Applied Biochemistry and Biotechnology (v.179, #5)

Chlorogenic Acids Biosynthesis in Centella asiatica Cells Is not Stimulated by Salicylic Acid Manipulation by E. N. Ncube; P. A. Steenkamp; N. E. Madala; I. A. Dubery (685-696).
Exogenous application of synthetic and natural elicitors of plant defence has been shown to result in mass production of secondary metabolites with nutraceuticals properties in cultured cells. In particular, salicylic acid (SA) treatment has been reported to induce the production of phenylpropanoids, including cinnamic acid derivatives bound to quinic acid (chlorogenic acids). Centella asiatica is an important medicinal plant with several therapeutic properties owing to its wide spectrum of secondary metabolites. We investigated the effect of SA on C. asiatica cells by monitoring perturbation of chlorogenic acids in particular. Different concentrations of SA were used to treat C. asiatica cells, and extracts from both treated and untreated cells were analysed using an optimised UHPLC-QTOF-MS/MS method. Semi-targeted multivariate data analyses with the aid of principal component analysis (PCA) and orthogonal projection to latent structures-discriminant analysis (OPLS-DA) revealed a concentration-dependent metabolic response. Surprisingly, a range of chlorogenic acid derivatives were found to be downregulated as a consequence of SA treatment. Moreover, irbic acid (3,5-O-dicaffeoyl-4-O-malonilquinic acid) was found to be a dominant CGA in C. asiatica cells, although the SA treatment also had a negative effect on its concentration. Overall SA treatment was found to be an ineffective elicitor of CGA production in cultured C. asiatica cells.
Keywords: Caffeoylquinic acids; Centella asiatica ; Chlorogenic acids; Elicitation; Neuroprotection; Salicylic acid

Identifying a Carotenoid Cleavage Dioxygenase 4a Gene and Its Efficient Agrobacterium-Mediated Genetic Transformation in Bixa orellana L. by Mohan Sankari; Hridya Hemachandran; Amirtha Anantharaman; Subramanian Babu; Renata Rivera Madrid; George Priya Doss C; Devanand P. Fulzele; Ramamoorthy Siva (697-714).
Carotenoids are metabolized to apocarotenoids through the pathway catalysed by carotenoid cleavage oxygenases (CCOs). The apocarotenoids are economically important as it is known to have therapeutic as well as industrial applications. For instance, bixin from Bixa orellana and crocin from Crocus sativus are commercially used as a food colourant and cosmetics since prehistoric time. In our present study, CCD4a gene has been identified and isolated from leaves of B. orellana for the first time and named as BoCCD4a; phylogenetic analysis was carried out using CLUSTAL W. From sequence analysis, BoCCD4a contains two exons and one intron, which was compared with the selected AtCCD4, RdCCD4, GmCCD4 and CmCCD4a gene. Further, the BoCCD4a gene was cloned into pCAMBIA 1301, transformed into Agrobacterium tumefaciens EHA105 strain and subsequently transferred into hypocotyledons and callus of B. orellana by agro-infection. Selection of stable transformation was screened on the basis of PCR detection by using GUS and hptII specific primer, which was followed by histochemical characterization. The percent transient GUS expression in hypocotyledons and callus was 84.4 and 80 %, respectively. The expression of BoCCD4a gene in B. orellana was confirmed through RT-PCR analysis. From our results, the sequence analysis of BoCCD4a gene of B. orellana was closely related to the CsCCD4 gene of C. sativus, which suggests this gene may have a role in various processes such as fragrance, insect attractant and pollination.
Keywords: Bixa orellana ; Carotenoid cleavage dioxygenase; CCD4a; RT-PCR; Apocarotenoids; Agro-infection

