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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.163, #5)

Single-Base Extension and ELISA-Based Approach for Single-Nucleotide Polymorphisms Genotyping by Guibo Liu; Yongxia Cheng; Wei Zhao; Zaishun Jin; Hongbo Shan; Gaolian Xu (pp. 573-576).

Single-nucleotide polymorphisms (SNPs) emerge as a fundamental tool in personalized medicine due to their association with drug responses or disease predisposition. Single-base extension (SBE) is a common method for characterizing known SNPs, but involves complicated procedures or requires costly analytical instruments. Here, we describe a novel SNP genotyping based on SBE and enzyme-linked immunosorbent assay (ELISA). During the SBE, the 5′ end fluorescein isothiocyanate-labeled allele-specific primer will extend with biotinylated dideoxynucleotides which are complementary to the SNP sites. The extension product will then be captured by streptavidin-coated nanoparticle and develop blue color in the ELISA assay. We validated this method by detecting SNPs for TP53 gene codon 273 from 68 individuals and the data were 100% in concordant with DNA sequencing. Thus, SBE and ELISA-based SNPs assay is a simple and accurate method for SNP genotyping.

Keywords: Single-nucleotide polymorphisms; Genotyping; ELISA; Single-base extension

Evaluation of Glycosyl Hydrolases in the Secretome of Aspergillus fumigatus and Saccharification of Alkali-Treated Rice Straw by Manju Sharma; Rohit Soni; Asiya Nazir; Harinder Singh Oberoi; Bhupinder Singh Chadha (pp. 577-591).

A thermotolerant Aspergillus fumigatus strain isolated from composting pile of mixed industrial waste was found to produce a spectrum of cellulase and hemicellulases when cultured on rice straw solidified substrate. The two-dimensional electrophoresis (2DE) resolved the secretome into 57 distinct protein spots. The zymograms developed against 2DE gels identified the presence of three β-glucosidases and five CBHI/EGI isoforms in the secretome. The peptide mass fingerprinting of 17 protein spots by liquid chromatography mass spectrometry characterized the secretome into different glycosyl hydrolase families. The enzyme cocktail produced by A. fumigatus was capable of efficient hydrolysis of alkali pretreated rice straw (at 7% and 10% w/v) resulting in 95% and 91% saccharification, respectively.

Keywords: Secretome; Peptide mass fingerprinting; Glycosyl hydrolases; Activity detection of CBHI/EGI and β-glucosidases in 2DE gels; Saccharification

Hydroxytyrosol Acyl Esters: Biosynthesis and Activities by Zouhaier Bouallagui; Mohamed Bouaziz; Salwa Lassoued; Jean Marc Engasser; Mohamed Ghoul; Sami Sayadi (pp. 592-599).

We previously reported the production of high yields of hydroxytyrosol through the bioconversion of tyrosol. In the present work, hydroxytyrosol was subjected to the lipase catalyzed acylation aiming for the recovery of more lipophilic esters that might be easily incorporated in cosmetic and food preparations. Hydroxytyrosyl acetate and hydroxytyrosyl oleate were produced with respective molar esterification yields of 98% and 78%. DPPH free radical quenching potency demonstrated that the acylation of hydroxytyrosol did not alter its antioxidant activity. The acylated esters were shown to be more effective than the natural antioxidant: caffeic acid and two synthetic ones as BHA and BHT. Antiproliferative activity on human cervical cells (HeLa) resulted in IC₅₀ values of 0.46, 0.42 and 0.33 mM for hydroxytyrosol and its acetyl and oleyl esters, respectively. Additionally, when used at a non-cytotoxic concentration (100 μM), these compounds showed significant effectiveness in preventing iron-induced oxidative stress, resulting in a reduction of 30%, 36% and 38% in thiobarbituric acid-reactive substance production, respectively.

Keywords: Hydroxytyrosol; Esterification; Novozym 435; Kinetics; Cytotoxicity; Oxidative stress

Rhamnolipid Production by Pseudomonas Aeruginosa GIM 32 Using Different Substrates Including Molasses Distillery Wastewater by An-hua Li; Mei-ying Xu; Wei Sun; Guo-ping Sun (pp. 600-611).

