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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.168, #5)
Role of TDZ in the Quick Regeneration of Multiple Shoots from Nodal Explant of Vitex trifolia L.—an Important Medicinal Plant by Md Rafique Ahmed; Mohammad Anis (pp. 957-966).
The effect of thidiazuron (TDZ) has been investigated in shoot multiplication for a simple, efficient, rapid, and commercially applicable regeneration protocol of an important medicinal plant, Vitex trifolia. Multiple shoots were induced in nodal explants obtained from a mature tree on Murashige and Skoog (MS) medium supplemented with TDZ in various concentrations (0.5, 1.0, 2.5, 5.0, 7.5, or 10.0 μM). Prolonged exposure of the culture to TDZ had an adverse affect. To avoid this, the cultures were transferred to TDZ-free MS medium or MS medium fortified with various concentrations of 6-benzyladenine (BA) alone or in combination with α-naphthalene acetic acid (NAA) to enhance multiplication, proliferation, and elongation of induced shoots. Optimum shoot multiplication and elongation was achieved when TDZ-exposed explants were repeatedly subcultured on MS media containing a combination of 1.0 μM BA and 0.5 μM NAA. The highest shoot regeneration frequency (90 %) and maximum number (22.3 ± 0.2) of shoots per explant with shoot length of (5.2 ± 0.2 cm) was recorded on MS medium fortified with 5.0 μM TDZ. In vitro rooting of isolated shoots was achieved best in half-strength MS medium containing 0.5 μM NAA. Properly rooted plantlets were successfully hardened off and acclimatized in thermocol cups containing sterile Soilrite. These plantlets were then transferred to pots containing different potting substrate; percentage survival of the plantlets was highest in vermiculite/garden soil mixture (1:1) and successfully transfer to greenhouse under sunlight.
Keywords: Verbenaceae; Shoot multiplication; In vitro rooting; Thidiazuron; Acclimatization
Selection of Single Chain Variable Fragments Specific for the Human-Inducible Costimulator Using Ribosome Display by Yangbin Pan; Weiping Mao; Xuanxuan Liu; Chong Xu; Zhijuan He; Wenqian Wang; Hao Yan (pp. 967-979).
We applied a ribosome display technique to a mouse single chain variable fragment (scFv) library to select scFvs specific for the inducible costimulator (ICOS). mRNA was isolated from the spleens of BALB/c mice immunized with ICOS protein. Heavy and κ chain genes (VH and κ) were amplified separately by reverse transcriptase polymerase chain reaction, and the anti-ICOS VH/κ chain ribosome display library was constructed with a special flexible linker by overlap extension PCR. The VH/κ chain library was transcribed and translated in vitro using a rabbit reticulocyte lysate system. Then, antibody–ribosome–mRNA complexes were produced and panned against ICOS protein under appropriate conditions. However, in order to isolate specific scFvs for ICOS, negative selection using CD28 was carried out before three rounds of positive selection on ICOS. After three rounds of panning, the selected scFv DNAs were cloned into pET43.1a and detected by SDS-PAGE. Then, enzyme-linked immunosorbent assay showed that we successfully constructed a native ribosome display library, and among seven clones, clone 5 had the highest affinity for the ICOS and low for the CD28. Anti-ICOS scFvs are assessed for binding specificity and affinity and may provide the potential for development of the humanized and acute and chronic allograft rejection.
Keywords: ICOS; Ribosome display; scFv; Prokaryotic expression
Detection and Confirmation of Alkaloids in Leaves of Justicia adhatoda and Bioinformatics Approach to Elicit Its Anti-tuberculosis Activity by Deepak Kumar Jha; Likun Panda; P. Lavanya; Sudha Ramaiah; Anand Anbarasu (pp. 980-990).
The extraction and determination of alkaloids was performed and confirmed by phytochemical analysis. Six different quinazoline alkaloids (vasicoline, vasicolinone, vasicinone, vasicine, adhatodine and anisotine) were found in the leaf of Justicia adhatoda (J. adhatoda). The presence of the peaks obtained through HPLC indicated the diverse nature of alkaloid present in the leaf. The enzyme β-ketoacyl-acyl-carrier protein synthase III that catalyses the initial step of fatty acid biosynthesis (FabH) via a type II fatty acid synthase has unique structural features and universal occurrence in Mycobacterium tuberculosis (M. tuberculosis). Thus, it was considered as a target for designing of anti-tuberculosis compounds. Docking simulations were conducted on the above alkaloids derived from J. adhatoda. The combination of docking/scoring provided interesting insights into the binding of different inhibitors and their activity. These results will be useful for designing inhibitors for M. tuberculosis and also will be a good starting point for natural plant-based pharmaceutical chemistry.
Keywords: J. adhatoda ; Tuberculosis; mtFabH; Docking; Vasicoline
Acetylcholinesterase Inhibitory Potential and Insecticidal Activity of an Endophytic Alternaria sp. from Ricinus communis by Bahaderjeet Singh; Abhinay Thakur; Sanehdeep Kaur; B. S. Chadha; Amarjeet Kaur (pp. 991-1002).
Keeping in view the vast potential of endophytic fungi to produce bioactive molecules, this study aimed at isolating and screening endophytes for the production of acetylcholinesterase inhibitors. Fifty-four endophytic fungi were isolated from Ricinus communis and screened for their AChE inhibitory activity using Ellman’s colorimetric assay method. Six isolates were found to possess AChE inhibitory activity with maximum inhibition of 78 % being evinced by culture Cas1 which was identified to be Alternaria sp. on the basis of molecular as well as microscopic methods. Optimization of inhibitor production was carried out using one factor at a time approach. Maximum production of inhibitor was obtained on potato dextrose broth after 10 days incubation. The IC50 of the chloroform extract was observed to be 40 μg/ml. The extract was purified on silica gel and eluted stepwise with a gradient of chloroform/methanol. The insecticidal potential of the extract was evaluated by feeding the larvae of Spodoptera litura on diet containing varying concentrations of the extract. It was observed that with increase in the concentration of the extract, mortality of the larvae increased. The culture has the potential of being exploited in medicine as well as a biocontrol agent.
