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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.167, #1)

Advances in Non-snake Venom l-Amino Acid Oxidase by Zhiliang Yu; Hua Qiao (pp. 1-13).

l-amino acid oxidase is widely found in diverse organisms and has different properties. It is thought to contribute to antimicrobial activity, amino acid catabolism, and so forth. The purpose of this communication is to summarize the advances in non-snake venom l-amino acid oxidase, including its enzymatic and structural properties, gene cloning and expression, and biological function. In addition, the mechanism of its biological function as well as its application is also discussed.

Keywords: l-amino acid oxidase; Non-snake venom; Enzymatic and structural properties; Gene cloning and expression; Biological function and application

Development of DNA-Designed Avian IgY Antibodies for Detection of Mycobacterium avium subsp. paratuberculosis Heat Shock Protein 70 (Hsp70) and Anti-Hsp70 Antibodies in the Serum of Normal Cattle by Gholamreza Nikbakht Brujeni; Darioush Gharibi (pp. 14-23).

Mycobacterium avium subsp. paratuberculosis heat shock protein 70 (MAPHsp70) is an immunodominant antigen, which can be used as a subunit vaccine against bovine paratuberculosis. In the present study, we evaluated the immunogenic activities of MAPHsp70 expressed by DNA vaccine in chicken and the use of prepared specific avian IgY antibodies for western blotting and ELISA methods. The gene encoding MAP Hsp70 was subcloned into the eukaryotic expression vector, pcDNA3.1, and the recombinant plasmid (pcDNA3.1-MAP Hsp70) transfected into COS-7 cells. Chickens were also immunized with pcDNA3.1-MAP Hsp70, and egg yolk antibodies extracted from eggs were collected after immunization. DNA-designed IgY antibody was used in Western blotting analysis to detect the expression of MAPHsp70, and in a sandwich ELISA to assess the prevalence of anti-MAPHsp70 antibodies in cattle serum. Western blotting results indicate the expression of rMAP hsp70 in COS-7 cells and sandwich ELISA could detect anti-MAPHsp70 antibodies in 7.5% of cows. Chicken immunization with pcDNA3.1-MAPHsp70 could demonstrate the effective production of anti-MAPHsp70 IgY antibodies. Monospecific anti-MAPHsp70 antibody generated in chickens is useful for detection of MAPHsp70 peptide in cell culture and MAP lysate.

Keywords: Mycobacterium avium subsp. paratuberculosis ; Hsp70; DNA-designed IgY; Sandwich ELISA

Heterologous Co-expression of accA, fabD, and Thioesterase Genes for Improving Long-Chain Fatty Acid Production in Pseudomonas aeruginosa and Escherichia coli by Sunhee Lee; Eunyoung Jeon; Yeontae Jung; Jinwon Lee (pp. 24-38).

The goal of the present study was to increase the content of intracellular long-chain fatty acids in two bacterial strains, Pseudomonas aeruginosa PA14 and Escherichia coli K-12 MG1655, by co-overexpressing essential enzymes that are involved in the fatty acid synthesis metabolic pathway. Recently, microbial fatty acids and their derivatives have been receiving increasing attention as an alternative source of fuel. By introducing two genes (accA and fabD) of P. aeruginosa into the two bacterial strains and by co-expressing with them the fatty acyl–acyl carrier protein thioesterase gene of Streptococcus pyogenes (strain MGAS10270), we have engineered recombinant strains that are efficient producers of long-chain fatty acids (C16 and C18). The recombinant strains exhibit a 1.3–1.7-fold increase in the production of long-chain fatty acids over the wild-type strains. To enhance the production of total long-chain fatty acids, we researched the carbon sources for optimized culture conditions and results were used for post-culture incubation period. E. coli SGJS17 (containing the accA, fabD, and thioesterase genes) produced the highest content of intracellular total fatty acids; in particular, the unsaturated fatty acid content was about 20-fold higher than that in the wild-type E. coli.

Keywords: Long-chain fatty acids; E. coli ; P. aeruginosa ; S. pyogenes ; Thioesterase gene

One Step Conversion of Wheat Straw to Sugars by Simultaneous Ball Milling, Mild Acid, and Fungus Penicillium simplicissimum Treatment by Li Yuan; Zhenhua Chen; Yonghua Zhu; Xuanming Liu; Hongdong Liao; Ding Chen (pp. 39-51).

