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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.168, #3)

Cloning, Sequencing, and Expression of Nitrile Hydratase Gene of Mutant 4D Strain of Rhodococcus rhodochrous PA 34 in E. coli by Amit Pratush; Amit Seth; T. C. Bhalla (pp. 465-486).

The NHase encoding gene of mutant 4D was isolated by PCR amplification. The NHase gene of mutant 4D was successfully cloned and expressed in Escherichia coli by using Ek/LIC Duet cloning kits (Novagen). For the active expression of the NHase gene, the co-expression of small cobalt transporter gene (P-protein gene) has also been co-expressed with NHase gene E. coli. The nucleotide sequence of this NHase gene revealed high homology with the H-NHase of Rhodococcus rhodochrous J1. The recombinant E. coli cells showed higher NHase activity (5.9 U/mg dcw) as compared to the wild (4.1 U/mg dcw) whereas it is less than the mutant strain (8.4 U/mg dcw). Addition of cobalt ion in Luria–Bertani medium is needed up to a very small concentration (0.4 mM) for NHase activity. The recombinant E. coli exhibited maximum NHase activity at 6 h of incubation and was purified with a yield of 56 % with specific activity of 37.1 U/mg protein.

Keywords: Mutant; Nitrile hydratase; Recombinant E. coli and Rhodococcus rhodochrous PA-34

Production of Triterpenoid Anti-cancer Compound Taraxerol in Agrobacterium-Transformed Root Cultures of Butterfly Pea (Clitoria ternatea L.) by Swasti S. Swain; Kedar K. Rout; Pradeep K. Chand (pp. 487-503).

Independent transformed root somaclones (rhizoclones) of butterfly pea (Clitoria ternatea L.) were established using explant co-cultivation with Agrobacterium rhizogenes. Rhizoclones capable of sustained growth were maintained under low illumination in auxin-free agar-solidified MS medium through subcultures at periodic intervals. Integration of T L-DNA rolB gene in the transformed rhizoclone genome was verified by Southern blot hybridization, and the transcript expression of T R-DNA ags and man2 genes was ascertained by reverse transcription polymerase chain reaction analysis. The major compound isolated and purified from the transformed root extracts was identified as the pentacyclic triterpenoid compound taraxerol using IR, ¹H-NMR, and ¹³C-NMR spectroscopy. The taraxerol yield in cultured hairy roots, as quantified by HPTLC analysis, was up to 4-fold on dry weight basis compared to that in natural roots. Scanning of bands from cultured transformed roots and natural roots gave super-imposable spectra with standard taraxerol, suggesting a remarkable homology in composition. To date, this is the first report claiming production of the cancer therapeutic phytochemical taraxerol in genetically transformed root cultures as a viable alternative to in vivo roots of naturally occurring plant species.

Keywords: Agrobacterium rhizogenes ; Clitoria ternatea L.; Rhizoclones; ¹H-NMR; ¹³C-NMR; HPTLC; Taraxerol

In Vivo Detection of Copper Ions by Magnetic Resonance Imaging Using a Prion-Based Contrast Agent by Satoshi Makino; Tomohiro Umemoto; Hiroshi Yamada; Eric M. Yezdimer; Ikuo Tooyama (pp. 504-518).

Abnormal distributions of transition metals inside the body are potential diagnostic markers for several diseases, including Alzheimer's disease, Parkinson's disease, Wilson’s disease, and cancer. In this article, we demonstrate that P57/Gd, a novel prion-based contrast agent, can selectively image tissues with excessive copper accumulation using magnetic resonance imaging (MRI). P57/Gd selectivity binds copper(II) over other physiologically relevant cations such as zinc, iron, manganese, and calcium. To simulate a metabolic copper disorder, we treated mice with an intraperitoneal injection of a CuSO₄ solution to induce a renal copper overload. The MRI signal intensities from the renal cortex and medulla of copper spiked animals that were administered P57/Gd were found to correlate with the ex vivo copper concentrations determined by inductively coupled plasma mass spectrometry.

Keywords: Peptide; Copper; Angiogenesis; In vivo test; MRI (magnetic resonance imaging)

Effects of Inoculum Physiological Stage on the Growth Characteristics of Chlorella sorokiniana Cultivated Under Different CO₂ Concentrations by Erico R. Mattos; Manjinder Singh; Miguel L. Cabrera; Keshav C. Das (pp. 519-530).