Keywords: Ricinus communis ; Endophytic fungi; Acetylcholinesterase inhibitors; Alternaria sp.; Insecticidal activity
Characterization and Phylogenetic Analysis of Microbial Surface Active Compound-Producing Bacteria by Atipan Saimmai; Onkamon Rukadee; Theerawat Onlamool; Vorasan Sobhon; Suppasil Maneerat (pp. 1003-1018).
Microbial surface active compounds (SACs) were isolated from various environmental sources in Thailand. Isolates were screened for SACs production in different carbon sources (crude glycerol, commercial sugar, decanter, glucose, molasses, used palm oil, and used lubricating oil) by using drop-collapsing test and emulsification activity. Molasses produced the highest number of positive results (23 of 25 isolates). Twenty-one isolate strains produced emulsions with xylene, and 15 exhibited high emulsion-stabilizing capacity, maintaining more than 50 % of the original emulsion volume for 24 h, and six isolate strains reduced the growth medium surface tension to 40 mN/m. The phylogenetic position of these 25 isolates was evaluated by 16S rRNA gene sequence analysis. The production of microbial SACs was determined for strains representative of 16 different bacterial genera, in which ten genera (Blastococcus, Erysipelothrix, Humicoccus, Methylophilus, Microlunatus, Nevskia, Pectinatus, Rubrimonas, Selenomonas, and Stenotrophomonas) were firstly reported as SAC-producing strains. Overall, the new SAC-producing strains isolated in this study display promising features for the future development and use in economically efficient industrial-scale biotechnological processes.
Keywords: Microbial surface active compounds; Phylogenetic; Drop-collapsing test; Emulsification activity
Solid-Phase Colorimetric Method for the Quantification of Fucoidan by Jung Min Lee; Z.-U. Shin; Gafurjon T. Mavlonov; Ibrokhim Y. Abdurakhmonov; Tae-Hoo Yi (pp. 1019-1024).
We described the simple, selective, and rapid method for determination of fucoidans using methylene blue staining of sulfated polysaccharides, immobilized into filter paper and consequent optic density (at A 663 nm) measurement of the eluted dye from filter paper. This solid-phase method allows selective determination of 1–20 μg fucoidan in presence of potentially interfering compounds (alginic acid, DNA, salts, proteins, and detergents). Further, we demonstrated the alternative way of using image processing software for fucoidan quantification without extraction of methylene blue dye from stained spots of fucoidan–dye complex.
Keywords: Sulfated polysaccharides; Fucoidan; Methylene blue binding; Solid-phase colorimetry; Spot quantitation; Quantity One® soft
Production of Acid-Stable and High-Maltose-Forming α-Amylase of Bacillus acidicola by Solid-State Fermentation and Immobilized Cells and Its Applicability in Baking by Archana Sharma; T. Satyanarayana (pp. 1025-1034).
Among matrices used for immobilizing Bacillus acidicola cells [calcium alginate, chitosan + alginate, scotch brite, and polyurethane foam (PUF)], α-amylase production was highest by PUF-immobilized cells (9.1 U ml−1), which is higher than free cells (7.2 U ml−1). The PUF-immobilized cells could be reused over seven cycles with sustained α-amylase production. When three variables (moisture, starch, and ammonium sulfate), which significantly affected enzyme production in solid-state fermentation (SSF), were optimized using response surface methodology, 5.6-fold enhancement in enzyme production was attained. The enzyme production in SSF is 3.8-fold higher than that in submerged fermentation. The bread made by supplementing dough with α-amylase of B. acidicola scored better than those with the xylanase of Bacillus halodurans and thermostable α-amylase of Geobacillus thermoleovorans.
Keywords: Immobilization; Polyurethane foam; Submerged fermentation; Solid-state fermentation; Baking
Optimisation of Batch Culture Conditions for Cell-Envelope-Associated Proteinase Production from Lactobacillus delbrueckii subsp. lactis ATCC® 7830™ by Dominic Agyei; Ravichandra Potumarthi; Michael K. Danquah (pp. 1035-1050).
Using a combination of conventional sequential techniques, the batch growth conditions for the production of cell-envelope-associated proteinases have for the first time been studied and optimised in Lactobacillus delbrueckii subsp. lactis 313 (ATCC 7830; LDL 313). Concentrations of inoculum (0.1 < X < 10 % vol/vol), agitation speed (0 < S < 200 rpm), varying incubation temperature (30 < T < 50 °C), starting pH (4.5 < pH < 7) and carbon/nitrogen ratio of production medium (0.2 < r < 5) had an individual effect on proteinase yield (p < 0.01). Optimal conditions for proteinase production included an initial pH of 6.0, 45 °C incubation temperature, 2 % (v/v) inoculum size of OD560 = 1, 150 rpm agitation speed, and growth medium carbon/nitrogen ratio of 1.0. Maximum proteinase activity obtained for whole cells was 0.99 U/ml after 8 h of incubation. The variables studied are very relevant due to their significance in improving the productivity of proteinase synthesis from LDL 313, under process and, likely, economic optimum conditions.
