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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.167, #4)

A Rapid and Simple Method for Preparing an Insoluble Substrate for Screening of Microbial Xylanase by Kyong-Cheol Ko; Yunjon Han; Bong Seok Shin; Jong Hyun Choi; Jae Jun Song (pp. 677-684).

Several types of enzymes, including cellulases and xylanases, are required to degrade hemicelluloses and cellulose, which are major components of lignocellulosic biomass. Such degradative processes can be used to produce various useful industrial biomaterials. Screening methods for detecting polysaccharide-degrading microorganisms include the use of dye-labeled substrates in growth medium and culture plate staining techniques. However, the preparation of screening plates, which typically involves chemical cross-linking to synthesize a dye-labeled substrate, is a complicated and time-consuming process. Moreover, such commercial substrates are very expensive, costing tenfold more than the natural xylan. Staining methods are also problematic because they may damage relevant microorganisms and are associated with contamination of colonies of desirable organisms with adjacent unwanted bacteria. In the present study, we describe a sonication method for the simple and rapid preparation of an insoluble substrate that can be used to screen for xylanase-expressing bacteria in microbial populations. Using this new method, we have successfully isolated a novel xylanase gene from a xylolytic microorganism termed Xyl02-KBRB and Xyl14-KBRB in the bovine rumen.

Keywords: Screening method; Xylanase; Dye substrate; Assay method

Effect of the Inducers Veratryl Alcohol, Xylidine, and Ligninosulphonates on Activity and Thermal Stability and Inactivation Kinetics of Laccase from Trametes versicolor by Jorge A. Saraiva; Ana P. M. Tavares; Ana M. R. B. Xavier (pp. 685-693).

Laccase production from Trametes versicolor was improved in the presence of the inducers ligninosulphonates, veratryl alcohol, and xylidine respectively two-, four-, and eightfold. The thermal inactivation of the produced laccase, after partial purification with ammonium sulfate was kinetically investigated at various temperatures (60–70 °C) and pH values (3.5, 4.5, and 5.5). The inactivation process followed first-order kinetics for all conditions tested, except for veratryl alcohol, for which a constant activity level was observed at the end of the inactivation, also after first-order decay. Enzyme thermostability was affected by the type of inducer used in the culture medium for the production of laccase and also by the pH of incubation mixture. Generally, laccase stability increased with pH increment, being more stable at pH 5.5, except with xylidine. At pHs 4.5 and 5.5, the three inducers significantly increased laccase thermal stability, with the higher effect being observed for pH 5.5 and ligninosulphonates, where increment of half-life times ranged from 3- to 20-fold, depending on the temperature.

Keywords: Laccase production; Inducers; Thermal inactivation; Trametes versicolor ; Kinetics

Influence of Free Ammonia on Completely Autotrophic Nitrogen Removal over Nitrite (CANON) Process by Shan Li; You-Peng Chen; Chun Li; Jin-Song Guo; Fang Fang; Xu Gao (pp. 694-704).

Free ammonia (FA) plays a significant role in the stable, long-term, completely autotrophic nitrogen removal over nitrite (CANON) system operation. The influence of FA on the CANON process in a sequencing batch biofilm reactor was explored. Under controlled FA concentrations of 5.0 mg L⁻¹ to 10.0 mg L⁻¹, nitrite-oxidizing bacteria (NOB) was inhibited and achieved partial nitrification, which was important for a successful and quick start-up of the CANON process from activated sludge. However, NOB was acclimated to the condition after the process start-up. Ammonia-oxidizing bacteria (AOB) and anaerobic ammonium-oxidizing bacteria (AnAOB) activities were unaffected when FA concentration was increased from 10 mg L⁻¹ to 17 mg L⁻¹, but NOB was completely inhibited only for a short time. The AOB and AnAOB activities were inhibited and the CANON system was deteriorated when FA concentration reached 30 mg L⁻¹ to 32.5 mg L⁻¹ at pH 8.5, whereas NOB activity was unaffected. Correlation analysis showed that FA concentration higher than 20 mg L⁻¹ resulted in the deterioration of the system.

Keywords: CANON; Free ammonia; AOB; AnAOB

Modeling the Biomass Growth and Enzyme Secretion by the White Rot Fungus Phanerochaete chrysosporium: a Stochastic-Based Approach by K. Sen; Kannan Pakshirajan; S. B. Santra (pp. 705-713).

