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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.166, #6)

Enhanced Ammonia Content in Compost Leachate Processed by Black Soldier Fly Larvae by Terrence R. Green; Radu Popa (pp. 1381-1387).

Black soldier fly (BSF) larvae (Hermetia illucens), feeding on leachate from decaying vegetable and food scrap waste, increase ammonia (NH ₄ ⁺ ) concentration five- to sixfold relative to leachate unprocessed by larvae. NH ₄ ⁺ in larva-processed leachate reached levels as high as ∼100 mM. Most of this NH ₄ ⁺ appears to have come from organic nitrogen within the frass produced by the larvae as they fed on leachate. In nitrate-enriched solutions, BSF larvae also facilitate dissimilatory nitrate reduction to ammonia. The markedly higher concentration of NH ₄ ⁺ recovered in leachates processed with BSF larvae and concomitant diversion of nutrients into insect biomass (itself a valuable feedstock) indicate that the use of BSF larvae in processing leachate of decaying organic waste could be advantageous in offsetting capital and environmental costs incurred in composting.

Keywords: Compost leachate; Black soldier fly larvae; Hermetia illucens ; Treatment; Ammonium

Modelling Growth and Bacteriocin Production by Pediococcus acidilactici PA003 as a Function of Temperature and pH Value by Jian Zhang; Ying Zhang; Shan-na Liu; Ye Han; Zhi-jiang Zhou (pp. 1388-1400).

To investigate the effect of pH and temperature on the cell growth and bacteriocin production of Pediococcus acidilactici PA003, a lactic acid bacterium isolated from traditionally fermented cabbage, the kinetic behaviour of P. acidilactici PA003 was simulated in vitro during laboratory fermentations by making use of MRS broth. Firstly, primary models were developed for cell growth, glucose consumption, lactic acid and bacteriocin production for a given set of environmental conditions. Based on primary models, further study was undertaken to fit secondary models to describe the influence of temperature and pH on microbial behaviour. The models were validated successfully for all components. The results from the cell yield coefficient for lactic acid production reflected the homofermentative nature of P. acidilactici PA003. Both cell growth and bacteriocin production were very much influenced by changes in temperature and pH. The optimal condition for specific growth rate and biomass concentration was almost the same at pH 6.5 and 35 °C. At 35 °C and pH 6.1, the maximal bacteriocin activity was also achieved. The kinetic models provide useful tools for elucidating the mechanisms of temperature and pH on the kinetic behaviour of P. acidilactici PA003. The information obtained in this paper may be very useful for the selection of suitable starter cultures for a particular fermentation process and is a first step in the optimization of food fermentation processes and technology as well.

Keywords: Model; Kinetic; Fermentation; Pediococcus acidilactici ; Bacteriocin

Atropa belladonna Hairy Roots: Orchestration of Concurrent Oxidation and Reduction Reactions for Biotransformation of Carbonyl Compounds by Vikas Srivastava; Arvind Singh Negi; P. V. Ajayakumar; Shamshad A. Khan; Suchitra Banerjee (pp. 1401-1408).

The biotransformation potential of a selected Atropa belladonna hairy root clone (AB-09) had been evaluated with regard to three different aromatic carbonyl compounds, i.e., 3,4,5-trimethoxybenzaldehyde (1), 3,4,5-trimethoxyacetophenone (2), and 3,4,5-trimethoxy benzoic acid (3). The results demonstrated for the first time the untapped potentials of the selected hairy root clone to perform simultaneous oxidation (34.49%) and reduction (32.68%) of 3,4,5-trimethoxy benzaldehyde (1) into 3,4,5-trimethoxy benzoic acid (3), and 3,4,5-trimethoxy benzyl alcohol (4), respectively, without any intermediate separation or addition of reagents. The same hairy root clone also demonstrated reduction (<5%) of a 3,4,5-trimethoxyacetophenone (2) into a secondary alcohol, i.e., 1-(3,4,5-trimethoxyphenyl) ethanol (5), while in the case of aromatic carboxylic acid substrate (3), no biotransformation could be obtained under the similar conditions. The current observations revealed oxidation and reduction of the formyl group of the aromatic ring, and only reduction of the carbonyl group of acetophenone through the specific hairy root clone. The concurrent oxidation and reduction reactions by the selected hairy root clone highlight the importance of this study, which, as per our observations, is the first of its kind relating the hairy root culture of A. belladonna.

