Applied Biochemistry and Biotechnology (v.184, #4)

Highly Efficient Deacidification of High-Acid Rice Bran Oil Using Methanol as a Novel Acyl Acceptor by Daoming Li; Muniba Faiza; Shahid Ali; Weifei Wang; Chin Ping Tan; Bo Yang; Yonghua Wang (1061-1072).
A highly efficient process for reducing the fatty acid (FA) content of high-acid rice bran oil (RBO) was developed by immobilized partial glycerides-selective lipase SMG1-F278N-catalyzed esterification/transesterification using methanol as a novel acyl acceptor. Molecular docking simulation indicated that methanol was much closer to the catalytic serine (Ser-171) compared with ethanol and glycerol, which might be one of the reasons for its high efficiency in the deacidification of high-acid RBO. Additionally, the reaction parameters were optimized to minimize the FA content of high-acid RBO. Under the optimal conditions (substrate molar ratio of methanol to FAs of 1.8:1, enzyme loading of 40 U/g, and at 30 °C), FA content decreased from 25.14 to 0.03% after 6 h of reaction. Immobilized SMG1-F278N exhibited excellent methanol tolerance and retained almost 100% of its initial activity after being used for ten batches. After purification by molecular distillation, the final product contained 97.86% triacylglycerol, 2.10% diacylglycerol, and 0.04% FA. The acid value of the final product was 0.09 mg KOH/g, which reached the grade one standard of edible oil. Overall, methanol was a superior acyl acceptor for the deacidification of high-acid RBO and the high reusability of immobilized SMG1-F278N indicates an economically attractive process.
Keywords: Deacidification; High-acid rice bran oil; Methanol; Molecular docking; Partial glycerides-selective lipase

Enhancement of Protective Effects of Radix Scutellariae on UVB-induced Photo Damage in Human HaCaT Keratinocytes by Yu-shuai Wang; Jin-Gyeong Cho; Eun-Son Hwang; Jung-Eun Yang; Wei Gao; Min-zhe Fang; Sheng-dao Zheng; Tae-Hoo Yi (1073-1093).
Radix Scutellariae (RS) has long been used in the treatment of inflammatory and allergic diseases. Its main flavonoids, baicalin (BG) and wogonoside (WG), can be hydrolyzed into their corresponding aglycones, baicalein (B) and wogonin (W). In this study, we developed a safe and effective method of transforming these glycosides using Peclyve PR. The transformation rate of BG and WG reached 98.5 and 98.1%, respectively, with 10% enzyme at 40 °C for 60 h. Furthermore, we compared the anti-photoaging activity of RS before and after enzyme treatment, as well as their respective main components, in UVB-irradiated HaCaT cells. Results found that enzyme-treated RS (ERS) appeared to be much better at preventing UVB-induced photoaging than RS. ERS significantly inhibited the upregulation of matrix metalloproteinase-1 and IL-6 caused by UVB radiation by inactivating the MAPK/AP-1 and NF-κB/IκB-α signaling pathways. ERS treatment also recovered UVB-induced reduction of procollagen type I by activating the TGF-β/Smad pathway. In addition, ERS exhibited an excellent antioxidant activity, which could increase the expression of cytoprotective antioxidants such as HO-1 and NQ-O1, by facilitating Nrf2 nuclear transfer. These findings demonstrated that the photoprotective effects of RS were significantly improved by enzyme-modified biotransformation.
Keywords: Radix Scutellariae ; Baicalin; Wogonoside; Peclyve PR; Biotransformation; Baicalein; Wogonin; Anti-skin photoaging

Molecular Characterization of Mosquitocidal Toxin (Surface Layer Protein, SLP) from Bacillus cereus VCRC B540 by Chinnasamy Mani; Jeyaperumal Selvakumari; YeonSoo Han; YongHun Jo; Krishnaraj Thirugnanasambantham; Somaiah Sundarapandian; Subbiah Poopathi (1094-1105).
A marine Bacillus cereus (VCRC B540) with mosquitocidal effect was recently reported from red snapper fish (Lutjanus sanguineous) gut and surface layer protein (S-layer protein, SLP) was reported to be mosquito larvicidal factor. In this present study, the gene encoding the surface layer protein was amplified from the genomic DNA and functionally characterized. Amplification of SLP-encoding gene revealed 1,518 bp PCR product, and analysis of the sequence revealed the presence of 1482 bp open reading frame with coding capacity for a polypeptide of 493 amino acids. Phylogenetic analysis revealed with homology among closely related Bacillus cereus groups of organisms as well as Bacillus strains. Removal of nucleotides encoding signaling peptide revealed the functional cloning fragment of length 1398 bp. Theoretical molecular weight (51.7 kDa) and isoelectric point (5.99) of the deduced functional SLP protein were predicted using ProtParam. The amplified PCR product was cloned into a plasmid vector (pGEM-T), and the open reading frame free off signaling peptide was subsequently cloned inpET-28a(+) and expressed in Escherichia coli BL21 (DE3). The isopropyl-β-D-thiogalactopyranoside (IPTG)-induced recombinant SLP was confirmed using western blotting, and functional SLP revealed mosquito larvicidal property. Therefore, the major findings revealed that SLP is a factor responsible for mosquitocidal activity, and the molecular characterization of this toxin was extensively studied.
Keywords: Surface layer protein; B. cereus ; Cloning; Mosquito; L. sanguineus ; Phylogenetic