The white rot fungus Phanerochaete chrysosporium has been identified to be an environmentally useful microorganism for the degradation of various hazardous pollutants, mainly because of its ligninolytic enzyme system, particularly the lignin peroxidase (LiP) secreted by the fungus. In the present work, the behavior of the fungus in liquid medium due to variation in physico-chemical parameters, i.e., glucose concentration, nitrogen concentration, agitation, etc., was studied. Increment of the initial concentration of glucose in the medium increases the biomass growth and LiP activity, when cultured under controlled conditions. The biomass growth and LiP activity by the fungus was modeled following stochastic approach. The behavior of growth and enzyme activity of the fungus observed from the model were found to be in agreement with the experiments qualitatively.

Keywords: Phanerochaete chrysosporium ; Biomass growth; Lignin peroxidase; Stochastic modeling

Power Generation Capabilities of Microbial Fuel Cells with Different Oxygen Supplies in the Cathodic Chamber by Der-Fong Juang; Chao-Hsien Lee; Shu-Chun Hsueh; Huei-Yin Chou (pp. 714-731).

Two microbial fuel cells (MFCs) inoculated with activated sludge of a wastewater treatment plant were constructed. Oxygen was provided by mechanical aeration in the cathodic chamber of one MFC, whereas it was obtained by the photosynthesis of algae in the other. Electrogenic capabilities of both MFCs were compared under the same operational conditions. Results showed that the MFC with mechanical aeration in the cathodic chamber displayed higher power output than the one with photosynthesis of algae. Good linear relationship between power density and chemical oxygen demand (COD) loading rate was obtained only on the MFC with mechanical aeration. Furthermore, the relationships between power density and effluent COD and between Coulombic efficiency and COD loading rate can only be expressed as binary quadratic equations for the MFC with mechanical aeration and not for the one with photosynthesis of algae.

Keywords: Anode; Cathode; Coulombic efficiency; Electron; Power density

Improvement of Hydrogen Productivity by Introduction of NADH Regeneration Pathway in Clostridium paraputrificum by Yuan Lu; Chong Zhang; Hongxin Zhao; Xin-Hui Xing (pp. 732-742).

To improve the hydrogen productivity and examine the hydrogen evolution mechanism of Clostridium paraputrificum, roles of formate in hydrogen evolution and effects of introducing formate-originated NADH regeneration were explored. The formate-decomposing pathway for hydrogen production was verified to exist in C. paraputrificum. Then NAD⁺-dependent formate dehydrogenase FDH1 gene (fdh1) from Candida boidinii was overexpressed, which regenerate more NADH from formate to form hydrogen by NADH-mediated pathway. With fdh1 overexpression, the hydrogen yield via NADH-involving pathway increased by at least 59 % compared with the control. Accompanied by the change of hydrogen metabolism, the whole cellular metabolism was redistributed greatly.

Keywords: Anaerobic fermentation; Clostridium paraputrificum ; Formate dehydrogenase; Hydrogen production; NADH regeneration

Investigation of Factors Affecting Controlled Release from Photosensitive DMPC and DSPC Liposomes by Aysegul Aygun; Kathryn Torrey; Ashok Kumar; Larry D. Stephenson (pp. 743-757).

An investigation of liposomes comprised of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) lipids with cholesterol and zinc phthalocyanine (ZnPC) revealed that several fundamental liposome properties are influenced by composition and by lipid-specific features. DMPC and DSPC liposomes were synthesized, and their compositional changes, encapsulation capacities, morphologies, and release properties were evaluated. In this research, liposome degradation, lysis, and content release were initiated by photolysis, i.e., rupture induced by exposure to light. A controlled release mechanism was created through the introduction of photosensitizers (i.e., ZnPC) embedded within the cholesterol-stabilized liposome membrane. The light wavelength and light exposure time accelerated photodegradation properties of DMPC liposomes compared to DSPC liposomes, which exhibited a slower release rate. Morphological changes in the liposomes were strongly influenced by light wavelength and light exposure time. For both the DMPC and DSPC liposomes, visible light with wavelengths in the red end of the spectrum and broad spectrum ambient lighting (400–700 nm) were more effective for lysis than UV-A light (365 nm). Heating liposomes to 100 °C decreased the stability of liposomes compared to liposomes kept at room temperatures. In addition, the optimal lipid-to-cholesterol-to-photoactivator ratio that produced the most stable liposomes was determined.

