Applied Biochemistry and Biotechnology (v.178, #3)

Evaluation of Suitable Reference Genes for Normalization of qPCR Gene Expression Studies in Brinjal (Solanum melongena L.) During Fruit Developmental Stages by Mogilicherla Kanakachari; Amolkumar U. Solanke; Narayanasamy Prabhakaran; Israr Ahmad; Gurusamy Dhandapani; Narayanasamy Jayabalan; Polumetla Ananda Kumar (433-450).
Brinjal/eggplant/aubergine is one of the major solanaceous vegetable crops. Recent availability of genome information greatly facilitates the fundamental research on brinjal. Gene expression patterns during different stages of fruit development can provide clues towards the understanding of its biological functions. Quantitative real-time PCR (qPCR) has become one of the most widely used methods for rapid and accurate quantification of gene expression. However, its success depends on the use of a suitable reference gene for data normalization. For qPCR analysis, a single reference gene is not universally suitable for all experiments. Therefore, reference gene validation is a crucial step. Suitable reference genes for qPCR analysis of brinjal fruit development have not been investigated so far. In this study, we have selected 21 candidate reference genes from the Brinjal (Solanum melongena) Plant Gene Indices database ( and studied their expression profiles by qPCR during six different fruit developmental stages (0, 5, 10, 20, 30, and 50 days post anthesis) along with leaf samples of the Pusa Purple Long (PPL) variety. To evaluate the stability of gene expression, geNorm and NormFinder analytical softwares were used. geNorm identified SAND (SAND family protein) and TBP (TATA binding protein) as the best pairs of reference genes in brinjal fruit development. The results showed that for brinjal fruit development, individual or a combination of reference genes should be selected for data normalization. NormFinder identified Expressed gene (expressed sequence) as the best single reference gene in brinjal fruit development. In this study, we have identified and validated for the first time reference genes to provide accurate transcript normalization and quantification at various fruit developmental stages of brinjal which can also be useful for gene expression studies in other Solanaceae plant species.
Keywords: Brinjal; Days post anthesis; Fruit development; Gene expression; Reference gene; Normalization; Quantitative real-time PCR

The Native Plasmid pML21 Plays a Role in Stress Tolerance in Enterococcus faecalis ML21, as Analyzed by Plasmid Curing Using Plasmid Incompatibility by Fang-Lei Zuo; Li-Li Chen; Zhu Zeng; Xiu-Juan Feng; Rui Yu; Xiao-Ming Lu; Hui-Qin Ma; Shang-Wu Chen (451-461).
To investigate the role of the native plasmid pML21 in Enterococcus faecalis ML21’s response to abiotic stresses, the plasmid pML21 was cured based on the principle of plasmid incompatibility and segregational instability, generating E. faecalis mutant strain ML0. The mutant and the wild strains were exposed to abiotic stresses: bile salts, low pH, H2O2, ethanol, heat, and NaCl, and their survival rate was measured. We found that curing of pML21 lead to reduced tolerance to stress in E. faecalis ML0, especially oxidative and osmotic stress. Complementation analysis suggested that the genes from pML21 played different role in stress tolerance. The result indicated that pML21 plays a role in E. faecalis ML21’s response to abiotic stresses.
Keywords: Enterococcus faecalis ; Native plasmid; Incompatibility; Plasmid curing; Stress tolerance

Low-Cost Synthesis of Smart Biocompatible Graphene Oxide Reduced Species by Means of GFP by Tiziana Masullo; Nerina Armata; Flavio Pendolino; Paolo Colombo; Fabrizio Lo Celso; Salvatore Mazzola; Angela Cuttitta (462-473).
The aim of this work is focused on the engineering of biocompatible complex systems composed of an inorganic and bio part. Graphene oxide (GO) and/or graphite oxide (GtO) were taken into account as potential substrates to the linkage of the protein such as Anemonia sulcata recombinant green fluorescent protein (rAsGFP). The complex system is obtained through a reduction process between GO/GtO and rAsGFP archiving an environmentally friendly biosynthesis. Spectroscopic measurements support the formation of reduced species. In particular, photoluminescence shows a change in the activity of the protein when a bond is formed, highlighted by a loss of the maximum emission signal of rAsGFP and a redshift of the maximum absorption peak of the GO/GtO species. Moreover, the hemolysis assay reveals a lower value in the presence of less oxidized graphene species providing evidence for a biocompatible material. This singular aspect can be approached as a promising method for circulating pharmaceutical preparations via intravenous administration in the field of drug delivery.
Keywords: Graphene oxide; Graphite oxide; GFP; Reduction; Biocompatibility; Hemolysis