Keywords: Atropa belladonna ; Hairy roots; Biotransformation; Aromatic carbonyl compounds; Oxidation; Reduction

In Vitro Gastric and Intestinal Digestions of Pulsed Light-Treated Shrimp Extracts by Wade W. Yang; Sandra K. Shriver; Si-yin Chung; Susan Percival; Melanie J. Correll; Taha M. Rababah (pp. 1409-1422).

Pulsed ultraviolet light (PUV), a novel technology most commonly used for microbial inactivation, has recently been employed to effectively mitigate food allergens in peanuts, soybean, shrimp, and almond. Putative mechanisms for the efficacy of PUV in reducing allergen reactivity include photothermal, photochemical, and photophysical effects. To date, there are no published data highlighting the effects of in vitro simulated gastric and intestinal digestion on the stability of PUV reduced allergen reactivity of food. In this study, PUV-treated shrimp extracts were subjected to simulated gastric fluid containing pepsin and simulated intestinal fluid containing trypsin and chymotrypsin, and then tested for changes in allergen potency. SDS-PAGE showed no major band deviation between undigested and digested PUV-treated shrimp extracts. IgE binding to tropomyosin remained markedly decreased as seen in Western blot analysis. Total shrimp allergen reactivity remained unchanged following in vitro peptic digestion and was markedly reduced following in vitro intestinal digestion as illustrated in indirect ELISA. The PUV reduced shrimp allergens remained at a low level under the in vitro simulated digestive conditions. The results inferred that PUV could be a potential method to create less allergenic shrimp products that would remain at a low allergen level under human gastric and intestinal digestive conditions.

Keywords: Shrimp; Allergen; Pulsed ultraviolet light; PUV; IgE binding; SGF; SIF

Effect of Physicochemical Characteristics of Cellulosic Substrates on Enzymatic Hydrolysis by Means of a Multi-Stage Process for Cellobiose Production by Caroline Vanderghem; Nicolas Jacquet; Sabine Danthine; Christophe Blecker; Michel Paquot (pp. 1423-1432).

The effect of two types of cellulose, microcrystalline cellulose and paper pulp, on enzymatic hydrolysis for cellobiose production was investigated. The particle size, the relative crystallinity index and the water retention value were determined for both celluloses. A previously studied multistage hydrolysis process that proved to enhance the cellobiose production was studied with both types of celluloses. The cellobiose yield exhibited a significant improvement (120% for the microcrystalline cellulose and 75% for the paper pulp) with the multistage hydrolysis process compared to continuous hydrolysis. The conversion of cellulose to cellobiose was greater for the microcrystalline cellulose than for the paper pulp. Even with high crystallinity, microcrystalline cellulose achieved the highest cellobiose yield probably due to its highest specific surface area accessible to enzymes and quantity of adsorbed protein.

Keywords: Cellobiose; Multistage process; Cellulase binding; Water retention value; Microcrystalline cellulose; Paper pulp

Covalent Immobilization of Cellulases onto a Water-Soluble–Insoluble Reversible Polymer by Yuanyuan Yu; Jiugang Yuan; Qiang Wang; Xuerong Fan; Ping Wang (pp. 1433-1441).

The covalent immobilization of a commercial preparation of cellulase on a reversibly soluble–insoluble enteric polymer Eudragit S-100 by carbodiimide coupling was carried out. The characteristics of covalent Eudragit cellulase were evaluated using Fourier transform infrared (FTIR) spectra, circular dichroism (CD) spectra, and fluorescence spectra. FTIR, CD, and fluorescence measurements also revealed that the cellulases were covalently bonded to the supports. Covalent Eudragit cellulase had binding efficiency of 81.08% which was higher than the noncovalent Eudragit cellulase 56.83%. The relative activity of the native cellulase and covalent Eudragit cellulase increased and reached the maximum (at pH 5.0, 50°C) and then decreased with further increases in pH and temperature. The covalent Eudragit cellulase shows higher stability especially at higher pH and temperature. The K _m value of covalent Eudragit cellulase (4.78 g·L⁻¹) was decreased compared to that of the native cellulase (2.89 g·L⁻¹). The affinity of the cellulase to its substrate was increased when it was immobilized on Eudragit S-100.