Keywords: Fermentation; Lactobacilli delbrueckii subsp. lactis 313; Optimisation; Process variables; Cell-envelope proteinases
Estimation of Fundamental Kinetic Parameters of Polyhydroxybutyrate Fermentation Process of Azohydromonas australica Using Statistical Approach of Media Optimization by Geeta Gahlawat; Ashok K. Srivastava (pp. 1051-1064).
Polyhydroxybutyrate or PHB is a biodegradable and biocompatible thermoplastic with many interesting applications in medicine, food packaging, and tissue engineering materials. The present study deals with the enhanced production of PHB by Azohydromonas australica using sucrose and the estimation of fundamental kinetic parameters of PHB fermentation process. The preliminary culture growth inhibition studies were followed by statistical optimization of medium recipe using response surface methodology to increase the PHB production. Later on batch cultivation in a 7-L bioreactor was attempted using optimum concentration of medium components (process variables) obtained from statistical design to identify the batch growth and product kinetics parameters of PHB fermentation. A. australica exhibited a maximum biomass and PHB concentration of 8.71 and 6.24 g/L, respectively in bioreactor with an overall PHB production rate of 0.75 g/h. Bioreactor cultivation studies demonstrated that the specific biomass and PHB yield on sucrose was 0.37 and 0.29 g/g, respectively. The kinetic parameters obtained in the present investigation would be used in the development of a batch kinetic mathematical model for PHB production which will serve as launching pad for further process optimization studies, e.g., design of several bioreactor cultivation strategies to further enhance the biopolymer production.
Keywords: Polyhydroxybutyrate; Azohydromonas australica ; Sucrose; Substrate inhibition; Central Composite Design; Bioreactor cultivation
Oil from the Tropical Marine Benthic-Diatom Navicula sp. by Zeily Nurachman; Dewi Susan Brataningtyas; Hartati; Lily Maria Goretty Panggabean (pp. 1065-1075).
The potential of the tropical marine benthic-diatom Navicula sp. for biodiesel feedstock was investigated. Growth profiles were analyzed by changing nutrient compositions in three different media (Walne, plain seawater, and modified seawater) and irradiance intensities. Navicula sp. cells showed significant growth in Walne and modified seawater medium but not in plain seawater medium. The microalgae grew well in a pH range of 7.8–8.4, and the cells were very sensitive to the intensity of direct sunlight exposure. The average cell concentration obtained from the cultures in plain seawater, Walne, and modified seawater media at the beginning of the stationary phase was 0.70, 2.17, and 2.54 g/L, respectively. Electron spray ionization-ion trap-mass spectrometry showed that the triacylglycerols of the algae oil were identified as POP (palmitic-oleic-palmitic), POO (palmitic-oleic-oleic), and OOLn (oleic-oleic-linoleic). The oil productivity of Navicula sp. cultivated in Walne and modified seawater media was 90 and 124 μL L−1 culture d−1. The Navicula sp. biodiesel exhibited a kinematic viscosity of 1.299 mm2/s, density of 0.8347 g/mL, and internal energy of 0.90 kJ/mL.
Keywords: Keywords; Benthic microalgae; Biodiesel; Modified seawater medium; Navicula sp.; Oil productivity; TAG; Tropical marine diatom
Kinetic Analysis, Expression Pattern, and Production of a Recombinant Fungal Protease Inhibitor of Tasar Silkworm Antheraea mylitta by Sobhan Roy; Venugopal Rao Ravipati; Suvankar Ghorai; Mrinmay Chakrabarti; Amit Kumar Das; Ananta Kumar Ghosh (pp. 1076-1085).
Antheraea mylitta, a tasar silk-producing insect of Saturniidae family, expresses a fungal protease inhibitor named as A. mylitta fungal protease inhibitor-1 (AmFPI-1). AmFPI-1 inhibits alkaline protease of Aspergillus oryzae but its mechanism of action is not known. To understand the mode of inhibition of AmFPI-1 against the fungal protease, it was purified from the hemolymph of A. mylitta larvae and inhibitory activity against A. oryzae protease was studied. Kinetic analysis of purified AmFPI-1 on alkaline protease of A. oryzae showed that AmFPI-1 acts as a canonical-type competitive inhibitor with equilibrium dissociation constant (K i ) of 60 nM. Expression of AmFPI-1 in different body tissues of fifth instar A. mylitta larvae was determined by real-time PCR, and the highest expression was observed in fat body followed by integument, silk gland, and gut, indicating that AmFPI-1 has pleiotropic functions including protection from invading fungi. The cDNA of AmFPI-1 was expressed in Escherichia coli, and recombinant His-tagged fusion protein was purified by Ni-NTA chromatography. Recombinant AmFPI-1 showed inhibitory activity against A. oryzae protease and suggested its use in various biological applications to prevent proteolysis.
Keywords: Alkaline protease; Antheraea mylitta ; Fungal protease inhibitor; Kinetic analysis; Expression
Switchgrass PviCAD1: Understanding Residues Important for Substrate Preferences and Activity by Aaron J. Saathoff; Mark S. Hargrove; Eric J. Haas; Christian M. Tobias; Paul Twigg; Scott Sattler; Gautam Sarath (pp. 1086-1100).
Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step in monolignol biosynthesis. Although plants contain numerous genes coding for CADs, only one or two CADs appear to have a primary physiological role in lignin biosynthesis. Much of this distinction appears to reside in a few key residues that permit reasonable catalytic rates on monolignal substrates. Here, several mutant proteins were generated using switchgrass wild type (WT) PviCAD1 as a template to understand the role of some of these key residues, including a proton shuttling HL duo in the active site. Mutated proteins displayed lowered or limited activity on cinnamylaldehydes and exhibited altered kinetic properties compared to the WT enzyme, suggesting that key residues important for efficient catalysis had been identified. We have also shown that a sorghum ortholog containing EW, instead of HL in its active site, displayed negligible activity against monolignals. These results indicate that lignifying CADs require a specific set of key residues for efficient activity against monolignals.
