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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.169, #3)

New Bacteria Bacillus nitroreducens PLC9 with Hydrogen Peroxide-Degrading Activity with High Survival Rate in Hydrogen Peroxide by Hyunjin Hong; Kyung Sook Bae; Yeonhee Lee (pp. 701-711).

Bacteria were isolated from wastewater containing highly concentrated hydrogen peroxide that had been used to clean the pure water delivery system in a semiconductor plant. One bacterium was selected for its high hydrogen peroxide degradation activity. In the presence of 1 % hydrogen peroxide, it degraded 72.5 % in 5 min. It showed 100 % viability after 6 h at 1 % hydrogen peroxide. Even at 3 % hydrogen peroxide, it survived for more than 6 h. This bacterium was named as Bacillus nitroreducens PLC9 since its 16S rRNA showed 100 % similarity with the recently reported new species B. nitroreducens. Purified catalase from B. nitroreducens PLC9 was characterized as a thermo-alkali-stable hydroperoxidase type II catalase, and it is suggested as a new type of catalase based on following: (1) it is stable over a broad pH range (pH 4–11); (2) it is consisted of homodimers with a molecular weight of 66 kDa (total molecular weight, 134 kDa); (3) its activity was not inhibited by 3-amino-1,2,4-triazole; and (4) its N-terminal sequence has never been reported before. Both B. nitroreducens PLC9 and the isolated catalase can be used for efficient degradation of hydrogen peroxide at high concentrations.

Keywords: Catalase; Hydrogen peroxide; Bacillus nitroreducens ; Wastewater treatment

Characterization of a Recombinant Flocculent Saccharomyces cerevisiae Strain that Co-ferments Glucose and Xylose: I. Influence of the Ratio of Glucose/Xylose on Ethanol Production by Akinori Matsushika; Shigeki Sawayama (pp. 712-721).

Glucose/xylose mixtures (90 g/L total sugar) were evaluated for their effect on ethanol fermentation by a recombinant flocculent Saccharomyces cerevisiae, MA-R4. Glucose was utilized faster than xylose at any ratio of glucose/xylose, although MA-R4 can simultaneously co-ferment both sugars. A high percentage of glucose can increase cell biomass production and therefore increase the rate of glucose utilization (1.224 g glucose/g biomass/h maximum) and ethanol formation (0.493 g ethanol/g biomass/h maximum). However, the best ratio of glucose/xylose for the highest xylose consumption rate (0.209 g xylose/g biomass/h) was 2:3. Ethanol concentration and yield increased and by-product (xylitol, glycerol, and acetic acid) concentration decreased as the proportion of glucose increased. The maximum ethanol concentration was 41.6 and 21.9 g/L after 72 h of fermentation with 90 g/L glucose and 90 g/L xylose, respectively, while the ethanol yield was 0.454 and 0.335 g/g in 90 g/L glucose and 90 g/L xylose media, respectively. High ethanol yield when a high percentage of glucose is available is likely due to decreased production of by-products, such as glycerol and acetic acid. These results suggest that ethanol selectivity is increased when a higher proportion of glucose is available and reduced when a higher proportion of xylose is available.

Keywords: Recombinant Saccharomyces cerevisiae ; Xylose; Glucose; Ethanol; Co-fermentation; Ratio of sugar

Eplt4 Proteinaceous Elicitor Produced in Pichia pastoris Has a Protective Effect Against Cercosporidium sofinum Infections of Soybean Leaves by Yun Wang; Jinzhu Song; Yingjie Wu; Margaret Odeph; Zhihua Liu; Barbara J. Howlett; Shuang Wang; Ping Yang; Lin Yao; Lei Zhao; Qian Yang (pp. 722-737).

A complementary DNA library was constructed from the mycelium of Trichoderma asperellum T4, and a highly expressed gene fragment named EplT4 was found. In order to find a more efficient and cost-effective way of obtaining EplT4, this study attempted to produce EplT4 using a Pichia pastoris expression system. The gene encoding EplT4, with an additional 6-His tag at the C-terminus, was cloned into the yeast vector pPIC9K and expressed in the P. pastoris strain GS115 to obtaining more protein for the further research. Transformants of P. pastoris were selected by PCR analysis, and the ability to secrete high levels of the EplT4 protein was determined. The optimal conditions for induction were assayed using the shake flask method and an enzyme-linked immunosorbent assay. The yield of purified EplT4 was approximately 20 mg/L by nickel affinity chromatography and gel-filtration chromatography. Western blot and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis revealed that the recombinant EplT4 was expressed in both its monomers and dimers. Soybean leaves treated with the EplT4 monomer demonstrated the induction of glucanase, chitinase III-A, cysteine proteinase inhibitor, and peroxidase genes. Early cellular events in plant defense response were also observed after incubation with EplT4. Soybean leaves protected by EplT4 against the pathogen Cercosporidium sofinum (Hara) indicated that EplT4 produced in P. pastoris was biologically active and would be potentially useful for improving food security.

Keywords: EplT4 elicitor; Trichoderma asperellum ; Pichia pastoris ; Soybean; Induced resistance

Review on the Angiotensin-I-Converting Enzyme (ACE) Inhibitor Peptides from Marine Proteins by Hai-Lun He; Dan Liu; Chang-Bei Ma (pp. 738-749).

Hypertension is now a major problem threatening people health in the world. Angiotensin-I-converting enzyme (ACE) plays an important physiological role in regulation of blood pressure via conversion of angiotensin I to angiotensin II. Inhibition of ACE may have an antihypertensive effect as a consequence of a decrease in blood pressure. A number of terrestrial-derived peptides have been reviewed about their in vitro and in vivo ACE-inhibitory activities. Marine organisms are potentially an untapped source of drugs and value-added food production. The aim of this review is to discuss the marine-derived ACE-inhibitory peptides from sources, production, structure aspects, and their future prospects as functional food or novel therapeutic drug candidates.