Keywords: Cellulase; Immobilization; Eudragit; Stability; Carbodiimide

A Novel endo-1,4-β-Mannanase from Bispora antennata with Good Adaptation and Stability over a Broad pH Range by Qiong Liu; Peilong Yang; Huiying Luo; Pengjun Shi; Huoqing Huang; Kun Meng; Bin Yao (pp. 1442-1453).

An endo-β-1,4-mannanase encoding gene, man5, was cloned from Bispora antennata CBS 126.38, which was isolated from a beech stump. The cDNA of man5 consists of 1,299 base pairs and encodes a 432-amino-acid protein with a theoretical molecular mass of 46.6 kDa. Deduced MAN5 exhibited the highest amino acid sequence identity of 58% to a β-mannanase of glycoside hydrolase family 5 from Aspergillus aculeatus. Recombinant MAN5 was expressed in Pichia pastoris and purified to electrophoretic homogeneity. The specific activity of the final preparation towards locust bean gum was 289 U mg⁻¹. MAN5 showed optimal activity at pH 6.0 and 70 °C and had good adaptation and stability over a broad range of pH values. The enzyme showed more than 60% of peak activity at pH 3.0–8.0 and retained more than 80% of activity after incubation at 37 °C for 1 h in both acid and alkaline conditions (pH 4.0–11.0). The K _m and V _max values were 1.33 mg ml⁻¹ and 444 μmol min⁻¹ mg⁻¹ and 1.17 mg ml⁻¹ and 196 μmol min⁻¹ mg⁻¹ for locust bean gum and konjac flour, respectively. Of all tested metal ions and chemical reagents, Co²⁺, Ni²⁺, and β-mercaptoethanol enhanced the enzyme activity at 1 mM, whereas other chemicals had no effect on or partially inhibited the enzyme activity. MAN5 was highly resistant to acidic and neutral proteases (trypsin, α-chymotrypsin, collagenase, subtilisin A, and proteinase K). By virtue of the favorable properties of MAN5, it is possible to apply this enzyme in the paper and food industries.

Keywords: Bispora antennata ; β-Mannanase; pH adaptation and stability

Nonthermal Effect of Microwave Irradiation in Nonaqueous Enzymatic Esterification by Hui-da Wan; Shi-yu Sun; Xue-yi Hu; Yong-mei Xia (pp. 1454-1462).

Microwave has nonthermal effects on enzymatic reactions, mainly caused by the polarities of the solvents and substrates. In this experiment, a model reaction with caprylic acid and butanol that was catalyzed by lipase from Mucor miehei in alkanes or arenes was employed to investigate the nonthermal effect in nonaqueous enzymatic esterification. With the comparison of the esterification carried by conventional heating and consecutive microwave irradiation, the positive nonthermal effect on the initial reaction rates was found substrate concentration-dependent and could be vanished ostensibly when the substrate concentration was over 2.0 mol L⁻¹. The polar parameter log P well correlates the solvent polarity with the microwave effect, comparing to dielectric constant and assayed solvatochromic solvent polarity parameters. The log P rule presented in conventional heating-enzymatic esterification still fits in the microwaved enzymatic esterification. Alkanes or arenes with higher log P provided positive nonthermal effect in the range of 2 ≤ log P ≤ 4, but yielded a dramatic decrement after log P = 4. Isomers of same log P with higher dielectric constant received stronger positive nonthermal effect. With lower substrate concentration, the total log P of the reaction mixture has no obvious functional relation with the microwave effect.

Keywords: Microwave; Nonthermal effect; Enzyme; Solvent; Esterification

Genetically Engineered Epidermal Growth Factor Conjugate Crosses Cell Membrane by Baoquan Zhao; Yanru Guo; Ailing Fu (pp. 1463-1471).

Epidermal growth factor (EGF) is a well-known pleiotropic growth factor in mammal, and has been attempted to be used in many different fields. However, the application of EGF is limited because of its poor cell permeation. In order to increase membrane permeation ability of EGF, a genetically modified recombinant EGF (GST-TAT-EGF) was prepared through conjugation of EGF with two protein transduction domains, glutathione-S-transferase and TAT47-57. The results showed that the GST-TAT-EGF fusion protein exhibited higher ability in biomembrane penetration than that of the EGF alone. The results also implied that two different mechanisms (EGF receptor-mediated endocytosis and direct penetration) might be involved in GST-TAT-EGF transmembrane delivery.