In order to maximize microalgae biomass production and reduce its overall costs, it is important to optimize inoculum conditions based on its physical and physiological characteristics. Chlorella sorokiniana cultures inoculated with inoculum at three different physiological stages (lag, exponential, and stationary) diluted to the same optical density were cultivated for 12 days under three different CO₂ concentrations (0.038, 5, or 10 % CO₂ v/v) and growth pattern and biomass production was observed. Samples inoculated with lag phase inoculum supplied with 5 % CO₂ achieved the maximum biomass production, whereas samples supplied with 0.038 % CO₂ never reached exponential growth. The better growth of samples inoculated with lag phase inoculum was attributed to its increased number of cells compared to the other two inocula.

Keywords: CO₂ ; Physiological stage; Chlorella ; Algae; Biomass

Improved Antimicrobial Potency through Synergistic Action of Chitosan Microparticles and Low Electric Field by Glareh Azadi; Matthew Seward; Mona Utne Larsen; Nina C. Shapley; Anubhav Tripathi (pp. 531-541).

Techniques to inhibit gram-negative bacteria such as Shiga toxin-producing Escherichia coli are valuable as the prevalence of large-scale industrial food preparation increases the likelihood of contamination. Chitosan, the deacetylated derivative of chitin, has been demonstrated to inhibit bacteria growth in acidic environments, but is significantly less effective in preventing bacteria grown at pH >7.0. Pulsed electric fields, constituting another method of bacteria inhibition, are difficult to generate at sufficient strength due to the high electric potentials required. This study utilizes adsorption of particulate chitosan in a very low electric field for an increased inhibition of gram-negative bacteria in neutral or alkaline pH conditions. Chitosan microparticles are demonstrated to flocculate E. coli, inhibit growth, and exhibit increased efficacy when combined with a low voltage electric field applied over 2-min intervals. Using sustained pulses of approximately 100 V/cm, it is demonstrated that bacteria viability is reduced by several orders of magnitude. The degree of bacterial inhibition is increased when chitosan microparticles are introduced to the system prior to imposing a small electric field.

Keywords: Chitosan microparticles; Synergistic antibacterial action; Bacteria inhibition; Chitosan and pulsed electric field

Degradation of Alkylphenols by White Rot Fungus Irpex lacteus and Its Manganese Peroxidase by Dong-Soo Moon; Hong-Gyu Song (pp. 542-549).

Alkylphenols are common endocrine disrupters that are produced from the degradation of widely used surfactants. Since they cause various harmful effects on aquatic life and in humans, they should be removed from the environments being contaminated. White rot fungus Irpex lacteus can completely degrade 100 mg/L of octylphenol, nonylphenol, and phenylphenol during 1 day of incubation in the complex YMG medium, which was the highest degrading capability among nine strains of white rot fungi tested. In the N-limited Kirk’s basal salts medium, I. lacteus could degrade almost 100 % of 100 mg/L octylphenol and nonylphenol in 1 h, and exhibited a high activity of manganese peroxidase (MnP; 1,790 U/L). MnP of I. lacteus was purified by ion exchange chromatography, and this degraded 99 % of 50 mg/L octylphenol and removed 80 % of estrogenic activity in 2 hours. In addition, the purified MnP (10 U/mL) degraded over 90 % of 50 mg/L nonylphenol in 1 h.

Keywords: Alkylphenols; White rot fungi; Irpex lacteus ; Manganese peroxidase; Estrogenic activity

Extracellular Production of Novel Halotolerant, Thermostable, and Alkali-Stable Carboxymethyl Cellulase by Marine Bacterium Marinimicrobium sp. LS-A18 by Kun Zhao; Li-Zhong Guo; Wei-Dong Lu (pp. 550-567).

