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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.165, #7-8)

Phage Displayed HBV Core Antigen with Immunogenic Activity by Aylin Ozdemir Bahadir; Bertan Koray Balcioglu; Kamil Serkan Uzyol; Ibrahim Hatipoglu; Ibrahim Sogut; Aynur Basalp; Berrin Erdag (pp. 1437-1447).

Hepatitis B is a major public health problem worldwide, which may lead to chronic liver diseases such as cirrhosis and hepatocellular carcinoma. The hepatitis B core antigen (HBcAg) is one of the major viral proteins, which forms the inner core of hepatitis B virus (HBV) particles. In this study, filamentous bacteriophage M13 was genetically modified to display the polypeptides of HBcAg in order to develop an alternative carrier system. HBcAg gene was inserted into the minor coat protein (pIII) gene of M13, and HBcAg was expressed on the phage surface as a whole protein. Antigenicity and immunogenicity of HBcAg were tested by immunizing BALB/c mice three times with HBcAg-displaying recombinant phages. After successful immunization, one of the mice with high antibody titer to HBcAg was selected for fusion, and four monoclonal antibodies specific for HBcAg were developed. This result showed that HBcAg-displaying recombinant bacteriophages are immunogenic and can potentially be used for the development of monoclonal antibodies.

Keywords: HBcAg; Phage display; M13 phage; Immune response; ELISA; Monoclonal antibody

Electrochemical Oxidation of Glucose Using Mutant Glucose Oxidase from Directed Protein Evolution for Biosensor and Biofuel Cell Applications by Eileen Hao Yu; Radivoje Prodanovic; Güray Güven; R. Ostafe; U. Schwaneberg (pp. 1448-1457).

In this study, electrochemical characterisation of glucose oxidation has been carried out in solution and using enzyme polymer electrodes prepared by mutant glucose oxidase (B11-GOx) obtained from directed protein evolution and wild-type enzymes. Higher glucose oxidation currents were obtained from B11-GOx both in solution and polymer electrodes compared to wt-GOx. This demonstrates an improved electrocatalytic activity towards electrochemical oxidation of glucose from the mutant enzyme. The enzyme electrode with B11-GOx also showed a faster electron transfer indicating a better electronic interaction with the polymer mediator. These encouraging results have shown a promising application of enzymes developed by directed evolution tailored for the applications of biosensors and biofuel cells.

Keywords: Directed protein evolution; Glucose oxidase; Glucose oxidation; Electron transfer; Biofuel cells; Biosensors

Purification, Characterization, and Molecular Cloning of a Novel Antifungal Lectin From the Roots of Ophioglossum pedunculosum by Xue-mei He; Na Ji; Xiao-cong Xiang; Ping Luo; Jin-ku Bao (pp. 1458-1472).

A novel mannan-specific lectin was isolated from the roots of a traditional Chinese herbal medicine, Ophioglossum pedunculosum through ion-exchange chromatography and gel filtration. With a molecular mass of 19,835.7 Da demonstrated by MALDI-TOF analysis, this novel agglutinin was designated as O. pedunculosum agglutinin (OPA), specifically agglutinating human O erythrocytes and rabbit erythrocytes. The hemagglutination could be strongly inhibited by mannan and thyroglobulin, the activity of which was stable in pH range of 4.0–8.0 and at temperatures below 50 °C. Chemical modification studies indicated that tryptophan and arginine residues were essential for its hemagglutinating activity. Meanwhile, it showed antifungal activities toward Sclerotium rolfsii and Fusarium graminearum. In addition, to amplify cDNA of OPA by 3′/5′-rapid amplification of cDNA ends (RACE), the N-terminal 30 amino acids sequence of OPA was determined, and degenerate primers were designed. The obtained full-length cDNA of OPA contained 885 bp with an open-reading frame of 600 bp encoding a precursor protein of 199 amino acids, while the mature protein had 170 amino acids.

Keywords: Ophioglossum pedunculosum agglutinin; Hemagglutination; Chemical modification; Antifungal activity; cDNA cloning

Thermostable Bacterial Endoglucanases Mined from Swiss-Prot Database by Li-Huan Qiu; Chun-Xiu Li; Jie Sun; Zhilong Wang; Qin Ye; Jian-He Xu (pp. 1473-1484).

As one critical enzyme in deconstructing complicated cellulose matrix, endoglucanase (EG) is needed to exhibit high activity and thermostability under severe industrial conditions. Driven by this purpose, EGtf1 (Q08166) and EGtf2 (Q7X2N2), with relatively high specific activities, were selected out of 43 putative EG genes from SWISS-PROT database. These distinguished EGs were successfully overexpressed in Escherichia coli and purified by one-step affinity chromatography. The maximal activity was shown at approximate pH 5.0 and 50 °C. It is worth noting that EGtf1 and EGtf2 displayed outstanding thermostability with a half-life of up to 1,386 h at 50 °C, which is almost 100-fold higher than other reported EGs. Furthermore, the presence of various metal ions (1 mM) or organic solvents (50%, v/v) did not cause significant effect on the activities of EGtf1 and EGtf2 and even showed 2.1- and 2.7-fold enhancement in the case of dodecanol. All these features, especially the excellent thermostability of EGtf1 and EGtf2, enable them to become a good candidate for further protein engineering to realize the ultimate practical application in biomass industry.

