ribeiro de Macedo; Luciana R. B. Gonçalves (pp. 929-943).

In this work, the potential of microwave-assisted alkali pretreatment in order to improve the rupture of the recalcitrant structures of the cashew able bagasse (CAB), lignocellulosic by-product in Brazil with no commercial value, is obtained from cashew apple process to juice production, was studied. First, biomass composition of CAB was determined, and the percentage of glucan and lignin was 20.54 ± 0.70% and 33.80 ± 1.30%, respectively. CAB content in terms of cellulose, hemicelluloses, and lignin, 19.21 ± 0.35%, 12.05 ± 0.37%, and 38.11 ± 0.08%, respectively, was also determined. Results showed that, after enzymatic hydrolysis, alkali concentration exerted influence on glucose formation, after pretreatment with 0.2 and 1.0 mo L⁻¹ of NaOH (372 ± 12 and 355 ± 37 mg g _glucan ⁻¹ ) when 2% (w/v) of cashew apple bagasse pretreated by microwave-assisted alkali pretreatment (CAB-M) was used. On the other hand, pretreatment time (15–30 min) and microwave power (600–900 W) exerted no significant effect on hydrolysis. On enzymatic hydrolysis step, improvement on solid percentage (16% w/v) and enzyme load (30 FPU g _CAB-M ⁻¹ ) increased glucose concentration to 15 g L⁻¹. The fermentation of the hydrolyzate by Saccharomyces cerevesiae resulted in ethanol concentration and productivity of 5.6 g L⁻¹ and 1.41 g L⁻¹ h⁻¹, respectively.

Keywords: Microwave-Assisted Alkali Pretreatment; Cashew Apple Bagasse; Enzymatic Hydrolysis; Saccharomyces cerevesiae and Ethanol

Purification and Properties of a Psychrotrophic Trichoderma sp. Xylanase and its Gene Sequence by Peng Zhou; Huifang Zhu; Qiaojuan Yan; Priti Katrolia; Zhengqiang Jiang (pp. 944-956).

A psychrotrophic fungus identified as Trichoderma sp. SC9 produced 36.7 U/ml of xylanase when grown on a medium containing corncob xylan at 20 °C for 6 days. The xylanase was purified 37-fold with a recovery yield of 8.2%. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of approximately 20.5 kDa. The enzyme had an optimal pH of 6.0, and was stable over pH 3.5–9.0. The optimal temperature of the xylanase was 42.5 °C and it was stable up to 35 °C at pH 6.0 for 30 min. The xylanase was thermolabile with a half-life of 23.9 min at 45 °C. The apparent K m values of the xylanase for birchwood, beechwood, and oat-spelt xylans were found to be 3, 2.1, and 16 mg/ml respectively. The xylanase hydrolyzed beechwood xylan and birchwood xylan to yield mainly xylobiose as end products. The enzyme-hydrolysed xylotriose, xylotetraose, and xylopentose to produce xylobiose, but it hardly hydrolysed xylobiose. A xylanase gene (xynA) with an open reading frame of 669 nucleotide base pairs (bp), encoding 222 amino acids, from the strain was cloned and sequenced. The deduced amino acid sequence of XynA showed 85% homology with Xyn2 from a mesophilic strain of Trichoderma viride.

Keywords: Pshychrotrophic; Xylanase; Characterization; Cloning; Trichoderma

Protein Refolding by N-Alkylpyridinium and N-Alkyl-N-methylpyrrolidinium Ionic Liquids by Etsushi Yamamoto; Satoshi Yamaguchi; Teruyuki Nagamune (pp. 957-967).

An important property of ionic liquids consisting of cations and anions is that the chemical structures can be easily tuned. To expand the repertoire of effective ionic liquid-based refolding additives, we focused on this tunable property and investigated the effects of new candidates such as N-alkylpyridinium chlorides and N-alkyl-N-methylpyrrolidinium chlorides on protein refolding. Denatured lysozyme (30 mg/mL) was used as a model protein and refolded by 30-fold dilution with various refolding buffers containing different ionic liquids consisting of a systematic variety of alkyl chains. Compared with the refolding yield without additives (lower than 10%), less hydrophobic ionic liquids such as N-ethyl, N-butyl and N-hexylpyridinium chlorides, and N-butyl-N-methylpyrrolidinium chloride were effective in enhancing the refolding yields (46–69%), because they primarily suppressed aggregation because of their chaotropic properties. N-alkylpyridinium cations were more hydrophobic than N-alkyl-N-methylpyrrolidinium cations according to the calculated log P values and prevented aggregation at lower concentrations because of their hydrophobicity. The results provide a range of new effective ionic liquid-based additives for higher protein refolding yields and the knowledge of the effect of chemical structures of additives on protein refolding.

Keywords: N-Alkylpyridinium chloride; N-Alkyl-N-methylpyrrolidinium chloride; Refolding; Aggregation; Protein stability; Ionic liquids

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.164, #6)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.164, #6)