Purification and Characterization of Bacteriocin Produced by a Strain of Enterococcus faecalis TG2 by Qianwen Xi; Jin Wang; Renpeng Du; Fangkun Zhao; Ye Han; Zhijiang Zhou (1106-1119).
The aims of this study were to purify and characterize a bacteriocin produced by a strain of Enterococcus faecalis TG2 and to test the safety of the strain. In this work, the active peptide was purified through precipitation with 70% saturated ammonium sulfate, cation-exchange chromatography, and gel filtration. The specific activity of purified bacteriocin was 30,073.42 AU/mg of protein, which corresponded to a 33.34-fold increase. The molecular mass of the purified bacteriocin was 6.3362 kDa determined by LC-MS/MS. The ten amino acid of N-terminal was MTRSKKLNLR and the ten amino acid of C-terminal was ATGGAAGWKS. The activity of the bacteriocin was unaffected by pH 2–10 and thermostable but was sensitive to proteolytic enzymes. The antimicrobial activity of the bacteriocin was not affected by metal ions. Tween-20, Tween-80, Triton X-100, and EDTA did not affect the bacteriocin activity and SDS was able to increase the activity of bacteriocin. Bacteriocin activity was not lost after treatment by < 8% NaCl. Inhibitory spectrum of the bacteriocin showed a wide range of activities against other lactic acid bacteria, food-spoilage, and food-borne pathogens. Ent. faecalis TG2 was sensitive to tetracycline and erythromycin but resistant to ampicillin, gentamicin, kanamycin, and chloramphenicol. Results from PCR indicated that Ent. faecalis TG2 did not harbor any virulence genes. The study suggests that Ent. faecalis TG2 and its bacteriocin might be used as bio-preservatives in food products.
Keywords: Bacteriocin; Enterococcus faecalis ; Purification; Characterization; Safety evaluation

Identification and Characterization of Anthocyanin Biosynthesis-Related Genes in Kohlrabi by Md Abdur Rahim; Arif Hasan Khan Robin; Sathishkumar Natarajan; Hee-Jeong Jung; Jeongyeo Lee; HyeRan Kim; Hoy-Taek Kim; Jong-In Park; Ill-Sup Nou (1120-1141).
Kohlrabi (Brassica oleracea var. gongylodes L.) is an important vegetable of the Brassicaceae family. The main edible part of kohlrabi is the swollen stem. The purple cultivars make anthocyanin mainly in the peel of the swollen stem, while in the leaf, it is limited to the midrib, but green cultivars do not. Anthocyanins are advantageous for both plants as well as humans. Two anthocyanin compounds were detected by high pressure liquid chromatography (HPLC) only in the peel of the purple kohlrabi cultivar. Three MYBs, three bHLHs, and one WD40 TF were identified as the candidate regulatory genes in kohlrabi. There was an abundance of transcript levels for the late biosynthetic genes more specifically for BoF3′H, BoDFR, BoLDOX, and BoGST in the purple peel while scarcely detectable in other tissues for both cultivars. The expression of BoPAP2 and BoTT8 was higher in the peel of the purple cultivar than the green cultivar. The expression of BoMYBL2.2 orthologue of Arabidopsis MYBL2, a negative regulator of anthocyanins, was dramatically decreased in the purple peel. The expression of BoACO1, a key gene for ethylene biosynthesis, and BoNCED3, an important gene of the ABA pathway, was down- and upregulated, respectively, in the peel of purple kohlrabi.
Keywords: Anthocyanin; MYB; bHLH; WD40 repeats; Negative regulator; Kohlrabi; Biosynthetic genes; HPLC; Ethylene; ABA