Keywords: Endoglucanase (EG); Carboxymethyl cellulose (CMC); Thermostability; Enzymatic properties; Data mining; Swiss-Prot

Production of Cyclodextrins from Cornstarch Granules in a Sequential Batch Mode and in the Presence of Ethanol by Luiza Pedrina Vilxenski Calsavara; Alessandro Rogério Dias da Cunha; Tiago Albertini Balbino; Gisella Maria Zanin; Flavio Faria de Moraes (pp. 1485-1493).

The influence of Toruzyme® cyclomaltodextrin glucanotransferase concentration and the presence of ethanol have been studied for the production of α-, β-, and γ-cyclodextrins (CDs) from 15% (w/v) cornstarch, at 65 °C and pH 6, with the aim of increasing CD yield. The selected concentrations for a single batch reactor were 10% (v/v) ethanol and 0.1% (v/v) enzyme, yielding after 12 h, 37% total CDs, of which 52.2% was α-CD, 38.8% β-CD, and 9.0% γ-CD. The enzyme specific activities per unit mass of protein for producing α-, β-, and γ-CD were 37.25, 19.61, and 8.63 U mg⁻¹, respectively. Total CD yield per milliliter of enzyme was 55 g. To increase CD yield per enzyme charge and thus reduce costs, the production of CDs was tested with two sequential batches in which a single enzyme charge was used. At the end of the first batch, the enzyme was adsorbed either on 65 °C pretreated starch granules or on raw starch, and a second batch was run with this material. The best result, in this case, was obtained for pretreated starch, increasing total CD produced by 57.4%, with 53.2% α-CD, 36.1% β-CD, and 10.7% γ-CD. CD yield per milliliter of enzyme was then 87 g.

Keywords: Cyclodextrin; Production; Starch; CGTase; Ethanol; Sequential batch

Large-Scale Production of Phospholipase D from Streptomyces racemochromogenes and Its Application to Soybean Lecithin Modification by Yozo Nakazawa; Yoshimasa Sagane; Shin-ichiro Sakurai; Masataka Uchino; Hiroaki Sato; Kazuki Toeda; Katsumi Takano (pp. 1494-1506).

Phospholipase D (PLD) catalyzes transphosphatidylation, causing inter-conversion of the polar head group of phospholipids and phospholipid hydrolysis. Previously, we cloned PLD103, a PLD with high transphosphatidylation activity, from Streptomyces racemochromogenes strain 10-3. Here, we report the construction of an expression system for the PLD103 gene using Streptomyces lividans as the host bacterium to achieve large-scale production. The phosphatidylcholine (PC) hydrolysis activity of S. lividans transformed with the expression plasmid containing the PLD103 gene was approximately 90-fold higher than that of the original strain. The recombinant PLD103 (rPLD103) found in the supernatant of the transformant culture medium was close to homogeneous. The rPLD103 was indistinguishable from the native enzyme in molecular mass and enzymatic properties. Additionally, rPLD103 had high transphosphatidylation activity on PC as a substrate in a simple aqueous one-phase reaction system and was able to modify the phospholipid content of soybean lecithin. Consequently, the expression system produces a stable supply of PLD, which can then be used in the production of phosphatidyl derivatives from lecithin.

Keywords: Phospholipase D; Lecithin; Phospholipid; Soybean; Transphosphatidylation; Phospholipid modification; Oil manufacture; Phosphatidylcholine; Phosphatidylserine; Phosphatidylglycerol

The Binding Sites of Class I Release Factor (eRF1) Toward Class II Release Factor (eRF3) in Euplotes octocarinatus by Jie Chen; Yue-jun Fu; Bing-sheng Yang; Yan-bo Wu; Ai-hua Liang (pp. 1507-1518).

The C domain of eRF1 interacts with the C domain of eRF3, and the binding of both factors is essential for fast kinetics of the termination of protein translation. Analysis by computational simulation demonstrated that several peptides involved in Eo-eRF1/Eo-eRF3 interaction directly. Among these peptides, the two motifs GVEDT and GFGG were highly conserved, while the fragment aa338-346 of Eo-eRF1a/b was variable. In additional, I290 and D293 of Eo-eRF1 were also highly conserved. By the site-directed mutagenesis and pull-down analysis, the amino acid D293 in Eo-eRF1bC domain was conformed playing an important role in eRF1–eRF3 interaction. Eo-eRF1a and Eo-eRF1b may select different manners to interact with Eo-eRF3. These studies contribute to the better understanding the mode of eRF1–eRF3 interaction.

Keywords: eRF1–eRF3 interaction; Binding sites; Computational simulation

Gluconacetobacter hansenii subsp. nov., a High-Yield Bacterial Cellulose Producing Strain Induced by High Hydrostatic Pressure by Han-Jing Ge; Shuang-Kui Du; De-Hui Lin; Jun-Na Zhang; Jin-Le Xiang; Zhi-Xi Li (pp. 1519-1531).