Cellulases which are active and stable under extreme conditions have attracted considerable attention because of their potential industrial applications. Marinimicrobium sp. LS-A18 showed high extracellular carboxymethylcellulase (CMCase) activity when grown on mineral salt medium containing carboxymethylcellulose as the sole carbon source. Maximum CMCase activity was obtained at 55°C and pH 7.0 in the absence of NaCl. Under the optimized fermentation conditions, the yield of CMCase was increased up to 2.5 U/ml, which was 3.1-fold higher than that before optimization. The enzyme retained 84 % of residual activity after incubation at 60°C for 1 h and more than 88 % of residual activity after incubation for 72 h in the presence of different pH (5–11) and NaCl concentrations (0–25 %, w/v), indicating it was halotolerant, thermostable and alkali-stable. These characteristics made the CMCase from Marinimicrobium sp. LS-A18 as a potentially novel biocatalyst in biotechnological and industrial applications.

Keywords: Marinimicrobium ; Carboxymethyl cellulase; Halotolerant; Thermostable; Alkali-stable

Increased Availability of Tryptophan in 5-Methyltryptophan-Tolerant Shoots of Catharanthus roseus and Their Postharvest in vivo Elicitation Induces Enhanced Vindoline Production by Priyanka Verma; Ajay K. Mathur; Karuna Shanker (pp. 568-579).

Ten 5-methyltryprophan (5-MT)-resistant multiple shoot culture lines in three genotypes of Catharanthus roseus were selected in vitro. The variant shoot lines displayed a differential threshold tolerance limit against the analogue stress, ranged from 20 to 70 mg/l 5-MT in the medium. The lines tolerant to 40 mg/l 5-MT stress were most stable and fast proliferating. All the selected lines in the presence of 5-MT stress recorded increased level of tryptophan in their free amino acid pool. Highest tryptophan accumulation occurred in lines P40, P30, D40, and N40 (i.e., 296.5, 241.0, 200.6, and 202.0 μg/g dry wt., respectively). A concomitant increase in the total alkaloid content (2.3–3.8 % dry wt.) under the analogue stress was also noticed in these lines when compared to 1.0–1.58 % dry wt. in the respective wild-type shoot maintained on a stress-free medium. The HPLC analysis of the alkaloid extracts of the 5-MT-tolerant lines grown under analogue stress also revealed vindoline as a major constituent with maximum accumulation in lines N40, N30, D30, D40, and P40 (0.046, 0.032, 0.034, and 0.022 % dry wt., respectively). The rooted shoots of 5-MT-tolerant lines were successfully acclimatized under glasshouse environment wherein they grew normally and set seeds. Flowering twigs or leaves excised from 1-year-old glasshouse-grown plants of 5-MT variant lines upon postharvest in vivo elicitation with 30 mg/l 5-MT or 5.0 mg/l tryptophan registered an eight-to-tenfold increment in their vindoline content within 24–48 h.

Keywords: Catharanthus roseus ; TIAs; Feedback inhibition; 5-Methyl tryptophan; Postharvest elicitation

Anacardium occidentale Bark Lectin: Purification, Immobilization as an Affinity Model and Influence in the Uptake of Technetium-99M by Rat Adipocytes by Maria Inês Sucupira Maciel; Maria do Socorro de Mendonça Cavalcanti; Thiago Henrique Napoleão; Patrícia Maria Guedes Paiva; Maria Teresa Jansem de Almeida Catanho; Luana Cassandra Breitenbach Barroso Coelho (pp. 580-591).

Lectins, proteins that recognize carbohydrates, have been immobilized on inert supports and used in the screening or purification of glycoproteins. Anacardium occidentale bark infusion has been used as a hypoglycemic agent in Brazil. The toxicity of natural products may be evaluated determining their capability to alter the biodistribution of technetium-99M (^99mTc). This work reports the isolation and characterization of a lectin from A. occidentale bark (AnocBL), its evaluation as an affinity support for glycoprotein isolation and lectin effect on the uptake of ^99mTc by rat adipocytes. AnocBL was isolated from 80 % ammonium sulphate supernatant by affinity chromatography on fetuin–agarose. SDS–PAGE showed a single protein band of 47 kDa. The monossacharide l-arabinose and the glycoproteins fetuin, asialofetuin, ovomucoid, casein, thyroglobulin, peroxidase, fetal bovine serum and IgG inhibited the activity. The lectin activity was stable until 70 °C and at a pH range of 3.0–7.5. AnocBL–Sepharose column bound fetuin indicating that the lectin matrix may be used to obtain glycoconjugates of biotechnological interest. In vitro assay revealed that glucose and insulin increase ^99mTc uptake by rat adipocytes. AnocBL decreases ^99mTc uptake, and this effect was not detected in the presence of glucose. Fetuin inhibited AnocBL effect in all insulin concentrations.