Bioproduction of d-Tagatose from d-Galactose Using Phosphoglucose Isomerase from Pseudomonas aeruginosa PAO1 by Manisha J. Patel; Arti T. Patel; Rekha Akhani; Samir Dedania; Darshan H. Patel (715-727).
Pseudomonas aeruginosa PAO1 phosphoglucose isomerase was purified as an active soluble form by a single-step purification using Ni-NTA chromatography that showed homogeneity on SDS-PAGE with molecular mass ∼62 kDa. The optimum temperature and pH for the maximum isomerization activity with d-galactose were 60 °C and 7.0, respectively. Generally, sugar phosphate isomerases show metal-independent activity but PA-PGI exhibited metal-dependent isomerization activity with aldosugars and optimally catalyzed the d-galactose isomerization in the presence of 1.0 mM MnCl2. The apparent Km and Vmax for d-galactose under standardized conditions were calculated to be 1029 mM (±31.30 with S.E.) and 5.95 U/mg (±0.9 with S.E.), respectively. Equilibrium reached after 180 min with production of 567.51 μM d-tagatose from 1000 mM of d-galactose. Though, the bioconversion ratio is low but it can be increased by immobilization and enzyme engineering. Although various l-arabinose isomerases have been characterized for bioproduction of d-tagatose, P. aeruginosa glucose phosphate isomerase is distinguished from the other l-arabinose isomerases by its optimal temperature (60 °C) for d-tagatose production being mesophilic bacteria, making it an alternate choice for bulk production.
Keywords: Pseudomonas aeruginosa PAO1; Phosphoglucose isomerase; d-Tagatose; d-Galactose

In this study, N-hexanoyl-homoserine lactone (C6-HSL), a member of the N-acyl-homoserine lactone class of microbial quorum sensing (QS) signaling molecules, was used to improve microbial flocculant production. After exogenous C6-HSL was added, exopolysaccharide concentration of microbial flocculants was improved by 1.6-fold and flocculation rate of microbial flocculants was increased by 10 %. Fermentation conditions with added C6-HSL were further optimized through response surface methodology. The obtained optimal fermentation conditions were as follows: added C6-HSL concentration of 0.45 μM, fermentation temperature of 30.4 °C, and initial fermentation pH of 7.25. Under these optimal fermentation conditions, the resulting exopolysaccharide concentration was improved by 1.75-fold and flocculation rate was increased by 10 % compared with that of the control group. The yield of microbial flocculants was also improved by 1.75-fold. Results demonstrated that the existence of QS system in Agrobacterium tumefaciens strain F2 played the important roles in the microbial flocculant production.
Keywords: Microbial flocculant; Quorum sensing; N-acyl-homoserine lactone; Production efficiency

As the second abundant natural carbohydrate, xylan is normally prepared through alkaline extraction and then used for xylo-oligosaccharides (XOS) production. However, the extracted xylan inevitably contains salt, ethanol, and pigment. In order to investigate the effects of these impurities on XOS production, the alkaline-extracted xylan with different kinds and concentrations of impurities was made and then hydrolyzed using alkaline xylanase (EC 3.2.1.8) to produce XOS. The results showed that a certain concentration of salt (NaCl) promoted the XOS production, while ethanol and pigment inhibited the enzymatic hydrolysis process significantly. The color value mainly ascribed to the phenolic compounds binding to xylan was a key restriction factor in the enzymatic hydrolysis later stage. Using optimal xylan sample (with 10 mg/mL NaCl, color value of 4.6 × 105, without ethanol) as substrate, the highest XOS yield of 58.58 % was obtained. As the substrate of XOS production, prepared xylan should contain colored materials and ethanol as less as possible, however, retains appropriate salt.
Keywords: Xylan; Enzymatic hydrolysis; Xylo-oligosaccharides; Sodium chloride; Ethanol; Pigment

