Applied Biochemistry and Biotechnology (v.178, #7)

Molecular Cloning, Expression Analysis, and Functional Characterization of the H+-Pyrophosphatase from Jatropha curcas by Yumei Yang; Zhu Luo; Mengru Zhang; Chang Liu; Ming Gong; Zhurong Zou (1273-1285).
H+-pyrophosphatase (H+-PPase) is a primary pyrophosphate (PPi)-energized proton pump to generate electrochemical H+ gradient for ATP production and substance translocations across membranes. It plays an important role in stress adaptation that was intensively substantiated by numerous transgenic plants overexpressing H+-PPases yet devoid of any correlated studies pointing to the elite energy plant, Jatropha curcas. Herein, we cloned the full length of J. curcas H+-PPase (JcVP1) complementary DNA (cDNA) by reverse transcription PCR, based on the assembled sequence of its ESTs highly matched to Hevea brasiliensis H+-PPase. This gene encodes a polypeptide of 765 amino acids that was predicted as a K+-dependent H+-PPase evolutionarily closest to those of other Euphorbiaceae plants. Many cis-regulatory elements relevant to environmental stresses, molecular signals, or tissue-specificity were identified by promoter prediction within the 1.5-kb region upstream of JcVP1 coding sequence. Meanwhile, the responses of JcVP1 expression to several common abiotic stresses (salt, drought, heat, cold) were characterized with a considerable accordance with the inherent stress tolerance of J. curcas. Moreover, we found that the heterologous expression of JcVP1 could significantly improve the salt tolerance in both recombinant Escherichia coli and Saccharomyces cerevisiae, and this effect could be further fortified in yeast by N-terminal addition of a vacuole-targeting signal peptide from the H+-PPase of Trypanosoma cruzi.
Keywords: H+-PPase; Jatropha curcas ; Gene cloning; Heterologous expression; Salt tolerance

Importance of Interaction between Integrin and Actin Cytoskeleton in Suspension Adaptation of CHO cells by Christa G. Walther; Robert Whitfield; David C. James (1286-1302).
The biopharmaceutical production process relies upon mammalian cell technology where single cells proliferate in suspension in a chemically defined synthetic environment. This environment lacks exogenous growth factors, usually contributing to proliferation of fibroblastic cell types such as Chinese hamster ovary (CHO) cells. Use of CHO cells for production hence requires a lengthy ‘adaptation’ process to select clones capable of proliferation as single cells in suspension. The underlying molecular changes permitting proliferation in suspension are not known. Comparison of the non-suspension-adapted clone CHO-AD and a suspension-adapted propriety cell line CHO-SA by flow cytometric analysis revealed a highly variable bi-modal expression pattern for cell-to-cell contact proteins in contrast to the expression pattern seen for integrins. Those have a uni-modal expression on suspension and adherent cells. Integrins showed a conformation distinguished by regularly distributed clusters forming a sphere on the cell membrane of suspension-adapted cells. Actin cytoskeleton analysis revealed reorganisation from the typical fibrillar morphology found in adherent cells to an enforced spherical subcortical actin sheath in suspension cells. The uni-modal expression and specific clustering of integrins could be confirmed for CHO-S, another suspension cell line. Cytochalasin D treatment resulted in breakdown of the actin sheath and the sphere-like integrin conformation demonstrating the link between integrins and actin in suspension-adapted CHO cells. The data demonstrates the importance of signalling changes, leading to an integrin rearrangement on the cell surface, and the necessity of the reinforcement of the actin cytoskeleton for proliferation in suspension conditions.
Keywords: Chinese hamster ovary cells; Suspension adaptation; Cell surface proteins; Integrin signalling; Cell-to-extracellular matrix interaction; Actin cytoskeleton