Keywords: Switchgrass; Cinnamyl alcohol dehydrogenase; Lignin; Protein mutagenesis
Effect of Additives on Freeze-Drying and Storage of Yarrowia lipolytica Lipase by Farshad Darvishi; Jacqueline Destain; Iraj Nahvi; Philippe Thonart; Hamid Zarkesh-Esfahani (pp. 1101-1107).
The extracellular lipase of Yarrowia lipolytica presents numerous potentialities for biotechnological applications. This work describes the development and storage of powders obtained from supernatants containing Y. lipolytica lipase by freeze-drying as downstream process that is important in obtaining a stable lipase powder with high enzymatic activity. Lipase was produced by Y. lipolytica U6 mutant strain in 20-L bioreactor. Non-concentrated cell-free culture supernatant samples were supplemented with different concentrations (0.5–1 %) of maltodextrin and glycerol as additives to freeze-drying. Effects of additives, temperature, pH, and storage time on lipase powders were determined. After addition of additives, freeze-drying yield increased 3.5-fold compared to supernatant without additive. Maltodextrin with 0.5 % concentration gave the best protection of lipase during dehydration treatment and its freeze-drying yield (77 %) is better than other formulations. Lipase powders were stored at 4 and 25 °C for 46 weeks without loss of lipase activity. A common impediment to the production of commercial enzyme is their low-stability aqueous solutions. The present study shows that freeze-dried lipase powders of Y. lipolytica have good stability for storage and various applications.
Keywords: Lipase; Yarrowia lipolytica ; Downstream processing; Formulation; Freeze-drying
Immobilization of Lecitase® Ultra onto a Novel Polystyrene DA-201 Resin: Characterization and Biochemical Properties by Ning Liu; Min Fu; Yong Wang; Qiangzhong Zhao; Weizheng Sun; Mouming Zhao (pp. 1108-1120).
A simple, rapid, and economic method of enzyme immobilization was developed for phospholipase Lecitase® ultra (LU) via interfacial adsorption. The effect of nature of the polystyrene supports and the kinetic behavior and stability of immobilized lecitase® ultra (IM-LU) were evaluated. Six macroporous resins (AB-8, X-5, DA-201, NKA-9, D101, D4006) and two anion resins (D318 and D201) were studied as the supports. DA-201 resin was selected because of its best immobilization effect for LU. Immobilization conditions were investigated, including immobilization time, pH, and enzyme concentration. IM-LU with a lipase activity of 1,652.4 ± 8.6 U/g was obtained. The adsorption process was modeled by Langmuir and Freundlich equations, and the experimental data were better fit for the former one. The kinetic constant (K m) values were found to be 192.7 ± 2.2 mM for the free LU and 249.3 ± 5.4 mM for the IM-LU, respectively. The V max value of free LU (169.5 ± 4.3 mM/min) was higher than that of the IM-LU (53.8 ± 1.5 mM/min). Combined strategies of scanning electron micrograph, thermogravimetric analysis, and Fourier transform infrared (FTIR) spectroscopy were employed to characterize the IM-LU. FTIR spectroscopy showed that the secondary conformation of the enzyme had changed after immobilization, through which a decrease of α-helix content and an increase of β-sheet content were observed. The IM-LU possessed an improved thermal stability as well as metal ionic tolerance when compared with its free form. The reusability of IM-LU was also evaluated through catalyzing esterification reaction between oleic acid and glycerol. It exhibited approximately 70 % of relative esterification efficiency after six successive cycles. This immobilized enzyme on hydrophobic support may well be used for the synthesis of structural lipids in lipid area.
Keywords: Lecitase® ultra; Immobilization; Adsorption; FTIR; Esterification
Kinetic Modeling of Solvent-Free Lipase-Catalyzed Partial Hydrolysis of Palm Oil by Fernando Augusto Pedersen Voll; Andreia Fatima Zanette; Vladimir Ferreira Cabral; Claudio Dariva; Rodrigo Octavio Mendonça Alves De Souza; Lucio Cardozo Filho; Marcos Lúcio Corazza (pp. 1121-1142).
This work reports the experimental data and kinetic modeling of diacylglycerol (DAG) production from palm oil using a commercial immobilized lipase (Lipozyme RM IM) in a solvent-free medium. The experiments were performed in batch mode, at 55 °C and 400 rpm, and the effects of enzyme concentration (0.68–2.04 wt% related to the mass of substrates), initial water concentration (5–15 wt% related to the mass of oil), and reaction time were evaluated. A novel kinetic model is presented based on the ordered-sequential bi–bi mechanism considering hydrolysis and esterification steps, in which a correlation between water-in-oil solubility and surfactant molecules concentration in the oil allowed the model to describe the induction period in the beginning of the hydrolysis reaction. Moreover, mass transfer limitations related to the enzyme concentration in the system were also taken into account. The proposed model presented a very satisfactory agreement with the experimental data, thus allowing a better understanding of the reaction kinetics. The best conditions obtained for the product (partially hydrolyzed palm oil) in terms of DAG yield (35.91 wt%) were 2.87 wt% enzyme/substrate, 2.10 wt% water/oil, and 72 h of reaction.
Keywords: Hydrolysis; Kinetic modeling; Lipase-catalyzed; Palm oil; Diacylglycerol
Visible-Light-Induced Bactericidal Activity of Vanadium-Pentoxide (V2O5)-Loaded TiO2 Nanoparticles by Yeon Seok Kim; Min Young Song; Eun Seuk Park; Sungmin Chin; Gwi-Nam Bae; Jongsoo Jurng (pp. 1143-1152).