The conversion of starchy sago (Metroxylon sagu) pith waste (SPW), a lignocellulosic biomass waste, to fermentable sugars under mild conditions had been successfully demonstrated. The optimum depolymerization of SPW was achieved at 2 wt% sample loading which was catalyzed by 100 mM of oxalic acid in the presence of 25 wt% NaCl solution at 110 °C for 3 h. Up to 97% SPW sample was being converted into fermentable sugars with limited formation of by-products after two sequential depolymerization cycles. Both reaction temperature and concentration of oxalic acid were crucial parameters for the depolymerization of SPW which exhibited a high selectivity for the production of glucose over other reducing sugars.
Keywords: Depolymerization; Inorganic salt; Dicarboxylic acid; Lignocellulosic biomass

Prostate apoptosis response-4 (Par-4), an anticancer protein that interacts with cell surface receptor GRP78, can selectively suppress proliferation and induce apoptosis of cancer cells. The core domain of Par-4 (aa 137–195), designated as SAC, is sufficient to inhibit tumor growth and metastasis without harming normal tissues and organs. Nevertheless, the anticancer effects of SAC have not been determined in ovarian cancer cells. Here, we developed a novel method for producing native SAC in Escherichia coli using a small ubiquitin-related modifier (SUMO) fusion system. This fusion system not only greatly improved the solubility of target protein but also enhanced the expression level of SUMO-SAC. After purified by Ni-NTA affinity chromatography, SUMO tag was cleaved from SUMO-SAC fusion protein using SUMO protease to obtain recombinant SAC. Furthermore, we simplified the purification process by combining the SUMO-SAC purification and SUMO tag cleavage into one step. Finally, the purity of recombinant SAC reached as high as 95% and the yield was 25 mg/L. Our results demonstrated that recombinant SAC strongly inhibited proliferation and induced apoptosis in ovarian cancer cells SKOV-3. Immunofluorescence analysis and competitive binding reaction showed that recombinant SAC could specifically induce apoptosis of SKOV-3 cells through combination with cell surface receptor, GRP78. Therefore, we have developed an effective strategy for expressing bioactive SAC in prokaryotic cells, which supports the application of SAC in ovarian cancer therapy.
Keywords: Prostate apoptosis response-4 (Par-4); SAC; Small ubiquitin-related modifier (SUMO); One-step purification; Ovarian cancer; GRP78

Production of 6-O-l-Ascorbyl Palmitate by Immobilized Candida antarctica Lipase B by Manish G. Yadav; Monali R. Kavadia; Rajeshkumar N. Vadgama; Annamma A. Odaneth; Arvind M. Lali (1168-1186).
l-ascorbyl palmitate (ASP) is an oil-soluble derivative of ascorbic acid which is used extensively in food, cosmetics industry, and medical hygiene. Enzymatic synthesis of ascorbyl palmitate in tert-butyl alcohol was carried out using indigenously immobilized lipase preparation PyCal with ascorbic acid and palmitic acid as starting material. The developed batch process under optimized reaction conditions resulted in conversion of 90% with relatively shorter reaction time of 6 h. Continuous process in packed bed reactor gave conversion of 50% with space time yield of 15.46 g/L/h which was found to be higher than the reported literature on enzymatic synthesis of ascorbyl palmitate. The immobilized lipase used in the present work showed good reusability. Characterization of formed ascorbyl palmitate was carried out by FTIR, MS/MS, H1-NMR, and C13-NMR. The enzymatic process resulted in selective synthesis of 6-O-l-ascorbyl palmitate with purity of 98.6% and no side product formation. The use of underivatized starting materials, high space time yield of 15.46 g L−1 h−1, high recyclability of catalyst, and no by-product formation make the overall process highly efficient and clean in terms of energy consumption and waste generation, respectively. The optimized reaction parameters for ascorbyl palmitate synthesis in the present study can be used as a useful reference for industrial synthesis of fatty acid esters of ascorbic acid by enzymatic route.
Keywords: l-ascorbyl palmitate; Immobilized lipase; tert-Butyl alcohol

Applicability of Microaerobic Technology to Enhance BTEX Removal from Contaminated Waters by Paulo Igor M. Firmino; Raquel S. Farias; Amanda N. Barros; Patrícia G. C. Landim; Gervina B. M. Holanda; Elisa Rodríguez; Alexandre C. Lopes; André B. dos Santos (1187-1199).
As the addition of low concentrations of oxygen can favor the initial degradation of benzene, toluene, ethylbenzene, and xylenes (BTEX) compounds, this work verified the applicability of the microaerobic technology to enhance BTEX removal in an anaerobic bioreactor supplemented with high and low co-substrate (ethanol) concentrations. Additionally, structural alterations on the bioreactor microbiota were assessed throughout the experiment. The bioreactor was fed with a synthetic BTEX-contaminated water (~ 3 mg L−1 of each compound) and operated at a hydraulic retention time of 48 h. The addition of low concentrations of oxygen (1.0 mL min−1 of atmospheric air at 27 °C and 1 atm) assured high removal efficiencies (> 80%) for all compounds under microaerobic conditions. In fact, the applicability of this technology showed to be viable to enhance BTEX removal from contaminated waters, especially concerning benzene (with a 30% removal increase), which is a very recalcitrant compound under anaerobic conditions. However, high concentrations of ethanol adversely affected BTEX removal, especially benzene, under anaerobic and microaerobic conditions. Finally, although bacterial community richness decreased at low concentrations of ethanol, in general, the bioreactor microbiota could deal with the different operational conditions and preserved its functionality during the whole experiment.
Keywords: Monoaromatics; Contaminated water; Anaerobic treatment; Microaerobic treatment; Microaeration