Wheat straw is one of the major lignocellulosic plant residues in many countries including China. An attractive alternative is the utilization of wheat straw for bioethanol production. This article mainly studies a simple one-step wet milling with Penicillium simplicissimum and weak acid to hydrolysis of wheat straw. The optimal condition for hydrolysis was ball milling 48 h in citrate solvent (pH = 4) with P. simplicissimum H5 at the speed of 500 rpm and the yield of sugar increased with increased milling time. Corresponding structure transformations before and after milling analyzed by X-ray diffraction, transmission Fourier transform infrared spectroscopy, and environmental scanning electron microscopy clearly indicated that this combined treatment could be attributed to the crystalline and chemical structure changes of cellulose in wheat straw during ball milling. This combined treatment of ball milling, mild acid, and fungus hydrolysis enabled the conversion of the wheat straw. Compared with traditional method of ball milling, this work showed a more simple, novel, and environmentally friendly way in mechanochemical treatment of wheat straw.

Keywords: Wheat straw; Ball milling; Citrate solvent; Penicillium simplicissimum ; Hydrolysis

Characteristics of the Toxin Extracted from Liquid Culture of Colletotrichum capsici F. nicotianae by Guang-min Zhang; Bao-hai Fang; Heng Chen; Xue-ling Li (pp. 52-61).

Colletotrichum capsici f. nicotianae is an important plant pathogen in tobacco-grown area of Weifang region of Shangdong Province, China. In this study, the toxicity of liquid culture media from different isolates was characterized, and some properties of the toxic ingredient were identified. The results indicated that the optimal toxin-producing conditions for C. capsici f. nicotianae were in potato dextrose broth under pH 6.0, at 25∼30 °C for 13 days. The liquid culture media from all isolates were toxic to tobacco plants and induced the wilting symptoms. The toxin from the liquid culture media has thermal, acid–base stability and a broad spectrum of toxicity to the plants. Furthermore, the direct bioassay for two components of the liquid filtrates precipitated by ethanol showed that the active ingredient of the toxin is a kind of nonprotein substance, which was further supported by the papain hydrolysis test.

Keywords: Colletotrichum capsici f. nicotianae ; Tobacco; Toxin; Extraction and characteristics

Improvement of Adenosylcobalamin Production by Metabolic Control Strategy in Propionibacterium freudenreichii by Peng Wang; Yunshan Wang; Zhiguo Su (pp. 62-72).

An efficient metabolic control approach was developed to improve the industrial anaerobic fermentation of adenosylcobalamin (ado-cbl) by Propionibacterium freudenreichii. The effects of 5,6-dimethylbenzimidazole (DMB) on cell growth and ado-cbl biosynthesis were investigated. Subsequently, the results obtained from the batch culture showed that an important intermediate of ado-cbl separated from the cell extract was identified as adenosylcobinamide (ado-cbi) by high-performance liquid chromatography coupled to an ultraviolet diode array detector and ESI mass spectrometry analysis. Ado-cbi can be converted to ado-cbl when linked to DMB, which is an essential compound for ado-cbi bioconversion. This key ado-cbi is useful not only in determining ado-cbl concentration in the fermentation process but also in serving as an effective compound to guide DMB incorporation for the harvest of the maximum ado-cbl concentration. Accordingly, with scaling up to 100 L fermentation, the experimental results showed that the discrepancy was less than 1 % using the developed prediction technique. Overall, the findings show that the method is efficient in evaluating the fermentation of ado-cbl by propionibacteria.

Keywords: Biosynthesis; Adenosylcobinamide; Metabolism control; 5, 6-Dimethylbenzimidazole; Propionibacterium freudenreichii

A Novel Bioassay for High-Throughput Screening Microorganisms with N-acyl Homoserine Lactone Degrading Activity by Pengfu Liu; Yang Gao; Wei Huang; Zongze Shao; Jiping Shi; Ziduo Liu (pp. 73-80).

A novel biosensor strain (Escherichia coli ALM403) that responded to N-acyl homoserine lactone (AHL) was constructed using a luxR-Plux cassette as a regulatory sequence and β-mannanase as a reporter gene. Dinitrosalicylic acid method was used to detect the response of the sensor strain to N-acyl homoserine lactone. By investigating the response to a range of concentrations of N-β-oxooctanoyl-l-homoserine lactone (OOHL), it was demonstrated that the expression of mannanase in E. coli ALM403 could be greatly enhanced by OOHL and resulted in an assayable phenotype. A high-throughput screening approach was developed to isolate AHL-degrading microorganisms, and a marine Halomonas sp. S66-4 showing a marked AHL-degrading ability was successfully isolated. In conclusion, the bioassay system provided a simple and efficient approach to isolate AHL-degrading bacteria.