Keywords: Liposome; DSPC; DMPC; Photo-induced destabilization; Controlled release; Lysis; Triggered liposomal release

MYMIV-AC2, a Geminiviral RNAi Suppressor Protein, Has Potential to Increase the Transgene Expression by Jamilur Rahman; Sumona Karjee; Sunil Kumar Mukherjee (pp. 758-775).

Gene silencing is one of the limiting factors for transgene expression in plants. But the plant viruses have learnt to suppress gene silencing by encoding the protein(s), called RNA silencing suppressor(s) (RSS). Hence, these proteins could be used to overcome the limitation for transgene expression. The RNAi suppressors, namely HC-Pro and P19, have been shown to enhance the transgene expression but other RSS proteins have not been screened for similar role. Moreover, none of RSSs from the DNA viruses are known for enhancing the expression of transgenes. The Mungbean Yellow Mosaic India Virus (MYMIV) belonging to the genus Begomovirus within the family of Geminiviridae encodes an RSS called the AC2 protein. Here, we used AC2 to elevate the expression of the transgenes. Upon introduction of MYMIV-AC2 in the silenced GFP transgenic tobacco lines, by either genetic hybridisation or transgenesis, the GFP expression was enhanced several fold in F1 and T0 lines. The GFP-siRNA levels were much reduced in F1 and T0 lines compared with those of the initial parental silenced lines. The enhanced GFP expression was also observed at the cellular level. This approach was also successful in enhancing the expression of another transgene, namely topoisomeraseII.

Keywords: RNAi; Viral suppressor; Post-transcriptional gene silencing; Molecular farming; Transgene expression

Biosynthesis of Silver Nanoparticles Using Latex from Few Euphorbian Plants and Their Antimicrobial Potential by Satish V. Patil; Hemant P. Borase; Chandrashekhar D. Patil; Bipinchandra K. Salunke (pp. 776-790).

The synthesis of well-dispersed and ultrafine metal nanoparticles has great interest due to their distinctive physicochemical properties and biomedical applications. This study is the first report of one-step solvent-free synthesis of AgNPs using Euphorbiaceae plant latex. Among evaluated eight latex-producing plants, four (Jatropha curcas, Jatropha gossypifolia, Pedilanthus tithymaloides, and Euphorbia milii) showed high potential to produce physicochemically distinct, small-sized and bactericidal AgNPs. Phytochemical screening showed presence of rich amount of biochemicals in these plants. J. gossypifolia showed uniformly dispersed comparatively small-sized AgNPs. Dose-dependent growth inhibition of bacterial pathogens Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermis, and Micrococcus luteus was observed for J. gossypifolia latex-synthesized AgNPs with minimum inhibitory concentration values 30, 40, 70, 60, and 60 ppm, respectively, after 24 h. Possible mode of action of AgNPs against pathogens was confirmed by analyzing enzymes and cell leakage.

Keywords: Euphorbiaceae; Latex; Nanoparticles; Antimicrobial; Jatropha gossypifolia ; Zeta potential

Nano-TiO₂-Induced Apoptosis by Oxidative Stress-Mediated DNA Damage and Activation of p53 in Human Embryonic Kidney Cells by Ramovatar Meena; Madhu Rani; Ruchita Pal; Paulraj Rajamani (pp. 791-808).

The aim of the present study is to explore the mechanism of cytotoxic and genotoxic effects of TiO₂ nanoparticles on human embryonic kidney (HEK-293) cells. Toxicity was evaluated using changes in various cellular parameters of HEK-293 cells like morphology, viability, metabolic activity, oxidative stress and apoptosis. Oxidative stress was measured by the level of reactive oxygen species (ROS), lipid peroxidation, superoxide dismutase, catalase and glutathione peroxidase. Apoptosis induced by nano-TiO₂ was characterized by PI staining and DNA ladder assay. Furthermore, apoptotic proteins such as p53 and Bax were analysed by western blot. Our results indicate that nano-TiO₂ induces cytotoxicity in a time- and dose-dependent manner. Oxidative stress and apoptosis were induced by exposure to nano-TiO₂. Moreover, the expression of p53, Bax and caspase-3 were increased in a dose-dependent pattern. In conclusion, ROS-mediated oxidative stress, the activation of p53, Bax, caspase-3 and oxidative DNA damage are involved in the mechanistic pathways of nano-TiO₂-induced apoptosis in HEK-293 cells.