A Novel Mesophilic Anaerobic Digestion System for Biogas Production and In Situ Methane Enrichment from Coconut Shell Pyroligneous by Jing-Rong Cheng; Xue-Ming Liu; Zhi-Yi Chen; You-Sheng Zhang; Ye-Hui Zhang (1303-1314).
A novel mesophilic anaerobic digestion process with detoxification-treated coconut shell pyroligneous was established, exhibiting an effective advantage in biogas production. The pyroligneous collected contained 166.2 g l−1 acetic acid, indicating great potential for biogas production. Detoxification was an effective way of simultaneously enriching biodegradable ingredients and removing inhibitors (mainly as phenols and organic acids) for digestion process. The digestion process lasted 96 h and fermentation characteristics (chemical oxygen demand (COD) removal ratio, volatile fatty acid (VFA) consumptions, pH, total gas, methane yield, and phenol removal efficiency) were measured. The experiments successfully explored the optimum detoxification parameters, oxidized with 10 % H2O2 followed by overliming, and demonstrated 89.3 % COD removal, 91.4 % methane content, 0.305 LCH4/g COD removed CH4 yield, and 88.81 % phenol removal ratio. This study provided clues to overcome the negative effects of inhibitors in pyroligneous on biogas production. The findings could contribute to significant process in detoxified pretreatment of pyroligneous and develop an economically feasible technology for treating pyroligneous after producing charcoal.
Keywords: Pyroligneous; Detoxification; Methane; Phenol removal; Anaerobic digestion

Redirection of Metabolic Flux into Novel Gamma-Aminobutyric Acid Production Pathway by Introduction of Synthetic Scaffolds Strategy in Escherichia Coli by Van Dung Pham; Sivachandiran Somasundaram; Seung Hwan Lee; Si Jae Park; Soon Ho Hong (1315-1324).
In general, gamma-aminobutyric acid (GABA) pathway involves the decarboxylation of glutamate, which is produced from sugar by Corynebacterium fermentation. GABA can be used for the production of pharmaceuticals and functional foods. Due to the increasing demand of GABA, it is essential to create an effective alternative pathway for the GABA production. In this study, Escherichia coli were engineered to produce GABA from glucose via GABA shunt, which consists of succinate dehydrogenase, succinate-semialdehyde dehydrogenase, and GABA aminotransferase. The three enzymes were physically attached to each other through a synthetic scaffold, and the Krebs cycle flux was redirected to the GABA pathway. By introduction of synthetic scaffold, 0.75 g/l of GABA was produced from 10 g/l of glucose at 30 °C and pH 6.5. The inactivation of competing metabolic pathways provided 15.4 % increase in the final GABA concentration.
Keywords: Gamma-aminobutyric acid; Glucose; Metabolic pathway; Recombinant DNA; Synthetic scaffold

Electrokinetic-Enhanced Remediation of Phenanthrene-Contaminated Soil Combined with Sphingomonas sp. GY2B and Biosurfactant by Weijia Lin; Chuling Guo; Hui Zhang; Xujun Liang; Yanfu Wei; Guining Lu; Zhi Dang (1325-1338).
Electrokinetic-microbial remediation (EMR) has emerged as a promising option for the removal of polycyclic aromatic hydrocarbons (PAHs) from contaminated soils. The aim of this study was to enhance degradation of phenanthrene (Phe)-contaminated soils using EMR combined with biosurfactants. The electrokinetic (EK) remediation, combined with Phe-degrading Sphingomonas sp. GY2B, and biosurfactant obtained by fermentation of Pseudomonas sp. MZ01, degraded Phe in the soil with an efficiency of up to 65.1 % at the anode, 49.9 % at the cathode after 5 days of the treatment. The presence of biosurfactants, electricity, and a neutral electrolyte stimulated the growth of the degrading bacteria as shown by a rapid increase in microbial biomass with time. The electrical conductivity and pH changed little during the course of the treatment, which benefitted the growth of microorganisms and the remediation of Phe-contaminated soil. The EMR system with the addition of biosurfactant had the highest Phe removal, demonstrating the biosurfactant may enhance the bioavailability of Phe and the interaction with the microorganism. This study suggests that the EMR combined with biosurfactants can be used to enhance in situ bioremediation of PAH-contaminated soils.
Keywords: Phenanthrene; Electrokinetics; Biosurfactants; Microorganism; Degradation

Lipoxygenase from Anabaena sp. PCC 7120 (Ana-LOX) was thermally unstable. So, improving the thermostability of the enzyme was quite essential. The target site of Ana-LOX selected for site-directed mutagenesis was based on computer-aided rational design. The thermostability and specific activity of Ana-LOX were improved with replacing valine with alanine at the target site 421 and the site 40. Compared to the wild-type enzyme which has a half-life (T 1/2) of inactivation of 3.8 min at 50 °C, the T 1/2 of mutant enzymes with V421A and V40A substitution increased to 4.4 and 7.0 min, respectively. The double mutant V421A/V40A showed a synergistic effect with a T 1/2 value of 8.3 min, resulting in a 1.18-fold improvement compared to the original Ana-LOX. V421A, V40A, and V421A/V40A also obtained 4.83, 41.58, and 80.07 % increase in specific activity, respectively. This study provides useful theoretical reference for enzyme molecular modification and computer-aided rational design.
Keywords: Lipoxygenase; Thermostability; Computer-aided rational design; Site-directed mutagenesis