Heterologous Expression, Purification, and Biochemical Characterization of α-Humulene Synthase from Zingiber zerumbet Smith by Semra Alemdar; Steffen Hartwig; Thore Frister; Jan Christoph König; Thomas Scheper; Sascha Beutel (474-489).
The α-humulene synthase from Zingiber zerumbet Smith was expressed as a polyhistidine-tagged protein in an E. coli BL21(DE3) strain. Induction time and inductor (isopropyl-β-D-thiogalactopyranoside) concentration were optimized. The enzyme was successfully purified directly from cell lysate by NTA affinity column chromatography and careful selection of coordinated metal ion and imidazole elution conditions. Bioactivity assays were conducted with the natural substrate farnesyl diphosphate (FDP) in a two-phase system with in situ extraction of products. The conversion of FDP to α-humulene (~94.5 %) and β-caryophyllene (~5.5 %) could be monitored by gas chromatography-flame ionization detection (GC-FID). Optimal pH and temperature as well as kinetic parameters K M and k cat were determined using a discontinuous kinetic assay.
Keywords: Terpene synthase; Sesquiterpene; Humulene; Recombinant expression; Purification; Enzyme activity

Eukaryotic Expression and Purification of Native Form of Mouse Midkine from Pichia pastoris by Jin Gao; Haixia Wang; Jingjing Li; Shixiang Jia; Wei Han; Yan Yu (490-503).
To confirm the treating effectiveness of midkine as an articular protective agent, mouse midkine (mMK) was produced for the pre-clinic long-term studies in mice. The protein was expressed under the control of the AOX1 gene promoter in Pichia pastoris, X-33 strain, and secreted into fermentation broth through high-density fermentation. Approximately 380 mg mMK, containing authentic and truncated forms, was secreted into 1 liter induction medium and 280 mg mMK was obtained after one-step purification on a 50 ml SP Sepharose Fast Flow column. The purified protein was characterized and identified to be the mature, authentic form of mMK. N-terminal five amino acid sequence was determined to be K-K-K-E-K. SDS-PAGE analysis indicated that the molecular weight of the product was about 13 KDa. The purity of the purified rmMK protein was determined to be 99 % by high performance liquid chromatography. The biological activity of final product was verified via migration assay on osteoblast-like UMR-106 cells.
Keywords: Midkine; Pichia pastoris ; Fermentation; Ion exchange chromatography; HPLC

The present study was undertaken to evaluate the effect of initial temperature treatment on phytochemical and antioxidant potential of commercially important plant Azadirachta indica A. Juss. The leaves were differentially treated after harvest at temperatures 4, −20, and 110 °C and at room temperature, separately. It was found that a quick drying process at 110 °C followed by air-drying helped in maximum retention of bioactive compounds and antioxidant activity, which was significantly higher than other processing methods. Correlation analysis revealed that total phenolic content (TPC) and total flavonoid content (TFC) can be a measure to assess the antioxidant potential using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing/antioxidant power (FRAP) assays. Nitric oxide scavenging assay (NOSA) was insignificantly related to DPPH and FRAP. Also, FRAP and DPPH can be a predictive assay for each other, but not with NOSA. Thus, a quick drying process at 110 °C using an oven can be a cost-effective venture, especially in developing countries, for retaining the nutritive value and antioxidant activity of A. indica.
Keywords: Azadirachta indica ; Temperature treatment; TPC; TFC; NOSA; Correlation

Insulin plant (Costus pictus D. Don) is an economically important medicinal plant for the content of its high value secondary metabolites, bioactive compounds, and remarkable flowering features. MicroRNAs are a class of short (∼21 nucleotides), endogenous, noncoding RNA molecules that play a vital role in regulating gene expression. Here, we used a computer-based homology approach to identify conserved miRNAs in Transcribed Sequence Assemblies (TSA) of C. pictus. It led us to identify 42 miRNAs of 13 different families in C. pictus for the first time. Using quantitative polymerase chain reaction (qPCR) assays, we further confirmed the expression of 8 miRNAs (miR394, miR159b, miR166k, miR172, miR159f, miR166, miR144, and miR858) in young and mature leaf tissues. A total of 109 potential target genes of the identified miRNAs were subsequently predicted in rice (Oryza sativa L.) genome. The target genes encode transcription factors, enzymes, and various functional proteins involved in the regulation of several metabolic pathways. The findings in the present study lay the foundation for further research on miRNAs and miRNA-mediated gene regulation in this important medicinal plant.
Keywords: Expression; Homolog; MicroRNA; Medicinal plant; Regulation; Targets