Keywords: Epidermal growth factor; Membrane penetration; Protein transduction domains; Cell proliferation; Transmembrane delivery

Design of Biosolvents Through Hydroxyl Functionalization of Compounds with High Dielectric Constant by Guangnan Ou; Biyan He; Youzhu Yuan (pp. 1472-1479).

We proposed basic principles for biosolvent design on the viewpoint of ionization. Two classes of biosolvents, based on cyclic carbonate moiety and amide moiety, were designed through hydroxyl functionalization of highly dielectric compound. The newly designed compounds, glycerol carbonate (GC) and N-hydroxymethyl formamide (HOF), were synthesized for the development of soluble enzymatic systems and characterized by ¹³C NMR and ¹H NMR. All the characterization data were consistent with the expected structures. Using conductance measurements, the pK _a values of trichloroacetic acid in GC and HOF were determined as 0.80 and 0.85 at 25.0 °C, which was very close to that in water (pK _a = 0.70), suggesting that the ionizing and dissociating abilities of GC and HOF are similar to those of water. The effects of various reaction parameters on activity and stability of Candida antarctica lipase B and lipase from Pseudomonas cepacia were investigated using the transesterification of ethyl butyrate with n-butanol as a model reaction. The activities of lipases in GC and HOF were comparable to those in water, indicating that the newly designed compounds were biocompactible. Biosolvent design is a promising and versatile method for developing new biosolvents.

Keywords: Biosolvent design; Hydroxyl functionalization; Ionization; Dissociation; Lipase; Activity

Improved Mass Multiplication of Rhodiola crenulata Shoots Using Temporary Immersion Bioreactor with Forced Ventilation by Yan Zhao; Wei Sun; Ying Wang; Praveen K. Saxena; Chun-Zhao Liu (pp. 1480-1490).

A temporary immersion bioreactor system was found to be suitable for mass shoot proliferation of Rhodiola crenulata. The shoot multiplication ratio and hyperhydration rate reached 46.8 and 35.4%, respectively, at a temporary immersion cycle of 3-min immersion every 300 min. Forced ventilation was employed in the temporary immersion bioreactor culture in order to decrease the hyperhydration rate, improve shoot quality and enhance the multiplication ratio. The highest multiplication ratio of 55.7 was obtained under a temporary immersion cycle of 3-min immersion every 180 min with the forced ventilation at an air flow rate of 40 l/h, and the hyperhydration rate was reduced to 26.1%. Forced ventilation also improved the subsequent elongation and rooting rate of these proliferated shoots, and the shoot cultures from the temporary immersion bioreactor formed complete plantlets when subcultured onto a rooting medium containing 5 μmol/l indole-3-acetic acid.

Keywords: Rhodiola crenulata ; Temporary immersion bioreactor; Forced ventilation; Shoot propagation

Isolation of Hemoglobin from Bovine Erythrocytes by Controlled Hemolysis in the Membrane Bioreactor by Radoslava Stojanović; Vesna Ilić; Verica Manojlović; Diana Bugarski; Marija Dević; Branko Bugarski (pp. 1491-1506).

In this work, we describe an optimized procedure based on gradual hemolysis for the isolation of hemoglobin derived from bovine slaughterhouse erythrocytes in a membrane bioreactor. The membrane bioreactor system that provided high yields of hemoglobin (mainly oxyhemoglobin derivate) and its separation from the empty erythrocyte membranes (ghosts) was designed at a pilot scale. Ten different concentrations of hypotonic media were assessed from the aspect of the extent of hemolysis, hematocrit values of the erythrocyte suspensions, cell swelling, and membrane deformations induced by decreased salt concentration. Effective gradual osmotic hemolysis with an extent of hemolysis of 88% was performed using 35 mM Na-phosphate/NaCl buffer of pH 7.2–7.4. Under these conditions most of the cell membranes presented the appearance of the normal ghosts under phase contrast microscope. The hemoglobin purity of >80% was confirmed by SDS-PAGE. Kinetic studies showed that maximal concentration of hemoglobin was reached after 40 min, but the process cycle at which recovery of 83% was achieved lasted for 90 min. The dynamics of both steps, (1) transport through the membrane of erythrocytes during process of hemolysis and (2) transport through the reactor filters, were evaluated.

