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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.168, #4)

Xylanase and Ultrasound Assisted Pulping of Wheat Straw by Bhavin S. Dedhia; Levente Csoka; Virendra K. Rathod (pp. 731-741).

In the present work, a novel approach to pretreat wheat straw pulping was investigated with ultrasound and xylanase to achieve maximum reduction in lignin content. Sequential xylanase pretreatment and alkaline pulping was found to reduce kappa number by 0.31 to 4.84 % compared with only alkaline pulping alone at different pulping conditions. Although Klason lignin of ultrasound-treated straw was found to be 7.37 % less compared with untreated straw, sequential ultrasound pretreatment and alkaline pulping could not show any significant reduction in kappa number compared with alkaline pulping alone. Also, sequential xylanase and ultrasound pretreatment could not show any significant reduction in kappa number. Total yield of the pulp was found to be less in ultrasound-assisted processing compared with both alkaline pulping alone and sequential xylanase pretreatment and alkaline pulping.

Keywords: Wheat straw; Lignin; Pulping; Kappa number; Xylanase; Ultrasound

Synthesis of Eugenol Esters by Lipase-Catalyzed Reaction in Solvent-Free System by V. Chiaradia; N. Paroul; R. L. Cansian; C. V. Júnior; M. R. Detofol; L. A. Lerin; J. Vladimir Oliveira; D. Oliveira (pp. 742-751).

Enzymatic esterification of eugenol is a matter of great scientific and technological interest due to the well-known drawbacks of the chemical-catalyzed route as well as the potential use of produced compounds as natural antimicrobials. This work reports the maximization of eugenil acetate production by esterification of eugenol and acetic anhydride in a solvent-free system using Novozym 435 as catalyst. The antimicrobial activity of eugenol and eugenil acetate was also determined. The operating conditions that maximized eugenil acetate production were 50 °C, eugenol to acetic anhydride of 1:3, 150 rpm, and 5.5 wt% of enzyme, with a conversion of 99 %. A kinetic study was performed to assess the influence of substrates molar ratio, enzyme concentration, and temperature on eugenil acetate yield. Results show that an excess of anhydride, low enzyme concentration (1 wt%), and 60 °C afforded nearly complete conversion after 6 h of reaction. The highest antimicrobial activity of eugenil acetate was observed against Acinetobacter sp. (48.66 mm) at concentration of 20 μL. Results indicate that the esterification of eugenol improved its antimicrobial properties. New experimental data on enzymatic esterification of eugenol and acetic anhydride are reported in this work, showing a promising perspective to overcome the inconvenient of the chemical-catalyzed route for obtaining antimicrobial natural compounds.

Keywords: Eugenil acetate; Novozym 435; Antimicrobial activity, eugenol, esterification; Eugenol; Esterification

Geno-Tox: Cell Array Biochip for Genotoxicity Monitoring and Classification by Joo-Myung Ahn; Man Bock Gu (pp. 752-760).

In vitro genotoxicity tests detect carcinogens that are thought to act primarily via a mechanism involving direct genetic damage. In this study, we constructed a Geno-Tox cell array chip for genotoxicity testing; eight recombinant bioluminescent bacteria were used to successfully fabricate a Geno-Tox cell array chip. Four well-characterized DNA damage chemicals were selected to determine the capabilities of this Geno-Tox array chip, and each strain of the chip was distinctly responsive, according to the specific mode of genotoxic action. Therefore, this Geno-Tox cell array chip could be implemented to characterize and understand the genotoxic modes of impact; thus, it could be used to provide the genotoxic mechanism of action for new drugs or unknown or newly synthesized chemicals in food and the environment.

Keywords: DNA damage response; Bioluminescence bacteria; Cell array biochip

Enhanced Green Fluorescent Protein Expression in Pleurotus ostreatus for In Vivo Analysis of Fungal Laccase Promoters by Antonella Amore; Yoichi Honda; Vincenza Faraco (pp. 761-769).