The presence of labile iron fractions in intravenous iron supplements compromises their safety. Poly(ethylene glycol) (PEG)-assisted silica xerogel was evaluated as a potential drug carrier for iron sucrose with the purpose of limiting labile iron available for in vitro uptake by transferrin. The drug entrapped xerogels were synthesized by the sol–gel process with varying amounts of PEG. In vitro release studies were conducted in simulated body fluid (SBF) at 37 ± 0.02 °C (pH 7.4). The results indicated that the cumulative release percentage increased with the increase in the amount of PEG in the matrix. The biphasic release profile followed first-order kinetics for the first 6 h and Higuchi model for the remaining time (up to 168 h). The sample showing highest percentage of cumulative release (the xerogel with 16 % PEG) was used for in vitro transferrin saturation studies in contrast with the plain drug. The xerogel formulation exhibited 7.25 ± 0.4 % transferrin saturation in 180 min as compared to 12.89 ± 0.2 % for the raw drug. These results indicate that encapsulation of iron sucrose in PEG-assisted silica xerogel and subsequent sustained release from the matrix can improve the safety of the drug when presence of labile iron is a major concern.
Keywords: PEG-assisted silica xerogel; Drug delivery; Iron sucrose; Transferrin saturation; Release kinetics

Tyrosinase-Mediated Construction of a Silk Fibroin/Elastin Nanofiber Bioscaffold by Yanqing Hong; Xueke Zhu; Ping Wang; Haitian Fu; Chao Deng; Li Cui; Qiang Wang; Xuerong Fan (1363-1376).
Elastin has characteristics of elasticity, biological activity, and mechanical stability. In the present work, tyrosinase-mediated construction of a bioscaffold with silk fibroin and elastin was carried out, aiming at developing a novel medical biomaterial. The efficiency of enzymatic oxidation of silk fibroin and the covalent reaction between fibroin and elastin were examined by spectrophotometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and size exclusion chromatography (SEC). The properties of composite air-dried and nanofiber scaffolds were investigated. The results reveal that elastin was successfully bonded to silk fibroins, resulting in an increase in molecular weight of fibroin proteins. ATR-FTIR spectra indicated that tyrosinase treatment impacted the conformational structure of fibroin-based membrane. The thermal behaviors and mechanical properties of the tyrosinase-treated scaffolds were also improved compared with the untreated group. NIH/3T3 cells exhibited optimum densities when grown on the nanofiber scaffold, implying that the nanofiber scaffold has enhanced biocompatibility compared to the air-dried scaffold. A biological nanofiber scaffold constructed from tyrosinase-treated fibroin and elastin could potentially be utilized in biomedical applications.
Keywords: Tyrosinase; Silk fibroin; Elastin; Bioscaffold; Biocompatibility

Discovery of novel secreted enzymes and proteins in Mycobacterium tuberculosis (M. tuberculosis) are imperative to understanding the pathogenic system for pathogenesis requires attention. Till date, the groups of these secreted enzymes are not meaningfully characterized in terms of M. tuberculosis. In this way, cutinase, a small lipolytic protein, exists in both bacteria and fungi as well which have a potential catalytic activity. During our search, we have found a few genes of M. tuberculosis demonstrating a same significant lipase action as fungi Fusarium solani cutinase contain. Genome sequencing of M. tuberculosis uncover a lot of proteins, wherein (Rv1758, Rv1984c, Rv2301, Rv3451, Rv3452, Rv3724A, Rv3724B, and Rv3802c) genes have been noted which are exhibiting a cutinase-like activity and closely homologous to that of F. solani cutinase and having the ability to hydrolyze model substrates including p-nitrophenyl butyrate (p-PNB), cutin, triacylglycerols (TAGs), and triolein (TO), yet their biological significance in pathogenesis stays subtle and uncharacterized. In a basic perspective, the measure of cutinase expressed by M. tuberculosis and part of these small lipolytic enzymes in the pathologic discipline require thorough characterization. So, through focusing on cutinase-encoding genes in M. tuberculosis and their active catalytic motif could help to build up a novel therapeutic approach.
Keywords: M. tuberculosis ; Cutinase-like protein; Macrophages; Triolein; Lipid droplets