A rhamnolipid production strain newly isolated from oil-contaminated soil was identified as Pseudomonas aeruginosa GIM32 by its morphology and 16S rDNA sequence analysis. The effect of carbon source and carbon to nitrogen (C/N) ratio on rhamnolipids production was investigated. Palm oil was favorable as a carbon source for rhamnolipid production. The maximum biomass and rhamnolipid concentration were 8.24 g/L and 30.4 g/L, respectively, with an optimization medium containing 50 g/L palm oil and 5 g/L sodium nitrate. Molasses distillery wastewater as an unconventional substrate for rhamnolipid production was investigated. It was found that 2.6 g/L of rhamnolipids was produced; this amount was higher than that of past reports using wastewater as a substrate. In addition, 44% of the chemical oxygen demand of wastewater was removed at the same time under the optimization condition. Eleven kinds of different molecular weight rhamnolipid homologues were identified in the rhamnolipids obtained from molasses distillery wastewater by P. aeruginosa GIM32 by LC–MS analysis.

Keywords: Rhamnolipids; Pseudomonas aeruginosa ; Molasses distillery wastewater; Biosurfactant

Kinetics of Lime Pretreatment of Sugarcane Bagasse to Enhance Enzymatic Hydrolysis by Laura L. G. Fuentes; Sarita C. Rabelo; Rubens Maciel Filho; Aline C. Costa (pp. 612-625).

The objective of this work was to determine the optimum conditions of sugarcane bagasse pretreatment with lime to increase the enzymatic hydrolysis of the polysaccharide component and to study the delignification kinetics. The first stage was an evaluation of the influence of temperature, reaction time, and lime concentration in the pretreatment performance measured as glucose release after hydrolysis using a 2³ central composite design and response surface methodology. The maximum glucose yield was 228.45 mg/g raw biomass, corresponding to 409.9 mg/g raw biomass of total reducing sugars, with the pretreatment performed at 90°C, for 90 h, and with a lime loading of 0.4 g/g dry biomass. The enzymes loading was 5.0 FPU/dry pretreated biomass of cellulase and 1.0 CBU/dry pretreated biomass of β-glucosidase. Kinetic data of the pretreatment were evaluated at different temperatures (60°C, 70°C, 80°C, and 90°C), and a kinetic model for bagasse delignification with lime as a function of temperature was determined. Bagasse composition (cellulose, hemicellulose, and lignin) was measured, and the study has shown that 50% of the original material was solubilized, lignin and hemicellulose were selectively removed, but cellulose was not affected by lime pretreatment in mild temperatures (60–90°C). The delignification was highly dependent on temperature and duration of pretreatment.

Keywords: Pretreatment of lignocellulosic biomass; Sugarcane bagasse; Kinetics of pretreatment; Lime pretreatment

Kinetics of Enzymatic High-Solid Hydrolysis of Lignocellulosic Biomass Studied by Calorimetry by Søren N. Olsen; Erik Lumby; Kc McFarland; Kim Borch; Peter Westh (pp. 626-635).

Enzymatic hydrolysis of high-solid biomass (>10% w/w dry mass) has become increasingly important as a key step in the production of second-generation bioethanol. To this end, development of quantitative real-time assays is desirable both for empirical optimization and for detailed kinetic analysis. In the current work, we have investigated the application of isothermal calorimetry to study the kinetics of enzymatic hydrolysis of two substrates (pretreated corn stover and Avicel) at high-solid contents (up to 29% w/w). It was found that the calorimetric heat flow provided a true measure of the hydrolysis rate with a detection limit of about 500 pmol glucose s⁻¹. Hence, calorimetry is shown to be a highly sensitive real-time method, applicable for high solids, and independent on the complexity of the substrate. Dose–response experiments with a typical cellulase cocktail enabled a multidimensional analysis of the interrelationships of enzyme load and the rate, time, and extent of the reaction. The results suggest that the hydrolysis rate of pretreated corn stover is limited initially by available attack points on the substrate surface (<10% conversion) but becomes proportional to enzyme dosage (excess of attack points) at later stages (>10% conversion). This kinetic profile is interpreted as an increase in polymer end concentration (substrate for CBH) as the hydrolysis progresses, probably due to EG activity in the enzyme cocktail. Finally, irreversible enzyme inactivation did not appear to be the source of reduced hydrolysis rate over time.