The laccase family of Pleurotus ostreatus has been widely characterized, and studies of the genes coding for laccase isoenzymes in P. ostreatus have so far led to the identification of four different genes and the corresponding cDNAs, poxc, pox1, poxa1b and poxa3. Analyses of P. ostreatus laccase promoters poxc, pox1, poxa1b and poxa3 have allowed identification of several putative response elements, and sequences of metal-responsive elements involved in the formation of complexes with fungal proteins have been identified in poxc and poxa1b promoters. In this work, development of a system for in vivo analysis of P. ostreatus laccase promoter poxc by enhanced green fluorescent protein expression is performed, based on a poly ethylene glycol-mediated procedure for fungal transformation. A quantitative measurement of fluorescence expressed in P. ostreatus transformants is hereby reported for the first time for this fungus.

Keywords: Fungi; Laccases; Transformation; Promoters; Fluorescence

Assessment of Performance Ability of Three Diagnostic Methods for Detection of Potato Leafroll Virus (PLRV) Using Different Visualizing Systems by Mohammad Amin Almasi; Aboubakr Moradi; Jaber Nasiri; Soraya Karami; Mojtaba Nasiri (pp. 770-784).

To diminish the time required for some diagnostic assays including reverse transcription PCR (RT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP; due to mainly RNA extraction step) and also DAS-ELISA into a minimum level, an innovative immunocapture RT-LAMP (IC–RT-LAMP) and immunocapture reverse transcription (IC/RT-PCR) protocol on the basis of Potato Leafroll virus (PLRV) genome were used and optimized. In this regard, all six IC–RT-LAMP primers (i.e. F3, B3, FIP, BIP, LF and LB) together with IC/RT-PCR primers were designed on the basis of the highly conserved sequence (ORF3) of coat protein gene (GenBank accession number: U73777) of PLRV genome. Even though DAS-ELISA, IC/RT-PCR and IC–RT-LAMP assays could successfully detect positive infected plant samples, considering the time, safety, sensitivity, cost and simplicity, the last one was overall superior. Meanwhile, among five different visual dyes to accurately detect IC–RT-LAMP products, both hydroxynaphthol blue and GeneFinder^TM could produce long stable colour change and brightness in a close tube-based approach to prevent cross-contamination risk, concluded eventually as the best ones. Altogether, as IC–RT-LAMP is sensitive, cost-effective, fairly user friendly and also can generate more accurate results than previous diagnostic procedures, we accordingly propose this colorimetric assay as a highly reliable alternative viral recognition system regarding PLRV recognition and probably other viral-based diseases.

Keywords: DAS-ELISA assay; IC–RT-LAMP assay; IC/RT-PCR assay; Potato; Potato Leafroll virus

Engineering of Human CYP3A Enzymes by Combination of Activating Polymorphic Variants by Ina Neunzig; Maria Widjaja; Călin-Aurel Drăgan; Frank T. Peters; Hans H. Maurer; Matthias Bureik (pp. 785-796).

The use of human cytochrome P450 (CYP) enzymes is increasing for the production of drug metabolites used for drug safety testing and doping analysis. Major challenges are high-priced cofactors, poor stability, and comparatively low activities. We have shown previously that production of specific metabolites in milligrams to gram scale is feasible using human CYPs recombinantly expressed in fission yeast. In this study, we sought to improve the activities of human CYP3A enzymes by genetic engineering. Two side chains (Pro293 and Arg409) of known activating human CYP3A polymorphic variants were—separately or together—introduced into the wild-type forms of each of the three enzymes CYP3A4, CYP3A5, and CYP3A7, respectively. Different effects of the two mutations and their combination on enzyme activity were monitored using both polar and nonpolar substrates. Interestingly, the CYP3A7 double mutant displayed a strong increase in activity with respect to testosterone 6β-hydroxylation (300 % of wild-type activity) and luciferin-6′-pentafluoro-benzyl ether turnover (400 % compared to wild type), while the single mutant CYP3A5^Pro293 showed 370 and 400 % of wild-type activity towards 6β-hydroxylation of testosterone and 16α-hydroxylation of dehydroepiandrosterone, respectively. Overall, six out of seven newly created mutants displayed increased activity with at least one of the tested substrates. These results support the notion that pharmacogenetic knowledge can directly contribute to the improvement of biotechnological processes.

Keywords: Enzyme engineering; Cytochrome P450; Rational design; Site-directed mutagenesis; Strain development; Whole-cell biotransformation

An Improved Method of Bisulfite Treatment and Purification to Study Precise DNA Methylation from as Little as 10 pg DNA by Sandeep K. Rajput; Sandeep Kumar; Vivek P. Dave; Ankita Rajput; Haushila P. Pandey; Tirtha K. Datta (pp. 797-804).