Soluble Expression and Characterization of a New scFv Directed to Human CD123 by Shima Moradi-Kalbolandi; Dariush Davani; Majid Golkar; Mahdi Habibi-Anbouhi; Mohsen Abolhassani; Mohammad Ali Shokrgozar (1390-1406).
Leukemic cancer stem cells (LSCs), as a unique cell population in acute myeloid leukemia (AML) marked by CD123 overexpression, are thought to play a key role in relapsed AML after chemotherapy. Thus, CD123 is considered as a particularly important target candidate for antibody-derived diagnosis and therapy. In the present work, we constructed an immunized murine antibody phage display library and isolated the functional anti-CD123 Single-chain fragment variable (scFv) clones. We also introduced fusing variable light (VL) and heavy (VH) chains with a new 18-amino acid residue linker as an alternative to conventional linkers. CD123-specific phage clones were progressively enriched through 4 rounds of biopanning, validated by phage ELISA, and anti-CD123 scFv clones with highest affinity were produced in Escherichia coli. The expression and purification of soluble scFv were verified by Western blot, and the results were indicative of the functionality of our proposed linker. The purified scFv specifically recognized CD123 by ELISA and flow cytometry, without any cross-reactivity with other related cell markers. Affinity of anti-CD123 scFv was measured to be 6.9 × 10−7 M, using the competitive ELISA. Our work, therefore, provides a framework for future studies involving biological functions and applications of our anti-CD123 scFv. It also reveals the feasibility of high throughput methods to isolate biomarker-specific scFvs.
Keywords: CD123; scFv; AML; Phage display; Leukemic cancer stem cell

Effects of Nitrogen and Phosphorus on Biochemical Composition of Microalgae Polyculture Harvested from Phycoremediation of Piggery Wastewater Digestate by William Michelon; Marcio Luis Busi Da Silva; Melissa Paola Mezzari; Mateus Pirolli; Jean Michel Prandini; Hugo Moreira Soares (1407-1419).
The effects of nitrogen (N) and/or phosphorus (P) starvation on the biochemical composition of native microalgae Chlorella spp. polyculture obtained from the phycoremediation of swine wastewaters were investigated. Microalgae-specific growth rate of 1.2 day−1 was achieved (30.3 mg L−1 day−1). PO4 −2 and NH3 were completely removed from swine digestate effluent after 3 and 11 days, respectively. Microalgae harvested immediately after nutrient removal showed high protein (56–59 %) and carbohydrate (25–34 %) but low lipid (1.8–3 %) contents. Depletion of N or P alone stimulated carbohydrate production at the expenses of proteins. Significant lipid accumulation from 3 % ± 0.5 to 16.3 % ± 0.8 was reached only after 25 days following N and P starvation as demonstrated by Nile red-stained cells. Regarding to the effects of harvesting methods on cellular biochemical composition, circumstantial evidences indicate that coagulation–flocculation with tannin may lead to lower protein and lipid amounts but increased carbohydrate content (p < 0.01) as compared to centrifugation.
Keywords: Carbohydrate; Lipid; Microalgae; Phycoremediation; Protein; Swine wastewater

Elucidating the Beneficial Effect of Corncob Acid Hydrolysate Environment on Lipid Fermentation of Trichosporon dermatis by Method of Cell Biology by Chao Huang; Can Wang; Lian Xiong; Xue-Fang Chen; Xiao-Qing Lin; Gao-Xiang Qi; Si-Lan Shi; Bo Wang; Xin-De Chen (1420-1429).
In present study, the beneficial effect of corncob acid hydrolysate environment on lipid fermentation of Trichosporon dermatis was elucidated by method of cell biology (mainly using flow cytometry and microscope) for the first time. Propidium iodide (PI) and rhodamine 123 (Rh123) staining showed that corncob acid hydrolysate environment was favorable for the cell membrane integrity and mitochondrial membrane potential of T. dermatis and thus made its lipid fermentation more efficient. Nile red (NR) staining showed that corncob acid hydrolysate environment made the lipid accumulation of T. dermatis slower, but this influence was not serious. Moreover, the cell morphology of T. dermatis elongated in the corncob acid hydrolysate, but the cell morphology changed as elliptical-like during fermentation. Overall, this work offers one simple and effective method to evaluate the influence of lignocellulosic hydrolysates environment on lipid fermentation.
Keywords: Corncob acid hydrolysate; Trichosporon dermatis ; Flow cytometry; Microbial oil; Cell biology