Keywords: Angiotensin-I-converting enzyme (ACE); Antihypertensive; ACE-inhibitory activity; Peptides; Marine organisms

Synthesis of Fructooligosaccharides from Aspergillus niger Commercial Inulinase Immobilized in Montmorillonite Pretreated in Pressurized Propane and LPG by Graciele de Oliveira Kuhn; Clarissa Dalla Rosa; Marceli Fernandes Silva; Helen Treichel; Débora de Oliveira; J. Vladimir Oliveira (pp. 750-760).

Commercial inulinase from Aspergillus niger was immobilized in montmorillonite and then treated in pressurized propane and liquefied petroleum gas (LPG). Firstly, the effects of system pressure, exposure time, and depressurization rate, using propane and LPG, on enzymatic activity were evaluated through central composite design 2³. Residual activities of 145.1 and 148.5 % were observed for LPG (30 bar, 6 h, and depressurization rate of 20 bar min⁻¹) and propane (270 bar, 1 h, and depressurization rate of 100 bar min⁻¹), respectively. The catalysts treated at these conditions in both fluids were then used for the production of fructooligosaccharides (FOS) using sucrose and inulin as substrates in aqueous and organic systems. The main objective of this step was to evaluate the yield and productivity in FOS, using alternatives for enhancing enzyme activity by means of pressurized fluids and also using low-cost supports for enzyme immobilization, aiming at obtaining a stable biocatalyst to be used for synthesis reactions. Yields of 18 % were achieved using sucrose as substrate in aqueous medium, showing the potential of this procedure, hence suggesting a further optimization step to increase the process yield.

Keywords: Inulinase; FOS; LPG; Propane; Fructooligosaccharides

Potential of Potassium Hydroxide Pretreatment of Switchgrass for Fermentable Sugar Production by Rajat Sharma; Vijaykumar Palled; Ratna R. Sharma-Shivappa; Jason Osborne (pp. 761-772).

Chemical pretreatment of lignocellulosic biomass has been extensively investigated for sugar generation and subsequent fuel production. Alkaline pretreatment has emerged as one of the popular chemical pretreatment methods, but most attempts thus far have utilized NaOH for the pretreatment process. This study aimed at investigating the potential of potassium hydroxide (KOH) as a viable alternative alkaline reagent for lignocellulosic pretreatment based on its different reactivity patterns compared to NaOH. Performer switchgrass was pretreated at KOH concentrations of 0.5–2 % for varying treatment times of 6–48 h, 6–24 h, and 0.25–1 h at 21, 50, and 121 °C, respectively. The pretreatments resulted in the highest percent sugar retention of 99.26 % at 0.5 %, 21 °C, 12 h while delignification up to 55.4 % was observed with 2 % KOH, 121 °C, 1 h. Six pretreatment conditions were selected for subsequent enzymatic hydrolysis with Cellic CTec2® for sugar generation. The pretreatment condition of 0.5 % KOH, 24 h, 21 °C was determined to be the most effective as it utilized the least amount of KOH while generating 582.4 mg sugar/g raw biomass for a corresponding percent carbohydrate conversion of 91.8 %.

Keywords: Switchgrass; Lignocelluloses; KOH; Enzymatic hydrolysis; AIL; Fermentable sugars

In Silico and Experimental Characterization of Chimeric Bacillus thermocatenulatus Lipase with the Complete Conserved Pentapeptide of Candida rugosa Lipase by Mostafa Hosseini; Ali Asghar Karkhane; Bagher Yakhchali; Mehdi Shamsara; Saeed Aminzadeh; Dena Morshedi; Kamahldin Haghbeen; Ibrahim Torktaz; Esmat Karimi; Zahra Safari (pp. 773-785).

Lipases are one of the highest value commercial enzymes as they have broad applications in detergent, food, pharmaceutical, and dairy industries. To provide chimeric Bacillus thermocatenulatus lipase (BTL2), the completely conserved pentapeptide (¹¹²Ala-His-Ser-Gln-Gly¹¹⁶) was replaced with similar sequences (²⁰⁷Gly-Glu-Ser-Ala-Gly²¹¹) of Candida rugosa lipase (CLR) at the nucleophilic elbow region. For this purpose, three mutations including A112G, H113E, and Q115A were inserted in the conserved pentapeptide sequence of btl2 gene. Based on the crystal structures of 2W22, the best structure of opened form of the chimeric lipases were garnered using the MODELLER v9.10 software. The native and chimeric lipases were docked to a set of ligands, and a trial version of Molegro Virtual Docker (MVD) software was used to obtain the energy values. Docking results confirmed chimeric lipase to be better than the native lipase. Following the in silico study, cloning experiments were conducted and expression of native and chimeric btl2 gene in Pichia pastoris was performed. The native and chimeric lipases were purified, and the effect of these mutations on characteristics of chimeric lipase studied and then compared with those of native lipase. Chimeric lipase exhibited 1.6-fold higher activity than the native lipase at 55 °C. The highest percentage of both lipases activity was observed at 60 °C and pH of 8.0. The ion Ca²⁺ slightly inhibited the activity of both lipases, whereas the organic solvent enhanced the lipase stability of chimeric lipase as compared with the native lipase. According to the results, the presence of two glycine residues at the conserved pentapeptide region of this chimeric lipase (¹¹² Gly-Glu-Ser-Ala-Gly ¹¹⁶) may increase the flexibility of the nucleophilic elbow region and affect the enzyme activity level.

Keywords: In silico; Bacillus thermocatenulatus ; Conserved pentapeptide; Docking

Effects of Alcohols and Compatible Solutes on the Activity of β-Galactosidase by Andrew N. W. Bell; Emma Magill; John E. Hallsworth; David J. Timson (pp. 786-794).

