Check out our New Publishers' Select for Free Articles

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.164, #4)

Improved Bioprocess with CHO-hTSH Cells on Higher Microcarrier Concentration Provides Higher Overall Biomass and Productivity for rhTSH by Daniella C. Ventini; Renata Damiani; Alvaro P. B. Sousa; João E. de Oliveira; Cibele N. Peroni; Maria T. C. P. Ribela; Paolo Bartolini; Aldo Tonso; Carlos R. J. Soares; Carlos A. Pereira (pp. 401-409).

Since the recombinant thyroid-stimulating hormone (rhTSH) is secreted by stably transfected Chinese hamster ovary (CHO-hTSH) cells, a bioprocess consisting of immobilizing the cells on a substrate allowing their multiplication is very suitable for rhTSH recovering from supernatants at relative high degree of purity. In addition, such a system has also the advantage of easily allowing delicate manipulations of culture medium replacement. In the present study, we show the development of a laboratory scale bioprocess protocol of CHO-hTSH cell cultures on cytodex microcarriers (MCs) in a 1 L bioreactor, for the preparation of rhTSH batches in view of structure/function studies. CHO-hTSH cells were cultivated on a fetal bovine serum supplemented medium during cell growth phase. For rhTSH synthesis phase, 75% of supernatant was replaced by animal protein-free medium every 24 h. Cell cultures were monitored for agitation (rpm), temperature (°C), dissolved oxygen (% DO), pH, cell concentration, MCs coverage, glucose consumption, lactate production, and rhTSH expression. The results indicate that the amount of MCs in the culture and the cell concentration at the beginning of rhTSH synthesis phase were crucial parameters for improving the final rhTSH production. By cultivating the CHO-hTSH cells with an initial cell seeding of four cells/MC on 4 g/L of MCs with a repeated fed batch mode of operation at 40 rpm, 37 °C, 20% DO, and pH 7.2 and starting the rhTSH synthesis phase with 3 × 10⁶ cells/mL, we were able to supply the cultures with enough glucose, to maintain low levels of lactate, and to provide high percent (∼80%) of fully covered MCs for a long period (5 days) and attain a high cell concentration (∼9 × 10⁵ cells/mL). The novelty of the present study is represented by the establishment of cell culture conditions allowing us to produce ∼1.6 mg/L of rhTSH in an already suitable degree of purity. Batches of produced rhTSH were purified and showed biological activity.

Keywords: rhTSH; CHO cells; Microcarriers; Animal cells; Protein expression; Bioreactor

A Solvent-Stable Metalloprotease Produced by Pseudomonas aeruginosa A2 Grown on Shrimp Shell Waste and Its Application in Chitin Extraction by Olfa Ghorbel-Bellaaj; Kemel Jellouli; Islem Younes; Laila Manni; Mohamed Ouled Salem; Moncef Nasri (pp. 410-425).

A solvent-stable protease-producing bacterium was isolated and identified as Pseudomonas aeruginosa A2. The strain was found to produce high level of protease activity when grown in media containing only fresh shrimp waste (FSW) or shrimp waste powder (SWP), indicating that it can obtain its carbon, nitrogen, and salts requirements directly from shrimp waste. Maximum protease activities 17,000 and 12,000 U/mL were obtained with 80 g/L SWP and 135 g/L FSW, respectively. The optimum temperature and pH for protease activity were 60 °C and 8.0, respectively. The crude protease, at different enzyme/substrate (E/S) ratio, was tested for the deproteinization of shrimp waste to produce chitin. The crude enzyme of P. aeruginosa A2 was found to be effective in the deproteinization of shrimp waste. The protein removals after 3 h hydrolysis at 40 °C with an E/S ratio of 0.5 and 5 U/mg protein were about 56% and 85%, respectively. ¹³C CP/MAS-NMR spectral analysis of the chitin prepared by treatment with the crude protease was carried out and was found to be similar to that of the commercial α-chitin. These results suggest that enzymatic deproteinization of shrimp waste by A2 protease could be applicable to the chitin production process.

Keywords: Shrimp shell; Waste valorization; Pseudomonas aeruginosa ; Protease; Enzymatic deproteinization; Chitin

Investigation of Nutrient Feeding Strategies in a Countercurrent Mixed-Acid Multi-Staged Fermentation: Experimental Data by Aaron Douglas Smith; Nur Ain Lockman; Mark T. Holtzapple (pp. 426-442).