Reliable Selection and Holistic Stability Evaluation of Reference Genes for Rice Under 22 Different Experimental Conditions by Zhaohai Wang; Ya Wang; Jing Yang; Keke Hu; Baoguang An; Xiaolong Deng; Yangsheng Li (753-775).
Stable and uniform expression of reference genes across samples plays a key role in accurate normalization of gene expression by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). For rice study, there is still a lack of validation and recommendation of appropriate reference genes with high stability depending on experimental conditions. Eleven candidate reference genes potentially owning high stability were evaluated by geNorm and NormFinder for their expression stability in 22 various experimental conditions. Best combinations of multiple reference genes were recommended depending on experimental conditions, and the holistic stability of reference genes was also evaluated. Reference genes would become more variable and thus needed to be critically selected in experimental groups of tissues, heat, 6-benzylamino purine, and drought, but they were comparatively stable under cold, wound, and ultraviolet-B stresses. Triosephosphate isomerase (TI), profilin-2 (Profilin-2), ubiquitin-conjugating enzyme E2 (UBC), endothelial differentiation factor (Edf), and ADP-ribosylation factor (ARF) were stable in most of our experimental conditions. No universal reference gene showed good stability in all experimental conditions. To get accurate expression result, suitable combination of multiple reference genes for a specific experimental condition would be a better choice. This study provided an application guideline to select stable reference genes for rice gene expression study.
Keywords: RT-qPCR; Tissue; Stress; geNorm; NormFinder; Rice (Oryza sativa)

Effect of Medium pH on Rhodosporidium toruloides NCYC 921 Carotenoid and Lipid Production Evaluated by Flow Cytometry by Carla Dias; Corália Silva; Claudia Freitas; Alberto Reis; Teresa Lopes da Silva (776-787).
The effect of the culture medium pH (3.5–6.0) on the carotenoid and lipid (as fatty acids) production by the yeast Rhodosporidium toruloides NCYC 921 was studied. Flow cytometry was used to evaluate the yeast’s physiological response to different culture medium pH values. The yeast biomass concentration and lipid content were maxima at pH 4.0 (5.90 g/L and 21.85 % w/w, respectively), while the maximum carotenoid content (63.37 μg/g) was obtained at pH 5.0. At the exponential phase, the yeast cell size and internal complexity were similar, at different medium pH. At the stationary phase, the yeast cell size and internal complexity decreased as the medium pH increased. At the exponential phase, the proportion of cells with polarized membranes was always high (>80 %) but at the stationary phase, the proportion of yeast cells with depolarized membranes was dominant (>65 %) and increased with the medium pH increase. The results here reported may contribute for yeast bioprocesses optimization. For the first time, multiparameter flow cytometry was used to evaluate the impact of medium pH changes on the yeast cell physiological status, specifically on the yeast membrane potential, membrane integrity, cell size and internal complexity.
Keywords: Yeast; Medium pH; Flow cytometry; Fatty acids; Carotenoids; Rhodosporidium toruloides ; Lipids

The intracellular proteomes of the Penicillium chrysogenum throughout pilot and industrial processes were investigated by using 2-DE combined with MALDI-TOF-TOF MS, respectively. We detected a total of 223 spots corresponding to 154 proteins and 231 spots corresponding to 157 proteins throughout pilot and industrial processes, respectively. The levels of glyceraldehyde-3-phosphate dehydrogenase increased (5.1- and 2.5-fold) under the pilot process, while its levels were no significant changes under the industrial process at 140 and 170 h when compared with that at 2 h. The levels of isocitrate lyase and fumarate hydratase were increased significantly under the industrial process, while their levels had no obvious changes after 20 h of fermentation throughout the pilot process. These results indicate that there were remarkable differences in carbohydrate metabolism (including glycolysis, gluconeogenesis, pentose phosphate pathway, and tricarboxylic acid cycle) of P. chrysogenum during the pilot and industrial fermentations, which likely result in alterations of the primary metabolism and penicillin biosynthesis. Moreover, the differences in the levels of proteins involved in amino acid metabolisms (including valine, cysteine, and α-aminoadipic acid biosynthesis) indicated that the pilot and industrial processes influenced the supplies of penicillin precursors. Compared with that at 2 h, the maximum levels of superoxide (6.9-fold, at 32 h) and catalase (9-fold, at 80 h) during the industrial process and the maximum levels of superoxide (1.2-fold, at 20 h) and catalase (7.7-fold at 128 h) during the pilot process revealed the significant difference in cell redox homeostasis and stress responses during scale-up fermentation. Particularly, 10 spots corresponding to isopenicillin N synthetase and 4 spots corresponding to isopenicillin N (IPN) acyltransferase in pilot and industrial processes were identified, respectively. The levels of IPN acyltransferase (spots 197 and 198) and CoA ligase at 80 h during the industrial process were around 2-fold of that during the pilot process, indicating that the industrial process with a higher penicillin production per cell might provide available environments to induce over-expression of IPN acyltransferase and accelerate penicillin formation. These results provide new insights into the globally potential responses of P. chrysogenum to variations of environments in different fermentation scales so as to consequently regulate the penicillin production.
Keywords: Proteome analysis; Penicillin; Industrial fermentation; Penicillium chrysogenum