Keywords: Cellulase kinetics; High solid; Isothermal calorimetry; Saccharification; Lignocellulose; Biomass

In Vivo Evaluation of Cerebral Transplantation of Resovist-Labeled Bone Marrow Stromal Cells in Parkinson’s Disease Rats Using Magnetic Resonance Imaging by Jing Guo; Jun-Kang Shen; Lan Wang; Li Xiao; Rong-Jun Zhang; Wei-Feng Luo; Zhi-Gang Gong; Jing Sun; Han Xu; Pierre Sirois; Kai Li (pp. 636-648).

The effectiveness of Resovist-labeled bone marrow stem cells (BMSCs) was evaluated in vivo following their cerebral transplantation in a model of Parkinson’s disease (PD) in rats using MRI, and the MRI findings were further compared with the behavior and histopathological manifestations of these rats. Forty PD rats were randomly assigned into five groups according to the cell doses injected into the rat brain site: control group (normal saline injection) and groups injected with 1 × 10⁵, 1.5 × 10⁵, 2 × 10⁵, and 2.5 × 10⁵ BMSCs. Gradient echo T2-weighted images were obtained immediately after cell transplantation and repeatedly taken 1, 4, 8, and 12 week(s) after cell transplantation. The rotational behavior of the animals was observed before and 1, 4, and 8 week(s) after transplantation. The rats were killed after the last MRI scanning, the brain tissues were analyzed by histopathology techniques, and RNAs were extracted for the expression analysis of selected genes using RT-PCR. One week following cell transplantation, all injected sites showed well-defined hypointense areas on MR images, with the most significant effect observed in rats injected with 2 × 10⁵ BMSCs. These MR findings in PD rats lasted up to 12 weeks. The effectiveness of BMSC transplantation revealed by MRI was well confirmed by the behavioral and histopathological observations as well as indirectly supported by gene expression analyses. With the use of SPIO labeling, MRI techniques provided a dynamic evaluation of the spatial and temporal changes following cell transplantation and allowed the association analysis among the imaging, functions, and gene expression analysis in rats. These data also suggest the therapeutic potential of transplanted BMSCs. It is reasonable to speculate that the use of MRI in in vivo evaluation of the effect and fate of transplanted cells in various disease models will be beneficial to developing new strategies of cell-based gene therapy.

Keywords: BMSCs; Cell transplantation; MRI; PD; Resovist; SPIO

Functional Annotation of Fibrobacter succinogenes S85 Carbohydrate Active Enzymes by Phillip Brumm; David Mead; Julie Boyum; Colleen Drinkwater; Krishne Gowda; David Stevenson; Paul Weimer (pp. 649-657).

Fibrobacter succinogenes is a cellulolytic bacterium that degrades plant cell wall biomass in ruminant animals and is among the most rapidly fibrolytic of all mesophilic bacteria. The complete genome sequence of Fisuc was completed by the DOE Joint Genome Institute in late 2009. Using new expression tools developed at Lucigen and C5-6 Technologies and a multi-substrate screen, 5,760 random shotgun expression clones were screened for biomass-degrading enzymes, representing 2× genome expression coverage. From the screen, 169 positive hits were recorded and 33 were unambiguously identified by sequence analysis of the inserts as belonging to CAZy family genes. Eliminating duplicates, 24 unique CAZy genes were found by functional screening. Several previously uncharacterized enzymes were discovered using this approach and a number of potentially mis-annotated enzymes were functionally characterized. To complement this approach, a high-throughput system was developed to clone and express all the annotated glycosyl hydrolases and carbohydrate esterases in the genome. Using this method, six previously described and five novel CAZy enzymes were cloned, expressed, and purified in milligram quantities.

Keywords: Fibrobacter succinogenes ; Biomass; Genome screening; Cellulase; Hemicellulase; Rumen; Screening

Dilute Acid Pretreatment of Corncob for Efficient Sugar Production by G. S. Wang; Jae-Won Lee; J. Y. Zhu; Thomas W. Jeffries (pp. 658-668).