Keywords: Slaughterhouse blood; Bovine erythrocytes; Gradual hemolysis; Membrane bioreactor; Erythrocyte ghosts

Proteomic Analysis of Plasma Proteins in Diabetic Rats by 2D Electrophoresis and MALDI-TOF-MS by D. Karthik; S. Ilavenil; B. Kaleeswaran; S. Sunil; S. Ravikumar (pp. 1507-1519).

Despite tremendous advances in our understanding of the molecular basis of diabetes mellitus, substantial gaps still remain in our understanding of disease pathogenesis and in the development of effective strategies for early diagnosis and treatment. The proteomic approach has offered many opportunities and challenges in identifying new marker proteins and therapeutic targets, i.e., using 2D-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. The differential protein expressions were analyzed in alloxan-induced diabetic rats treated with Cynodon dactylon leaf extract. The plant extract was administered for 15 days that resulted in a significant increase in plasma insulin and C-peptide levels. We have also identified four differentially expressed proteins from rat plasma. These four diabetes-associated proteins were broadly classified into three groups as per their function: (1) lipid metabolism-associated protein (Apo A-IV), (2) antioxidant activity-related proteins [preprohaptoglobin and heat shock proteins B8 (HspB8)], and (3) muscle function-related protein (TPM3). Apo A-IV, HspB8, and preprohaptoglobin may play a key role in the recovery of diabetes mellitus and also prevent the diabetes-associated complications such as prevention of oxidative stress due to free radical and free hemoglobin. These results show the value of proteomic approach in identifying the potential markers that may eventually serve as diagnostic markers or therapeutic targets.

Keywords: Alloxan diabetes; Proteomics; Rat Plasma; 2D electrophoresis; MALDI-TOF-MS/MS; Phylogenetic tree

Neuroprotective Effect of Phlorotannin Isolated from Ishige okamurae Against H₂O₂-Induced Oxidative Stress in Murine Hippocampal Neuronal Cells, HT22 by Soo-Jin Heo; Seon-Heui Cha; Kil-Nam Kim; Seung-Hong Lee; Ginnae Ahn; Do-Hyung Kang; Chulhong Oh; Young-Ung Choi; Abu Affan; Daekyung Kim; You-Jin Jeon (pp. 1520-1532).

The present study is designed to investigate the neuroprotective effect of a kind of phlorotannins, diphlorethohydroxycarmalol (DPHC) isolated from Ishige okamurae against hydrogen peroxide (H₂O₂)-induced oxidative stress in murine hippocampal neuronal cells, HT22. H₂O₂ treatment induced neurotoxicity, whereas DPHC prevented cells from H₂O₂-induced damage then restoring cell viability was significantly increased. DPHC slightly reduced the expression of Bax induced by H₂O₂ but recovered the expression of Bcl-xL as well as caspase-9 and -3 mediated PARP cleavage by H₂O₂. Intracellular reactive oxygen species (ROS) and lipid peroxidation was overproduced as the result of the addition of H₂O₂; however, these ROS generations and lipid peroxidation were effectively inhibited by addition of DPHC in a dose-dependent manner. Moreover, DPHC suppressed the elevation of H₂O₂-induced Ca²⁺ release. These findings indicate that DPHC has neuroprotective effects against H₂O₂-induced damage in neuronal cells, and that an inhibitory effect on ROS production may contribute to the underlying mechanisms.

Keywords: Neuroprotection; HT22; Oxidative stress; Hydrogen peroxide; Ishige okamurae

Gold Nanomaterials: Preparation, Chemical Modification, Biomedical Applications and Potential Risk Assessment by Xiu-Mei Jiang; Li-Ming Wang; Jing Wang; Chun-Ying Chen (pp. 1533-1551).