Methylation of vertebrate DNA is one of the most important epigenetic alterations which have become a center of scientific attraction, especially because of its important role in the regulation of transcription, genomic imprinting, developmental process, and pathogenesis of various diseases. Currently, there are wide ranges of methods available to produce quantitative and qualitative information on genomic DNA methylation. The vast majority of these methods rely on the optimization of the efficient bisulfite treatment. However, all the available methods for bisulfite treatment suffer from major disadvantages, such as large amount of starting material, poor conversion efficiency as well as low recovery and integrity of DNA after bisulfite treatment. Here, we developed a simple, rapid, and convenient column-based bisulfite treatment method by improving the several critical steps, which leads to consistent C-to-U conversion rate 99–100 %, >75 % recovery of DNA after bisulfite treatment. In addition, it is commercially viable and requires very less amount (∼10 pg) of DNA.

Keywords: DNA methylation; Bisulfite treatment; Epigenetic study

Molecular Modeling of Metabolism for Allergen-Free Low Linoleic Acid Peanuts by Godson O. Osuji; Tassine K. Brown; Sanique M. South; Dwiesha Johnson; Shanique Hyllam (pp. 805-823).

It is necessary to eliminate linoleic acid and allergenic arachins from peanuts for good health reasons. Virginia-type peanuts, harvested from plots treated with mineral salts combinations that mimic the subunit compositions of glutamate dehydrogenase (GDH) were analyzed for fatty acid and arachin compositions by HPLC and polyacrylamide gel electrophoresis, respectively. Fatty acid desaturase and arachin encoding mRNAs were analyzed by Northern hybridization using the homologous RNAs synthesized by peanut GDH as probes. There were 70–80 % sequence similarities between the GDH-synthesized RNAs and the mRNAs encoding arachins, fatty acid desaturases, glutamate synthase, and nitrate reductase, which similarities induced permutation of the metabolic pathways at the mRNA level. Modeling of mRNAs showed there were 210, 3,150, 1,260, 2,520, and 4,200 metabolic permutations in the control, NPKS-, NS-, Pi-, NH₄Cl-, and PK-treated peanuts, respectively. The mRNA cross-talks decreased the arachin to almost zero percent in the NPKS- and PK-treated peanuts, and linoleate to ∼18 % in the PK-treated peanut. The mRNA cross-talks may account for the vastly reported environmentally induced variability in the linoleate contents of peanut genotypes. These results have quantitatively unified molecular biology and metabolic pathways into one simple biotechnology for optimizing peanut quality and may encourage small-scale industry to produce arachin-free low linoleate peanuts.

Keywords: Glutamate dehydrogenase-synthesized RNA; Partial downregulation; HPLC; Northern hybridization; mRNA permutation

Kinetic Involvement of Acetaldehyde Substrate Inhibition on the Rate Equation of Yeast Aldehyde Dehydrogenase by Matthew W. Eggert; Mark E. Byrne; Robert P. Chambers (pp. 824-833).

In order to evaluate the effectiveness of aldehyde dehydrogenase (ALDH) from Saccharomyces cerevisiae as a catalyst for the conversion of acetaldehyde into its physiologically and biologically less toxic acetate, the kinetics over broad concentrations were studied to develop a suitable kinetic rate expression. Even with literature accounts of the binding complexations, the yeast ALDH currently lacks a quantitative kinetic rate expression accounting for simultaneous inhibition parameters under higher acetaldehyde concentrations. Both substrate acetaldehyde and product NADH were observed as individual sources of inhibition with the combined effect of a ternary complex of acetaldehyde and the coenzyme leading to experimental rates as little as an eighth of the expected activity. Furthermore, the onset and strength of inhibition from each component were directly affected by the concentration of the co-substrate NAD. While acetaldehyde inhibition of ALDH is initiated below concentrations of 0.05 mM in the presence of 0.5 mM NAD or less, the acetaldehyde inhibition onset shifts to 0.2 mM with as much as 1.6 mM NAD. The convenience of the statistical software package JMP allowed for effective determination of experimental kinetic constants and simplification to a suitable rate expression: $$ v = frac{{Vmaxleft( {{ ext A}{ ext B}} ight)}}{{KiaKb + KbA + KaB + AB + frac{{{B^2}}}{{KI-Ald}} + frac{{{B^2}Q}}{{KI-Ald-NADH}} + frac{{BQ}}{{KI-NADH}}}} $$ where the last three terms represent the inhibition complex terms for acetaldehyde, acetaldehyde–NADH, and NADH, respectively. The corresponding values of K _I–Ald, K _{I–Ald–NADH}, and K _I–NADH for yeast ALDH are 2.55, 0.0269, and 0.162 mM at 22 °C and pH 7.8.