Anaerobic Digestion of Cattle Manure Influenced by Swirling Jet Induced Hydrodynamic Cavitation by Michela Langone; Mariangela Soldano; Claudio Fabbri; Francesco Pirozzi; Gianni Andreottola (1200-1218).
In this work, a modified swirling jet-induced cavitation has been employed for increasing anaerobic digestion efficiency of cattle manure. The hydrodynamic cavitation (HC) treatment improved the organic matter solubilization and the anaerobic biodegradability of cattle manure. The degree of disintegration increased by 5.8, 8.9, and 15.8% after the HC treatment at 6.0, 7.0, and 8.0 bars, respectively. However, the HC treatment at 7.0 bars had better results in terms of methane production. This result may be attributed to the possible formation of toxic and refractory compounds at higher inlet pressures, which could inhibit the methanization process. Further, total Kjeldahl nitrogen content was found to decrease with increasing inlet pressures, as the pH and the turbulent mixing favored the ammonia stripping processes. HC treatment decreased the viscosity of the treated cattle manure, favoring the manure pumping and mixing. Considerations on the energy input due to the HC pre-treatment and the energy output due to the enhanced methane yield have been presented. A positive energy balance can be obtained looking at the improved operational practices in the anaerobic digesters after the implementation of the HC pre-treatment.
Keywords: Ammonia stripping; Anaerobic biodegradability; Cattle manure; Hydrodynamic cavitation; Viscosity

Effect of Feedstock Concentration on Biogas Production by Inoculating Rumen Microorganisms in Biomass Solid Waste by Na Li; Fenglin Yang; Huining Xiao; Jian Zhang; Qingwei Ping (1219-1231).
A methane production system with continuous stirred-tank reactor, rumen liquid as inoculate microorganisms, and paper mill excess sludge (PES) as feedstock was studied. The work mainly focused on revealing the effect of feedstock concentration on the biogas production, which was seldom reported previously for the current system. The optimal fermentation conditions were found as follows: pH = 7, T = 39 ± 1 °C, sludge retention time is 20 days, sludge with total solids (TS) are 1, 2, 3.5, 5, 10, and 13% in weight. Daily gas yields were measured, and biogas compositions were analyzed by gas chromatograph. Under such conditions, the optimum input TS was 10 wt%, and the biogas yield and volume gas productivity were 280.2 mL/g·TS and 1188.4 mL L−1·d−1, respectively. The proportions of CH4 and CO2 in the biogas were 65.1 and 34.2%. The CH4 yield reached 182.7 mL/g VS (volatile suspended solid), which was higher than previously reported values. The findings of this work have a significant effect on promoting the application of digesting PES by rumen microorganisms and further identified the technical parameter.
Keywords: Excess sludge; Rumen microorganisms; Lignocellulose biodegradation; Biogas production; Feedstock concentration

High xylanase activity and stability toward alkaline pH is strongly desired for pulping and bleaching processes. We previously enhanced thermal stability of Bacillus circulans xylanase (BCX) by inserting into a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) (the resulting complex named as PfMBP-BCX165). In the present study, we aimed to evolve the inserted BCX domain within PfMBP-BCX165 for greater xylanase activity toward alkaline pH while maintaining enhanced thermal stability. No BCX sequence variation was required for the thermal stabilization, thus allowing us to explore the entire BCX sequence space for the evolution. Specifically, we randomized the BCX sequence within PfMBP-BCX165 and then screened the resulting libraries to identify a PfMBP-BCX165 variant, PfMBP-BCX165T50R. The T50R mutation enhanced xylanase activity of PfMBP-BCX165 toward alkaline pH without compromising thermal stability. When compared to PfMBP-BCX165T50R, the corresponding unfused BCX mutant, BCXT50R, exhibited similar pH dependence of xylanase activity, yet suffered from limited thermal stability. In summary, we showed that one can improve thermal stability and xylanase activity of BCX toward alkaline pH by inserting into PfMBP followed by sequence variation of the BCX domain. Our study also suggested that insertional fusion to PfMBP would be a useful stabilizing platform for evolving many proteins.
Keywords: Protein engineering; Thermal stabilization; Insertional fusion; Bacillus circulans xylanase; Maltodextrin-binding protein from Pyrococcus furiosus