Strain M₄₃₈, deposited as CGMCC3917 and isolated from inoculums of bacterial cellulose (BC) producing strain screened in homemade vinegar and then induced by high hydrostatic pressure treatment (HHP), has strong ability to produce BC more than three times as that of its initial strain. It is the highest yield BC-producing strain ever reported. In this paper, M₄₃₈ was identidied as Gluconacetobacter hansenii subsp. nov. on the basis of the results obtained by examining it phylogenetically, phenotypically, and physiologically–biochemically. Furthermore, the genetic diversity of strain M₄₃₈ and its initial strain was examined by amplified fragment length polymorphism. The results indicated that strain M₄₃₈ was a deletion mutant induced by HHP, and the only deleted sequence showed 99% identity with 24,917–24,723 bp in the genome sequence of Ga. hansenii ATCC23769, and the complement gene sequence was at 24,699–25,019 bp with local tag GXY_15142, which codes small multidrug resistance (SMR) protein. It can be inferred that SMR might be related to inhibiting BC production to a certain extent.

Keywords: Phenotypically; Physiologically–biochemically; Phylogenetically; 16S rRNA genes; Gluconacetobacter hansenii subsp. nov.; Genetic diversity

Enhanced Reducing Equivalent Generation for 1,3-Propanediol Production Through Cofermentation of Glycerol and Xylose by Klebsiella pneumoniae by Ping Jin; Sheng-guo Lu; He Huang; Fang Luo; Shuang Li (pp. 1532-1542).

1,3-Propanediol (1,3-PD) biosynthesis plays a key role in NADH consumption to regulate the intracellular reducing equivalent balance of Klebsiella pneumoniae. This study aimed to increase reducing equivalent for enhancing 1,3-PD production through cofermentation of glycerol and xylose. Adding xylose as cosubstrate resulted in more reducing equivalent generation and higher cell growth. In batch fermentation under microaerobic condition, the 1,3-PD concentration, conversion from glycerol, and biomass (OD₆₀₀) relative to cofermentation were increased significantly by 9.1%, 20%, and 15.8%, respectively. The reducing equivalent (NADH) was increased by 1–3 mg/g (cell dry weight) compared with that from glycerol alone. Furthermore, 2,3-butannediol was also doubly produced as major byproduct. In fed-batch fermentation with xylose as cosubstrate, the final 1,3-PD concentration, conversion from glycerol, and productivity were improved evidently from 60.78 to 67.21 g/l, 0.52 to 0.63 mol/mol, and 1.64 to1.82 g/l/h, respectively.

Keywords: 1,3-Propanediol; Cofermentation; Klebsiella pneumoniae ; Reducing equivalent; Xylose

Influence of Operational Parameters and Low Nickel Concentrations on Partial Nitrification in a Submerged Biofilter by Sukru Aslan; Burhanettin Gurbuz (pp. 1543-1555).

The effect of Ni²⁺ concentrations on ammonium oxidation was studied in a batch and partial bionitrification reactor (PBNR). The nitrification rates up to the concentration of 0.1 mg Ni²⁺/l were close to those without Ni²⁺. After testing the operational conditions in the PNBR, the highest NO₂–N/NO x–N ratio was achieved at the DO concentrations of 2.0 mg/l and pH 9.00. The PNBR was operated at steady state (NH₄–N loading rate and NO₂–N/NO x–N ratio were 405 g m⁻³ day⁻¹ and 0.74, respectively) before exposure to Ni²⁺. The removal efficiency of NH₄–N and NO₂–N/NO x–N ratio in the effluent waters was increased by adding low concentrations of heavy metals to the PBNR. The average number of aerobic mesophilic bacteria at the biofilm surface and in the water in the void volume of PNBR were 1.0 × 10⁴ CFU/g and 1.4 × 10⁵ CFU/ml, respectively.

Keywords: Partial nitrification; NO₂–N/NO x–N ratio; Ni²⁺

A New Nitrilase-Producing Strain Named Rhodobacter sphaeroides LHS-305: Biocatalytic Characterization and Substrate Specificity by Chunsheng Yang; Xuedong Wang; Dongzhi Wei (pp. 1556-1567).

The characteristics of the new nitrilase-producing strain Rhodobacter sphaeroides LHS-305 were investigated. By investigating several parameters influencing nitrilase production, the specific cell activity was ultimately increased from 24.5 to 75.0 μmol g⁻¹ min⁻¹, and hereinto, the choice of inducer proved the most important factor. The aromatic nitriles (such as 3-cyanopyridine and benzonitrile) were found to be the most favorable substrates of the nitrilase by analyzing the substrate spectrum. It was speculated that the unsaturated carbon atom attached to the cyano group was crucial for this type of nitrilase. The value of apparent K _m, substrate inhibition constant, and product inhibition constant of the nitrilase against 3-cyanopyridine were 4.5 × 10⁻², 29.2, and 8.6 × 10⁻³ mol L⁻¹, respectively. When applied in nicotinic acid preparation, the nitrilase is able to hydrolyze 200 mmol L⁻¹ 3-cyanopyridine with 93% conversion rate in 13 h by 6.1 g L⁻¹ cells (dry cell weight).

Keywords: Rhodobacter sphaeroides ; Nitrilase; 3-Cyanopyidine; Nicotinic acid; Biocatalyst

Byssus Thread: A Novel Support Material for Urease Immobilization by Neelam Mishra; Kespi Pithawala; Anita Bahadur (pp. 1568-1576).