Antifouling Activity of Lipidic Metabolites Derived from Padina tetrastromatica by Murugan Suresh; Palanisamy Iyapparaj; Perumal Anantharaman (805-818).
An attempt has been made to identify the potential seaweed for antifouling property due to the growing need for environmentally safe antifouling systems. The antibacterial, antimicroalgal, and antimussel foot adherence potentials of methanol, dichloromethane, and hexane extracts of the chosen seaweeds such as Padina tetrastromatica, Caulerpa taxifolia, and Amphiroa fragilissima have been compared against copper sulfate. Among the extracts, the maximum antibacterial activities were exhibited by the methanol extract of P. tetrastromatica. The minimum inhibitory concentration (MIC) of the methanolic extract of P. tetrastromatica was found to be 10 and 1 μg/ml against test biofilm bacteria and diatoms, respectively. The antimussel foot adherence assay indicated that the extract had inhibited the foot adherence of the green mussels Perna viridis with the effective concentration (EC50) of 25.51 ± 0.03 μg/ml, and lethal concentration for 50 % mortality (LC50) was recorded at 280.22 ± 0.12 μg/ml. Based on the prolific results, the crude methanolic extract of P. tetrastromatica was subjected to purification using silica gel column and thin-layer chromatography (TLC). Then, the active compounds of the bioassay-guided fraction (F13) were identified using gas chromatography coupled with mass spectroscopy (GC-MS), and it was observed that fatty acids were the major components, which may be responsible for the antifouling properties.
Keywords: P. tetrastromatica ; Antifouling; Methanol; TLC; GC-MS; Copper sulfate

Whole Body Vibration Improves Insulin Resistance in db/db Mice: Amelioration of Lipid Accumulation and Oxidative Stress by Ying Liu; Mingming Zhai; Fan Guo; Tengrui Shi; Jiangzheng Liu; Xin Wang; Xiaodi Zhang; Da Jing; Chunxu Hai (819-829).
Insulin resistance (IR) is the hallmark of type 2 diabetes mellitus (T2DM), which is one of the most important chronic noncommunicable diseases. Effective and feasible strategies to treat IR are still urgently needed. Previous research studies reported that whole body vibration (WBV) was beneficial for IR in clinical; however, its underlying mechanisms remains unknown. In the present study, db/db mice were treated with WBV administration 60 min/day for 12 weeks and the impaired insulin sensitivity was improved. Besides, liver steatosis was also ameliorated. Further explorations revealed that WBV could reduce the expression of SREBP1c and increase the expression of GSH-Px and consequently suppress oxidative stress. In conclusion, WBV attenuates oxidative stress to ameliorate liver steatosis and thus improves insulin resistance in db/db mice. Therefore, WBV administration is a promising treatment for individuals who suffered from central obesity and IR.
Keywords: Insulin resistance; Liver steatosis; Oxidative stress; Glutathione peroxidases; Whole body vibration