During alcoholic fermentation, the products build up and can, ultimately, kill the organism due to their effects on the cell’s macromolecular systems. The effects of alcohols on the steady-state kinetic parameters of the model enzyme β-galactosidase were studied. At modest concentrations (0 to 2 M), there was little effect of methanol, ethanol, propanol and butanol on the kinetic constants. However, above these concentrations, each alcohol caused the maximal rate, V _max, to fall and the Michaelis constant, K _m, to rise. Except in the case of methanol, the chaotropicity of the solute, rather than its precise chemical structure, determined and can, therefore, be used to predict inhibitory activity. Compounds which act as compatible solutes (e.g. glycerol and other polyols) generally reduced enzyme activity in the absence of alcohols at the concentration tested (191 mM). In the case of the ethanol- or propanol-inhibited β-galactosidase, the addition of compatible solutes was unable to restore the enzyme’s kinetic parameters to their uninhibited levels; addition of chaotropic solutes such as urea tended to enhance the effects of these alcohols. It is possible that the compatible solutes caused excessive rigidification of the enzyme’s structure, whereas the alcohols disrupt the tertiary and quaternary structure of the protein. From the point of view of protecting enzyme activity, it may be unwise to add compatible solutes in the early stages of industrial fermentations; however, there may be benefits as the alcohol concentration increases.

Keywords: Protein denaturation; Chaotropic solute; Model enzyme; Steady-state kinetics; Polyol; Enzyme flexibility

Analysis of Nitrification in Agricultural Soil and Improvement of Nitrogen Circulation with Autotrophic Ammonia-Oxidizing Bacteria by Toshihide Matsuno; Sachie Horii; Takanobu Sato; Yoshiki Matsumiya; Motoki Kubo (pp. 795-809).

Accumulations of inorganic nitrogen (NH₄ ⁺, NO₂ ⁻, and NO₃ ⁻) were analyzed to evaluate the nitrogen circulation activity in 76 agricultural soils. Accumulation of NH₄ ⁺ was observed, and the reaction of NH₄ ⁺ → NO₂ ⁻ appeared to be slower than that of NO₂ ⁻ → NO₃ ⁻ in agricultural soil. Two autotrophic and five heterotrophic ammonia-oxidizing bacteria (AOB) were isolated and identified from the soils, and the ammonia-oxidizing activities of the autotrophic AOB were 1.0 × 10³–1.0 × 10⁶ times higher than those of heterotrophic AOB. The relationship between AOB number, soil bacterial number, and ammonia-oxidizing activity was investigated with 30 agricultural soils. The ratio of autotrophic AOB number was 0.00032–0.26 % of the total soil bacterial number. The soil samples rich in autotrophic AOB (>1.0 × 10⁴ cells/g soil) had a high nitrogen circulation activity, and additionally, the nitrogen circulation in the agricultural soil was improved by controlling the autotrophic AOBs.

Keywords: Nitrogen circulation; Autotrophic ammonia-oxidizing bacteria; Heterotrophic ammonia-oxidizing bacteria; Ammonia nitrogen; Nitrite nitrogen; Nitrate nitrogen

Pulse Respirometry in Two-Phase Partitioning Bioreactors: Case Study of Terephthalic Acid Biodegradation by Alberto Ordaz; Guillermo Quijano; Frederic Thalasso; Claudio Garibay-Orijel (pp. 810-820).

Two-phase partitioning bioreactors (TPPBs) are based on the addition of an organic phase, often called vector, to a bioreactor in order to increase mass transfer of oxygen or gaseous substrates from the gaseous phase to the aqueous phase. In TPPBs, like in any other reactor design, the characterization of the bioprocess is often required for design, control, and operation purposes. Pulse respirometry is a method that allows for microbial processes characterization through the determination of several stoichiometric and kinetic parameters with relatively little experimental effort. Despite its interest and its previous application in countless applications, pulse respirometry has never been applied in TPPBs. In this work, pulse respirometry was assessed in a model TPPB degrading terephthalic acid and using Elvax™ as solid vector to enhance oxygen transfer. The results indicated that the addition of 10 to 20 % Elvax increased oxygen transfer by up to 97 %, compared to control with no vector. Pulse respirometry was successfully applied and allowed for the determination of the growth yield, the substrate affinity constant, and the maximum growth rate, within other. It is concluded that pulse respirometry is a useful method, not only for the characterization of processes in TPPBs but also for the selection of a vector within several brands commercially available.

Keywords: Aerobic bioprocesses; Mass transfer; Respirometry; Terephthalic acid; Two-phase partitioning bioreactor

Use of NAD(P)H Fluorescence Measurement for On-Line Monitoring of Metabolic State of Azohydromonas australica in Poly(3-hydroxybutyrate) Production by Geeta Gahlawat; Ashok K. Srivastava (pp. 821-831).

Culture fluorescence measurement is an indirect and non-invasive method of biomass estimation to assess the metabolic state of the microorganism in a fermentation process. In the present investigation, NAD(P)H fluorescence has been used for on-line in situ characterization of metabolic changes occurring during different phases of batch cultivation of Azohydromonas australica in growth associated poly(3-hydroxybutyrate) or PHB production. A linear correlation between biomass concentration and net NAD(P)H fluorescence was obtained during early log phase (3–12 h) and late log phase (24–39 h) of PHB fermentation. After 12 h (mid log phase) cultivation PHB accumulation shot up and a drop in culture fluorescence was observed which synchronously exhibited continuous utilization of NAD(P)H for the synthesis of biomass and PHB formation simultaneously. A decrease in the observed net fluorescence value was observed again towards the end of fermentation (at 39 h) which corresponded very well with the culture starvation and substrate depletion towards the end of cultivation inside the bioreactor. It was therefore concluded that NAD(P)H fluorescence measurements could be used for indication of the time of fresh nutrient (substrate) feed during substrate limitation to further enhance the PHB production.

Keywords: Poly (3-hydroxybutyrate); Azohydromonas australica ; NAD(P)H fluorescence; Bioprocess monitoring; Bioreactor; Batch fermentation

Proteomic Identification Network Analysis of Haptoglobin as a Key Regulator Associated with Liver Fibrosis by Aihua Zhang; Hui Sun; Wejun Sun; Yuan Ye; Xijun Wang (pp. 832-846).