Growth of Cyanobacteria: Optimization for Increased Carbohydrate Content by Deepika Kushwaha; S. N. Upadhyay; Pradeep Kumar Mishra (1247-1262).
Growths of Lyngbya limnetica and Oscillatoria obscura were investigated at varying pH, light intensity, temperature, and trace element concentration with a view to optimize these parameters for obtaining the maximum carbohydrate content. The maximum growth for both strains was obtained at pH 9.0 and temperature 20 ± 3 °C using a light intensity of 68.0 μmol m−2 s−1 with continuous shaking. Growth under the nitrogen starvation condition affected the carbohydrate content more compared to the phosphorus starvation, and maximum concentrations were found as 0.660 and 0.621 g/g of dry biomass for L. limnetica and O. obscura, respectively. Under the optimized nitrogen-rich conditions, the specific growth rates for the two strains were found to be 0.187 and 0.215 day−1, respectively. The two-stage growth studies under nitrogen-rich (stage I) followed by nitrogen starvation (stage II) conditions were performed, and maximum biomass and carbohydrate productivity were found as 0.088 and 0.423 g L−1 day−1 for L. limnetica. This is the first ever attempt to evaluate and optimize various parameters affecting the growth of cyanobacterial biomass of L. limnetica and O. obscura as well as their carbohydrate contents.
Keywords: Cyanobacteria; Growth optimization; Carbohydrate; Specific growth rate; Lyngbya limnetica ; Oscillatoria obscura

Effect of the Presence of Surfactants and Immobilization Conditions on Catalysts’ Properties of Rhizomucor miehei Lipase onto Chitosan by Ulisses M. F. de Oliveira; Leonardo J. B. Lima de Matos; Maria Cristiane M. de Souza; Bruna B. Pinheiro; José C. S. dos Santos; Luciana R. B. Gonçalves (1263-1285).
Lipase from Rhizomucor miehei (RML) was immobilized onto chitosan support in the presence of some surfactants added at low levels using two different strategies. In the first approach, the enzyme was immobilized in the presence of surfactants on chitosan supports previously functionalized with glutaraldehyde. In the second one, after prior enzyme adsorption on chitosan beads in the presence of surfactants, the complex chitosan beads-enzyme was then cross-linked with glutaraldehyde. The effects of surfactant concentrations on the activities of free and immobilized RML were evaluated. Hexadecyltrimethylammonium bromide (CTAB) promoted an inhibition of enzyme activity while the nonionic surfactant Triton X-100 caused a slight increase in the catalytic activity of the free enzyme and the derivatives produced in both methods of immobilization. The best derivatives were achieved when the lipase was firstly adsorbed on chitosan beads at 4 °C for 1 h, 220 rpm followed by cross-link the complex chitosan beads-enzyme with glutaraldehyde 0.6% v.v−1 at pH 7. The derivatives obtained under these conditions showed high catalytic activity and excellent thermal stability at 60° and 37 °C. The best derivative was also evaluated in the synthesis of two flavor esters namely methyl and ethyl butyrate. At non-optimized conditions, the maximum conversion yield for methyl butyrate was 89%, and for ethyl butyrate, the esterification yield was 92%. The results for both esterifications were similar to those obtained when the commercial enzyme Lipozyme® and free enzyme were used in the same reaction conditions and higher than the one achieved in the absence of the selected surfactant.
Keywords: RML hyperactivation; Surfactants; Immobilization; Chitosan; Ester synthesis

Electroinduced Extraction of Human Ferritin Heavy Chain Expressed in Hansenula polymorpha by Valentina Ganeva; Bojidar Galutzov; Boyana Angelova; Manfred Suckow (1286-1307).
А protocol for the efficient and selective recovery of human ferritin heavy chain (FTH1) expressed intracellularly in Hansenula polymorpha was developed. It was based on electropermeabilisation and an increase in the cell wall porosity by pulsed electric field (PEF) treatment and subsequent incubation with a low concentration of a lytic enzyme. Irreversible plasma membrane permeabilisation was induced by applying rectangular electric pulses in the flow mode. The electrical treatment itself did not cause the release of the recombinant protein but induced the sensitisation of H. polymorpha cells to the lytic enzyme. Consequently, the subsequent incubation of the permeabilised cells with lyticase led to the recovery of approximately 90% of the recombinant protein, with a purification factor of 1.8. A similar efficiency was obtained by using the industrial lytic enzyme Glucanex. The released FTH1 appears in the form of an oligomer with a molecular mass of approximately 480 kDa, which is able to bind iron. The possibility for scaling the proposed protocol is discussed.
Keywords: Hansenula polymorpha ; PEF treatment; Electropermeabilisation; Extraction; FTH1; Lyticase; Glucanex