Byssus threads are tough biopolymer produced by mussels (Mytilus viridis) to attach themselves to rocks. These were collected from mussels in their natural habitat (N) and from animals maintained in laboratory condition (L) as a novel support. Byssus thread surfaces were characterized by SEM analysis, chemically modified and used for adsorption of urease. The efficiency of the immobilization was calculated by examining the relative enzyme activity of free and the immobilized urease. The pH stabilities of immobilized urease were higher (0.5 unit) than free enzyme. Immobilized enzymes on byssus (both N and L) when stored at 6 °C retained 50% of its activity after 30 days, but they were more stable in dry condition. The optimum temperature of immobilized enzymes was found to increase (25 °C). A Michaelis–Menten constant (K _m) value for immobilized urease was also elevated (2.08 mol).

Keywords: Immobilization; Urease; Byssus thread; Mytilus ; Adsorption; SEM

Proteome Analysis of Abundant Proteins Extracted from the Leaf of Gynura procumbens (Lour.) Merr by Chaw-Sen Hew; Lay-Harn Gam (pp. 1577-1586).

Gynura procumbens (Lour.) Merr. is a traditionally used medicinal plant to decrease cholesterol level, reduce high blood pressure, control diabetics, and for treatment of cancer. In our present study, a proteomic approach was applied to study the proteome of the plant that had never analyzed before. We have identified 92 abundantly expressed proteins from the leaves of G. procumbens (Lour.) Merr. Amongst the identified proteins was miraculin, a taste-masking agent with high commercial value. Miraculin made up ∼0.1% of the total protein extracted; the finding of miraculin gave a great commercial value to G. procumbens (Lour.) Merr. as miraculin’s natural source is limited while the production of recombinant miraculin faced challenges of not being able to exhibit the taste-masking effect as in the natural miraculin. We believe the discovery of miraculin in G. procumbens (Lour.) Merr., provides commercial feasibility of miraculin in view of the availability of G. procumbens (Lour.) Merr. that grow wildly and easily in tropical climate.

Keywords: Gynura procumbens (Lour.) Merr; Proteomics; Leaf extract; Protein profiling; Miraculin

Batch Biodegradation of Para-Nitrophenol Using Arthrobacter chlorophenolicus A6 by Naresh K. Sahoo; Kannan Pakshirajan; P. K. Ghosh (pp. 1587-1596).

The present study reports the kinetics of p-nitrophenol (PNP) biodegradation by Arthrobacter chlorophenolicus A6 in batch shake flasks for initial PNP concentrations in the range of 25–225 mg l⁻¹. Results of batch growth kinetics of A. chlorophenolicus A6 at various initial PNP concentrations revealed that the culture followed substrate inhibition kinetics with estimated decay coefficient value of 0.0132 h⁻¹. Biokinetic constants involved in the process were estimated by fitting the experimental data to several substrate inhibition kinetics models available from the literature. Among the models tested, Webb model fitted the experimental data best with the least root mean square error value, and the estimated model constants values were μ = 0.161 h⁻¹, K _i = 128 mg l⁻¹, K _s = 60.15 mg l⁻¹, and K = 100 mg l⁻¹. In addition, observed and theoretical yield coefficients, maintenance energy, and specific growth rate of the culture at various initial PNP concentrations were also investigated in the study.

Keywords: p-Nitrophenol; Arthrobacter chlorophenolicus A6; Biokinetic constants; Substrate inhibition kinetics; Webb model

Isolation, Purification, and Characterization of Two Thermostable Endo-1,4-β-d-glucanase Forms from Opuntia vulgaris by Sivalingam Shyamala; Sambandam Ravikumar; Jeyaraman Vikramathithan; Kotteazeth Srikumar (pp. 1597-1610).

Four endoglucanase temperature isoforms (T ₃₀, T ₅₀, T ₇₀, and T ₉₀) were identified and purified from the cladodes of the xerophytic plant Opuntia vulgaris. These isoforms exhibited optimum catalytic activity at 30 °C, 50 °C, 70 °C, and 90 °C and yielded an apparent molecular mass of 150, 20, 74, and 45 kDa, respectively, on gel filtration chromatography. These isoforms were purified 24-, 25-, 29-, and 27-fold with a yield of 15%, 12%, 17%, and 19% and having a specific activity of 120, 125, 144, and 136 U/mg, respectively. The thermostable T ₇₀ and T ₉₀ isoforms exhibited optimum activity at pH 4.5 and 7 and also yielded a molecular weight of 66 and 36 kDa, respectively, as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The T ₇₀ had a K _m of 43 mM and a V _max of 12.5 μmol min⁻¹ μg⁻¹ of protein, and the T ₉₀ isoform had a K _m of 40 mM, with an apparent V _max of 10 μmol min⁻¹ μg⁻¹ of protein. Western blot, immunodiffusion, and in vitro inhibition assays established the reactivity of the T ₉₀ isoform with polyclonal anti-T ₉₀ antibody raised in rabbit. Cross-reactivity of this antibody with the T ₇₀ endoglucanase isoform was also noted.