Keywords: N-Acyl homoserine lactone; Quorum sensing; Quorum quenching; Bioassay; AHL-degrading bacterium

Extrusion Pretreatment of Pine Wood Chips by C. Karunanithy; K. Muthukumarappan; W. R. Gibbons (pp. 81-99).

Pretreatment is the first step to open up lignocellulose structure in the conversion of biomass to biofuels. Extrusion can be a viable pretreatment method due to its ability to simultaneously expose biomass to a range of disruptive conditions in a continuous flow process. Extruder screw speed, barrel temperature, and feedstock moisture content are important factors that can influence sugar recovery from biomass. Hence, the current study was undertaken to investigate the effects of these parameters on extrusion pretreatment of pine wood chips. Pine wood chip at 25, 35, and 45 % wb moisture content were pretreated at various barrel temperatures (100, 140, and 180 °C) and screw speeds (100, 150, and 200 rpm) using a screw with compression ratios of 3:1. The pretreated pine wood chips were subjected to standard enzymatic hydrolysis followed by sugar and byproducts quantification. Statistical analyses revealed the existence of significant differences in sugar recovery due to independent variables based on comparing the mean of main effects and interaction effects. Pine wood chips pretreated at a screw speed of 150 rpm and a barrel temperature of 180 °C with a moisture content of 25 % resulted in a maximum cellulose, hemicellulose, and total sugar recoveries of 65.8, 65.6, and 66.1 %, respectively, which was about 6.7, 7.9, and 6.8 fold higher than the control (unpretreated pine chips). Furthermore, potential fermentation inhibitors such as furfural, hydroxyl methyl furfural, and acetic acid were not found in any of the treatment combinations.

Keywords: Biomass; Sugar recovery; Screw speed; Barrel temperature; Moisture content; Byproducts

Effect of Microwave Irradiation on Xylanase Production from Wheat Bran and Biobleaching of Eucalyptus Kraft Pulp by Fantahun Woldesenbet; Antar Puneet Virk; Naveen Gupta; Prince Sharma (pp. 100-108).

Microwave irradiation (MWI) was used as pretreatment of wheat bran and eucalyptus kraft pulp to examine its effect on xylanase production by Bacillus halodurans FNP 135 using solid state fermentation and biobleaching with xylanase, respectively. Irradiation of wheat bran under optimized conditions (600 W, 6 min, and 20 % consistency) resulted in 56.8, and 31.7 % increase in xylanase yield and water absorbance of wheat bran and 17.3 % reduction in reducing sugars content. Optimized MWI of kraft pulp at 850 W, 2 min, and 20 % consistency led to 0.9 % increase in brightness, 10 % decrease in kappa number, 7.7 % increase in water absorbance, 4.6 % decrease in tear factor, 0.9 % increase in burst factor, and 7.5 % increase in viscosity. Also, MWI enhanced xylanase-mediated biobleaching by increasing brightness (1.1 %) and decreasing kappa number (14.3 %) and leading to a total of about 20 % reduction in chlorine consumption. MWI is an economical, efficient, and environment-friendly pretreatment of wheat bran and pulp for enhanced enzyme yield and rapid heating, respectively.

Keywords: Microwave irradiation; Wheat bran; Xylanase; Kraft pulp; Biobleaching

Evaluation of Enzyme Activity and Fiber Content of Soybean Cotyledon Fiber and Distiller’s Dried Grains with Solubles by Solid State Fermentation by Shengli Yang; JunYi Lio; Tong Wang (pp. 109-121).

To increase the value of coproducts from corn ethanol fermentation and soybean aqueous processing, distiller’s dried grains with solubles (DDGS) and soybean cotyledon fiber were used as the substrates for solid state fermentation (SSF) to improve feed digestibility. Aspergillus oryzae, Trichoderma reesei, and Phanerochaete chrysosporium were chosen as they produce desirable enzymes and are widely used in SSF for feed. The results showed that the cellulase and xylanase activities were significantly increased after 7 days of fermentation, and cellulose and hemicellulose degradation was also greatly increased. When soybean fiber was used as SSF substrate, the maximum activities of the cellulase and xylanase were 10.3 and 84.2 IU/g substrate (dry weight basis) after SSF treatment, respectively. However, the enzyme activities were relatively low in DDGS, and the growth of the three fungi was poor. The fungi grew better when soybean cotyledon fiber was added to DDGS, and cellulase and xylanase activity increased with the increase of soybean fiber content. Porosity was identified as an important factor for SSF because the addition of inert soybean hull alone improved the fungi growth significantly. These data suggest that the nutritional value of DDGS and soybean cotyledon fiber as monogastric animal feed could be greatly enhanced by SSF treatment.