Keywords: Nano-TiO₂ ; Cytotoxicity; Oxidative stress; p53; Apoptosis

Purification and Characterization of Cytotoxin Produced by a Clinical Isolate of Vibrio cholerae O54 TV113 by Sree Renjini Isac; Gopinath Balakrish Nair; Durg Vijai Singh (pp. 809-823).

Vibrio cholerae O54 TV113 isolated from a diarrheal patient produces an extracellular cytotoxin that caused alteration in the morphology of Chinese hamster ovary cells manifested as cell shrinkage with intact cell boundaries and finally causing cell death. Syncase medium supplemented with lincomycin (50 μg/ml), pH 7.2, and 18 h incubation with shaking at 37 °C supported optimal cytotoxin production. We isolated and purified this cytotoxin to homogeneity by ultrafiltration, 40–80 % ammonium sulfate precipitation, gradient–anion exchange chromatography, stepwise-anion exchange chromatography, and size exclusion chromatography increasing the specific activity by 866-fold. The cytotoxin is heat-labile, sensitive to protease and papain, and has a molecular weight of 64 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and enterotoxic activity in rabbit ileal loop assay. Both cytotoxic and enterotoxic activity could be inhibited or neutralized by antiserum raised against purified cytotoxin but not by preimmune serum. Immunodiffusion test between purified cytotoxin and its antiserum gave a single well-defined precipitin band showing reaction of complete identity and a well-defined single band in an immunoblot assay. This study thus indicate that the cytotoxin expressed by strain TV113 has both cytotoxic and enterotoxic activity and appears to contribute in pathogenesis of non-O1, non-O139 strains.

Keywords: Purification; Characterization; Cytotoxin; Chromatography; SDS-PAGE; Vibrio cholerae ; O54 TV113

Cryopreservative Effects of the Recombinant Ice-Binding Protein from the Arctic Yeast Leucosporidium sp. on Red Blood Cells by Sung Gu Lee; Hye Yeon Koh; Jun Hyuck Lee; Sung-Ho Kang; Hak Jun Kim (pp. 824-834).

Antifreeze proteins (AFPs) have important functions in many freeze-tolerant organisms. The proteins non-colligatively lower the freezing point and functionally inhibit ice recrystallization in frozen solutions. In our previous studies, we found that the Arctic yeast Leucosporidium sp. produces an AFP (LeIBP), and that the protein could be successfully produced in Pichia expression system. The present study showed that recombinant LeIBP possesses the ability to reduce the damage induced to red blood cells (RBCs) by freeze thawing. In addition to 40 % glycerol, both 0.4 and 0.8 mg/ml LeIBPs significantly reduced freeze–thaw-induced hemolysis at either rapid- (45 °C) or slow-warming (22 °C) temperatures. Post-thaw cell counts of the cryopreserved RBCs were dramatically enhanced, in particular, in 0.8 mg/ml LeIBP. Interestingly, the cryopreserved cells in the presence of LeIBP showed preserved cell size distribution. These results indicate that the ability of LeIBP to inhibit ice recrystallization helps the RBCs avoid critically damaging electrolyte concentrations, which are known as solution effects. Considering all these data, LeIBP can be thought of as a key component in improving RBC cryopreservation efficiency.

Keywords: Antifreeze protein; Recombinant; LeIBP; Red blood cells; Cryopreservation; Cell size distribution

Cross-Linked Enzyme Aggregates of Phenylalanine Ammonia Lyase: Novel Biocatalysts for Synthesis of L-Phenylalanine by Jian-Dong Cui; Si Zhang; Li-Mei Sun (pp. 835-844).