Gold nanomaterials (Au NMs) have attracted increasing attention in biomedicine due to their facile preparation, multifunctional modifications, unique optical and electrical properties, and good biocompatibility. The physicochemical properties of Au NMs at nanoscale, like size, shape, surface chemistry, and near field effects, are rendering Au NMs potent candidates in biomedicine. Thus, risk assessment of negative effects of Au NMs on biological systems is becoming urgent and necessary for future applications. In this review, we summarize up-to-date progresses on the preparation and modification of Au NMs and their biomedical applications, including biosensor, bioimaging and phototherapy, gene/drug delivery. Finally, we discuss the potential risk of Au NMs to biological systems, which is instructive for rationally designing and preparing nanomaterials for safe applications in nanomedicine.

Keywords: Preparation; Chemical modification; Biosensor; Imaging; Phototherapy; Gene/drug delivery; Risk assessment

Induction of Apoptosis by Ribosome Inactivating Proteins by Mrinal Kumar Das; Radhey Shyam Sharma; Vandana Mishra (pp. 1552-1561).

Apoptotic cell death is a fundamental process in the development and physiological homeostasis of multicellular organisms. It is associated with control of cell numbers in tissues and organs during development, with cell turnover, and with response to infection. Molecules that trigger this process in continuously proliferating cancer cells can be used as chemotherapeutic agents. Ribosome inactivating proteins (RIPs) that inhibit translation in a cell by depurinating (N-glycosidase activity) the 28S rRNA are known to serve as apoptosis inducers. However, the role of depurination activity of the RIPs in apoptosis induction is still controversial. Presently, there are three different hypotheses which propose that depurination is: (1) essential, (2) essential but not the sole factor, or (3) not essential for apoptosis induction. This article reviews various experimental outcomes on the importance of N-glycosidase activity of RIPs in the induction of apoptosis.

Keywords: Apoptosis; Ribosome inactivating protein; N-glycosidase activity

Effects of Wort Gravity and Nitrogen Level on Fermentation Performance of Brewer’s Yeast and the Formation of Flavor Volatiles by Hongjie Lei; Haifeng Zhao; Zhimin Yu; Mouming Zhao (pp. 1562-1574).

Normal gravity wort and high gravity wort with different nitrogen levels were used to examine their effects on the fermentation performance of brewer’s yeast and the formation of flavor volatiles. Results showed that both the wort gravity and nitrogen level had significant impacts on the growth rate, viability, flocculation, and gene expression of brewer’s yeast and the levels of flavor volatiles. The sugar (glucose, maltose, and maltotriose) consumption rates and net cell growth decreased when high gravity worts were used, while these increased with increasing nitrogen level. Moreover, high gravity resulted in lower expression levels of ATF1, BAP2, BAT1, HSP12, and TDH, whereas the higher nitrogen level caused higher expression levels for these genes. Furthermore, the lower nitrogen level resulted in increases in the levels of higher alcohols and esters at high wort gravity. All these results demonstrated that yeast physiology and flavor balance during beer brewing were significantly affected by the wort gravity and nitrogen level.

Keywords: Wort gravity; Nitrogen level; Yeast physiology; Fermentation performance; Flavor volatiles

Production of Thermophilic Endo-β-1,4-xylanases by Aspergillus fumigatus FBSPE-05 Using Agro-industrial By-products by D. T. Souza; A. S. R. Bispo; E. P. S. Bon; R. R. R. Coelho; R. P. Nascimento (pp. 1575-1585).

In the present paper, endo-β-1,4-xylanase production by Aspergillus fumigatus was evaluated in solid-state fermentation using low-cost substrates such as sugarcane bagasse (SCB), brewer’s spent grain (BSG), and wheat bran (WB). The partial characterization of the crude enzyme was also performed. In the experimental conditions, the highest levels of endo-β-1,4-xylanase production by A. fumigatus FBSPE-05 occurred within 8 days incubation when using SCB/liquid medium at 1:2 ratio (219.5 U g⁻¹) and 4 days incubation when using WB/liquid medium at 1:1 ratio (215.6 U g⁻¹). Crude enzyme from this last condition was used to enzyme characterization, showing best enzyme activity at 60 °C and pH 6.0, which suggests a thermophilic endoxylanase. The crude enzyme retained 73% of its activity after 1 h at 60 °C, and zymogram has shown three bands of endo-β-1,4-xylanase activity, with different molecular masses. A. fumigatus FBSPE-05 was able to grow and produce good levels of endo-β-1,4-xylanase using agro-industrial by-products, making this strain worthy for further investigation. To our knowledge, this is the first study reporting the use of SCB and/or BSG as sole substrates for endoxylanase production by solid-state fermentation using A. fumigatus.