Keywords: Cellulases; Endoglucanase; Thermophilic; Metal ion; Opuntia vulgaris

Thermodynamics of Chitinase Partitioning in Soy Lecithin Liposomes and Their Storage Stability by Lucía F. Cano-Salazar; Antonio J. Juárez-Ordáz; Karla M. Gregorio-Jáuregui; José L. Martínez-Hernández; Jesús Rodríguez-Martínez; Anna Ilyina (pp. 1611-1627).

The goal of this study was to define the partitioning behavior of chitinase from Trichoderma spp. in soy lecithin liposomes, using a thermodynamic approach based on the partitioning variation with temperature. An effort has been made to define the liposomes, as well as free and immobilized enzyme stability during storage at 4 and 25 °C. The partition coefficients (K _o/w) were greater than 1; therefore, the standard free energies of the enzyme transfer were negative, indicating an affinity of the enzymes for encapsulation in liposomes. The enthalpy calculation led to the conclusion that the process is exothermic. The presence of enzyme decreased the liposome storage stability from 70 days to an approximately 20 days at 25 °C and 30 days at 4 °C. Monitoring of the liposome’s diameter demonstrated that their size and concentration decreased during storage. The liposome’s diameters ranged from 1.06 to 3.30 μm. The higher percentage of liposome corresponded to a diameter range from 1.06 to 1.34 μm. This percentage increased during storage. There were no evidences for liposome fusion process. The stability of immobilized enzyme was increased in comparison with free chitinase.

Keywords: Chitinase; Microencapsulation; Soy lecithin liposomes; Storage stability; Immobilized enzyme

Theranostic Applications of Nanomaterials in Cancer: Drug Delivery, Image-Guided Therapy, and Multifunctional Platforms by Alicia Fernandez-Fernandez; Romila Manchanda; Anthony J. McGoron (pp. 1628-1651).

Successful cancer management depends on accurate diagnostics along with specific treatment protocols. Current diagnostic techniques need to be improved to provide earlier detection capabilities, and traditional chemotherapy approaches to cancer treatment are limited by lack of specificity and systemic toxicity. This review highlights advances in nanotechnology that have allowed the development of multifunctional platforms for cancer detection, therapy, and monitoring. Nanomaterials can be used as MRI, optical imaging, and photoacoustic imaging contrast agents. When used as drug carriers, nanoformulations can increase tumor exposure to therapeutic agents and result in improved treatment effects by prolonging circulation times, protecting entrapped drugs from degradation, and enhancing tumor uptake through the enhanced permeability and retention effect as well as receptor-mediated endocytosis. Multiple therapeutic agents such as chemotherapy, antiangiogenic, or gene therapy agents can be simultaneously delivered by nanocarriers to tumor sites to enhance the effectiveness of therapy. Additionally, imaging and therapy agents can be co-delivered to provide seamless integration of diagnostics, therapy, and follow-up, and different therapeutic modalities such as chemotherapy and hyperthermia can be co-administered to take advantage of synergistic effects. Liposomes, metallic nanoparticles, polymeric nanoparticles, dendrimers, carbon nanotubes, and quantum dots are examples of nanoformulations that can be used as multifunctional platforms for cancer theranostics. Nanomedicine approaches in cancer have great potential for clinically translatable advances that can positively impact the overall diagnostic and therapeutic process and result in enhanced quality of life for cancer patients. However, a concerted scientific effort is still necessary to fully explore long-term risks, effects, and precautions for safe human use.

Keywords: Nanocarriers; Multifunctional nanoparticles; Theranostics; Cancer; Nanomaterials; Image-guided therapy

Studies on the Zymogram Method for the Detection of Pectinolytic Activities Using CTAB by Noomen Hadj-Taieb; Hajer Tounsi; Amina Chabchoub; Najla Abid; Ali Gargouri (pp. 1652-1660).

Zymogram analysis is a useful tool for the identification of several enzymes. The present study was undertaken to investigate the efficiency gains from the characterization of pectic enzymes on zymograms by staining of pectin–agarose overlays using cetyl trimethyl ammonium bromide also known as cetrimide or CTAB. The method is based on the fact that the enzymatic hydrolysis of the pectic substrates included in the agarose matrix gel inhibited their precipitation by CTAB, leading to the appearance of cleared zones in front of the pectin hydrolases and lyases. Conversely, esterases led to the increase of pectin precipitation. Fungal pectinolytic enzymes were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to the zymogram detection technique, using two pectin substances, namely citrus pectin and polygalacturonic acid. Overall, the findings presented in the current study indicate that several elements (ions, salts, pH, temperature, chelators, and reducing agents) may significantly affect the results of zymogram analysis and can, therefore, be employed to enhance the discriminatory and operational potential of the analysis in terms of accurate discrimination between several pectinolytic activities involved and effective implementation of the purification procedures required in the process.

Keywords: Zymogram; Pectinase activities; Cetyl trimethyl ammonium bromide (CTAB); CT1 mutant: Penicillium occitanis

Endo-inulinase Stabilization by Pyridoxal Phosphate Modification: A Kinetics, Thermodynamics, and Simulation Approach by Homa Torabizadeh; Mehran Habibi-Rezaei; Mohammad Safari; Ali Akbar Moosavi-Movahedi; Ahmad Sharifizadeh; Homa Azizian; Massoud Amanlou (pp. 1661-1673).