Enzymatic Hydrolysis of Pretreated Sugarcane Straw: Kinetic Study and Semi-Mechanistic Modeling by Bruna Pratto; Renata Beraldo Alencar de Souza; Ruy Sousa Jr; Antonio Jose Gonçalves da Cruz (1430-1444).
Although there are already commercial-scale productions of second generation (2G) ethanol, focusing efforts on process optimization can be of key importance to make the production cost-effective in large scale. In this scenario, mathematical models may be useful in design, scale-up, optimization, and control of bioreactors. For this reason, the aim of this work was to study the kinetics of the enzymatic hydrolysis of cellulose from sugarcane straw. Experiments using hydrothermally pretreated sugarcane (HPS) straw (195 °C, 10 min, 200 rpm) with and without alkaline delignification (4 % NaOH m/v, 30 min, 121 °C) were carried out in shake flasks (50 °C, pH 5.0, 200 rpm). Solid load was varied in a range of 0.8 to 10 % (m/v), in initial velocity and long-term assays. Enzyme concentration (Cellic®CTec2) was varied from 5 to 80 filter paper unit (FPU) gcellulose −1. It was possible to fit Michaelis–Menten (MM), modified MM, with and without competitive inhibition by glucose, and Chrastil models. Chrastil model and modified MM with inhibition (both suitable for heterogeneous system, with high resistance to internal diffusion) showed more appropriate than pseudo-homogeneous MM model. The fitted models were able to identify key features of the hydrolysis process and can be very useful within the perspective of bioreactors engineering.
Keywords: Sugarcane straw; Enzymatic hydrolysis; Kinetic study; Mathematical modeling

ε-Poly-l-lysine (ε-PL) is a widely used natural food preservative. To test the effects of the Vitreoscilla hemoglobin (VHb) and S-adenosylmethionine (SAM) on ε-PL synthesis in Streptomyces albulus NK660, the heterologous VHb gene (vgb) and SAM synthetase gene (metK) were inserted into the S. albulus NK660 chromosome under the control of the constitutive ermE* promoter. CO-difference spectrum analysis showed S. albulus NK660-VHb strain could express functional VHb. S. albulus NK660-VHb produced 26.67 % higher ε-PL and 14.57 % higher biomass than the wild-type control, respectively. Reversed-phase high-pressure liquid chromatography (RP-HPLC) results showed the overexpression of the metK gene resulted in increased intracellular SAM synthesis in S. albulus NK660-SAM, which caused increases of biomass as well as the transcription level of ε-PL synthetase gene (pls). Results indicated that the expression of vgb and metK gene improved on ε-PL synthesis and biomass for S. albulus NK660, respectively.
Keywords: ε-poly-l-lysine; Streptomyces albulus NK660; Vitreoscilla hemoglobin gene; S-adenosylmethionine synthetase gene

Efficient Production of Hydroxylated Human-Like Collagen Via the Co-Expression of Three Key Genes in Escherichia coli Origami (DE3) by Yunping Tang; Xiuliang Yang; Baojian Hang; Jiangtao Li; Lei Huang; Feng Huang; Zhinan Xu (1458-1470).
Mature collagen is abundant in human bodies and very valuable for a range of industrial and medical applications. The biosynthesis of mature collagen requires post-translational modifications to increase the stability of collagen triple helix structure. By co-expressing the human-like collagen (HLC) gene with human prolyl 4-hydroxylase (P4H) and d-arabinono-1, 4-lactone oxidase (ALO) in Escherichia coli, we have constructed a prokaryotic expression system to produce the hydroxylated HLC. Then, five different media, as well as the induction conditions were investigated with regard to the soluble expression of such protein. The results indicated that the highest soluble expression level of target HLC obtained in shaking flasks was 49.55 ± 0.36 mg/L, when recombinant cells were grew in MBL medium and induced by 0.1 mM IPTG at the middle stage of exponential growth phase. By adopting the glucose feeding strategy, the expression level of target HLC can be improved up to 260 mg/L in a 10 L bench-top fermentor. Further, HPLC analyses revealed that more than 10 % of proline residues in purified HLC were successfully hydroxylated. The present work has provided a solid base for the large-scale production of hydroxylated HLC in E. coli.
Keywords: Human like collagen; Hydroxylation; Post-translation modification; Prolyl 4-hydroxylase; E. coli