Keywords: Aldehyde dehydrogenase; Enzyme kinetics; Acetaldehyde substrate inhibition; Saccharomyces cerevisiae ; Statistical software kinetic modeling

Proteomic Analysis for Low and High Nitrogen-Responsive Proteins in the Leaves of Rice Genotypes Grown at Three Nitrogen Levels by Khalid Rehman Hakeem; Ruby Chandna; Altaf Ahmad; Mohd. Irfan Qureshi; Muhammad Iqbal (pp. 834-850).

Nitrogen (N) is an essential nutrient for plants. Increase in crop production is associated with increase in N fertilizers. Excessive use of N fertilizers and the low nitrogen utilization efficiency by crop plants is a major cause for environmental damage. Therefore, to reduce the N-fertilizer pollution, there is an urgent need to improve nitrogen use efficiency. Identification and/or development of genotypes which can grow and yield well at low nitrogen levels may provide a solution. Understanding the molecular mechanism of differential nitrogen use efficiency of the genotypes may provide some clues. Keeping the above facts in mind, in this study we have identified the high N-responsive and low N-responsive contrasting rice genotypes, out of 20 genotypes that were grown at low (1 mM), moderate (10 mM), and high (25 mM) levels of N (KNO₃). Proteome analysis of leaves revealed that the proteins involved in the energy production/regulation and metabolism in plant leaf tissues are differentially expressed under N treatments. Moreover, some disease-resistant and stress-induced proteins were found to be overexpressed at high levels of N. The present study could be useful in identifying proteins responding to different levels of nitrogen fertilization, which may open new avenues for a better understanding of N use efficiency, and for developing new strategies to enhance N efficiency in cereal crops.

Keywords: 2D Electrophoresis; Mass spectrometry; N-use efficiency; Proteomics; Rice genotypes

Purification and Properties of a Thermostable Xylanase GH 11 from Penicillium occitanis Pol6 by Dorra Driss; Fatma Bhiri; Mariem Siela; Raoudha Ghorbel; Semia Ellouz Chaabouni (pp. 851-863).

An extracellular, endo-β-1,4-xylanase was purified to homogeneity from the culture filtrate of the filamentous fungus Penicillium occitanis Pol6, grown on oat spelt xylan. The purified enzyme (PoXyn2) showed a single band on SDS–PAGE with an apparent molecular weight of 30 kDa. The xylanase activity was optimal at pH 3.0 and 65 °C. The specific activity measured for oat spelt xylan was 2,368 U mg⁻¹. The apparent K _m and V _max values were 8.33 mg ml⁻¹ and 58.82 μmol min⁻¹ ml⁻¹, respectively, as measured on oat spelt xylan. Thin-layer chromatography experiments revealed that purified PoXyn2 degrades xylan in an endo-fashion releasing xylobiose as main end product. The genomic DNA and cDNA encoding this protein were cloned and sequenced. This PoXyn2 presents an open reading frame of 962 bp, not interrupted by any introns and encoding for a mature protein of 320 amino acids and 29.88 kDa.

Keywords: Penicillium occitanis pol6; Endo-1,4-β-xylanase; Purification; Characterization; DNA sequence

Laccase-Mediated Transformations of Endocrine Disrupting Chemicals Abolish Binding Affinities to Estrogen Receptors and Their Estrogenic Activity in Zebrafish by Cristina Torres-Duarte; María Teresa Viana; Rafael Vazquez-Duhalt (pp. 864-876).