Liver fibrosis (LF) is the final stage of liver dysfunction, characterized by diffuse fibrosis which is the main response to the liver injury. Haptoglobin (HP) protein, produced as an acute phase reactant during LF, preventing liver damage, may be potential molecular targets for early LF diagnostics and therapeutic applications. However, protein networks associated with the HP are largely unknown. To address this issue, we used a pathological mouse model of LF that was induced by treatment with carbon tetrachloride for 8 days. HP protein was separated and identified by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. HP protein was subjected to functional pathway analysis using STRING and Cytoscape software for better understanding of the protein–protein interaction (PPI) networks in biological context. Bioinformatics analyses revealed that HP expression associated with fibrosis was upregulated, and suggested that HP responsible for fibrosis may precede the onset and progression of LF. Using the web-based database, functional pathway analysis suggested the modulation of multiple vital physiological pathways, including antioxidation immunity, signal transduction, metabolic process, energy production, cell apoptosis, oxidation reduction, DNA repair process, cell communication, and regulation of cellular process. The generation of protein interaction networks clearly enhances the interpretation and understanding of the molecular mechanisms of HP. HP protein represents targets for further experimental investigation that will provide biological insight and potentially could be exploited for novel therapeutic approaches to combat LF.

Keywords: Proteomics; Liver fibrosis; Protein–protein interaction network; Two-dimensional gel electrophoresis; Mass spectrometry

Molecular Cloning, Expression, and Enzymatic Analysis of Cathepsin X from Starfish (Asterina pectinifera) by Hye Jin Bak; Moo-Sang Kim; Na Young Kim; Hye-Jin Go; Jin Woo Han; Hyae In Jo; Sang Jung Ahn; Nam Gyu Park; Joon Ki Chung; Hyung Ho Lee (pp. 847-861).

Cathepsin X, also known as cathepsin Z, is referred to as a “lysosomal proteolytic enzyme” and a member of the peptidase C1 family, which is involved in various biological processes such as immune response, cell adhesion, and proliferation. In the present study, the cDNA of starfish (Asterina pectinifera), which is known to cause serious damage to commercial shellfish mariculture, cathepsin X (ApCtX) was isolated through the combination of homology molecular cloning and rapid amplification of cDNA ends (RACE) methods for the application to find a way to reduce/control starfish densities. The full-length of ApCtX gene was determined to consist of the 2,240 bp nucleotide sequence, which encoded for a preproprotein of 296 amino acids with a molecular mass of about 32.7 kDa. The tissue type expression of ApCtX was determined in various tissues of A. pectinifera and was shown most abundantly in the liver. The cDNA encoding pro-mature enzyme of ApCtX was expressed in Escherichia coli BL21 (DE3) using the pGEX-4T-1 expression vector. Its activity was quantified by cleaving the synthetic peptide Z-Phe-Arg-AMC. The optimal pH for the protease activity was 6.5. The enzymatic activity of proApCtX was reduced by antipain, NEM, EDTA, EGTA, and 1,10-phenanthroline, and the proApCtX enzyme was significantly inhibited by CuSO₄, HgCl₂, CoCl₂, and SDS whereas Triton X-100 and Brij 35 might have potentially acted as an activator. Here, we demonstrated for the first time that the structural features and enzymatic characteristics of Echinoderms cathepsin X are similar to those of the other mammalian and piscine cathepsin X except its pH optimum, and the results of tissue-specific expression might explain their importance in food digestion by hepatic cecain starfish.

Keywords: Cathepsin X; Cysteine protease; cDNA cloning; mRNA expression; Starfish (Asterina pectinifera)

Cloning, Expression and Characterization of NADP-Dependent Isocitrate Dehydrogenase from Staphylococcus aureus by U. Venkateswara Prasad; D. Vasu; Y. Nanda Kumar; P. Santhosh Kumar; S. Yeswanth; V. Swarupa; B. V. Phaneendra; Abhijit Chaudhary; P. V. G. K. Sarma (pp. 862-869).

The Krebs cycle dictates oxidative and reductive conditions in Staphylococcus aureus and is mainly regulated by isocitrate dehydrogenase (IDH) which plays pivotal role in the growth and pathogenesis of the bacteria. In the present study, IDH gene from S. aureus ATCC12600 was cloned in the Sma I site of pQE 30 vector; the resultant clone was named as UVIDH1. The insert in the clone was sequenced (accession number HM067707), and the sequence showed complete homology with IDH sequence of other S. aureus strains reported in the database indicating presence of single enzyme in S. aureus, and considerable sequence homology with other bacteria was observed; however, only 24 % homology was found with NADP-dependent human IDH. Phylogenetically, the S. aureus IDH showed close identity with Bacillus subtilis and high degree of variability with other bacteria and human IDH. The expression of IDH in the clone UVIDH1 was induced with 1 mM IPTG, and the recombinant IDH was purified by passing through nickel metal chelate column; the purified recombinant IDH showed a single band in SDS–PAGE with a molecular weight of 40 kDa; K _m and V _max for isocitrate are 8.2 ± 0.28 and 525 ± 25 μM NADPH/mg/min, respectively, and for cofactor NADP 67.5 ± 2.82 μM and V _max 50.5 ± 2.12 μM NADPH/mg/min.

Keywords: Isocitrate dehydrogenase; Krebs cycle; K _m ; V _max

Two-Step Purification of a Novel β-Glucosidase with High Transglycosylation Activity and Another Hypothetical β-Glucosidase in Aspergillus oryzae HML366 and Enzymatic Characterization by Haiyan He; Yongling Qin; Guiguang Chen; Nan Li; Zhiqun Liang (pp. 870-884).