Keywords: Aspergillus fumigatus ; Endo-1,4-xylanase; Sugarcane bagasse; Brewer’s spent grain; Solid-state fermentation

Characterization of Cellulolytic Extract from Pycnoporus sanguineus PF-2 and Its Application in Biomass Saccharification by Daniel Luciano Falkoski; Valéria Monteze Guimarães; Maíra Nicolau de Almeida; Acelino Couto Alfenas; Jorge Luiz Colodette; Sebastião Tavares de Rezende (pp. 1586-1603).

The aim of this work was to evaluate the biochemical features of the white-rot fungi Pycnoporus sanguineus cellulolytic complex and its utilization to sugarcane bagasse hydrolysis. When cultivated under submerged fermentation using corn cobs as carbon source, P. sanguineus produced high FPase, endoglucanase, β-glucosidase, xylanase, mannanase, α-galactosidase, α-arabinofuranosidase, and polygalacturonase activities. Cellulase activities were characterized in relation to pH and temperature. β-Glucosidase and FPase activities were higher at 55 °C, pH 4.5, and endoglucanase activity was higher at 60 °C, in a pH range of 3.5–4.0. All cellulase activities were highly stable at 40 and 50 °C through 48 h of pre-incubation. Crude enzymatic extract from P. sanguineus was applied in a saccharification experiment using acid-treated and alkali-treated sugarcane bagasse as substrate, and the hydrolysis yields were compared to that obtained by a commercial cellulase preparation. Reducing sugar yields of 60.4% and 64.0% were reached when alkali-treated bagasse was hydrolyzed by P. sanguineus extract and commercial cellulase, respectively. Considering the glucose production, it was observed that P. sanguineus extract and commercial cellulase ensured yields of 22.6% and 36.5%, respectively. The saccharification of acid-treated bagasse was lower than that of alkali-treated bagasse regardless of the cellulolytic extract. The present work showed that P. sanguineus has a great potential as an enzyme producer for biomass saccharification.

Keywords: Pycnoporus sanguineus ; Cellulases; Hemicellulases; Saccharification; Biomass

Highly Sensitive Chemiluminescent Analysis of Residual Bovine Serum Albumin (BSA) Based on a Pair of Specific Monoclonal Antibodies and Peroxyoxalate–glyoxaline–PHPPA Dimer Chemiluminescent System in Vaccines by Pan Xue; Kui Zhang; Zhujun Zhang; Yun Li; Feng Liu; Yuanjie Sun; Xiaoming Zhang; Chaojun Song; Aihua Fu; Boquan Jin; Kun Yang (pp. 1604-1614).

Enzyme-linked immunosorbent assay (ELISA), horseradish peroxidase (HRP)-catalyzed fluorescent reaction, and oxalate chemiluminescence analysis have been combined to develop a highly sensitive, simple, and rapid method for analysis of bovine serum albumin (BSA) based on a pair of specific monoclonal antibodies in vaccines. A typical “sandwich type” immunoassay was used. Reaction of 3-(4-hydroxyphenyl propionate) (PHPPA) with hydrogen peroxide-urea, catalyzed by HRP, produced fluorescence of 3-(4-hydroxyphenyl propionate) dimer, which was detected by chemiluminescence analysis with the bis(2,4,6-trichlorophenyl)oxalate (TCPO)–H₂O₂–glyoxaline–PHPPA dimer chemiluminescent system. This method exhibited high performance with a linear correlation between response and amount of bovine serum albumin (BSA) in the range 0.1 to 100.0 ng mL⁻¹ (r = 0.9988), and the detection limit was 0.03 ng mL⁻¹ (S/N = 3). Intra- and interassay coefficient variations were all lower than 9.0% at three concentrations (1.0, 20.0, and 80.0 ng mL⁻¹). The proposed method has been used for successful analysis of the amount of residual BSA in vaccines. The results obtained compared well with those obtained by conventional colorimetric ELISA and luminol chemiluminescent ELISA.

Keywords: Bovine serum albumin (BSA); Enzyme-linked immunosorbent assays; Chemiluminescence analysis

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.166, #6)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.166, #6)