Efficient Malic Acid Production in Escherichia coli Using a Synthetic Scaffold Protein Complex by Sivachandiran Somasundaram; Gyeong Tae Eom; Soon Ho Hong (1308-1318).
Recently, malic acid has gained attention due to its potential application in food, pharmaceutical, and medical industries. In this study, the synthetic scaffold complex strategy was employed between the two key enzymes pyruvate kinase (PykF) and malic enzyme (SfcA); SH3 ligand was attached to PykF, and the SH3 domain was attached to the C-terminus of ScfA. Synthetic scaffold systems can organize enzymes spatially and temporally to increase the local concentration of intermediates. In a flask culture, the recombinant strain harboring scaffold complex produced a maximum concentration of 5.72 g/L malic acid from 10 g/L glucose. The malic acid production was significantly increased 2.1-fold from the initial culture period. Finally, malic acid production was elevated to 30.2 g in a 5 L bioreactor from recombinant strain XL-1 blue.
Keywords: Malic acid; Co-localization; Scaffold complex; Malic enzyme; Pathway flux

(R)-3, 5-Bis(trifluoromethyl)phenyl ethanol is a key chiral intermediate for the synthesis of aprepitant. Through a genome mining approach, an NADPH-dependent short-chain dehydrogenases derived from Burkholderia cenocepacia (Bc-SDR) was discovered with excellent anti-Prelog’s stereoselectivity of reducing 3, 5-bis(trifluoromethyl) acetophenone. The enzyme with 247 amino acids was successfully expressed in Escherichia coli and the molecular weight was about 26 kDa. Optimization of reaction conditions showed that the optimum temperature and pH of the enzyme was 25 °C and pH 7.0, respectively. Strong enhancement of enzyme activity was observed in the presence of 1 mM Mn2+. In addition, Bc-SDR exhibited (R)-selective enantioselectivity toward acetophenone derivatives, which makes it a potential catalyst for obtaining aromatic chiral alcohols as useful blocks in pharmaceutical applications.
Keywords: 3, 5-Bis(trifluoromethyl) acetophenone; Carbonyl reductase; Anti-Prelog’s; Burkholderia cenocepacia ; Aprepitant

Harnessing the Effect of pH on Lipid Production in Batch Cultures of Yarrowia lipolytica SKY7 by Mathiazhakan Kuttiraja; Ayed Dhouha; Rajeshwar Dayal Tyagi (1332-1346).
The objective of this research was to investigate the kinetics of lipid production by Yarrowia lipolytica SKY7 in the crude glycerol-supplemented media with and without the control of pH. Lipid and citric acid production were improved with the pH control condition. There was no significant difference observed in the biomass concentration with or without the pH control. In the pH-controlled experiments, the biomass and lipid concentration reached 18 and 7.78 g/L, (45.5% w/w), respectively, with lipid yield (Yp/s) of 0.179 g/g at 60 h of fermentation. The lipid production was directly correlated with growth and the process was defined as growth associated. After 60 h of fermentation, the lipid degradation was noticed in the pH-controlled reactor whereas it occurred after 84 h in the pH-uncontrolled reactor. Apart from lipid, citric acid was produced as the major extracellular product in both fermentations but the much lower concentration in uncontrolled pH. Based on the experimental results, it is evident that controlling the pH will enhance the lipid production by 15% compared to pH-uncontrolled fermentation.
Keywords: Y. lipolytica ; Crude glycerol; Biodiesel; Microbial lipid; C/N ratio; Effect of pH

Cloning, Expression, and Production of Xylo-Oligosaccharides by Using a Newly Screened Xylanase Isolated from Bovine Rumen by J. M. Lim; J. H. Choi; J. W. Choi; J. W. Yun; T. J. Park; J. P. Park (1347-1357).