Nutrients are essential for microbial growth and metabolism in mixed-culture acid fermentations. Understanding the influence of nutrient feeding strategies on fermentation performance is necessary for optimization. For a four-bottle fermentation train, five nutrient contacting patterns (single-point nutrient addition to fermentors F1, F2, F3, and F4 and multi-point parallel addition) were investigated. Compared to the traditional nutrient contacting method (all nutrients fed to F1), the near-optimal feeding strategies improved exit yield, culture yield, process yield, exit acetate-equivalent yield, conversion, and total acid productivity by approximately 31%, 39%, 46%, 31%, 100%, and 19%, respectively. There was no statistical improvement in total acid concentration. The traditional nutrient feeding strategy had the highest selectivity and acetate-equivalent selectivity. Total acid productivity depends on carbon–nitrogen ratio.

Keywords: Countercurrent mixed-acid fermentation; MixAlco process; Fermentation optimization; Nitrogen behavior; Carbon–nitrogen (C/N) ratio; Nutrient feeding strategies

Effects of Pulsed Electric Field on Secondary Metabolism of Vitis vinifera L. cv. Gamay Fréaux Suspension Culture and Exudates by Zhenzhen Cai; Heidi Riedel; Nay Min Min Thaw Saw; Onur Kütük; Inga Mewis; Henry Jäger; Dietrich Knorr; Iryna Smetanska (pp. 443-453).

Plant cell cultures provide a large potential for the production of secondary metabolites. Through the application of different physical and chemical cell stress factors, we investigated the production of the secondary metabolites in plant cell cultures. The effects of pulsed electric field (PEF) and ethephon on growth and secondary metabolism, particularly anthocyanins and phenolic acids synthesis, were investigated by using suspension culture of Vitis vinifera L. cv. Gamay Fréaux as a model system. Anthocyanins were measured by spectrophotometer and extracellular phenolic acids were determined by high-performance liquid chromatography. The compounds were identified by liquid chromatography–mass spectrometry and nuclear magnetic resonance. After the treatments with PEF and ethephon, the concentrations of anthocyanins and phenolic acids in cell culture were higher than in the control, without loss of biomass. The combination of PEF treatment and ethephon improved secondary metabolites formation. Production levels of extracellular phenolic acids, 3-O-glucosyl-resveratrol were increased by PEF and ethephon treatments. The results show that PEF induced a defense response of plant cells and may have altered the cell/membrane’s dielectric properties. PEF, an external stimulus or stress, is proposed as a promising new abiotic elicitor for stimulating secondary metabolites biosynthesis in plant cell cultures.

Keywords: Pulsed electric field (PEF); Ethephon, cell culture; Vitis vinifera ; Phenolic acid; Anthocyanin; Resveratrol; Exudate; Medium

Investigating the Structural and Functional Effects of Mutating Asn Glycosylation Sites of Horseradish Peroxidase to Asp by Sedigheh Asad; Khosro Khajeh; Nasser Ghaemi (pp. 454-463).

Horseradish peroxidase (HRP) has long attracted intense research interest and is used in many biotechnological fields, including diagnostics, biosensors, and biocatalysis. Enhancement of HRP catalytic activity and/or stability would further increase its applications. One of the problems with heterologus expression of HRP especially in prokaryotic host is lack of glycosylation that affects it's stability toward H₂O₂ and thermal inactivation. In this study, two asparagine residues which constitute two of the eight glycosylation sites in native HRP (Asn 13 and 268) with respectively 83% and 65% surface accessibility were substituted with aspartic acid in recombinant HRP. Both mutant proteins expressed in Escherichia coli showed increased stabilities against heat (increase in t _1/2 from 20 min in native rHRP to 32 and 67 min in N13D and N268D) and H₂O₂ (up to threefold). Unexpectedly, despite the distance of the mutated positions from the active site, notable alterations in steady-state k _cat and K _m values occurred with phenol/4-aminoantipyrine as reducing substrate which might be due to conformational changes. No significant alteration in flexibility was detected by acrylamide quenching analyses, but ANS binding experiments purposed lesser binding of ANS to hydrophobic patches in mutated HRPs. Double mutation was non-additive and non-synergistic.