Keywords: Anacardium occidentale ; Lectin; Affinity matrix; Technetium-99M

Fluorescence Spectroscopic Analysis of the Interaction of Papain with Ionic Liquids by Yunchang Fan; Jinlong Yan; Sheli Zhang; Junhai Li; Dong Chen; Peigao Duan (pp. 592-603).

The interaction between papain and two typical ionic liquids (ILs), 1-octyl-3-methylimidazolium chloride ([C₈mim]Cl) and 1-butyl-3-methylimidazolium chloride ([C₄mim]Cl), was investigated by using fluorescence spectroscopy technique at a pH value of 7.4. The results suggested that ILs could quench the intrinsic fluorescence of papain probably via a static quenching mechanism. The binding constants were determined by employing the fluorescence quenching method. They were very small compared with that of volatile organic solvents, indicating that only very weak interaction between ILs and papain existed. The Gibbs free energy change (∆G), enthalpy change (∆H), and entropy change (∆S) during the interaction of papain and ILs were estimated. Negative values of these parameters indicated that the interaction between ILs and papain was a spontaneous process, also implying that hydrogen bonding and van der Waals forces played important roles in the interaction processes. Figure Three-dimensional fluorescence spectrum of papain (0.2 g L^-1)

Keywords: Ionic liquids (ILs); Fluorescence spectroscopy; Binding mechanism; Papain; Thermodynamic parameters

Improvement of Thermostability and Activity of Firefly Luciferase Through [TMG][Ac] Ionic Liquid Mediator by Mehdi Ebrahimi; Saman Hosseinkhani; Akbar Heydari; Ramazan Ali Khavari-Nejad; Jafar Akbari (pp. 604-615).

Firefly luciferase catalyzes production of light from luciferin in the presence of Mg²⁺–ATP and oxygen. This enzyme has wide range of applications in biotechnology and development of biosensors. The low thermal stability of wild-type firefly luciferase is a limiting factor in most applications. Improvements in activity and stability of few enzymes in the presence of ionic liquids were shown in many reports. In this study, kinetic and thermal stability of firefly luciferase from Photinus pyralis in the presence of three tetramethylguanidine-based ionic liquids was investigated. The enzyme has shown improved activity in the presence of [1, 1, 3, 3-tetramethylguanidine][acetate], but in the presence of [TMG][trichloroacetate] and [TMG][triflouroacetate] activity, it decreased or unchanged significantly. Among these ionic liquids, only [TMG][Ac] has increased the thermal stability of luciferase. Incubation of [TMG][Ac] with firefly luciferase brought about with decrease of K _m for ATP.

Keywords: Photinus pyralis ; Luciferase; Ionic liquids; Tetramethylguanidine; Thermostability

Production of Mixed-Linkage Beta-Oligosaccharides from Lichenan Using Immobilized Bacillus licheniformis UEB CF Lichenase by Fatma Chaari; Monia Blibech; Fatma Bhiri; Sameh Maktouf; Raoudha Ellouz-Ghorbel; Semia Ellouz-Chaabouni (pp. 616-628).

Lichenase from Bacillus licheniformis UEB CF was immobilized on Amberlite IR120 H. The immobilization yield and lichenase activity were 87 and 92.81 % of initial activity, respectively. The immobilized enzyme exhibited a shift in the optimal pH from 5.0 to 3.0, but the activity optimal temperature was not affected. The immobilized enzyme showed a residual activity of 50 % after five uses. It also exhibited high storage stability and retained 50 % of its initial activity after 120 days at 4 °C. The main hydrolysis products yielded from lichenan were trisaccharide and tetrasaccharide. The resulting mixed-linkage beta-oligosaccharides could be used as a special nutriment for lactic bacteria.

Keywords: Lichenase; Amberlite; Immobilization; Oligosaccharides

Animal Lectins: Potential Antitumor Therapeutic Targets in Apoptosis by Zhe Liu; Qian Zhang; Hao Peng; Wen-zhi Zhang (pp. 629-637).