Keywords: Cellulase; DDGS; Enzyme activity; Fungi; Solid state fermentation; Soybean cotyledon fiber; Xylanase

Sago Pith Residue as an Alternative Cheap Substrate for Fermentable Sugars Production by S. Linggang; L. Y. Phang; M. H. Wasoh; S. Abd-Aziz (pp. 122-131).

Sago pith residue is one of the most abundant lignocellulosic biomass which can serve as an alternative cheap substrate for fermentable sugars production. This residue is the fibrous waste left behind after the starch extraction process and contains significant amounts of starch (58%), cellulose (23%), hemicellulose (9.2%) and lignin (3.9%). The conversion of sago pith residue into fermentable sugars is commonly performed using cellulolytic enzymes or known as cellulases. In this study, crude cellulases were produced by two local isolates, Trichoderma asperellum UPM1 and Aspergillus fumigatus, UPM2 using sago pith residue as substrate. A. fumigatus UPM2 gave the highest FPase, CMCase and β-glucosidase activities of 0.39, 23.99 and 0.78 U/ml, respectively, on day 5. The highest activity of FPase, CMCase and β-glucosidase by T. asperellum UPM1 was 0.27, 12.03 and 0.42 U/ml, respectively, on day 7. The crude enzyme obtained from A. fumigatus UPM2 using β-glucosidase as the rate-limiting enzyme (3.9, 11.7 and 23.4 IU) was used for the saccharification process to convert 5% (w/v) sago pith residue into reducing sugars. Hydrolysis of sago pith residue using crude enzyme containing β-glucosidase with 23.4 IU, produced by A. fumigatus UPM2 gave higher reducing sugars production of 20.77 g/l with overall hydrolysis percentage of 73%.

Keywords: Sago pith residue; Crude enzyme; Trichoderma asperellum UPM1; Aspergillus fumigatus UPM2; Saccharification

Identification of Cellulase Gene from the Metagenome of Equus burchelli Fecal Samples and Functional Characterization of a Novel Bifunctional Cellulolytic Enzyme by Matam Chandrasekharaiah; Appoothy Thulasi; Madiajagan Bagath; Duvvuri Prasanna Kumar; Sunil Singh Santosh; Chenniappan Palanivel; Vazhakkala Lyju Jose; Koratokare Thirumalachar Sampath (pp. 132-141).

The metagenomic approach has been used successfully to isolate novel biocatalyst gene from uncultured microorganisms. The gene encoding exo-1,4-β-glucanase avicelase was amplified from the metagenome of the Equus burchelli fecal sample and cloned. The gene was found to be of 1,007 bp of nucleotide which encodes a protein of 318 amino acids with a calculated MW of 36 kDa. The deduced amino acid sequence was homologous with cellulases belonging to the glycosyl hydrolases 6 superfamily. The expressed protein was active towards the substrates avicel and carboxymethyl cellulose, indicating that it has bifunctional cellulolytic enzyme activity. The recombinant protein showed an activity of 5.23 U with specific activity of 6.8 U mg⁻¹ protein with the substrate avicel, while when CMC was used, an activity of 3.0 U with a specific activity of 4.2 U mg⁻¹ protein was achieved. Its optimum pH was determined to be 7.0 and optimum temperature of 35°C.

Keywords: Exo-1,4-β-glucanase; Avicelase; Metagenome; Enzyme activity; Cloning

Kinetic Modeling of Fructooligosaccharide Production Using Aspergillus oryzae N74 by Felipe Guio; Luz D. Rugeles; Sonia E. Rojas; María P. Palomino; María C. Camargo; Oscar F Sánchez (pp. 142-163).

In this study, the kinetic for the bioconversion of sucrose to fructooligosaccharides (FOS) by free cells of Aspergillus oryzae N74 was modeled. In addition, the effect of immobilized glucose isomerase (IGI) on FOS production yield was evaluated and considered in the kinetic model. The selected kinetic models were based on a proposed reaction mechanism described by elementary rate equations and modified Michaelis–Menten kinetic equations. The use of IGI allowed to increase the FOS production yield (FOS_Yield) and to decrease the glucose/fructose (G/F) ratio. At shake flask scale, the FOS_Yield was increased in 4.7 % (final yield 58.3 %), while the G/F ratio was reduced 6.2-fold. At bench scale, the FOS_Yield was increased in 2.2 % (final yield 57.3 %), while the G/F ratio was reduced 4.5-fold. The elementary rate equation model was the one that best adjusted experimental data for FOS production using either the fungus biomass or the mixture fungus biomass–IGI, with an overall average percentage error of 7.2. Despite that FOS production yield was not highly improved by the presence of IGI in the reaction mixture, it favored the reduction of residual glucose in the mixture, avoiding the loss of material owe to glucose transformation to fructose that can be used in situ for FOS production by the fructosyltransferase.