Endocrine disrupting chemicals (EDCs) are known to mainly affect aquatic organisms, producing negative effects in aquaculture. Transformation of the estrogenic compounds 17β-estradiol (E2), bisphenol-A (BPA), nonylphenol (NP), and triclosan (TCS) by laccase of Coriolopsis gallica was studied. Laccase is able to efficiently transform them into polymers. The estrogenic activity of the EDCs and their laccase transformation products was evaluated in vitro as their affinity for the human estrogen receptor alpha (hERα) and for the ligand binding domain of zebrafish (Danio rerio) estrogen receptor alpha (zfERαLBD). E2, BPA, NP, and TCS showed higher affinity for the zfERαLBD than for hERα. After laccase treatment, no affinity was found, except a marginal affinity of E2 products for the zfERαLBD. Endocrine disruption studies in vivo on zebrafish were performed using the induction of vitellogenin 1 as a biomarker (VTG1 mRNA levels). The use of enzymatic bioreactors, containing immobilized laccase, efficiently eliminates the endocrine activity of BPA and TCS, and significantly reduces the effects of E2. The potential use of enzymatic reactors to eliminate the endocrine activity of EDCs in supply water for aquaculture is discussed.

Keywords: Bisphenol-A; Endocrine disruption; Estrogen receptor; Zebrafish; Laccase; Nonylphenol; Triclosan; Immobilized laccase; Vitellogenin

Antibacterial, Anti-Chlamydial, and Cytotoxic Activities of a Marine Snail (Hexaplex trunculus) Phospholipase A2: an In Vitro Study by Zied Zarai; Houda Gharsallah; Adnane Hammami; Hafedh Mejdoub; Sofiane Bezzine; Youssef-Talel Gargouri (pp. 877-886).

In order to report pharmacological characterization of marine snail (Hexaplex trunculus) hepatopancreatic phospholipase A₂ (mSDPLA₂), we have talked for the first time the antimicrobial activity against different pathogenic bacterial strains, anti-chlamydial activity as well as its cytotoxic activity against McCoy cell lines. mSDPLA₂, showed a high level of activity towards Gram-positive bacteria as Staphylococcus aureus and Staphylococcus epidermidis. Whereas Gram-negative bacteria, unfortunately, exhibited a higher resistance, mSDPLA₂ was also found to have a strong cytotoxic activity, causing significant morphological alterations of the McCoy cell lines surfaces and to be a hinder to the proliferation. Moreover, mSDPLA₂ proved to have a very potent anti-chlamydial activity. Over 95 % inhibition of chlamydial inclusions were obtained at a concentration of 10 μg/ml of mSDPLA₂ after 24 h postinfection. Interestingly, at a concentration of 10 μg/ml of mSDPLA₂, the proliferation of McCoy cells was not affected. Approximately 50 % inhibition of cell growth was obtained with a concentration of 37 μg/mL of mSDPLA₂. mSDPLA₂ could be considered as an excellent candidate for the development of a new anti-infective agent. This enzyme showed significant antimicrobial activities.

Keywords: Phospholipase A₂ ; Hexaplex trunculus ; Antimicrobial activity; Anti-chlamydial activity; Cytotoxic activity

Dietary Supplementation with Bovine Lactoferrampin–Lactoferricin Produced by Pichia pastoris Fed-batch Fermentation Affects Intestinal Microflora in Weaned Piglets by Xiang-Shan Tang; Hua Shao; Tie-Jun Li; Zhi-Ru Tang; Rui-Ling Huang; Sheng-Ping Wang; Xiang-Feng Kong; Xin Wu; Yu-Long Yin (pp. 887-898).

This work is aimed at investigating the effects of recombinant bovine lactoferrampin–lactoferricin (LFA-LFC) instead of chlortetracycline on intestinal microflora in weaned piglets. The high cost of peptide production from either native digestion or chemical synthesis limits the clinical application of antimicrobial peptides. The expression of recombinant peptides in yeast may be an effective alternative. In the current study, recombinant LFA-LFC was produced via fed-batch fermentation in recombinant strain Pichia pastoris (KM71) XS10. Uniform design U6(6⁴) was used to optimize the fermentation conditions. The target peptide purified via cation-exchange and size-exclusion chromatography was added into the dietary of weaned piglets. After 21 days, the Lactobacilli, Bifidobacteria, and Enterobacteria in the chyme of the gut were quantified using real-time polymerase chain reaction. The results showed that approximately 82 mg of LFA-LFC was secreted into 1 L of medium under optimized conditions. Moreover, purified peptide showed strong antimicrobial activities against all the tested microorganisms. Compared with the control group, the LFA-LFC group increased the amount of Lactobacilli and Bifidobacteria (P < 0.05) in the chyme of the stomach, duodenum, jejunum, ileum, colon, and caecum. These results show that dietary supplementation with LFA-LFC can affect intestinal microflora in weaned piglets.