The bactericidal activity of TiO2 nanoparticles under visible light is very important in regards to its practical applications. In this paper, we synthesized vanadium-pentoxide-loaded TiO2 nanoparticles (V2O5–TiO2) using a chemical vapor condensation method, followed by the impregnation method, and characterized its physicochemical properties through X-ray diffraction patterning, X-ray photoelectron spectroscopy analysis, Raman spectra analysis, and Fourier transform infrared analysis. In addition, the antibacterial activity of V2O5–TiO2 nanoparticles against E. coli was evaluated and compared with pure TiO2 nanoparticles. In these experiments, the population of E. coli was shown to be significantly reduced by V2O5–TiO2 nanoparticles under illumination with fluorescent light, whereas pure TiO2 nanoparticles showed about 3.3-fold lower antibacterial activity than the V2O5–TiO2 nanoparticles. This result was most likely due to the change in surface conditions of the TiO2 nanoparticles, which was due to the loading of vanadium pentoxide on the TiO2 nanoparticles. Furthermore, both photocatalysts showed similar antibacterial activity under UV-A (352 nm) irradiation.
Keywords: Bactericidal activity; Photocatalysis; TiO2 nanoparticle; Visible light
Synthesis and Biological Evaluation of a Novel Series of Chalcones Incorporated Pyrazole Moiety as Anticancer and Antimicrobial Agents by Magda F. Mohamed; Mervat S. Mohamed; Samia A. Shouman; Mohamed M. Fathi; Ismail Abdelshafy Abdelhamid (pp. 1153-1162).
A newly synthesized series of chalcone derivatives containing pyrazole rings were synthesized and evaluated for their cytotoxic activities in vitro against several human cancer cell lines. Most of the prepared compounds showed potential cytotoxicity against human breast cancer cell lines MCF-7, HEPG-2, and HCT-116. Also the compounds were evaluated as antimicrobial agents. The three compounds 3, 4, and 5 were proved to be better anticancer agents than the positive standard doxorubicin with IC50 values (4.7, 4.4, and 3.9 μg/ml) against the same human cancer cell lines, whereas compounds 5 and 6 showed the most active antimicrobial compounds in comparison to the other chalcones.
Keywords: Chalcones; Cancer cell lines (MCF-7, HEPG-2 and HCT-116); Potential pharmacological activity
New Tools for Exploring “Old Friends—Microbial Lipases” by Saisubramanian Nagarajan (pp. 1163-1196).
Fat-splitting enzymes (lipases), due to their natural, industrial, and medical relevance, attract enough attention as fats do in our lives. Starting from the paper that we write, cheese and oil that we consume, detergent that we use to remove oil stains, biodiesel that we use as transportation fuel, to the enantiopure drugs that we use in therapeutics, all these applications are facilitated directly or indirectly by lipases. Due to their uniqueness, versatility, and dexterity, decades of research work have been carried out on microbial lipases. The hunt for novel lipases and strategies to improve them continues unabated as evidenced by new families of microbial lipases that are still being discovered mostly by metagenomic approaches. A separate database for true lipases termed LIPABASE has been created recently which provides taxonomic, structural, biochemical information about true lipases from various species. The present review attempts to summarize new approaches that are employed in various aspects of microbial lipase research, viz., screening, isolation, production, purification, improvement by protein engineering, and surface display. Finally, novel applications facilitated by microbial lipases are also presented.
Keywords: Microbial lipases; Metagenomic approaches; Laboratory evolution; Surface display
Galacto-oligosaccharides Synthesis from Lactose and Whey by β-Galactosidase Immobilized in PVA by Ruzica Jovanovic-Malinovska; Pedro Fernandes; Eleonora Winkelhausen; Luis Fonseca (pp. 1197-1211).
The synthesis of galacto-oligosaccharides (GOS) by β-galactosidase immobilized in both polyvinyl alcohol (PVA) lenses and sol–gel carriers was studied and compared with the performance of the free enzyme. PVA-immobilized β-galactosidase retained 95 % of the initial activity after seven repeated uses and retained 51 % of the initial activity after 3 months of storage, while sol–gel-immobilized β-galactosidase only retained 39 % of the initial activity under storage. Lactose conversion takes place at a higher rate in the PVA-immobilized β-galactosidase, while the lowest rate of lactose conversion was noticed with immobilized β-galactosidase in sol–gel. Continuous production of GOS from either lactose or whey, with PVA-immobilized β-galactosidase, was performed in a packed-bed reactor. A maximum GOS production of 30 % of total sugars was attained for a 40-% lactose feed solution with a feed rate of 10.8 ml/h, at pH 4.5 and 40 °C, corresponding to a productivity of 117 g/l h. The maximum GOS productivity of 344 g/l h was obtained at a flow rate of 28.7 ml/h. 3-OS and 4-OS were the major types of GOS formed. Conversion of whey in continuous mode resulted in GOS production of 15 % of total sugars and formation of 45 % 3-OS, 40 % 4-OS, and 15 % 5-OS.
Keywords: β-Galactosidase; Entrapment; Galacto-oligosaccharides; Whey; Lactose; Continuous operation
Production of the Antimicrobial Protein Weisselin A by Weissella paramesenteroides DX in Batch Fermentations: the Type of Carbohydrate Used as the C-Source in the Substrate Affects the Association of Production with Growth by Maria Papagianni; Emmanuel M. Papamichael (pp. 1212-1222).