Keywords: Fructosyltransferase; Fructooligosaccharides; Aspergillus oryzae ; Immobilized glucose isomerase

Development and Characterization of a Solid-Phase Biocatalyst Based on Cyclodextrin Glucantransferase Reversibly Immobilized onto Thiolsulfinate-Agarose by Santiago Einar Viera; Francisco Batista-Viera; Karen Ovsejevi (pp. 164-176).

Reduction of disulfide bonds and introduction of “de novo” thiol groups in cyclodextrin glucantransferase from Thermoanaerobacter sp. were assessed in order to perform reversible covalent immobilization onto thiol-reactive supports (thiolsulfinate-agarose). Only the thiolation process dramatically improved the immobilization yield, from 0 % for the native and reduced enzyme, up to nearly 90 % for the thiolated enzyme. The mild conditions of the immobilization process (pH 6.8–7.0 and 22 °C) allowed the achievement of 100 % coupling efficiencies when low loads were applied. Ionic strength was a critical parameter for the immobilization process; for high activity recoveries, 50 mM phosphate buffer supplemented with 0.15 M NaCl was required. The kinetic parameters, pH and thermal stabilities for the immobilized biocatalyst were similar to those for the native enzyme. For β-cyclization activity, optimal pH range and temperature were 4.0–5.4 and 85 °C. The possibility of reusing the support was demonstrated by the reversibility of enzyme–support binding.

Keywords: Thermoanaerobacter sp.; Cyclodextrin glucantransferase; Cyclodextrins; Enzyme immobilization; Covalent reversible immobilization; β-Cyclodextrin synthesis

Defense-Related Polyphenol Oxidase from Hevea brasiliensis Cell Suspension: Purification and Characterization by Nisaporn Muhamad; Nion Chirapongsatonkul; Nunta Churngchow (pp. 177-189).

Polyphenol oxidase (PPO) was examined from the extract of leaf, seed, and cell suspension of Hevea brasiliensis, a rubber plant. The defense-related isozyme from Hevea cell suspension induced by culture filtrate of Phytophthora palmivora or by agitation stress was isolated through anion exchange and affinity chromatography, respectively. A 104-purification fold, migrated as a single band of 70 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis of PPO, was obtained after further purified by the preparative gel electrophoresis. Based on reaction with catechol and dopamine but not with p-cresol and guaiacol, it is a diphenol-type PPO. The values of V _max /K _m ratio indicated that catechol was the most specific substrate. The optimal activity of the purified PPO was observed at pH 6.0. The PPO activity was retained at pH 4.0–10.0 and temperature 10–60 °C. The inhibitors which completely inhibited the activity were ascorbic acid, dithiothreitol, and β-mercaptoethanol while sodium azide was a poor inhibitor. The PPO obtained from Hevea cell suspension possesses high specific activity and is stable at wide range of pH and temperature. It is therefore suitable for extreme condition uses and may lead to an alternative source of PPO in various industrial applications.

Keywords: Cell suspension; Characterization; Defense response; Hevea brasiliensis ; Polyphenol oxidase

A Simplified Filter Paper Assay Method of Cellulase Enzymes Based on HPLC Analysis by Deqiang Chu; Hongbo Deng; Xiaoxi Zhang; Jian Zhang; Jie Bao (pp. 190-196).

A simplified filter paper assay (FPA) method of cellulase enzymes was proposed based on high-performance liquid chromatography (HPLC) measurement. The method was according to the sum of glucose and cellobiose concentrations measured by HPLC that was able to be correlated with filter paper units (FPU) of the cellulase enzymes assayed by the traditional FPA method, regardless of the differences in the sources, activities, and components of the cellulases. This simple and quick assay method for the cellulase enzymes provided another parameter of the ratio of glucose to cellobiose (G/C ratio) representing the capacity of cellulase enzymes degrading cellulose into fermentable monomeric sugars.

Keywords: Filter paper activity; Cellulase; Simplified method; HPLC measurement; Corn stover

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.167, #1)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.167, #1)