Cross-linked enzyme aggregates of phenylalanine ammonia lyase (PAL-CLEAs) from Rhodotorula glutinis were prepared. The effects of the type of aggregating agent, its concentration, and that of cross-linking agent were studied. PAL-CLEAs production was most effective using ammonium sulfate (40 % saturation), followed by cross-linking for 1 h with 0.2 % (v/v) glutaraldehyde. Moreover, the storage and operational stability of the resulting PAL-CLEAs were also investigated. Compared to the free enzyme, the PAL-CLEAs exhibited the expected increased stability of the enzyme against various deactivating conditions such as pH, temperature, denaturants, and organic solvents and showed higher storage stability than its soluble counterpart. Additionally, the reusability of PAL-CLEAs with respect to the biotransformation of l-phenylalanine was evaluated. PAL-CLEAs could be recycled at least for 12 consecutive batch reactions without dramatic activity loss, which should dramatically increase the commercial potential of PAL for synthesis of l-phenylalanine. To the best of our knowledge, this is the first report of immobilization of PAL as cross-linked enzyme aggregates.

Keywords: Phenylalanine ammonia lyase; Rhodotorula glutinis ; Cross-linked enzyme aggregates; l-phenylalanine; Biocatalysis

In Silico Analyses of COMT, an Important Signaling Cascade of Dopaminergic Neurotransmission Pathway, for Drug Development of Parkinson’s Disease by Chiranjib Chakraborty; Soumen Pal; C. George Priya Doss; Zhi-Hong Wen; Chan-Shing Lin (pp. 845-860).

Catechol-O-methyltransferase (COMT) has a vital role for degradation of dopamine, a neurotransmitter, and this dopamine performs an important function in our mental and physical health. The scarcity of dopamine may lead to Parkinson’s disease, and inhibition of COMT can stop dopamine metabolism. Here, we have carried out genomics and proteomics analyses of COMT in order to facilitate new inhibitors of COMT. For genomics analyses, we performed codon composition investigation of COMT gene which shows A+T content which is 53.3 %. For proteomics analyses, conservation patterns and residues (highly conserved amino acids GLU64, LEU65, GLY66, CYS69, GLY70, ALA77, GLU90, THR99, SER119, ASP136, LEU140, ASP141, THR164, ASN170, VAL171, and ILE172), binding grooves, binding pockets, binding and conformation with substrate, evaluation of amino acid composition (15 % leucine rich), high scoring hydrophobic segments, high scoring transmembrane segments, tandem and periodic repeats, and disulfide bonds (three numbers), sequence logos (maximum stack height of 3 b and minimum stack height of <0.5 b) have been investigated for COMT protein. Furthermore, using COMT sequences of different species (class Mammalia, class Amphibia, and class Pisces), a phylogenetic tree has been constructed to examine the evolutionary relationship among different species.

Keywords: Catechol-O-methyltransferase; Dopaminergic neurotransmitter; Parkinson’s disease; In silico analysis

The Anticancer Activity and HSA Binding Properties of the Structurally Related Platinum (II) Complexes by Reza Yousefi; Sadaf Aghevlian; Fatemeh Mokhtari; Hamidreza Samouei; Mehdi Rashidi; S. Masoud Nabavizadeh; Zohreh Tavaf; Zahra Pouryasin; Ali Niazi; Reza Faghihi; Mohammad Mehdi Papari (pp. 861-872).

The development of resistance and unwanted harmful interaction with other biomolecules instead of DNA are the major drawbacks for application of platinum (Pt) complexes in cancer chemotherapy. To conquer these problems, much works have been done so far to discover innovative Pt complexes. The objective of the current study was to evaluate the anti cancer activities of a series of four and five-coordinated Pt(II) complexes, having deprotonated 2-phenyl pyridine (abbreviated as C^N), biphosphine moieties, i.e., dppm = bis(diphenylphosphino) methane (Ph₂PCH₂PPh₂) and dppa = bis(diphenylphosphino)amine (Ph₂PNHPPh₂), as the non-leaving carrier groups. The growth inhibitory effect of the Pt complexes [Pt(C^N)(dppm)]PF₆: C 1, [Pt(C^N)(dppa)]PF₆: C 2, and [Pt(C^N)I(dppa)]: C 3, toward the cancer cell lines was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. In addition, the florescence quenching experiments of the interaction between human serum albumin (HSA) and the Pt complexes were performed in order to obtain the binding parameters and to evaluate the denaturing properties of these complexes upon binding to the general carrier protein of blood stream. The structure–activity relationship studies reveal that four-coordinated Pt complexes C 1 and C 2 with both significant hydrophobic and charge characteristics, not only exhibit strong antiproliferation activity toward the cancer cell lines, but also they display lower denaturing effect against carrier protein HSA. On the other hand, five-coordinated C 3 complex with the unusual intermolecular NH…Pt hydrogen binding and the intrinsic ability for oligomerization, exhibits poor anticancer activity and strong denaturing property. The current study reveals that the balance between charge and hydrophobicity of the Pt complexes, also their hydrogen binding abilities and coordination mode are important for their anticancer activities. Moreover, this study may suggest C 1 and C 2 as the potential template structures for synthesis of new generation of four-coordinated Pt complexes with strong anticancer activities and weak denaturing effects against proteins.