Two novel β-glucosidases (BGHG1 and BGHG2) were purified from the enzyme extract of Aspergillus oryzae HML366 through nondenaturing gel electrophoresis and anion-exchange chromatography. The molecular weights for BGHG1 and BGHG2 were 93 and 138 kDa, respectively. The amino acid sequences were determined by matrix-assisted laser desorption/ionization tandem time of flight. The Mascot and Blast analyses indicated that BGHG1 has the same sequence as the hypothetical protein XP_001816831 from A. oryzae RIB40. Sequence comparison suggested that both enzymes belong to the glycosyl hydrolase family 3. Results from thin layer chromatography and high performance liquid chromatography showed that BGHG2 has relatively high transglycosylation activity, and after preliminary optimization, it was able to convert glucose to produce 52.48 mg/ml gentiobiose. This is the first report of production of hypothetical protein XP_001816831 and β-glucosidase with high transglycosylation activity in A. oryzae. Results provide a valuable reference for potential applications in food industry, biomass power generating industry, and many others.

Keywords: β-Glucosidase; Transglycosylation; Purification; MALDI-TOF MS; Hypothetical protein; Aspergillus oryzae HML366

Physicochemical and Biochemical Profiling of Diphenyl Diselenide by Marina Prigol; Cristina W. Nogueira; Gilson Zeni; Maria Rosário Bronze; Luís Constantino (pp. 885-893).

The objective of the present study was to evaluate the physicochemical and biochemical profiling of diphenyl diselenide (PhSe)₂, a selenoorganic compound with biological activity. Experimental protocols were established for chemical stability in isotonic phosphate buffer (PBS) pH 7.4 and in simulated gastric and intestinal fluids, biological stability (bovine serum albumin (BSA) and plasma), solubility in PBS pH 7.4, distribution coefficient (Log D) in octanol/PBS, and determination of free (PhSe)₂ concentrations in BSA and plasma by using liquid chromatography with ultraviolet detection and tandem mass spectrometry. (PhSe)₂ was found to be chemically stable and not susceptible to degradation in plasma. The aqueous solubility was 0.98 ± 0.072 μM and the Log D in octanol/PBS system was found to be 3.13. The percentage of unbound fractions of (PhSe)₂ obtained by equilibrium dialysis from BSA and plasma incubated with 100 μM (PhSe)₂ were 0.69 ± 0.12 and 0.44 ± 0.09 %, respectively. The findings indicated that (PhSe)₂ presents chemical and biological stability. Though, the compound showed low aqueous solubility, high Log D value and high binding to plasmatic protein. These data contribute to the knowledge of the toxicokinetic properties of (PhSe)₂ and further explain its low bioavailability in experimental models.

Keywords: Selenium; Protein binding; Solubility; Toxicology

In Vitro Plant Propagation of Catharanthus roseus and Assessment of Genetic Fidelity of Micropropagated Plants by RAPD Marker Assay by Ajay Kumar; Krishna Prakash; Rajesh Kumar Sinha; Nitish Kumar (pp. 894-900).

An investigation was carried out to develop an efficient micropropagation protocol for Catharanthus roseus. Experiments were conducted to optimize suitable media for in vitro shoot multiplication and root induction. Out of the different media compared for in vitro shoot multiplication, Murashige and Skoog (MS) medium supplemented with 1 mg/l of 6-benzylaminopurine and 0.2 mg/l α-naphthaleneacetic acid showed better response in terms of the emergence of shoots from axillary buds as well as proliferation and multiplication of shoots. The shoots when placed on half strength of MS medium having 1 mg/l indole 3-butyric acid and 0.25 % charcoal showed cent percent root induction with maximum number of roots per shoot (4.2) as well as maximum root length (1.72 cm). Further, clonal fidelity of the in vitro-raised plants was carried out using randomly amplified polymorphic DNA marker and results indicated that all the tissue culture-derived plants are true-to-type and there were no somaclonal variations among these plants.

Keywords: Catharanthus roseus ; Genetic fidelity; Micropropagation; RAPD

Aldehyde Oxidase Activity and Stability in Water-Miscible Organic Solvents by Mohammad-Reza Rashidi; Moharam Dehghany; Golamreza Dehghan; Abolghasem Jouyban; Akram Faridi (pp. 901-910).

In this study, the catalytic activity of aldehyde oxidase was investigated in various water-miscible organic solvents for the first time. The enzyme was partially purified from rabbit liver, and its activity was determined in the absence/presence of nine hydro-organic mixtures. The effects of pH and temperature on the enzyme activity were also investigated. The activity was reduced in the presence of all nine organic solvents. However, in some cases, the residual activity remained almost unchanged throughout the incubation of enzyme at 35 °C for 24 h. Considering potential advantages of doing reactions in the presence of organic solvents, these results could be of value. The enzyme showed different behavior in the reaction solutions making it difficult to formulate results in a single comprehensive model. The results indicated that binding and cleavage of the substrate are influenced in the presence of organic solvents.

Keywords: Aldehyde oxidase; Water-miscible organic solvents; Enzyme activity; Thermal stability; Kinetic constants

In Silico and in Vitro Physicochemical Screening of Rigidoporus sp. Crude Laccase-assisted Decolorization of Synthetic Dyes—Approaches for a Cost-effective Enzyme-based Remediation Methodology by S. Sridhar; V. Chinnathambi; P. Arumugam; P K Suresh (pp. 911-922).

The objective of this paper is to compare in silico data with wet lab physicochemical properties of crude laccase enzyme isolated from Rigidoporus sp. using wheat bran as solid substrate support towards dye decolorization. Molecular docking analysis of selected nine textile and non-textile dyes were performed using laccase from Rigidoporus lignosus as reference protein. Enzyme-based remediation methodology using crude enzyme enriched from solid state fermentation was applied to screen the effect of four influencing variables such as pH, temperature, dye concentration, and incubation time toward dye decolorization. The extracellular crude enzyme decolorized 69.8 % Acid Blue 113, 45.07 % Reactive Blue 19, 36.61 % Reactive Orange 122, 30.55 % Acid Red 88, 24.59 % Direct Blue 14, 18.48 % Reactive Black B, 16.49 % Reactive Blue RGB, and 11.66 % Acid Blue 9 at 100 mg/l dye concentration at their optimal pH at room temperature under static and dark conditions after 1 h of incubation without addition of any externally added mediators. Our wet lab studies approach, barring other factors, validate in silico for screening and ranking textile dyes based on their proximity to the T1 site. We are reporting for the first time a combinatorial approach involving in silico methods and wet lab-based crude laccase-mediated dye decolorization without any external mediators.