The structural and storage and functional thermostabilization of endo-inulinase (EC 3.2.1.7) through semi-rational modification of surface accessible lysine residues by pyridoxal-5′-phosphate (PLP) and ascorbate reduction have been explored. Improved stability was observed on modifications in the absence or presence of inulin, which indicates storage or functional thermostabilization, respectively. Comparisons have been made between non-modified and modified enzyme by the determination of Tm as an indicator of structural stability, temperature-dependent half-lives (t1/2), energy barrier of the inactivation process, and thermodynamic parameters (ΔH*, ΔG*, and ΔS*) in a storage thermostability approach. These parameters coincided well with the observed stabilization of the engineered enzyme. Moreover, relative activities with sucrose and inulin were determined for non-modified and modified endo-inulinases at different temperatures. A comparison of the sucrose-to-inulin ratios of the initial rate of hydrolysis as an indicator of substrate specificity revealed about twofold improvement in inulinase versus sucrose activity by enzyme modification. Molecular dynamics simulations and molecular docking approaches were employed to explain the observed structural and functional thermostabilization of endo-inulinase upon modification. We hypothesize the establishment of intramolecular interactions between the covalently attached PLP–Lys381 and Arg526 and Ser376 residues as a representative of modification-originated intramolecular contacts in the modified enzyme.

Keywords: Endo-inulinase; Pyridoxal 5′-phosphate; Molecular dynamics; Chemical modification; Thermostability

Construction of Copper Removing Bacteria Through the Integration of Two-Component System and Cell Surface Display by Sambandam Ravikumar; Ik-keun Yoo; Sang Yup Lee; Soon Ho Hong (pp. 1674-1681).

Synthetic biological systems are becoming more and more feasible for commercial and medical purposes through the genetic engineering of several components. The simple assembly of a genetic circuit was shown to stimulate the removal of copper by bacteria through the engineering of a two-component system. The CusSR two-component systems is a regulator of Escherichia coli copper homeostatic system. In this system, genetic circuits of CusSR were fused to a cell surface display system for metal adsorption; this system is suitable for the display of a copper binding peptide through outer membrane protein C (OmpC). E. coli ompC codes for an outer membrane pore protein (porin) are induced at high osmolarity and temperature, which can also be used as an anchoring motif to accept the passenger proteins. The bacteria that produce the chimeric OmpC containing the copper binding peptide adsorbed maximum concentrations of 92.2 μmol of Cu²⁺/gram dry weight of bacterial cells. This synthetic bacterial system senses the specific heavy metal and activates a cell surface display system that acts to remove the metal.

Keywords: E. coli ; Copper; Two-component system; Cell surface display

Influence of Agitation Speed on Tannase Production and Morphology of Aspergillus niger FETL FT3 in Submerged Fermentation by I. Darah; G. Sumathi; K. Jain; S. H. Lim (pp. 1682-1690).

Agitation speed was found to influence the tannase production and fungal growth of Aspergillus niger FETL FT3. The optimal agitation speed was at 200 rpm which produced 1.41 U/ml tannase and 3.75 g/l of fungal growth. Lower or higher agitation speeds than 200 rpm produced lower enzyme production and fungal growth. Based on the SEM and TEM micrograph observation, there was a significant correlation between agitation speed and the morphology of the fungal mycelia. The results revealed an increase of the enzyme production with the change of the fungal growth morphology from filamentous to pelleted growth forms. However, the exposure to higher shear stress with an increasing agitation speed of the shaker also resulted in lower biomass yields as well as enzyme production.

Keywords: Agitation speed; Tannase; Aspergillus niger FETL FT3; Fungal morphology

Activity and Stability of Chloroperoxidase in the Presence of Small Quantities of Polysaccharides: A Catalytically Favorable Conformation Was Induced by Chaonan Li; Limin Wang; Yucheng Jiang; Mancheng Hu; Shuni Li; Quanguo Zhai (pp. 1691-1707).

Chloroperoxidase (CPO) is thought to be the most versatile heme-containing enzyme with enormous applications in organic synthesis, biotransformation, pharmaceutical production, and detoxification of environmental pollutants. Any improvement in the stability of this enzyme will greatly enhance its application in the mentioned areas. In the present study, the effects of three polysaccharides (soluble starch, β-cyclodextrin, and dextrin) on the stability of CPO at elevated temperatures (20, 30, 35, 40, and 50 °C) or in aqueous–organic solvents media (methanol, dioxane, DMSO, and DMF) were investigated. An improved catalytic performance of CPO was observed in the presence of a small amount of the three polysaccharides, where dextrin provided the most effective promotion. The changes of enzyme structure and microenvironment around heme in the presence of additives were studied by fluorescence, circular dichroism, and UV–vis spectra analyses, as well as kinetic parameters measurement. A catalytically favorable structure of CPO was induced, including the strengthening of the α-helix structure and more exposure of heme for easy access of the substrate, resulting in an increase of catalytic turnover frequency (k _cat) and the improvement of affinity and selectivity of CPO to substrate. The results revealed that the introduction of trace soluble starch, β-cyclodextrin, and dextrin (<10 μmol/L) in reaction media was an effective strategy for the enhancement of the thermodynamic and the operational stability of the enzyme, which are promising in view of the industrial applications of this versatile biological catalyst.