Keywords: Platinum complexes; 2-Pheynyl pyridine; Biphosphine ligand; Anticancer activity; Fluorescence study

Improved Bioethanol Production Using Fusants of Saccharomyces cerevisiae and Xylose-Fermenting Yeasts by Rajni Kumari; K. Pramanik (pp. 873-884).

The present research deals with the development of a hybrid yeast strain with the aim of converting pentose and hexose sugar components of lignocellulosic substrate to bioethanol by fermentation. Different fusant strains were obtained by fusing protoplasts of Saccharomyces cerevisiae and xylose-fermenting yeasts such as Pachysolen tannophilus, Candida shehatae and Pichia stipitis. The fusants were sorted by fluorescent-activated cell sorter and further confirmed by molecular characterization. The fusants were evaluated by fermentation of glucose–xylose mixture and the highest ethanol producing fusant was used for further study to ferment hydrolysates produced by acid pretreatment and enzymatic hydrolysis of cotton gin waste. Among the various fusant and parental strains used under present study, RPR39 was found to be stable and most efficient strain giving maximum ethanol concentration (76.8 ± 0.31 g L⁻¹), ethanol productivity (1.06 g L⁻¹ h⁻¹) and ethanol yield (0.458 g g⁻¹) by fermentation of glucose–xylose mixture under test conditions. The fusant has also shown encouraging result in fermenting hydrolysates of cotton gin waste with ethanol concentration of 7.08 ± 0.142 g L⁻¹, ethanol yield of 0.44 g g⁻¹, productivity of 0.45 g L⁻¹ h⁻¹ and biomass yield of 0.40 g g⁻¹.

Keywords: S. cerevisiae ; Protoplast; Fusant; Ethanol fermentation; Cotton gin waste; FACS; RAPD

Morindone, an Anthraquinone, Intercalates DNA Sans Toxicity: a Spectroscopic and Molecular Modeling Perspective by Dipita Bhakta; Ramamoorthy Siva (pp. 885-896).

For the first time, interaction between non-toxic anthraquinone morindone with both natural and synthetic DNA duplexes has been demonstrated in this paper. Detailed analyses of the binding of morindone with DNA via UV–vis, FTIR, and circular dichroism spectroscopies were carried out. In addition, bioinformatics tools have been employed to scrutinize the binding of the dye with DNA in silico. Results represent morindone to be a better binder with a score of −5.79 compared to EtBr (known mutagenic intercalator) recorded at −5.02. Further interaction is accentuated by the microscope-assisted evidence of nuclear specific staining of tissues by morindone. The electrophoretic analysis reveals the efficacy endowed within morindone dye in rendering protection to DNA exposed to H₂O₂ damage and thereby conferring it safe to the nucleic acid. As DNA is often the target for majority of anticancer and antibiotic drugs, study on the interaction between molecules like morindone and DNA has relevance and implications in several biological applications including cancer therapy. Thus, we propose that morindone can also be harnessed as a diagnostic probe for DNA structure in addition to DNA-directed therapeutics.

Keywords: Morindone; Interaction; Intercalating agent; Therapeutic; Spectroscopy

Strategies for Enhancing Extracellular Secretion of Recombinant Cyclodextrin Glucanotransferase in E. coli by Berhane Teklehaimanot Tesfai; Dan Wu; Sheng Chen; Jian Chen; Jing Wu (pp. 897-908).