Keywords: Lactoferrampin–lactoferricin; Antimicrobial peptide; Pichia pastoris ; Piglet; Gut; Microflora; Nutrition; Digestion

A Novel Alkaliphilic Xylanase from the Newly Isolated Mesophilic Bacillus sp. MX47: Production, Purification, and Characterization by Won-Jae Chi; Da Yeon Park; Yong-Keun Chang; Soon-Kwang Hong (pp. 899-909).

A newly isolated bacterial strain, Bacillus sp. MX47, was actively producing extracellular xylanase only in xylan-containing medium. The xylanase was purified from the culture broth by two chromatographic steps. The xylanase had an apparent molecular weight of 26.4 kDa with an NH₂-terminal sequence (Gln-Gly-Gly-Asn-Phe) distinct from that of reported proteins, implying it is a novel enzyme. The optimum pH and temperature for xylanase activity were 8.0 and 40 °C, respectively. The enzyme activity was severely inhibited by many divalent metal ions and EDTA at 5 mM. The xylanase was highly specific to beechwood and oat spelt xylan, however, not active on carboxymethyl cellulose (CMC), avicel, pectin, and starch. Analysis of the xylan hydrolysis products by Bacillus sp. MX47 xylanase indicated that it is an endo-β-1,4-xylanase. It hydrolyzed xylan to xylobiose as the end product. The K _m and V _max values toward beechwood xylan were 3.24 mg ml⁻¹ and 58.21 μmol min⁻¹ mg⁻¹ protein, respectively.

Keywords: Xylanase; Endo-β-1,4-xylanase; Xylobiose; Bacillus sp.; Purification; Chromatography

Ovarian Cancer: Biomarker Proteomic Diagnosis in Progress by Yong Zhang; Bin Guo; Ran Bi (pp. 910-916).

Ovarian cancer lacks clear syndromes at an early stage and could result in serious problem in woman's health status. The current diagnostic approach relies on physical examination, ultrasound examination, and blood test for CA125. These approaches could not diagnose early stage ovarian cancer with high sensitivity and specificity. The present paper reviewed the efforts in screening the proteomic biomarkers for ovarian cancer. The sources of biomarkers were discussed. Then, the current techniques in proteomics were introduced. Finally, the biomarkers for ovarian cancer were summarized.

Keywords: Ovarian cancer; Biomarker; CA125; Proteomics; HE4

Three-Stage Extraction of Gelatines from Tendons of Abattoir Cattle: 1—Reaction Conditions by Pavel Mokrejs; Dagmar Janacova; Petr Svoboda (pp. 917-927).

Short and long tendons of abattoir cattle are collagen by-products of the meat industry. They offer no utilisation at present, being a raw material source of over 90 % protein characteristic. This contribution deals with the three-stage extraction of gelatine from short cattle tendons. The principle of treatment consists in processing degreased tendons in the first processing stage in an environment resulting in the swelling of the starting material. In the second stage, the material is treated with a proteolytic enzyme to produce such disruption of the collagen substrate that makes gelatine extraction when boiling possible in the third stage of the process. In order to study the influence of the significant parameters during the extraction process on gelatine yield, experiments were planned using a factor experiment of 2³ types. The variables under study were the duration of the second processing stage (5–25 h), temperature in the first and second processing stages (10–40 °C) and the addition of a proteolytic enzyme (1–5 %) on the quantity of the extracted gelatine. The results were processed statistically, and statistical significance of the studied factors was thus found. Contour graphs were plotted to easily survey the influence of the observed factors on gelatine yield. The process achieves up to 71 % efficiency, runs under atmospheric pressure and mild reaction conditions, and is conducive to preparing quality gelatines.

Keywords: By-products; Cattle tendons; Enzymatic treatment; Food industry; Gelatine; Extraction

Effects of Holdfast of Laminaria japonica on Listeria Invasion on Enterocyte-Like Caco-2 Cells and NO Production of Macrophage RAW 264.7 Cells by Takashi Kuda; Shinsuke Nakamura; Choa An; Hajime Takahashi; Bon Kimura; Makoto Nishizawa (pp. 928-935).