Aqueous dilute acid pretreatments of corncob were conducted using cylindrical pressure vessels in an oil bath. Pretreatments were conducted in a temperature range of 160–190 °C with acid-solution-to-solid-corncob ratio of 2. The acid concentration (v/v) in the pretreatment solution was varied from 0% to 0.7%, depending on temperature. This gives acid charge on ovendry-weight corncob of 0–2.58%. It was found that optimal pretreatment temperature is between 160 and 170 °C based on total xylose and glucose yields and thermal energy consumption in pretreatment. At 170 °C and acid charge of 2.2% on cob, total glucose yield and xylose recovery were 97% and 75%, respectively, which resulted in an overall monomeric sugar recovery of about 88%. Xylose concentration in the hydrolysate was about 12%, with xylose-to-acetic-acid ratio of 8 and to furan (furfural and hydroxymethylfurfural) of about 15.

Keywords: Pretreatment; Cellulosic ethanol; Enzymatic hydrolysis; Corncob; Inhibitors

An Exopolysaccharide from Cultivated Cordyceps sinensis and its Effects on Cytokine Expressions of Immunocytes by Lu Sheng; Jiaping Chen; Jing Li; Weiyun Zhang (pp. 669-678).

The exopolysaccharide (EPS) is a polysaccharide from cultivated Cordyceps sinensis, which possesses immunomodulatory and antitumor effects, was purified by DEAE-32 cellulose and Sephadex G-200 gel. The preliminary characters of EPS were analyzed by IR and GC, and the molecular weight was estimated by gel filtration. The effect of EPS on proliferation ability of lymphocytes from ICR mice was assayed by MTT method. The mRNA and protein expression levels of several cytokines in spleen and thymus cells were detected by RT-PCR and ELISA. The results showed that EPS consists of mannose, glucose, and galactose in a ratio of 23:1:2.6. Its molecular weight is about 1.04 × 10⁵. EPS elevated proliferation ability of spleen lymphocytes only at 100 μg/ml after 48 h treatment. Tumor necrosis factor alpha (TNF-α), interferon-α (IFN-γ), and interleukin-2 (IL-2) mRNA levels in splenocytes and thymocytes were increased after EPS treatment for 2, 4, 8, or 20 h. EPS also significantly elevated splenic TNF-α and IFN-γ protein expressions at 100 μg/ml and increased thymic TNF-α and IFN-γ protein levels at 50 and 100 μg/ml. These data indicated that EPS may stimulate cytokine expressions of immunocytes.

Keywords: Cultivated Cordyceps sinensis ; Cytokine; Exopolysaccharide; Immunocyte

Molecular Cloning and Expression of Two Cytosolic Copper–Zinc Superoxide Dismutases Genes from Nelumbo nucifera by Chen Dong; Xingfei Zheng; Guolin Li; Honglin Zhu; Mingquan Zhou; Zhongli Hu (pp. 679-691).

Two cytosolic copper–zinc superoxide dismutase (cytCuZnSOD) complementary deoxyribonucleic acid were achieved in Nelumbo nucifera (Elian). The active sites and common characteristics of cytCuZnSOD family were showed by homology modeling. The two recombinant proteins expressed by PET-32a vector showed the similar SOD activity (89.94 ± 0.54 U/mg) and could maintain more than 90% activity after incubation at 65°C. The subcellular location by green fluorescent protein revealed that these two isoforms were all located in cytosol and nucleus. The cytCuZnSODs were expressed in various parts of N. nucifera, which were expressed highest in the leafstalks and young leaves and lowest in the roots. The cytCuZnSOD messenger ribonucleic acids isolated from wounded leaves significantly increased at 1.5 h after treatment (HAT) with the highest expression at 3 HAT, after which the level decreased.

Keywords: CuZnSODs; Recombinant proteins; Real-time PCR

Erratum: Functional Annotation of Fibrobacter succinogenes S85 Carbohydrate Active Enzymes by Phillip Brumm; David Mead; Julie Boyum; Colleen Drinkwater; Jan Deneke; Krishne Gowda; David Stevenson; Paul Weimer (pp. 692-692).

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.163, #5)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.163, #5)