Development of Adenosine Deaminase-Specific IgY Antibodies: Diagnostic and Inhibitory Application by Agnieszka Łupicka-Słowik; Mateusz Psurski; Renata Grzywa; Kamila Bobrek; Patrycja Smok; Maciej Walczak; Andrzej Gaweł; Tadeusz Stefaniak; Józef Oleksyszyn; Marcin Sieńczyk (1358-1374).
Adenosine deaminase (ADA) is currently used as a diagnostic marker for tuberculous pleuritis. Although ADA has been suggested as a potential marker for several types of cancer, the importance of each of ADA isoforms as well as their levels and enzymatic activities in tumors need to be further investigated. Herein we developed avian immunoglobulin Y highly specific to human ADA via hens immunization with calf adenosine deaminase. The obtained antibodies were used for the development of a sensitive double-egg yolk immunoglobulin (IgY) sandwich ELISA assay with an ADA detection limit of 0.5 ng/ml and a linearity range of up to 10 ng/ml. Specific, affinity-purified IgYs were able to recognize human recombinant ADA and ADA present in human cancer cell lines. In addition, antigen-specific IgY antibodies were able to inhibit catalytic activity of calf ADA with an IC50 value of 47.48 nM. We showed that generated IgY antibodies may be useful for ADA detection, thus acting as a diagnostic agent in immunoenzymatic assays.
Keywords: Egg yolk immunoglobulin (IgY); Adenosine deaminase (ADA); Anti-adenosine deaminase antibody; Affinity purification; Enzyme inhibition; ELISA

Stryphnodendron adstringens (“Barbatimão”) Leaf Fraction: Chemical Characterization, Antioxidant Activity, and Cytotoxicity Towards Human Breast Cancer Cell Lines by A. P. L. Sabino; L. M. S. Eustáquio; A. C. F. Miranda; C. Biojone; T. N. Mariosa; Cibele Marli Cação Paiva Gouvêa (1375-1389).
We evaluated the chemical composition, antioxidant activity, and antitumor potential of a fraction that was isolated from Stryphnodendron adstringens (barbatimão) leaf aqueous extract. Fraction is composed by gallic acid, procyanidin dimer B1, and (−)-epicatechin-3-O-gallate and it exhibits antioxidant and cytotoxic activities. Fraction was cytotoxic against two human breast cancer cell lines, ER (+) and MCF-7 and the triple-negative, MDA-MB-435. The sulforhodamine B assay showed that, as compared to normal control cells, the fraction significantly (P < 0.05) decreased cancer cell viability. The morphological alterations noted in the treated cancer cells were cell rounding-up, shrinkage, and nuclear condensation reduction of cell diameter and length. Treatment with fraction increased cancer cell expression of Bax, caspase-9, active caspase-3, caspase-8, LC-3, and beclin-1 and decreased Bcl-2, caspase-3, and pro-caspase-8 expression. Altogether, fraction is cytotoxic to both breast cancer cell lines, induces cell death, and its mechanism of action seems to include the induction of apoptosis. Our data support a positive role of the fraction as a chemopreventive agent for antineoplastic drug development.
Keywords: Anticancer; Cell death; DNA fragmentation; MCF-7; MDA-MB-435; Plant extract; Western blot

Chronic kidney disease may lead to subsequent tissue fibrosis. However, many factors can combat injurious stimuli in these tissues aiming to repair, heal, and alleviate any disturbance. Chemokines release, migration of inflammatory cells to the affected site, and activation of fibroblasts for the production of extracellular matrix are commonly observed in this disease. In the last years, many studies have focused on spironolactone (SPL), a mineralocorticoid receptor antagonist, and its pharmacological effects. In the present study, SPL was selected as an anti-inflammatory agent to combat nephrotoxicity and renal fibrosis induced by cisplatin. Mesenchymal stem cells (MSCs) were also selected in addition as a referring agent. Renal fibrosis induced by cisplatin intake significantly increased creatinine, urea, nuclear factor kappa B, insulin-like growth factor-1, fibroblast growth factor-23, and kidney malondialdehyde (MDA) content. Hepatocyte growth factor and renal content of reduced glutathione demonstrated a significant decrease. Histopathological examination of kidney tissues demonstrated marked cellular changes which are correlated with the biochemical results. Oral SPL intake (20 mg/kg/body weight) daily for 4 weeks and MSCs administration (3 × 106 cell/rat) intravenous to the experimental rats resulted in a significant improvement of both the biomarkers studied and the histopathological profile of the renal tissue. Individual administration of spironolactone and MSCs exhibited a marked anti-inflammatory potential and alleviated to a great extent the nephrotoxicity and renal fibrotic pattern induced by cisplatin.
Keywords: Nephrotoxicity; Cisplatin; Renal fibrosis; Spironolactone; Nuclear factor kappa B; Fibroblast growth factor-23