Listeria monocytogenes (Lm) causes food poisoning in humans mainly through consumption of ready-to-eat foods. Immunocompromised persons are at the highest risk for infection. We investigated effects of crude soluble polysaccharides (SPS) and ethanolic extract (EE) fractions of frond (kombu) and holdfast (ganiashi) parts of Laminaria japonica on Lm invasion into human enterocyte-like Caco-2 cells and immune and/or inflammatory reactions of murine macrophage RAW 264.7 cells. Recovery and viscosity were high in kombu SPS. Total phenolic content and antioxidant activities (2,2-diphenyl-1-picrylhydrazyl radical scavenging capacity and Fe-reducing power) were higher in ganiashi EE. EE of ganiashi, rather than kombu, suppressed the Lm invasion into the differentiated Caco-2 cells, though the inhibitory effect of SPS was not significant. Ganiashi SPS increased the nitric oxide (NO) production of intact RAW 264.7 cells. On the other hand, the NO production from Escherichia coli O111 lipopolysaccharide-activated cells was suppressed by kombu SPS and ganiashi EE. These results suggest that L. japonica, particularly ganiashi, might suppress the invasion and infection of Lm and also the inflammation.

Keywords: Laminaria japonica ; Holdfast; Listeria monocytogenes ; Caco-2; RAW 264.7

Extraction and Purification of Purple Membrane for Photochromic Thin Film Development: Application in Photoelectrochemical Investigation by P. C. Pandey; Digvijay Pandey; Richa Singh (pp. 936-946).

Purple membrane (PM) has been extracted and purified from archaebacteria for thin film development. The purified purple membrane is isolated in 1 % polyvinyl alcohol solution for making thin film within gelatin and organically modified silicate matrices. For thin film within gelatin matrix, homogenized purple membrane suspension is mixed with 8 % gelatin and poured into a specially designed block with desired thickness of spacer having hydrophobicity followed by gelatinization of the same over home-made thermostatic control unit at 38 °C. The gelatinized matrix is then allowed to dry under controlled conditions of humidity and temperature. The films of varying thicknesses ranging between 40, 50, and 60 μ are used for photo-electrochemical measurements. The results on photo-electrochemistry of non-oriented purple membrane film provides valuable information on the generation of forward (light on) and backward (light off) photocurrent as a function of: (a) applied potential and (b) film thickness. An increase in applied negative potential increases the amplitude of photocurrent whereas decrease in film thickness facilitates the reversibility of photocurrent response.

Keywords: Halobacterium; Photoelectrochemistry; Purple membrane; Thin film of purple membrane

The Cellulase-Mediated Saccharification on Wood Derived from Transgenic Low-Lignin Lines of Black Cottonwood (Populus trichocarpa) by Douyong Min; Quanzi Li; Hasan Jameel; Vincent Chiang; Hou-min Chang (pp. 947-955).

Downregulated lignin transgenic black cottonwood (Populus trichocarpa) was used to elucidate the effect of lignin and xylan content on enzymatic saccharification. The lignin contents of three transgenic samples (4CL1-1, 4CL1-4, and CH8-1-4) were 19.3, 16.7, and 15.0 %, respectively, as compared with the wild type (21.3 %). The four pretreatments were dilute acid (0.1 % sulfuric acid, 185 °C, 30 min), green liquor (6 % total titratable alkali, 25 % sulfidity based on TTA, 185 °C, and 15 min.), autohydrolysis (185 °C, 30 min), and ozone delignification (25 °C, 30 min). Following the pretreatment, enzymatic saccharification was carried out using an enzyme charge of 5 FPU/g of substrates. The removal of lignin and hemicellulose varies with both the types of pretreatments and the lignin content of the transgenic trees. Due to the greatest removal of lignin, green liquor induced the highest sugar production and saccharification efficiency, followed by acid, ozone, and autohydrolysis in descending order. The results indicated that lignin is the main recalcitrance of biomass degradation. At a given lignin content, pretreatment with ozone delignification had lower saccharification efficiency than the other pretreatment methods due to higher xylan content.

Keywords: Transgenic hardwood; Downregulated lignin; Pretreatment; Enzymatic saccharification

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.168, #4)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.168, #4)