Transcriptomic Analysis of 3-Hydroxypropanoic Acid Stress in Escherichia coli by Tu Wang Yung; Sudhakar Jonnalagadda; Balaji Balagurunathan; Hua Zhao (527-543).
The stress response of Escherichia coli to 3-hydroxypropanoic acid (3-HP) was elucidated through global transcriptomic analysis. Around 375 genes showed difference of more than 2-fold in 3-HP-treated samples. Further analysis revealed that the toxicity effect of 3-HP was due to the cation and anion components of this acid and some effects-specific to 3-HP. Genes related to the oxidative stress, DNA protection, and repair were upregulated in treated cells due to the lowered cytoplasmic pH caused by accumulated cations. 3-HP-treated E. coli used the arginine acid tolerance mechanism to increase the cytoplasmic pH. Additionally, the anion effects were manifested as imbalance in the osmotic pressure. Analysis of top ten highly upregulated genes suggests the formation of 3-hydroxypropionaldehyde under 3-HP stress. The transcriptomic analysis shed light on the global genetic reprogramming due to 3-HP stress and suggests strategies for increasing the tolerance of E. coli toward 3-HP.
Keywords: 3-Hydroxypropanoic acid; Transcriptomic analysis; Microarray; Escherichia coli ; Acid tolerance

Physicochemical Biomolecular Insights into Buffalo Milk-Derived Nanovesicles by Vijay Simha Baddela; Varij Nayan; Payal Rani; Suneel Kumar Onteru; Dheer Singh (544-557).
Milk is a natural nutraceutical produced by mammals. The nanovesicles of milk play a role in horizontal gene transfer and confer health-benefits to milk consumers. These nanovesicles contain miRNA, mRNA, and proteins which mediate the intercellular communication. In this work, we isolated and characterized the buffalo milk-derived nanovesicles by dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), scanning electron microscopy (SEM), Western probing, and Fourier transform infrared (FTIR) spectroscopy. The DLS data suggested a bimodal size distribution with one mode near 50 nm and the other around 200 nm for the nanovesicles. The NTA and SEM data also supported the size of nanovesicles within a range of 50–200 nm. The FTIR measurements of nanovesicles identified some prominent absorption bands attributable to the proteins (1300–1700 cm−1, amide A and amide B bands), lipids (2800–3100 cm−1), polysaccharides, and nucleic acids (900–1200 cm−1). The comparative expression profiles of immune miRNA signatures (miR-15b, miR-21, miR-27b, miR-125b, miR-155, and miR-500) in nanovesicles isolated from milk, serum, and urine revealed that these miRNAs are present abundantly (P < 0.05) in milk-derived nanovesicles. Milk miRNAs (miR-21 and 500) that were also found stable under different household storage conditions indicated that these could be biologically available to milk consumers. Overall, nanovesicles are a new class of bioactive compounds from buffalo milk with high proportion of stable immune miRNAs compared to urine and plasma of same animals.
Keywords: Milk; Nanovesicles; Exosomes; Dynamic light scattering; Nanoparticle tracking analysis; FTIR; miRNA

A novel microbial esterase BSE01281 identified from the Indian Ocean was cloned, expressed, and functionally characterized. Esterase BSE01281 could enanoselectively resolve (±)-1-phenylethanol and (±)-1-phenylethyl acetate through two types of enzymatic reactions. After the optimization of enzymatic reactions, BSE01281 could efficiently generate (R)-1-phenylethyl acetate with high enantiomeric excess (>99 %) and high conversion (42 %) after 96 h trans-esterification reactions. Additionally, BSE01281 could also produce (R)-1-phenylethanol (e.e. > 99 %) and (S)-1-phenylethyl acetate (e.e. > 95 %) at a conversion of 49 % through direct hydrolysis of inexpensive racemic 1-phenylethyl acetate for 8 h. Optically pure (R)-1-phenylethanol generated from direct enzymatic hydrolysis of racemic 1-phenylethyl acetate by BSE01281 is not easily prepared by dehydrogenases, which generally follow the “Prelog’s rule” and give (S)-1-phenylethanol instead.
Keywords: Esterase; Kinetic resolution; Chiral 1-phenylethanol; Chiral 1-phenylethyl acetate; High enantiomeric excess