Substrate Scope of O-Methyltransferase from Streptomyces peucetius for Biosynthesis of Diverse Natural Products Methoxides by Prakash Parajuli; Ramesh Prasad Pandey; Thi Huyen Trang Nguyen; Dipesh Dhakal; Jae Kyung Sohng (1404-1420).
Methylation is a common post-modification reaction that is observed during the biosynthesis of secondary metabolites produced by plants and microorganisms. Based on the sequence information from Streptomyces peucetius ATCC27952, a putative O-methyltransferase (OMT) gene SpOMT7740 was polymerase chain reaction amplified and cloned into E. coli BL21 (DE3) host to test the substrate promiscuity and conduct functional characterization. In vitro and in vivo reaction assays were carried out over various classes of substrates: flavonoids (flavonol, flavones, and isoflavonoid), chalcones, anthraquinones, anthracyclines, and sterol molecules, and the applications in synthesizing diverse classes of O-methoxy natural products were also illustrated. SpOMT7740 catalyzed the O-methylation reaction to form various natural and non-natural O-methoxides, includes 7-hydroxy-8-O-methoxy flavone, 3-O-methoxy flavone, three mono-, di-, and tri-O-methoxy genistein, mono-O-methoxy phloretin, mono-O-methoxy luteolin, 3-O-methoxy β-sitosterol, and O-methoxy anthraquinones (emodin and aloe emodin) and O-methoxy anthracycline (daunorubicin) exhibiting diverse substrate flexibility. Daunorubicin is a native secondary metabolite of S. peucetius. Among the compounds tested, 7,8-dihydroxyflavone was the best substrate for bioconversion to 7-hydroxy-8-O-methoxy flavone, and it was structurally elucidated. This enzyme showed a flexible catalysis over the given ranges of temperature, pH, and divalent cationic conditions for O-methylation.
Keywords: O-methyltransferase; SpOMT7740; Streptomyces ; Methylated natural products; Substrate flexibility

Neuraminidase (NA), a surface protein, facilitates the release of nascent virus and thus spreads infection. It has been renowned as a potential drug target for influenza A virus infection. The drugs such as oseltamivir, zanamivir, peramivir, and laninamivir are approved for the treatment of influenza infection. Additionally, investigational drugs namely MK2206, tamiphosphor, crenatoside, and dehydroepiandrosterone (DHEA) are also available for the treatment. However, recent outbreaks of highly pathogenic and drug-resistant influenza A strains highlighted the need to discover novel NA inhibitor. Keeping this in mind, in the current investigation, an effort was made to ascertain potent inhibitors using pharmacophore-based virtual screening and docking approach. A 3D pharmacophore model was generated based on the chemical features of approved and investigational NA inhibitors using PHASE module of Schrödinger suite. The model consists of two hydrogen bond acceptors (A), one hydrogen bond donor (D), and one positively charged group (P), AADP. Subsequently, molecules with same pharmacophoric features were screened from among the two million compounds available in the ZINC database using the generated pharmacophore hypothesis. Ligand filtration was also done to obtain an efficient collection of hit molecules by employing Lipinski “rule of five” using Qikprop module. Finally, the screened molecule was subjected to docking and molecular dynamic simulations to examine the inhibiting activity of the compounds. The results of our analysis suggest that “acebutolol hydrochloride” (156792) could be the promising candidates for the treatment of influenza A virus infection.
Keywords: Neuraminidase; Pharmacophore model; Virtual screening; ZINC database; Molecular simulation

Comparison of One-Stage Batch and Fed-Batch Enzymatic Hydrolysis of Pretreated Hardwood for the Production of Biosugar by Liang He; Qiang Han; Hasan Jameel; Hou-min Chang; Richard Phillips; Ziyu Wang (1441-1452).
Fed-batch method has shown a great promise in debottlenecking the high-solid enzymatic hydrolysis for the commercialization of cellulosic biosugar conversion for biofuel/biochemical production. To further improve enzymatic hydrolysis efficiency at high solid loading, fed-batch methods of green liquor-pretreated hardwood were performed to evaluate their effects on sugar recovery by comparing with one-stage batch method in this study. Among all the explored conditions, the fed-batch at 15% consistency gave higher sugar recovery on green liquor-pretreated hardwood compared to that of one-stage batch. By using general linear model analysis, the percentage of enzymatic sugar recovery in fed-batch consistency method (increasing consistency from the initial 10.7 to 15% at intervals of 24 and 48 h) was higher than that of batch hydrolysis at higher density of 15% consistency. Under that best fed-batch condition, the total sugar recovery of pretreated hardwood in enzymatic hydrolysate reached approximately 48.41% at Cellic® enzyme loading of 5 filter-paper unit (FPU)/g and 58.83% at Cellic® enzyme loading of 10 FPU/g with a hydrolysis time of 96 h.
Keywords: Hardwood; Pretreatment; Fed-batch methods; Enzymatic hydrolysis; Sugar recovery