Metabolite and Mechanistic Basis of Antifungal Property Exhibited by Endophytic Bacillus amyloliquefaciens BmB 1 by B. Jasim; Rosemin Benny; Rohini Sabu; Jyothis Mathew; E. K. Radhakrishnan (830-845).
Plants are ubiquitously colonized by endophytic microorganisms which contribute significantly to plant health through production of plant growth regulators or disease suppression. In the present study, an endophytic bacterial isolate designated as BmB 1 with significant antifungal and plant growth promoting properties was isolated from the stem tissue of Bacopa monnieri (L.) Pennell. The isolate was studied in detail for the molecular and chemical basis of its bioactivity which proved it to have the presence of surfactin, iturin, and type I polyketide synthase (PKS) genes. For the analysis of the chemical basis of antifungal property, extract of the isolate was initially checked for its activity on test pathogens and LC-MS/MS based analysis further confirmed the presence of bacillomycin (m/z (M+H+) 1031.8) and surfactin (m/z (M+H+) 1008.6 and 1022.6) in the extract prepared. The light microscopic and SEM analysis of the treated and untreated mycelia of the pathogens clearly revealed the hypal destruction caused by the compounds produced by the selected isolate. This confirms the ability of the organism to directly inhibit the growth of the tested pathogens. The GC-MS analysis also confirmed the isolate to have the presence of volatile compounds with the expected role to induce induced systemic resistance (ISR) of the plant. Because of the multitargeted antifungal property, the isolate which was identified as Bacillus amyloliquefaciens can have potential biocontrol applications.
Keywords: Endophytic B. amyloliquefaciens ; Peptide antibiotics; PCR screening; LC-MS/MS; GC-MS analysis; SEM analysis; B. monnieri

Anaerobic Mesophilic Codigestion of Rice Straw and Chicken Manure: Effects of Organic Loading Rate on Process Stability and Performance by Zili Mei; Xiaofeng Liu; Xianbo Huang; Dong Li; Zhiying Yan; Yuexiang Yuan; Yajun Huang (846-862).
To investigate the effects of organic loading rate (OLR) on performance and stability of mesophilic co-digestion of rice straw (RS) and chicken manure (CM), benchtop experiments (40 L) were carried out at OLRs of 3.0, 3.6, 4.2, 4.8, 6.0, 8.0, and 12.0 kg volatile solid (VS)/(m3·day) with volatile solid (VS) ratio of 1:1 (RS/CM) which was based on batch tests. Anaerobic co-digestion was slightly and severely inhibited by the accumulation of ammonia when the digester was overloaded at an OLR of 6 and 12 kg VS/(m3·day), respectively. The recommended OLR for co-digestion is 4.8 kg VS/(m3·day), which corresponds to average specific biogas production (SBP) of 380 L/kg VS and volumetric biogas production rate (VBPR) of 1.8 m3/(m3·day). An OLR of 6–8 kg VS/(m3·d) with SBP of 360–440 L/kg VS and VBPR of 2.1–3.5 m3/(m3·day) could be considered, if an Anaerobic digestion (AD) system assisted by in situ removal of ammonia was adopted.
Keywords: Biogas; Co-digestion; Rice straw; Chicken manure; Process stability