Keywords: Chloroperoxidase; Activity; Polysaccharide; Spectroscopy analysis; Kinetic parameters

Multienzymatic Sucrose Conversion into Fructose and Gluconic Acid through Fed-Batch and Membrane-Continuous Processes by Fadi Antoine Taraboulsi Jr.; Ester Junko Tomotani; Michele Vitolo (pp. 1708-1724).

Multienzymatic conversion of sucrose into fructose and gluconic acid was studied through fed-batch and continuous (in a membrane reactor) processes. The law of substrate addition (sucrose or glucose) for the fed-batch process which led to a yield superior to 80% was the decreasing linear type, whose feeding rate (ϕ; L/h) was calculated through the equation: ϕ = ϕ_o − k.t, where ϕ_o (initial feeding rate, L/h), k (linear addition constant, L/h ²), and t (reaction time, h). In the continuous process, the yield of conversion of sucrose (Y) was superior to 70% under the following conditions: dilution rate = 0.33 h⁻¹, total duration of 15 h, pH 5.0, 37 °C and initial sucrose concentration of 64 g/L (Y = 92%), 100 g/L (Y = 83%), or 150 g/L (Y = 76%).

Keywords: Invertase; Glucose oxidase; Catalase; Gluconic acid; Membrane reactor

Metabolic Flux Analysis and Principal Nodes Identification for Daptomycin Production Improvement by Streptomyces roseosporus by Di Huang; Xiaoqiang Jia; Jianping Wen; Guoying Wang; Guanghai Yu; Qinggele Caiyin; Yunlin Chen (pp. 1725-1739).

In the present work, a comprehensive metabolic network of Streptomyces roseosporus LC-54-20 was proposed for daptomycin production. The analysis of extracellular metabolites throughout the batch fermentation was evaluated in addition to daptomycin and biomass production. Metabolic flux distributions were based on stoichiometrical reaction as well as the extracellular metabolites fluxes. Experimental and calculated values for both the specific growth rate and daptomycin production rate indicated that the in silico model proved a powerful tool to analyze the metabolic behaviors based on the analysis under different initial glucose concentrations throughout the fermentation. Through manipulating different pH values, the production rates of various extracellular metabolites were also presented in this paper. Flux distribution variations revealed that the daptomycin production could be significantly influenced by the branch points of glucose 6-phosphate, 3-phosphoglycerate, phosphoenolpyruvate, pyruvate, and oxaloacetate. The five principal metabolites were certified as the flexible nodes and could form potential bottlenecks for a further enhancement of daptomycin production. Furthermore, various concentrations of the five precursors were added into the batch fermentation and led to the enhancement of daptomycin concentration and production rate.

Keywords: Daptomycin production; Streptomyces roseosporus ; Initial glucose concentrations; pH control; Metabolic flux analysis; Principal nodes identification; Precursor feeds; Batch fermentation

Kinetic Stability Modelling of Keratinolytic Protease P45: Influence of Temperature and Metal Ions by Daniel Joner Daroit; Voltaire Sant’Anna; Adriano Brandelli (pp. 1740-1753).

The activity and kinetic stability of a keratinolytic subtilisin-like protease from Bacillus sp. P45 was investigated in 100 mM Tris-HCl buffer (pH 8.0; control) and in buffer with addition of Ca²⁺ or Mg²⁺ (1–10 mM), at different temperatures. Addition of 3 mM Ca²⁺ or 4 mM Mg²⁺ resulted in a 26% increment on enzyme activity towards azocasein when compared to the control (100%; without added Ca²⁺ or Mg²⁺) at 55 °C. Optimal temperature for activity in the control (55 °C) was similar with Mg²⁺; however, temperature optimum was increased to 60 °C with 3 mM Ca²⁺, displaying an enhancement of 42% in comparison to the control at 55 °C. Stability of protease P45 in control buffer and with Mg²⁺ addition was assayed at 40–50 °C, and at 55–62 °C with Ca²⁺ addition. Data were fitted to six kinetic inactivation models, and a first-order equation was accepted as the best model to describe the inactivation of protease P45 with and without metal ions. The kinetic and thermodynamic parameters obtained showed the crucial role of calcium ions for enzyme stability. As biocatalyst stability is fundamental for commercial/industrial purposes, the stabilising effect of calcium could be exploited aiming the application of protease P45 in protein hydrolysis.

Keywords: Protease; Kinetic modelling; Thermostability; Enzyme inactivation; Calcium

Screening and Production of Ligninolytic Enzyme by a Marine-Derived Fungal Pestalotiopsis sp. J63 by Hui-Ying Chen; Dong-Sheng Xue; Xiao-Yu Feng; Shan-Jing Yao (pp. 1754-1769).