Keywords: Molecular docking; Synthetic dyes; Rigidoporus sp.; Crude laccase-based decolorization; Physicochemical properties

Application of Enzymatic Method in the Extraction and Transformation of Natural Botanical Active Ingredients by Yan Lu; Jian-Guo Jiang (pp. 923-940).

Active components from traditional Chinese medicine (TCM) are the material basis for disease treatment. Extraction, identification, and transformation are the critical processes to analyze and use the active components of TCM. Botanic TCM takes up 90 % of total Chinese crude drugs. Some active components are complex and of lower level; moreover, most of them are enveloped under plant cell wall. Traditional extraction methods such as lixiviate method, decoction, and others are often hindered by cell wall, leading to low extraction efficiency, low clearance of impurity, and other problems, which have restricted the development of TCM. This paper reviews both domestically and internationally published literatures in recent years on application of enzymatic methods in the extraction and transformation of active ingredients from TCM. Principles of enzymatic method and its application in extraction and transformation of active ingredients and in dreg recycles of TCM are introduced in detail. With the development of TCM modernization, enzymatic method applied in the domain of TCM has achieved prominent benefits, not only improving the extraction and separation rate of active ingredients from TCM and elevating the transformation level and production, but also reducing costs in the transformation of active ingredients.

Keywords: Biotransformations; Bioseparations; Enzymes; Cellulose; Enzymatic methods; Natural active ingredients

Molecular Cloning and Expression of a Novel β-Glucosidase Gene from Phialophora sp. G5 by Xuejun Li; Junqi Zhao; Pengjun Shi; Peilong Yang; Yaru Wang; Huiying Luo; Bin Yao (pp. 941-949).

A novel β-glucosidase gene, bgl1G5, was cloned from Phialophora sp. G5 and successfully expressed in Pichia pastoris. Sequence analysis indicated that the gene consists of a 1,431-bp open reading frame encoding a protein of 476 amino acids. The deduced amino acid sequence of bgl1G5 showed a high identity of 85 % with a characterized β-glucosidase from Humicola grisea of glycoside hydrolase family 1. Compared with other fungal counterparts, Bgl1G5 showed similar optimal activity at pH 6.0 and 50 °C and was stable at pH 5.0–9.0. Moreover, Bgl1G5 exhibited good thermostability at 50 °C (6 h half-life) and higher specific activity (54.9 U mg^–1). The K _m and V _max values towards p-nitrophenyl β-d-glucopyranoside (pNPG) were 0.33 mM and 103.1 μmol min^–1 mg^–1, respectively. The substrate specificity assay showed that Bgl1G5 was highly active against pNPG, weak on p-nitrophenyl β-d-cellobioside (pNPC) and p-nitrophenyl-β-d-galactopyranoside (ONPG), and had no activity on cellobiose. This result indicated Bgl1G5 was a typical aryl β-glucosidase.

Keywords: Phialophora sp. G5; Aryl β-glucosidase; Pichia pastoris ; Heterologous expression

Selective n-Butanol Production by Clostridium sp. MTButOH1365 During Continuous Synthesis Gas Fermentation Due to Expression of Synthetic Thiolase, 3-Hydroxy Butyryl-CoA Dehydrogenase, Crotonase, Butyryl-CoA Dehydrogenase, Butyraldehyde Dehydrogenase, and NAD-Dependent Butanol Dehydrogenase by Vel Berzin; Michael Tyurin; Michael Kiriukhin (pp. 950-959).

Acetogen Clostridum sp. MT1962 produced 287 mM acetate (p < 0.005) and 293 mM ethanol (p < 0.005) fermenting synthesis gas blend 60 % CO and 40 % H₂ in single-stage continuous fermentation. This strain was metabolically engineered to the biocatalyst Clostridium sp. MTButOH1365. The engineered biocatalyst lost production of ethanol and acetate while initiated the production of 297 mM of n-butanol (p < 0.005). The metabolic engineering comprised Cre-lox66/lox71-based elimination of phosphotransacetylase and acetaldehyde dehydrogenase along with integration to chromosome synthetic thiolase, 3-hydroxy butyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase, and NAD-dependent butanol dehydrogenase. This is the first report on elimination of acetate and ethanol production genes and expression of synthetic gene cluster encoding n-butanol biosynthesis pathway in acetogen biocatalyst for selective fuel n-butanol production with no antibiotic support for the introduced genes.

Keywords: Acetogens; Gene elimination; n-Butanol; Syngas; Cell energy pool

Purification, Characterization, and Heterologous Expression of a Thermostable β-1,3-1,4-Glucanase from Bacillus altitudinis YC-9 by Shurui Mao; Zhaoxin Lu; Chong Zhang; Fengxia Lu; Xiaomei Bie (pp. 960-975).

Purification, characterization, gene cloning, and heterologous expression in Escherichia coli of a thermostable β-1,3-1,4-glucanase from Bacillus altitudinis YC-9 have been investigated in this paper. The donor strain B. altitudinis YC-9 was isolated from spring silt. The native enzyme was purified by ammonium sulfate precipitation, diethylaminoethyl-cellulose anion exchange chromatography, and Sephadex G-100 gel filtration. The purified β-1,3-1,4-glucanase was observed to be stable at 60 °C and retain more than 90 % activity when incubated for 2 h at 60 °C and remain about 75 % and 44 % activity after incubating at 70 °C and 80 °C for 10 min, respectively. Acidity and temperature optimal for this enzyme was pH 6 and 65 °C. The open reading frame of the enzyme gene was measured to be 732 bp encoding 243 amino acids, with a predicted molecular weight of 27.47 kDa. The gene sequence of β-1,3-1,4-glucanase showed a homology of 98 % with that of Bacillus licheniformis. After being expressed in E. coli BL21, active recombinant enzyme was detected both in the supernatants of the culture and the cell lysate, with the activity of 102.7 and 216.7 U/mL, respectively. The supernatants of the culture were used to purify the recombinant enzyme. The purified recombinant enzyme was characterized to show almost the same properties to the wild enzyme, except that the specific activity of the recombinant enzyme reached 5392.7 U/mg, which was higher than those ever reported β-1,3-1,4-glucanase from Bacillus strains. The thermal stability and high activity make this enzyme broad prospect for industry application. This is the first report on β-1,3-1,4-glucanase produced by B. altitudinis.