Lectins, a group of carbohydrate-binding proteins ubiquitously distributed into plants and animals, are well-known to have astonishing numerous links to human cancers. In this review, we present a brief outline of the representative animal lectins such as galectins, C-type lectins, and annexins by targeting programmed cell death (or apoptosis) pathways, and also summarize these representative lectins as possible anti-cancer drug targets. Taken together, these inspiring findings would provide a comprehensive perspective for further elucidating the multifaceted roles of animal lectins in apoptosis pathways of cancer, which, in turn, may ultimately help us to exploit lectins for their therapeutic purposes in future drug discovery.

Keywords: Lectin; Cancer; Apoptosis; Galectin; C-type lectin; Annexin

Directed Evolution of Penicillium janczewskii zalesk α-Galactosidase Toward Enhanced Activity and Expression in Pichia pastoris by Yiqun Chen; Bo Zhang; Honglei Pei; Junnan Lv; Wenhan Yang; Yunhe Cao; Bing Dong (pp. 638-650).

In this study, the activity of an α-galactosidase obtained from Penicillium janczewskii zalesk was improved via modifying its gene by error-prone PCR and DNA shuffling. The mutated DNA was ligated to pBGP1, an autonomous-replicating vector, which was subsequently transformed into Pichia pastoris X-33. The expressed enzyme activities were measured after single colonies were cultured in yeast–peptone–dextrose medium in deep-well plates. After two rounds of screening, two mutants with higher activity were obtained. By PCR analysis, four mutation sites (S167G, P455L, N637S, and P490L/P490H) were found in these two variants (mutant-59 and mutant-8). Mutant-59 showed the highest activity at pH 5.0 and 40 °C with an increased V _max value of 769 μmol/min and the specific activity of 667 U/mg against p-nitrophenyl α-D-galactopyranoside. The two mutant enzymes also showed similar resistance to the metal ions of Cu²⁺, Fe²⁺, and Zn²⁺. In a 10-L fermenter, the supernatant enzyme activity reached the maximum of 550.2 U/mL upon the methanol induction for 96 h. This fermentation activity of the mutant was improved approximately two more folds than the wild type α-galactosidase. This mutant of α-galactosidase is prospective in feed manufacturing as feed additives to improve nutrient digestibility in monogastric animals.

Keywords: Directed evolution; α-Galactosidase; Improved activity; Pichia pastoris

Hydrogen and Methane Production, Energy Recovery, and Organic Matter Removal from Effluents in a Two-Stage Fermentative Process by Guilherme Peixoto; Jorge Luis Rodrigues Pantoja-Filho; José Augusto Bolzan Agnelli; Marlei Barboza; Marcelo Zaiat (pp. 651-671).

This study evaluates the potential for using different effluents for simultaneous H₂ and CH₄ production in a two-stage batch fermentation process with mixed microflora. An appreciable amount of H₂ was produced from parboiled rice wastewater (23.9 mL g⁻¹ chemical oxygen demand [COD]) and vinasse (20.8 mL g⁻¹ COD), while other effluents supported CH₄ generation. The amount of CH₄ produced was minimum for sewage (46.3 mL g⁻¹ COD), followed by parboiled rice wastewater (115.5 mL g⁻¹ COD) and glycerol (180.1 mL g⁻¹ COD). The maximum amount of CH₄ was observed for vinasse (255.4 mL g⁻¹ COD). The total energy recovery from vinasse (10.4 kJ g⁻¹ COD) corresponded to the maximum COD reduction (74.7 %), followed by glycerol (70.38 %, 7.20 kJ g⁻¹ COD), parboiled rice wastewater (63.91 %, 4.92 kJ g⁻¹ COD), and sewage (51.11 %, 1.85 kJ g⁻¹ COD). The relatively high performance of vinasse in such comparisons could be attributed to the elevated concentrations of macronutrients contained in raw vinasse. The observations are based on kinetic parameters of H₂ and CH₄ production and global energy recovery of the process. These observations collectively suggest that organic-rich effluents can be deployed for energy recovery with sequential generation of H₂ and CH₄.