Keywords: Recombinant horseradish peroxidase; Site-directed mutagenesis; Glycosylation site; Protein stability; H₂O₂ inactivation

Nitrate as an Oxidant in the Cathode Chamber of a Microbial Fuel Cell for Both Power Generation and Nutrient Removal Purposes by Cheng Fang; Booki Min; Irini Angelidaki (pp. 464-474).

Nitrate ions were used as the oxidant in the cathode chamber of a microbial fuel cell (MFC) to generate electricity from organic compounds with simultaneous nitrate removal. The MFC using nitrate as oxidant could generate a voltage of 111 mV (1,000 Ω) with a plain carbon cathode. The maximum power density achieved was 7.2 mW m⁻² with a 470 Ω resistor. Nitrate was reduced from an initial concentration of 49 to 25 mg (NO ₃ ⁻ −N) L⁻¹ during 42-day operation. The daily removal rate was 0.57 mg (NO ₃ ⁻ –N) L⁻¹ day⁻¹ with a voltage generation of 96 mV. In the presence of Pt catalyst dispersed on cathode, the cell voltage was significantly increased up to 450 mV and the power density was 117.7 mW m⁻², which was 16 times higher than the value without Pt catalyst. Significant nitrate removal was also observed with a daily removal rate of 2 mg (NO ₃ ⁻ –N) L⁻¹ day⁻¹, which was 3.5 times higher compared with the operation without catalyst. Nitrate was reduced to nitrite and ammonia in the liquid phase at a ratio of 0.6% and 51.8% of the total nitrate amount. These results suggest that nitrate can be successfully used as an oxidant for power generation without aeration and also nitrate removal from water in MFC. However, control of the process would be needed to reduce nitrate to only nitrogen gas, and avoid further reduction to ammonia.

Keywords: Nitrate removal; Microbial fuel cell; Power generation; Cathode chamber; Electron acceptor

Extracellular Production and Characterization of Streptomyces X-prolyl Dipeptidyl Aminopeptidase by Tadashi Hatanaka; Akihiro Yamasato; Jiro Arima; Hirokazu Usuki; Yukihiro Yamamoto; Yuya Kumagai (pp. 475-486).

X-prolyl dipeptidyl aminopeptidases (X-PDAPs) are useful in various food industries. In this study, we performed sequence-based screening to obtain a stable X-PDAP enzyme from thermophilic Streptomyces strains. We found three genes that encoded X-PDAP from Streptomyces thermoluteus subsp. fuscus NBRC 14270 (14270 X-PDAP), Streptomyces thermocyaneoviolaceus NBRC 14271 (14271 X-PDAP), and Streptomyces thermocoerulescens NBRC 14273, which were subsequently cloned and sequenced. The deduced amino acid sequences of these genes showed high similarity, with ~80% identity with each other. The isolated X-PDAPs and an X-PDAP from Streptomyces coelicolor were expressed in Streptomyces lividans using a hyperexpression vector: pTONA5a. Among these genes, only 14270 and 14271 X-PDAPs caused overexpression and extracellular production without artificial signal peptides. We also characterized the biochemical properties of purified 14271 X-PDAP. In addition, we found that, in peptide synthesis via an aminolysis reaction, this enzyme recognized d-amino acid derivatives as acyl acceptors, similar to l-amino acid derivatives.

Keywords: Peptidase; Streptomyces ; Extracellular production; Dipeptidyl aminopeptidase; Aminolysis

Effects of Pluronic F68 on Manganese Peroxidase Production by Pelletized Phanerochaete chrysosporium by Zhi-min Li; Yan Liu; Zhan-you Chi; Shu-lin Chen (pp. 487-496).

In this study, a new process was developed for manganese peroxidase (MnP) production by Phanerochaete chrysosporium under an agitated and aerated cultivation condition. It was found that change of the inoculum from spore suspension to pellets resulted in enhanced MnP production of 200 U/L in rotated shake flasks. Several additives, including Pluronic F68, Tween 80, and PEG8000, significantly increased the enzyme production. With an optimal concentration in 125 mL flasks, Pluronic F68 increased MnP productivity by 180%. Moreover, successful enzyme production was achieved in a 5-L fermentor at an agitation speed of 300 rpm with the addition of 0.1% Pluronic F68.