The effect of the type of carbohydrate (glucose, fructose, sucrose or galactose) used as the carbon source in the substrate on weissellin A production by Weissella paramesenteroides DX was evaluated on a solid-state cultivation procedure and conventional batch fermentations. Solid-state cultivation was done on M17-based medium over 3- and 6-h incubation periods. Experimental data showed that glucose supports the highest production levels on a per cell basis. Stirred tank bioreactor fermentations carried out at 50 % dissolved oxygen tension revealed the superiority of glucose over the other carbohydrates. Glucose supports growth-associated production and increased production rates and productivities (1,120 AU/ml). Growth-associated production was maintained with fructose but with lower fermentation rates and productivities. Sucrose cannot support this type of production. Fermentations with sucrose were characterized of lower sugar uptake rates, and lower specific growth and production rates, with bacteriocin titres not exceeding 630 AU/ml, while product formation kinetics were of the intermediate type.
Keywords: Weissellin A; Weissella paramesenteroides DX; Bacteriocin; Fermentation kinetics; Growth-associated production
Comparative Metabolomic Study of Penicillium chrysogenum During Pilot and Industrial Penicillin Fermentations by Ming-Zhu Ding; Hua Lu; Jing-Sheng Cheng; Yao Chen; Jing Jiang; Bin Qiao; Bing-Zhi Li; Ying-Jin Yuan (pp. 1223-1238).
Comparative metabolomics was carried out to investigate the metabolic differences of Penicillium chrysogenum in the pilot and industrial fermentations that resulted from the scale-up. By principal component analysis, the early stages of two fermentation processes were clearly distinguished, whereas the middle and final stages were clustered together. It indicated that the different metabolisms of cells in the pilot and industrial fermentations mainly existed during the early stage. Furthermore, the levels of polyamines, polyols, glycolysis, and tricarboxylic acid cycle intermediates, which changed more dramatically during the pilot process, were all higher in the pilot than in the industrial fermentation during the early stage. This indicated that the fermentation conditions of the early stage should be the focus of process management which is aimed at increasing penicillin production. Additionally, the comparative accumulations of the precursors of penicillin (valine, cysteine, and lysine) revealed that penicillin biosynthesis in the industrial process was more affected during the middle stage of fermentation. These findings provide new insights to further regulate the industrial process and improve the production of penicillin. More generally, this study attempts to address the scarcity of studies that contrast the metabolic outcomes between commercial- and pilot-scale conditions.
Keywords: Penicillin; Scale-up; Pilot fermentation; Industrial fermentation; Metabolomics
Acceleration of Adventitious Shoots by Interaction Between Exogenous Hormone and Adenine Sulphate in Althaea Officinalis L. by Ruphi Naz; M. Anis (pp. 1239-1255).
In the current study attempts were made to investigate the effects of three different phases of callus induction followed by adventitious regeneration from leaf segments (central and lateral vein). Callus induction was observed in Murashige and Skoog’s (MS) medium supplemented with 15.0 μM 2,4-dichloro phenoxy acetic acid (2,4-D). Adventitious shoot buds formation was achieved on MS medium supplemented with 7.5 μM 2,4-D and 20.0 μM AdS in liquid medium as it induced 19.2 ± 0.58 buds in central vein explants. Addition of different growth regulators (cytokinins—6-benzyladenine, kinetin and 2-isopentenyl adenine alone or in combination with auxins—indole-3-acetic acid, indole-3-butyric acid and α-naphthalene acetic acid, improved the shoot regeneration efficiency, in which 5.0 μM 6-benzyl adenine along with 0.25 μM α-naphthalene acetic acid was shown to be the most effective medium for maximum shoot regeneration (81.3 %) with 24.6 number of shoots and 4.4 ± 0.08 cm shoot length per explant. Leaf culture of central veins led to better shoot formation capacity in comparison to lateral vein. Rooting was readily achieved on the differentiated shoots on 1/2 MS medium augmented with 20.0 μM indole-3-butyric acid. The plants were successfully hardened off in sterile soilrite followed by their establishment in garden soil with 80 % survival rate.
Keywords: Dedifferentiated tissue; Regeneration; Growth regulators; Acclimatization
A Novel Dextran Dextrinase from Gluconobacter oxydans DSM-2003: Purification and Properties by Xiangzhao Mao; Shu Wang; Feifei Kan; Dongzhi Wei; Fuli Li (pp. 1256-1264).
Dextran has already been widely applied in food, pharmaceutical, and chemical industries. In this study, a novel intracellular dextran dextrinase (DDase, EC 2.4.1.2) from Gluconobacter oxydans DSM-2003 exhibiting catalytic activity to synthesize dextran from maltodextrin was purified to homogeneity by ultrasonic cell disruption, ion exchange chromatography, and gel filtration. This procedure showed 187.5-fold purification from the cell-free extract with 41.9 % yield. And the apparent molecular weight was estimated to be 62 kDa by SDS-PAGE. It was different from the reported literatures, which found that the molecular weight of intracellular and extracellular DDase of G. oxydans ATCC-11894 was 300 and 152 kDa, respectively. Otherwise, it showed different physicochemical characteristics (optimal temperature and pH, thermal, pH stability, effect of metal ions) from the DDase of G. oxydans ATCC-11894. This indicated that DDase of G. oxydans DSM-2003 was a novel one compared to the reported literatures.
Keywords: Gluconobacter oxydans ; Dextran dextrinase; Enzyme purification; Catalytic property
Appraisal of Conjugated Linoleic Acid Production by Probiotic Potential of Pediococcus spp. GS4 by Vinay Dubey; Asit Ranjan Ghosh; Badal Kumar Mandal (pp. 1265-1276).