Cyclodextrin glycosyltransferase (CGTase) is an enzyme that produces cyclodextrins from starch by an intramolecular transglycosylation reaction. Due to the increasing industrial application of cyclodextrins in many fields such as pharmacy, agriculture, biotechnology, food, environment and cosmetics, CGTases have attracted the attention of many scientific researches. Undoubtedly, due to its well-known genetic properties, simplicity and capacity to accommodate many foreign proteins, Escherichia coli remains the most widely used host for recombinant proteins production and thus for CGTases. Like all other proteins, CGTases are originally produced in the cytoplasm, but expressing them out into the periplasm or further to the culture media is preferred due to several advantages such as simplified downstream processing and high expression level which otherwise would be costly, complicated and time consuming. Since E. coli, other than some of its degradative enzymes and toxins, does not normally secrete proteins extracellularly, many strategies have been tried to overcome this drawback using the recombinant technologies. Unfortunately, oversecretion of the recombinant proteins most of the time results in the formation of inactive protein aggregates, called inclusion bodies, which result as a consequence of the burden caused by the methods meant to enhance the secretion. Thus, in this mini-review, the few but most commonly used strategies which offered a solution to the enhancement of extracellular secretion of CGTase in its native state are discussed.

Keywords: Cyclodextrin glucanotransferase; Recombinant proteins; E. coli ; Strategies; Periplasm; Extracellular secretion

Effect of Microaerobic Fermentation in Preprocessing Fibrous Lignocellulosic Materials by Manar Arica Alattar; Terrence R. Green; Jordan Henry; Vitalie Gulca; Mikias Tizazu; Robby Bergstrom; Radu Popa (pp. 909-917).

Amending soil with organic matter is common in agricultural and logging practices. Such amendments have benefits to soil fertility and crop yields. These benefits may be increased if material is preprocessed before introduction into soil. We analyzed the efficiency of microaerobic fermentation (MF), also referred to as Bokashi, in preprocessing fibrous lignocellulosic (FLC) organic materials using varying produce amendments and leachate treatments. Adding produce amendments increased leachate production and fermentation rates and decreased the biological oxygen demand of the leachate. Continuously draining leachate without returning it to the fermentors led to acidification and decreased concentrations of polysaccharides (PS) in leachates. PS fragmentation and the production of soluble metabolites and gases stabilized in fermentors in about 2–4 weeks. About 2 % of the carbon content was lost as CO₂. PS degradation rates, upon introduction of processed materials into soil, were similar to unfermented FLC. Our results indicate that MF is insufficient for adequate preprocessing of FLC material.

Keywords: Lignocellulose; Microaerobic fermentation; Bokashi; Polysaccharides; Carbon recycling; Soil fertilizers

Characterization and Activity Determination of the Human Protein Phosphatase 2A Catalytic Subunit α Expressed in Insect Larvae by J. A. Rubiolo; H. López-Alonso; A. Alfonso; F. V. Vega; M. R. Vieytes; L. M. Botana (pp. 918-928).

Protein phosphatase 2A is the major enzyme that dephosphorylates the serine/threonine residues of proteins in the cytoplasm of animal cells. This phosphatase is most strongly inhibited by okadaic acid. Besides okadaic acid, several other toxins and antibiotics have been shown to inhibit protein phosphatase 2A, including microsystin-LR, calyculin-A, tautomycib, nodularin, cantharidine, and fostriecin. This makes protein phosphatase 2A a valuable tool for detecting and assaying these toxins. High-scale production of active protein phosphatase 2A requires processing kilograms of animal tissue and involves several chromatographic steps. To avoid this, in this work we report the recombinant expression and characterization of the active catalytic subunit α of the protein phosphatase 2A in Trichoplusia ni insect larvae. Larvae were infected with baculovirus carrying the coding sequence for the catalytic subunit α of protein phosphatase 2A under the control of the polyhedrin promoter and containing a poly-His tag in the carboxyl end. The catalytic subunit was identified in the infected larvae extracts, and it was calculated to be present at 250 μg per gram of infected larvae, by western blot. Affinity chromatography was used for protein purification. Protein purity was determined by western blot. The activity of the enzyme, determined by the p-nitrophenyl phosphate method, was 94 μmol/min/mg of purified protein. The catalytic subunit was further characterized by inhibition with okadaic acid and dinophysis toxin 2. The results presented in this work show that this method allows the production of large quantities of the active enzyme cost-effectively. Also, the enzyme activity was stable up to 2 months at −20 °C.

Keywords: Recombinant protein phosphatase 2A; Trichoplusia ni; Okadaic acid; Protein phosphatase 2A inhibition; Enzyme stability

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.167, #4)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.167, #4)