High costs of natural cellulose utilization and cellulase production are an industrial challenge. In view of this, an isolated soil actinobacterium identified as Promicromonospora sp. VP111 showed potential for production of major cellulases (CMCase, FPase, and β-glucosidase) utilizing untreated agricultural lignocellulosic wastes. Extensive disintegration of microcrystalline cellulose and adherence on it during fermentation divulged true cellulolytic efficiency of the strain. Conventional optimization resulted in increased cellulase yield in a cost-effective medium, and the central composite design (CCD) analysis revealed cellulase production to be limited by cellulose and ammonium sulfate. Cellulase activities were enhanced by Co+2 (1 mM) and retained up to 60 °C and pH 9.0, indicating thermo-alkaline tolerance. Cellulases showed stability in organic solvents (25 % v/v) with log P ow  ≥ 1.24. Untreated wheat straw during submerged fermentation was particularly degraded and yielded about twofold higher levels of cellulases than with commercial cellulose (Na-CMC and avicel) which is especially economical. Thus, this is the first detailed report on cellulases from an efficient strain of Promicromonospora that was non-hemolytic, alkali-halotolerant, antibiotic (erythromycin, kanamycin, rifampicin, cefaclor, ceftazidime) resistant, multiple heavy metal (Mo+6 = W+6 > Pb+2 > Mn+2 > Cr+3 > Sn+2), and organic solvent (n-hexane, isooctane) tolerant, which is industrially and environmentally valuable.
Keywords: Actinobacteria; Alkali halo tolerant; Lignocellulose; Organic solvent tolerant; Promicromonospora sp. VP111

Efficient Secretory Overexpression of Endoinulinase in Escherichia coli and the Production of Inulooligosaccharides by Peipei Wang; Jiangfeng Ma; Yue Zhang; Min Zhang; Mingke Wu; Zhongxue Dai; Min Jiang (880-894).
Endoinulinase production was achieved by heteroexpression of endoinulinase-encoding gene from Aspergillus ficuum which is an eukaryotic organism in Escherichia coli BL21 (DE3). Further analysis demonstrated that the native signal peptide existed in inu2 gene lowered the enzyme expression level. To realize extracellular accumulation of target protein and improve its expression level, native signal peptide was substituted with pelB, ompC, and pelB fusing with the native signal peptides; then, the effects on endoinulinase production were investigated. As a result, E. coli A606-3, with replacement of pelB as its signal peptide, showed the highest endoinulinase enzyme activity (75.22 U/mg). Also, it suggested that eukaryotic signal peptides have an inhibition on enzyme expression in prokaryotic organism. Moreover, the condition for inulooligosaccharide (IOS) production from inulin was optimized, and an IOS yield of 94.41 % was achieved under the condition of 15 % (w/v) inulin, purified endoinulinase dosage of 5 U/g inulin, 55 °C, and pH 4.6 for 24 h. The major products of hydrolysis of inulin were identified as DP3 to DP7.
Keywords: Endoinulinase; Aspergillus ficuum ; Escherichia coli ; Signal peptide; Inulooligosaccharides

Optimization of the Production of Inactivated Clostridium novyi Type B Vaccine Using Computational Intelligence Techniques by P. L. M. Aquino; F. S. Fonseca; O. D. Mozzer; R. C. Giordano; R. Sousa Jr. (895-909).
Clostridium novyi causes necrotic hepatitis in sheep and cattle, as well as gas gangrene. The microorganism is strictly anaerobic, fastidious, and difficult to cultivate in industrial scale. C. novyi type B produces alpha and beta toxins, with the alpha toxin being linked to the presence of specific bacteriophages. The main strategy to combat diseases caused by C. novyi is vaccination, employing vaccines produced with toxoids or with toxoids and bacterins. In order to identify culture medium components and concentrations that maximized cell density and alpha toxin production, a neuro-fuzzy algorithm was applied to predict the yields of the fermentation process for production of C. novyi type B, within a global search procedure using the simulated annealing technique. Maximizing cell density and toxin production is a multi-objective optimization problem and could be treated by a Pareto approach. Nevertheless, the approach chosen here was a step-by-step one. The optimum values obtained with this approach were validated in laboratory scale, and the results were used to reload the data matrix for re-parameterization of the neuro-fuzzy model, which was implemented for a final optimization step with regards to the alpha toxin productivity. With this methodology, a threefold increase of alpha toxin could be achieved.
Keywords: Clostridium novyi ; Neuro-fuzzy; Simulated annealing; Mathematical modeling of bioprocesses; Culture medium optimization