Probiotics with ability to produce conjugated linoleic acid (CLA) is considered as an additive health benefit property for its known role in colon cancer mitigation. The conversion involves the biohydrogenation of the unsaturated fatty acid into conjugated form. Probiotic strain Pediococcus spp. GS4 was efficiently able to biohydrogenate linoleic acid (LA) into its conjugated form within 48 h of incubation. Quantum of CLA produced with a concentration of 121 μg/ml and sustained cell viability of 8.94 log cfu/ml maximally. Moreover, antibacterial effect of LA on the strain ability for biohydrogenation was examined at different concentrations and concluded to have a direct relationship between LA and amount of CLA produced. The efficiency of the strain for CLA production at different pH was also estimated and found maximum at pH 6.0 with 149 μg/ml while this ability was reduced at pH 9.0 to 63 μg/ml. Sesame oil, which is rich in the triacylglycerol form of LA, was also found to act as a substrate for CLA production by Pediococcus spp. GS4 with the aid of lipase-catalyzed triacylglycerol hydrolysis and amount of CLA produced was 31 μg/ml at 0.2 % while 150 μg/ml at 1.0 % of lipolysed oil in skim milk medium. Conjugated form was analyzed using UV scanning, RP-HPLC, and GC-MS. This study also focused on the alternative use of lipolysed sesame oil instead of costly LA for biohydrogenation and could be a potential source for the industrial production of CLA.
Keywords: Probiotic; Linoleic acid; CLA; Biohydrogenation; Lipolysed oil
Antibacterial Properties of Intestinal Phospholipase A2 from the Common Stingray Dasyatis pastinaca by Abir Ben Bacha; Islem Abid; Habib Horchani (pp. 1277-1287).
Stingray phospholipase A2 group IIA (SPLA2-IIA) was recently isolated and purified to homogeneity from the intestine of the common stingray Dasyatis pastinaca, suggesting that this enzyme plays an important role in systemic bactericidal defense. The present study showed that SPLA2-IIA was highly bactericidal against Gram-positive bacteria with inhibition zones and minimal inhibitory concentration values in the range of 13–25 mm and 2–8 μg/ml, respectively, whereas Gram-negative bacteria exhibited a much higher resistance. The bactericidal efficiency of SPLA2-IIA was shown to be unaffected by high protein and salt concentrations, but dependent upon the presence of calcium ions, and then correlated to the hydrolytic activity of membrane phospholipids. Importantly, we showed that stingray phospholipase A2 group IIA presents no cytotoxicity after its incubation with MDA-MB-231 cells. SPLA2-IIA may be considered as a future therapeutic agent against bacterial infections.
Keywords: Stingray Dasyatis pastinaca ; Phospholipase A2-IIA; Bactericidal effect; Cytotoxicity
Improved Covalent Immobilization of Horseradish Peroxidase on Macroporous Glycidyl Methacrylate-Based Copolymers by Olivera Prodanović; Miloš Prokopijević; Dragica Spasojević; Željko Stojanović; Ksenija Radotić; Zorica D. Knežević-Jugović; Radivoje Prodanović (pp. 1288-1301).
A macroporous copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate, poly(GMA-co-EGDMA), with various surface characteristics and mean pore size diameters ranging from 44 to 200 nm was synthesized, modified with 1,2-diaminoethane, and tested as a carrier for immobilization of horseradish peroxidase (HRP) by two covalent methods, glutaraldehyde and periodate. The highest specific activity of around 35 U g−1 dry weight of carrier was achieved on poly(GMA-co-EGDMA) copolymers with mean pore diameters of 200 and 120 nm by the periodate method. A study of deactivation kinetics at 65 °C and in 80 % dioxane revealed that periodate immobilization also produced an appreciable stabilization of the biocatalyst, while stabilization factor depended strongly on the surface characteristics of the copolymers. HRP immobilized on copolymer with a mean pore diameter of 120 nm by periodate method showing not only the highest specific activity but also good stability was further characterized. It appeared that the immobilization resulted in the stabilization of enzyme over a broader pH range while the Michaelis constant value (K m) of the immobilized HRP was 10.8 mM, approximately 5.6 times higher than that of the free enzyme. After 6 cycles of repeated use in a batch reactor for pyrogallol oxidation, the immobilized HRP retained 45 % of its original activity.
Keywords: Macroporous polymers; Copolymerization; Enzymes; Horseradish peroxidase; Covalent immobilization; Morphology; Biological applications of polymers
Identification of New Dominant-Negative Mutants of Anthrax Protective Antigen Using Directed Evolution by Gaobing Wu; Chunfang Feng; Sha Cao; Aizhen Guo; Ziduo Liu (pp. 1302-1310).
The anthrax toxin is composed of three proteins: protective antigen (PA), lethal factor (LF), and edema toxin (EF). The PA moiety carries EF and LF into the cytosol of mammalian cells via a mechanism that depends on the oligomerization of PA and transmembrane pore formation by the PA oligomer. Certain mutants of PA, termed dominant-negative (DN) mutants, can co-oligomerize with wild-type PA and disrupt the translocation ability of the pore. Here, we constructed a PA mutant library by introducing random mutations into domain II of PA and screened three new DN mutants of PA: V377E, T380S, and I432C. All the mutants inhibited the anthrax toxin action against sensitive cells. V377E had the strongest inhibitory effect and was further confirmed to be able to protect mice against a challenge with anthrax lethal toxin. Furthermore, we functionally characterized these mutants. The result showed that these mutations did not impair proteolytic activation or oligomer formation of PA, but impeded the prepore–pore conversion of the oligomer. These DN mutants of PA identified in our study may provide valuable information for elucidating the structure–function relationship of PA and for designing therapeutics for anthrax treatment.