Keywords: β-1,3-1,4-Glucanase; Characterization; Heterologous expression; Bacillus altitudinis YC-9

High-Level Expression, Purification and Production of the Fungal Immunomodulatory Protein-Gts in Baculovirus-Infected Insect Larva by Tzong-Yuan Wu; Hsin-An Chen; Feng-Yin Li; Ching-Ting Lin; Chi-Ming Wu; Feng-Chia Hsieh; Jason Tze-Cheng Tzen; Sheng-Kuo Hsieh; Jiunn-Liang Ko; Tzyy-Rong Jinn (pp. 976-989).

Fip-gts, a fungal immunomodulatory protein (Fip) isolated from Ganoderma tsugae (gts), has been reported to possess therapeutic effects in the treatment of cancer and autoimmune disease. To cost-effectively produce Fip-gts and bypass the bottleneck involved in its time-consuming purification from G. tsugae, in this study, we incorporated the SP_bbx secretion signal into recombinant baculovirus for expressing glycosylated and bioactive rFip-gts in baculovirus-infected insect cells and Trichoplusia ni larva. This is the first study to employ the aerosol infecting T. ni larva with recombinant baculovirus for economical and high-level production of foreign proteins. In this study, one purification could yield 10 mg of rFip-gts protein merely from ∼100 infected T. ni larvae by aerosol inoculation, corresponding to 5 L (5 × 10⁹ cells) of the infected Sf21 culture. In addition, the rFip-gts purified from T. ni larvae could induce the expression of interleukin-2 in murine splenocytes with an immunoresponsive level similar to that induced by LZ-8 (a known potent immunomodulatory protein purified from Ling zhi, Ganoderma lucidum). Thus, our results demonstrated that the larva-based baculovirus expression system can successfully express rFip-gts with the assembling capability required for maintaining immunomodulatory and anticancer activity. Our approach will open a new avenue for the production of rFip-gts and facilitate the immunoregulatory activity of rFip-gts available in the future.

Keywords: Aerosol infection; Baculovirus expression system; Fungal immunomodulatory protein; Glycoprotein; Ling zhi; Trichoplusia ni larva

Kinetic and Physiological Evaluation of Ammonium and Nitrite Oxidation Processes in Presence of 2-Chlorophenol by J. E. Pérez-Alfaro; G. Buitrón; J. Gomez; A.-C. Texier; F. M. Cuervo-López (pp. 990-1000).

The effect of 2-chlorophenol (2-CP) on ammonium and nitrite-oxidizing processes was kinetically evaluated in batch cultures with nitrifying sludge at steady state. Assays with ammonium or nitrite as energy source and 2.5, 5.0, or 10.0 mg 2-CP-C/l were conducted. Control assays without 2-CP were also performed. Ammonium-oxidizing activity was completely inhibited at the different 2-CP concentrations, whereas nitrite-oxidizing activity was present as nitrite was completely consumed and converted to nitrate irrespectively of 2-CP concentration. In the presence of 2.5 and 5.0 mg 2-CP-C/l, no significant effect on specific rates of nitrite consumption and nitrate production was observed, but a significant decrease on these parameters was observed at 10.0 mg 2-CP-C/l. The nitrifying sludge previously exposed to 2-CP was unable to completely recover its ammonium and nitrite oxidation capacity. Nevertheless, complete 2-CP consumption was achieved in all assays. The effect of 2-CP on ammonium oxidation was observed at kinetic and metabolic pathway level, whereas the effect on nitrite oxidation was observed only at kinetic level. The results obtained in this work evidenced that in order to achieve a successful nitrification process the presence in wastewater of even 2.5 mg 2-CP-C/l should be avoided.

Keywords: Ammonium oxidation; 2-Chlorophenol; Inhibition; Nitrite oxidation; Membrane; Nitrification

Synthesis and Functional Characterization of Antibiofilm Exopolysaccharide Produced by Enterococcus faecium MC13 Isolated from the Gut of Fish by Paulraj Kanmani; K. Suganya; R. Satish kumar; N. Yuvaraj; V. Pattukumar; K. A. Paari; Venkatesan Arul (pp. 1001-1015).

The synthesis and functional characterization of an antibiofilm exopolysaccharide (EPS) from a probiotic Enterococcus faecium MC13 were investigated. The temperature of 35 °C, pH of 6.5, and salinity of 1–2 % were found to be optimum for EPS production. The sucrose (30 g l⁻¹) and yeast extract (20 g l⁻¹) acted as suitable carbon and nitrogen sources, respectively, which strongly influenced EPS production with yield of 11.33 and 11.91 g l⁻¹. Based on the thin layer chromatography, EPS of E. faecium MC13 was found to be a heteropolysaccharide, composed of galactose and glucose sugar units with a molecular mass of 2.0 × 10⁵ Da. Fourier transform infrared spectrum analysis of the EPS revealed many predominant functional groups including hydroxyl, carboxyl, and amide groups. EPS exhibited better emulsifying and flocculating activities which is relatively similar to those of commercial polysaccharides. In vitro antioxidant inspect of EPS showed lesser antioxidant activity than that of the control ascorbic acid. Thermal behavior of EPS was different from the other EPS produced by other lactic acid bacteria. In vitro antibiofilm assay of EPS exhibited significant biofilm inhibition, especially with Listeria monocytogenes. To the best of our knowledge, this is the first report on EPS of E. faecium with strong emulsifying and flocculating activities.