Keywords: Hydrogen; Methane; Glycerol; Parboiled rice wastewater; Vinasse; Sewage

Experimental Investigation and Optimization of Process Variables Affecting the Production of Extracellular Lipase by Kluyveromyces marxianus IFO 0288 by Panagiota-Yiolanda Stergiou; Athanasios Foukis; Helen Sklivaniti; Paraskevi Zacharaki; Maria Papagianni; Emmanuel M. Papamichael (pp. 672-680).

In this study, the production and optimization of extracellular lipase from Kluyveromyces marxianus IFO 0288 was investigated by using optimized nutritional and cultural conditions in a yeast medium containing glucose as the carbon source in fully aerobic batch fermentation (150 rpm). The influence of four fermentation parameters (type of lipidic source, initial culture pH, temperature, and length of fermentation) on growth and lipase production was investigated and evaluated using the conventional “one variable at a time” approach and response surface methodology. An 18-fold increase in lipase production during 65 h of fermentation was obtained with optimized nutritional (0.5 % olive oil) and cultivation (pH 6.5, 35 °C) conditions by employing the conventional optimization method. By applying the response surface methodology technique the initial pH value of 6.4 and temperature of 32.5 °C were identified as optimal and led to further improvements (up to 18-fold) of extracellular lipase production. The results provide, for the first time, evidence that K. marxianus has the potential to be used as an efficient producer of extracellular lipase with prospective application in a variety of industrial and biotechnological areas.

Keywords: Extracellular lipase; Kluyveromyces marxianus ; Optimization; Response surface methodology; Factorial design

Optimization of Elicitation Conditions with Methyl Jasmonate and Salicylic Acid to Improve the Productivity of Withanolides in the Adventitious Root Culture of Withania somnifera (L.) Dunal by Ganeshan Sivanandhan; Muthukrishnan Arun; Subramanian Mayavan; Manoharan Rajesh; Murugaraj Jeyaraj; Gnanajothi Kapil Dev; Markandan Manickavasagam; Natesan Selvaraj; Andy Ganapathi (pp. 681-696).

Adventitious root cultures derived from leaf derived callus of Withania somnifera (L.) Dunal were treated with methyl jasmonate and salicylic acid independently. Biomass accumulation, culture age, elicitation period, and culture duration were optimized for higher withanolides production in the two best-responding varieties collected from Kolli hills (Eastern Ghats) and Cumbum (Western Ghats) of Tamil Nadu, India. Between the two elicitors, salicylic acid (SA) improved the production of major withanolides (withanolide A, withanolide B, withaferin A, and withanone) as well as minor constituents (12-deoxy withastramonolide, withanoside V, and withanoside IV) in the Kolli hills variety. Treatment of root biomass (11.70 g FW) on 30-day-old adventitious root cultures with 150 μM SA for 4 h elicitor exposure period resulted in the production of 64.65 mg g^−l dry weight (DW) withanolide A (48-fold), 33.74 mg g^−l DW withanolide B (29-fold), 17.47 mg g^−l DW withaferin A (20-fold), 42.88 mg g^−l DW withanone (37-fold), 5.34 mg g^−l DW 12-deoxy withastramonolide (nine fold), 7.23 mg g^−l DW withanoside V (seven fold), and 9.45 mg g^−l DW withanoside IV (nine fold) after 10 days of elicitation (40th day of culture) when compared to untreated cultures. This is the first report on the use of elicitation strategy on the significant improvement in withanolides production in the adventitious root cultures of W. somnifera.

Keywords: Adventitious root culture; Methyl jasmonate; Salicylic acid; Solanaceae; Withania somnifera ; Withanolides

Immobilization of Pseudomonas fluorescens Lipase onto Magnetic Nanoparticles for Resolution of 2-Octanol by Er-na Xun; Xiao-li Lv; Wei Kang; Jia-xin Wang; Hong Zhang; Lei Wang; Zhi Wang (pp. 697-707).