Keywords: Anthrax; Protective antigen; Dominant-negative mutant; Directed evolution
Animal Bone Char Solubilization with Itaconic Acid Produced by Free and Immobilized Aspergillus terreus Grown on Glycerol-Based Medium by Nikolay Vassilev; Almudena Medina; Bettina Eichler-Löbermann; Elena Flor-Peregrín; Maria Vassileva (pp. 1311-1318).
Cells of Aspergillus terreus, free and immobilized in polyurethane foam, were employed in itaconic acid fermentation processes on glycerol-based media. The purpose was to assess their suitability for animal bone char solubilization and the development of a biotechnological alternative to P fertilizers chemically produced from rock phosphate. Animal bones constitute a renewable source of P that can replace the traditionally used finite, nonrenewable rock phosphate as a P source. Glycerol was an excellent substrate for growth (10.2 g biomass L−1) and itaconic acid production (26.9 g L−1) by free fungal cells after 120-h fermentation. Simultaneously, A. terreus solubilized the insoluble phosphate to a yield of 23 to 50 %, depending on the particle size and concentration. Polyurethane foam cut into cubes of 0.5–0.6 cm per side, with 0.3 mm pore size and applied at 2.0 g L−1 proved to be an excellent cell carrier. In repeated batch fermentation, the immobilized mycelium showed a high capacity to solubilize animal bone char, which resulted on average in 168.8 mg L–1 soluble phosphate per 48-h cycle and 59.4 % yield (percent of total phosphate) registered in the fourth batch.
Keywords: Immobilized cells; Aspergillus terreus ; Glycerol; Animal bone char; Solubilization
Biodecolorization of Azo Dye Remazol Orange by Pseudomonas aeruginosa BCH and Toxicity (Oxidative Stress) Reduction in Allium cepa Root Cells by Shekhar B. Jadhav; Shripad N. Surwase; Dayanand C. Kalyani; Ranjit G. Gurav; Jyoti P. Jadhav (pp. 1319-1334).
In this report a textile azo dye Remazol orange was degraded and detoxified by bacterium Pseudomonas aeruginosa BCH in plain distilled water. This bacterial decolorization performance was found to be pH and temperature dependent with maximum decolorization observed at pH 8 and temperature 30 °C. Bacterium tolerated higher dye concentrations up to 400 mg l−1. Effect of initial cell mass showed that higher cell mass concentration can accelerate decolorization process with maximum of 92 % decolorization observed at 2.5 g l−1 cell mass within 6.5 h. Effect of various metal ions showed Mn has inducing effect whereas Zn strongly inhibited the decolorization process at 5 mM concentration. Analysis of biodegradation products carried out with UV–vis spectroscopy, HPTLC and FTIR confirmed the decolorization and degradation of Remazol orange. Possible route for the degradation of dye was proposed based on GC-MS analysis. During toxicological scrutiny in Allium cepa root cells, induction in the activities of superoxide dismutase (SOD), guaiacol peroxidase (GPX) and inhibition of catalase (CAT) along with raised levels of lipid peroxidation and protein oxidation in dye treated samples were detected which conclusively indicated the generation of oxidative stress. Less toxic nature of the dye degraded products was observed after bacterial treatment.
Keywords: Biodecolorization; HPTLC; Oxidative stress; Pseudomonas aeruginosa BCH; Remazol orange; Superoxide dismutase
Immunomodulatory Response of Mice Splenocytes Induced by RcaL, a Lectin Isolated from Cobia Fish (Rachycentron canadum) Serum by Marília Cavalcanti Coriolano; Cynarha Daysy Cardoso da Silva; Cristiane Moutinho Lagos de Melo; Ranilson de Souza Bezerra; Athiê Jorge Guerra Santos; Valéria Rêgo Alves Pereira; Luana Cassandra Breitenbach Barroso Coelho (pp. 1335-1348).
This work reports the isolation of a serum lectin from cobia fish (Rachycentron canadum) named RcaL. Immunomodulatory activity on mice splenocyte experimental cultures through cytotoxic assays and cytokine production were also performed. RcaL was obtained through precipitation with ammonium sulphate and affinity chromatography on a Concanavalin A-Sepharose 4B column. The ammonium sulphate fraction F3 showed the highest specific hemagglutinating activity and was applied to affinity chromatography. The lectin was eluted with methyl-α-d-mannopyranoside. RcaL showed highest affinity for methyl-α-d-mannopyranoside and d-mannose; eluted fractions of RcaL agglutinated rabbit erythrocytes (titre, 128−1) retained 66 % of chromatographed lectin activity, and the obtained purification factor was 1.14. Under reducing conditions, a polypeptide band of 19.2 kDa was revealed in sodium dodecyl sulphate polyacrylamide gel electrophoresis (PAGE). PAGE confirmed RcaL as an acidic protein revealed in a single band. Cytotoxic and immunomodulatory assays with RcaL in mice splenocyte cultures showed that the lectin was not cytotoxic and induced higher interferon gamma and nitric oxide production in splenocyte cultures. Purified RcaL induced preferential Th1 response, suggesting that it acts as an immunomodulatory compound.
Keywords: Rachycentron canadum ; Cobia; Lectin purification; Immunomodulatory activity
Erratum to: Production of XynX, a Large Multimodular Protein of Clostridium thermocellum, by Protease-Deficient Bacillus subtilis Strains
by Nguyen Dinh Phuong; Yu Seok Jeong; Thangaswamy Selvaraj; Sung Kyum Kim; Yong Ho Kim; Kyung Hwa Jung; Jungho Kim; Han Dae Yun; Sui-Lam Wong; Jung-Kul Lee; Hoon Kim (pp. 1349-1350).