Keywords: Phanerochaete chrysosporium ; Pluronic F68; Enzyme; Biomass; Submerged; Optimization

Early In Vitro Transcription Termination in Human H5 Influenza Viral RNA Synthesis by Matthew B. Kerby; Aartik A. Sarma; Madhukar S. Patel; Andrew W. Artenstein; Steven M. Opal; Anubhav Tripathi (pp. 497-513).

Rapid diagnostic identification of the human H5 influenza virus is a strategic cornerstone for outbreak prevention. We recently reported a method for direct detection of viral RNA from a highly pathogenic human H5 influenza strain (A/Hanoi/30408/2005(H5N1)), which necessarily was transcribed in vitro from non-viral sources. This article provides an in-depth analysis of the reaction conditions for in vitro transcription (IVT) of full-length influenza H5 RNA, which is needed for diagnostic RNA production, for the T7 and SP6 phage promoter systems. Gel analysis of RNA transcribed from plasmids containing the H5 sequence between a 5′ SP6 promoter and 3′ restriction site (BsmBI) showed that three sequence-verified bands at 1,776, 784, and 591 bases were consistently produced, whereas only one 1,776-base band was expected. These fragments were not observed in H1 or H3 influenza RNA transcribed under similar conditions. A reverse complement of the sequence produced only a single band at 1,776 bases, which suggested either self-cleavage or early termination. Aliquots of the IVT reaction were quenched with EDTA to track the generation of the bands over time, which maintained a constant concentration ratio. The H5 sequence was cloned with T7 and SP6 RNA polymerase promoters to allow transcription in either direction with either polymerase. The T7 transcription product from purified, restricted plasmids in the vRNA direction only produced the 1,776-base full-length sequence and the 784-base fragment, instead of the three bands generated by the SP6 system, suggesting an early termination mechanism. Additionally, the T7 system produced a higher fraction of full-length vRNA transcripts than the SP6 system did under similar reaction conditions. By sequencing we identified a type II RNA hairpin loop terminator, which forms in a transcription direction-dependent fashion. Variation of the magnesium concentration produced the greatest impact on termination profiles, where some reaction mixtures were unable to produce full-length transcripts. Optimized conditions are presented for the T7 and SP6 phage polymerase systems to minimize these early termination events during in vitro transcription of H5 influenza vRNA.

Keywords: Termination; Influenza; H5; In vitro transcription; SP6; T7

Optimization of α-Terpineol Production by the Biotransformation of R-(+)-Limonene and (−)-β-Pinene by Ieda Rottava; Priscila F. Cortina; Eduarda Martello; Rogerio L. Cansian; Geciane Toniazzo; Octavio A. C. Antunes; Enrique G. Oestreicher; Helen Treichel; Debora de Oliveira (pp. 514-523).

The main objective of this work is to present the optimization of the biotransformation of R-(+)-limonene and (−)-β-pinene aiming at the production of α-terpineol by strains of fungal and yeasts previously isolated by our research group using the methodology of experimental design. New optimized experimental data on α-terpineol production by the biotransformation of R-(+)-limonene and (−)-β-pinene using newly isolated microorganisms are reported in this work. Conversion of about 1,700 mg/L was achieved when R-(+)-limonene was used as substrate and the newly isolated strain 05.01.35 as microorganism at the central point of the experimental design, corresponding to a substrate concentration of 1.75%, mass of inoculum of 2 g, and substrate to ethanol volume ratio of 1:1. The same experimental condition led to higher conversions when (−)-β-pinene was used as substrates and the strains coded as 04.05.08 and 01.04.03 as microorganism. Here, conversions of about 770 mg/L were achieved.

Keywords: Experimental design; α-Terpineol; R-(+)-Limonene; (−)-β-Pinene

Study of the Alkyl Chain Length on Laccase Stability and Enzymatic Kinetic with Imidazolium Ionic Liquids by Oscar Rodríguez; Raquel O. Cristóvão; Ana P. M. Tavares; Eugénia A. Macedo (pp. 524-533).