Aspergillus niger Enhance Bioactive Compounds Biosynthesis As Well As Expression of Functional Genes in Adventitious Roots of Glycyrrhiza uralensis Fisch by Jing Li; Juan Wang; Jinxin Li; Dahui Liu; Hongfa Li; Wenyuan Gao; Jianli Li; Shujie Liu (576-593).
In the present study, the culture conditions for the accumulation of Glycyrrhiza uralensis adventitious root metabolites in balloon-type bubble bioreactors (BTBBs) have been optimized. The results of the culture showed that the best culture conditions were a cone angle of 90° bioreactor and 0.4–0.6–0.4-vvm aeration volume. Aspergillus niger can be used as a fungal elicitor to enhance the production of defense compounds in plants. With the addition of a fungal elicitor (derived from Aspergillus niger), the maximum accumulation of total flavonoids (16.12 mg g−1) and glycyrrhetinic acid (0.18 mg g−1) occurred at a dose of 400 mg L−1 of Aspergillus niger resulting in a 3.47-fold and 1.8-fold increase over control roots. However, the highest concentration of polysaccharide (106.06 mg g−1) was achieved with a mixture of elicitors (Aspergillus niger and salicylic acid) added to the medium, resulting in a 1.09-fold increase over Aspergillus niger treatment alone. Electrospray ionization tandem mass spectrometry (ESI-MSn) analysis was performed, showing that seven compounds were present after treatment with the elicitors, including uralsaponin B, licorice saponin B2, liquiritin, and (3R)-vestitol, only identified in the mixed elicitor treatment group. It has also been found that elicitors (Aspergillus niger and salicylic acid) significantly upregulated the expression of the cinnamate 4-hydroxylase (C4H), β-amyrin synthase (β-AS), squalene epoxidase (SE) and a cytochrome P450 monooxygenase (CYP72A154) genes, which are involved in the biosynthesis of bioactive compounds, and increased superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activity.
Keywords: Glycyrrhiza uralensis Fisch; Adventitious root; Aspergillus niger ; HPLC-ESI-MSn ; Anti-oxidant enzymes; Gene

Using pharmacological and biochemical approaches, Ca2+ involved in the betulin production in mycelia of Phellinus linteus induced by hydrogen sulfide (H2S) were investigated. The results showed that 2 mM H2S donor NaHS or 10 mM CaCl2 was found to enhance the betulin content in the mycelia of Phellinus to the maximum, which were 112.43 and 93.24 % higher than that in the control, respectively. Further, NaHS and CaCl2 co-treatment also showed positive outcome, which were 128.95 or 24.52 % higher than that in the control or NaHS treatment. At the same time, NaHS also enhanced the content of Ca2+ and CaM. But, the above positive inductive effects for Ca2+, CaM, and betulin production can be blocked with either Ca2+ channel blocker (LaCl3, 2-aminoethoxydiphenyl borate) or Ca2+ chelator (ethylenediaminetetraacetic acid (EDTA)). Among of them, betulin content was reduced 35.06 % by NaHS and EGTA to the minimum, and this reduction could be reversed by the application of CaCl2 (NaHS + EGTA + CaCl2). From above results, it can be concluded that endogenous and exogenous calcium involved in the betulin production from submerged culture of P. linteus induced by hydrogen sulfide.
Keywords: Hydrogen sulfide (H2S); Ca2+ ; Betulin; Phellinus linteus ; Submerged culture

The budC gene encoding a meso-2,3-butanediol dehydrogenase (BlBDH) from Bacillus licheniformis was cloned and overexpressed in Escherichia coli BL21(DE3). Sequence analysis reveals that this BlBDH belongs to short-chain dehydrogenase/reductase (SDR) superfamily. In the presence of NADH, BlBDH catalyzes the reduction of diacetyl to (3S)-acetoin (97.3 % ee), and further to (2S,3S)-2,3-butanediol (97.3 % ee and 96.5 % de). Similar to other meso-2,3-BDHs, it shows oxidative activity to racemic 2,3-butanediol whereas no activity toward racemic acetoin in the presence of NAD+. For diacetyl reduction and 2,3-butanediol oxidation, the pH optimum of BlBDH is 5.0 and 10.0, respectively. Unusually, it shows relatively high activity over a wide pH range from 5.0 to 8.0 for racemic acetoin reduction. BlBDH shows lower K m and higher catalytic efficiency toward racemic acetoin (K m = 0.47 mM, k cat /K m = 432 s–1·mM–1) when compared with 2,3-butanediol (K m = 7.25 mM, k cat /K m = 81.5 s–1·mM–1), indicating its physiological role in favor of reducing racemic acetoin into 2,3-butanediol. The enzymatic characterization of BlBDH provides evidence for the directed engineering of B. licheniformis for producing enantiopure 2,3-butanediol.
Keywords: Bacillus licheniformis ; meso-2,3-Butanediol dehydrogenase; Expression; Characterization