The lipase from Pseudomonas fluorescens (Lipase AK, AKL) was immobilized onto the magnetic Fe₃O₄ nanoparticles via hydrophobic interaction. Enzyme loading and immobilization yield were determined as 21.4 ± 0.5 mg/g and 49.2 ± 1.8 %, respectively. The immobilized AKL was successfully used for resolution of 2-octanol with vinyl acetate used as acyl donor. Effects of organic solvent, water activity, substrate ratio, and temperature were investigated. Under the optimum conditions, the preferred isomer for AKL is the (R)-2-octanol and the highest enantioselectivity (E = 71.5 ± 2.2) was obtained with a higher enzyme activity (0.197 ± 0.01 μmol/mg/min). The results also showed that the immobilized lipase could be easily separated from reaction media by the magnetic steel and remained 89 % of its initial activity as well as the nearly unchanged enantioselectivity after five consecutive cycles, indicating a high stability in practical operation.

Keywords: Magnetic nanoparticles; Immobilization; Lipase; 2-Octanol; Resolution

Efficiency and Stability Enhancement of Cis-epoxysuccinic Acid Hydrolase by Fusion with a Carbohydrate Binding Module and Immobilization onto Cellulose by Shan Wang; Gu-Zhen Cui; Xiang-Fei Song; Yingang Feng; Qiu Cui (pp. 708-717).

Cis-epoxysuccinic acid hydrolase (CESH) is an enzyme that catalyzes cis-epoxysuccinic acid to produce enantiomeric L(+)-tartaric acid. The production of tartaric acid by using CESH would be valuable in the chemical industry because of its high yield and selectivity, but the low stability of CESH hampers its application. To improve the stability of CESH, we fused five different carbohydrate-binding modules (CBMs) to CESH and immobilized the chimeric enzymes on cellulose. The effects of the fusion and immobilization on the activity, kinetics, and stability of CESH were compared. Activity measurements demonstrated that the fusion with CBMs and the immobilization on cellulose increased the pH and temperature adaptability of CESH. The chimeric enzymes showed significantly different enzyme kinetics parameters, among which the immobilized CBM30-CESH exhibited twofold catalytic efficiency compared with the native CESH. The half-life measurements indicated that the stability of the enzyme in its free form was slightly increased by the fusion with CBMs, whereas the immobilization on cellulose significantly increased the stability of the enzyme. The immobilized CBM30-CESH showed the longest half-life, which is more than five times the free native CESH half-life at 30 °C. Therefore, most CBMs can improve enzymatic properties, and CBM30 is the best fusion partner for CESH to improve both its enzymatic efficiency and its stability.

Keywords: Cis-epoxysuccinic acid hydrolase; Carbohydrate binding module; Fusion enzyme; Enzyme stability; Affinity immobilization

Characterization of Glycosyltransferase Activity of Wild-Type Leuconostoc mesenteroides Strains from Bulgarian Fermented Vegetables by Tonka Vasileva; Ilia Iliev; Myriam Amari; Veselin Bivolarski; Marie-Sophie Bounaix; Hervé Robert; Sandrine Morel; Philippe Rabier; Iskra Ivanova; Bruno Gabriel; Catherine Fontagné-Faucher; Valérie Gabriel (pp. 718-730).

Glycosyltransferase activity of 13 Leuconostoc mesenteroides strains isolated from Bulgarian fermented vegetables was investigated. All the strains displayed a mucoid phenotype on sucrose-containing agar media. Strains were characterized according to carbohydrate fermentation, species-specific multiple PCR using several primers, repetitive element-PCR fingerprinting using (GTG)₅ primers and glycosyltransferase activity. Level of activity and cellular localization (soluble or cell-associated) were variable among strains. Precipitation of exopolysaccharides produced from sucrose by the soluble fractions from these strains allowed recovery of only glucans and further characterization by ¹H and ¹³C NMR analysis and enzymatic digestion with dextranase revealed dextran production. However, levans could be detected in presence of raffinose as fructosyl donor. Both fructosyltransferase and glucosyltransferase encoding genes were detected by PCR and both active enzymes were detected after functional characterization by SDS-PAGE electrophoresis and in situ polymer production after incubation with sucrose. This work therefore showed that concomitant production of glucosyltransferase and fructosyltransferase is widespread in L. mesenteroides strains.

Keywords: Glucosyltransferase; Fructosyltransferase; Leuconostoc mesenteroides ; Fermented vegetables; Dextran; Fructan; Glucansucrase; Fructansucrase

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.168, #3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.168, #3)