Keywords: Enterococcus faecium MC13; Exopolysaccharide; Bioflocculation; Emulsification; Antibiofilm activity

Development of Industrial Brewing Yeast with Low Acetaldehyde Production and Improved Flavor Stability by Jinjing Wang; Nan Shen; Hua Yin; Chunfeng Liu; Yongxian Li; Qi Li (pp. 1016-1025).

Higher acetaldehyde concentration in beer is one of the main concerns of current beer industry in China. Acetaldehyde is always synthesized during beer brewing by the metabolism of yeast. Here, using ethanol as the sole carbon source and 4-methylpyrazole as the selection marker, we constructed a new mutant strain with lower acetaldehyde production and improved ethanol tolerance via traditional mutagenesis strategy. European Brewery Convention tube fermentation tests comparing the fermentation broths of mutant strain and industrial brewing strain showed that the acetaldehyde concentration of mutant strain was 81.67 % lower, whereas its resistant staling value was 1.0-fold higher. Owing to the mutation, the alcohol dehydrogenase activity of the mutant strain decreased to about 30 % of the wild-type strain. In the meantime, the fermentation performance of the newly screened strain has little difference compared with the wild-type strain, and there are no safety problems regarding the industrial usage of the mutant strain. Therefore, we suggest that the newly screened strain could be directly applied to brewing industry.

Keywords: Industrial brewing yeast; Acetaldehyde; Traditional mutagenesis; 4-Methylpyrazole

Chemical Modification of Ethyl Cellulose-Based Highly Porous Membrane for the Purification of Immunoglobulin G by H. V. Adikane; G. J. Iyer (pp. 1026-1038).

The chemical modification of developed ethyl cellulose-based membrane was carried out to make it suitable for bioseparation. The different reagents were used for the modification of membrane to couple protein A (PA) to study the purification of immunoglobulin G (IgG) from blood. The chemical modification was carried out using relatively simple and mild reaction conditions. The attenuated total reflectance Fourier transform infrared analysis of chemically modified membrane showed new peak at 1,596.06 and 1,716.49 cm⁻¹. The scanning electron microscopy of PA-coupled membrane, which was used for IgG purification showed open pores and 950 ± 21.5 LMH (L m⁻² h⁻¹) operational flux at 0.5-bar out pressure. The flux of unmodified membrane was 1,746 ± 18.5 LMH at 0.5-bar out pressure. The equilibrium adsorption concentration (318.5 ± 5.9 μg cm⁻²) was obtained at 3 h. The adsorption character of PA-coupled membrane was consistent with the Langmuir adsorption model and the non-specific binding was 67.08 ± 1.3 μg cm⁻². The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed similar purification pattern for purified IgG from human serum and commercial preparation of IgG. All the results have suggested a high potential of PA-coupled ethyl cellulose-based membrane for large-scale purification of IgG.

Keywords: Affinity bioseparation; Ethyl cellulose; Polyethylenimine; Glutaraldehyde; Protein A; Immunoglobulin G

Effect of Different Variables on the Efficiency of the Baker's Yeast Cell Disruption Process to Obtain Alcohol Dehydrogenase Activity by Martina Sudar; Davor Valinger; Zvjezdana Findrik; Đurđa Vasić-Rački; Želimir Kurtanjek (pp. 1039-1055).

Cell disruption process of dry baker's yeast was studied in this work to obtain maximum activity of alcohol dehydrogenase (ADH). Disruption by ultrasonication, glass beads, and combination of these two methods was compared. A 1.8-fold increase of ADH activity can be achieved by combining glass beads with ultrasonication in comparison to ultrasonication. To achieve maximum volume activity of ADH, the effect of different variables on the cell disruption process was investigated (time, glass bead diameter, mass of glass beads, and ultrasound amplitude). Using the Design-Expert© software, 2⁴ factorial experimental design was performed. Two ultrasound probes were tested: MS 73 and KE 76. Optimal conditions (process variables) for cell disruption process were obtained. Optimal ADH activities after cell disruption with MS 73 and KE 76 probes were 1,890.9 and 1,531.7 U cm⁻³, respectively. Necessary ultrasonication time and ultrasound amplitude should be at the maximum values in the investigated variable range (30 min and 62 %). Bead size should be at maximum (4 mm) when using MS 73 probe and at minimum (0.3 mm) when using KE 76 probe. Partial purification of the enzyme was carried out and it was kinetically characterized using several oxidation and reduction systems.

Keywords: Bioseparation; Downstream processing; Protein; Cell disruption; Enzyme; Design of experiments (DoE)

Dehydration of Ethanol by Facile Synthesized Glucose-Based Silica by Baokun Tang; Wentao Bi; Kyung Ho Row (pp. 1056-1068).

Bioethanol is considered a potential liquid fuel that can be produced from biomass by fermentation and distillation. Although most of the water is removed by distillation, the purity of ethanol is limited to 95–96 % due to the formation of a low-boiling point, water–ethanol azeotrope. To improve the use of ethanol as a fuel, many methods, such as dehydration, have been proposed to avoid distillation and improve the energy efficiency of extraction. Glucose-based silica, as an adsorbent, was prepared using a simple method, and was proposed for the adsorption of water from water–ethanol mixtures. After adsorption using 0.4 g of adsorbent for 3 h, the initial water concentration of 20 % (water, v/v) was decreased to 10 % (water, v/v). For water concentrations less than 5 % (water, v/v), the adsorbent could concentrate ethanol to 99 % (ethanol, v/v). The Langmuir isotherms used to describe the adsorption of water on an adsorbent showed a correlation coefficient of 0.94. The separation factor of the adsorbent also decreased with decreasing concentration of water in solution.

Keywords: Glucose-based silica; Dehydration; Bioethanol; Synthesis

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.169, #3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.169, #3)