The activity and stability of laccase and their kinetic mechanisms in water soluble ionic liquids (ILs): 1-butyl-3-methyl imidazolium chloride [C₄mim][Cl], 1-octyl-3-methyl imidazolium chloride [C₈mim][Cl], and 1-decyl-3-methyl imidazolium chloride [C₁₀mim][Cl] were investigated. The results show that an IL concentration up to 10% is satisfactory for initial laccase activity at pH 9.0. The laccase stability was well maintained in [C₄mim][Cl] IL when compared to the control. The inactivation of laccase increases with the length of the alkyl chain in the IL: [C₁₀mim][Cl] > [C₈mim][Cl] > [C₄mim][Cl]. The kinetic studies in the presence of ABTS as substrate allowed calculating the Michaelis–Menten parameters. Among the ILs, [C₄mim][Cl] was the suitable choice attending to laccase activity and stability. Alkyl chains in the ions of ILs have a deactivating effect on laccase, which increases strongly with the length of the alkyl chain.

Keywords: Ionic liquids; Laccase; Stability; Activity; Kinetic parameters

Consolidated Conversion of Hulled Barley into Fermentable Sugars Using Chemical, Thermal, and Enzymatic (CTE) Treatment by Tae Hyun Kim; Nhuan P. Nghiem; Frank Taylor; Kevin B. Hicks (pp. 534-545).

A novel process using chemical, thermal, and enzymatic treatment for conversion of hulled barley into fermentable sugars was developed. The purpose of this process is to convert both lignocellulosic polysaccharides and starch in hulled barley grains into fermentable sugars simultaneously without a need for grinding and hull separation. In this study, hulled barley grains were treated with 0.1 and 1.0 wt.-% sulfuric acid at various temperatures ranging from 110 to 170 °C in a 63-ml flow-through packed-bed stainless steel reactor. After sulfuric acid pretreatment, simultaneous conversion of lignocellulose and starch in the barley grains into fermentable sugars was performed using an enzyme cocktail, which included α-amylase, glucoamylase, cellulase, and β-glucosidase. Both starch and non-starch polysaccharides in the pre-treated barley grains were readily converted to fermentable sugars. The treated hulled barley grains, including their hull, were completely hydrolyzed to fermentable sugars with recovery of almost 100% of the available glucose and xylose. The pretreatment conditions of this chemical, thermal, and enzymatic (CTE) process for achieving maximum yield of fermentable sugars were 1.0 wt.% sulfuric acid and 110 °C. In addition to starch, the acid pretreatment also retained most of the available proteins in solid form, which is essential for subsequent production of fuel ethanol and high protein distiller’s dried grains with solubles co-product.

Keywords: Hulled barley; Dilute sulfuric acid pretreatment; Fermentable sugar production; Enzyme hydrolysis; Lignocellulosic biomass conversion

Effects of Radiofrequency Electromagnetic Wave Exposure from Cellular Phones on the Reproductive Pattern in Male Wistar Rats by Kavindra Kumar Kesari; Sanjay Kumar; Jitendra Behari (pp. 546-559).

The present study investigates the effect of free radical formation due to mobile phone exposure and effect on fertility pattern in 70-day-old male Wistar rats (sham exposed and exposed). Exposure took place in Plexiglas cages for 2 h a day for 35 days to mobile phone frequency. The specific absorption rate was estimated to be 0.9 W/kg. An analysis of antioxidant enzymes glutathione peroxidase (P < 0.001) and superoxide dismutase (P < 0.007) showed a decrease, while an increase in catalase (P < 0.005) was observed. Malondialdehyde (P < 0.003) showed an increase and histone kinase (P = 0.006) showed a significant decrease in the exposed group. Micronuclei also show a significant decrease (P < 0.002) in the exposed group. A significant change in sperm cell cycle of G₀–G₁ (P = 0.042) and G₂/M (P = 0.022) were recorded. Generation of free radicals was recorded to be significantly increased (P = 0.035). Our findings on antioxidant, malondialdehyde, histone kinase, micronuclei, and sperm cell cycle are clear indications of an infertility pattern, initiated due to an overproduction of reactive oxygen species. It is concluded that radiofrequency electromagnetic wave from commercially available cell phones might affect the fertilizing potential of spermatozoa.

Keywords: Microwave radiation; Histone kinase; Cell cycle; Flow cytometry; Reactive oxygen species; Apoptosis

Sections

Personal tools

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.164, #4)

Sections

Personal tools

Local resources

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.164, #4)