Marine-derived fungi are prone to produce structurally unique secondary metabolites, a considerable number of which display the promising biological properties and/or industrial applications. Among those, ligninolytic enzymes have attracted great interest in recent years. In this work, about 20 strains were isolated from sea mud samples collected in the East China Sea and then screened for their capacity to produce lignin-degrading enzymes. The results showed that a strain, named J63, had a great potential to secrete a considerable amount of laccase. Using molecular method, it was identified as an endophytic fungus, Pestalotiopsis sp. which was rarely reported as ligninolytic enzyme producer in the literature. The production of laccase by Pestalotiopsis sp. J63 was investigated under submerged fermentation (SF) and solid state fermentation (SSF) with various lignocellulosic by-products as substrates. The SSF of rice straw powder accumulated the highest level of laccase activity (10,700 IU/g substrate), whereas the SF of untreated sugarcane bagasse provided the maximum amount of laccase activity (2,000 IU/ml). The value was far higher than those reported by other reports. In addition, it produced 0.11 U/ml cellulase when alkaline-pretreated sugarcane bagasse was used as growth substrate under SF. Meanwhile, the growth of fungi and laccase production under different salinity conditions were also studied. It appeared to be a moderately halo-tolerant organism.

Keywords: Marine-derived fungi; Laccase; Salinity; Submerged fermentation; Solid state fermentation

Expression and Characterization of a Novel Propionyl-CoA Dehydrogenase Gene from Candida rugosa in Pichia pastoris by Feng-li Zhou; Yong-guang Zhang; Ru-bing Zhang; Wei Liu; Mo Xian (pp. 1770-1778).

The propionyl-CoA dehydrogenase (PACD) gene was firstly cloned from Candida rugosa by the cDNA RACE technique. The 6× His-tagged recombinant PACD gene was expressed in Pichia pastoris GS115 and purified with Ni-NTA affinity chromatography. SDS-PAGE analysis and Western blotting revealed that the molecular mass of the purified PACD was 49 kDa. The results showed that the recombinant protein had the activity of catalyzing propionyl-CoA to acrylyl-CoA. The K _m, k _cat, and V _max values of the purified PACD were calculated to be 40.86 μM, 0.566 s⁻¹ and 0.693 U mg⁻¹ min⁻¹. The optimal temperature and pH of the purified PACD were 30 °C and 7.0, respectively. The recombinant PACD maintained 76.3%, 30.1%, and 4.3% of its original activity after 2 h incubation in standard buffer at 30, 40, and 50 °C, respectively. Mg²⁺ had an activating effect on the enzyme, while Mn²⁺, Ca²⁺, Zn²⁺, and Cu²⁺ had weak inhibition. Since PACD catalyzed the key step (from propionyl-CoA to acrylyl-CoA) in the modified β-oxidation pathway from glucose to 3-hydroxypropionic acid (3-HP), the integration of recombinant PACD could benefit the engineered strains for effective production of 3-HP from the most abundant biomass–sugars.

Keywords: Propionyl-CoA dehydrogenase; Gene expression; Characterization; Candida rugosa ; Pichia pastoris

An Apolipophorin III Protein from the Hemolymph of Desert Locust, Schistocerca gregaria by Zulfiqar A. Malik; Sumaira Amir (pp. 1779-1788).

Apolipophorin III (apoLp-III) from insects and apolipoprotein A-I from humans, are major component of the lipoprotein and share various properties. ApoLp-III is an abundant hemolymph protein. Besides its crucial role in lipid transport, apoLp-III is able to associate with fungal and bacterial membranes and stimulate cellular immune responses. ApoLp-III was isolated and purified from the hemolymph of desert locust Schistocerca gregaria by ion-exchange and reversed-phase chromatography. The purity and the molecular weight of apoLp-III were determined at ∼19,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. According to similarities in the amino terminal sequence, molar mass and retention on reversed-phase analytical HPLC column, this protein is a Schistocerca gregaria homologue of Locusta migratoria apoLp-III.

Keywords: Apolipophorin III; Locusta migratoria ; Schistocerca gregaria ; Hemolymph

Immobilized Laccase on a New Cryogel Carrier and Kinetics of Two Anthraquinone Derivatives Oxidation by Michaela Dina Stanescu; Andreia Sanislav; Roman V. Ivanov; Anca Hirtopeanu; Vladimir I. Lozinsky (pp. 1789-1798).

A coordinatively immobilized laccase was prepared using a new cryogel type carrier. The support has a wide-pore texture facilitating diffusion of different substrates to the enzyme reaction center. The biocatalyst proved to be efficient in decolorization of two anthraquinone derivatives, namely Acid Blue 62 and bromaminic acid. After 24 h over 80% of the two substrates have been oxidated. The kinetic data (K _m and V _max) for the oxidation of the two anthraquinone derivatives, with the free and immobilized enzyme, have been determined and compared. Other parameters, like k _cat and the specificity constant have been calculated and analyzed. The influence of substrate properties (hydrophobicity, polarity, etc.) has been discussed.

Keywords: Laccase coordinative immobilization; Functionalized polyamide–cryogel; Free and immobilized enzyme kinetics; Acid Blue 62; Bromaminic acid

Cellulase Hydrolysis of Unsorted MSW by Jacob Wagner Jensen; Claus Felby; Henning Jørgensen (pp. 1799-1811).

A recent development in waste management and engineering has shown that the cellulase can be used for the liquefaction of organic fractions in household waste. The focus of this study was to optimize the enzyme hydrolysis of thermally treated municipal solid waste (MSW) by the addition of surfactant. Concurrently, the enzyme performance was analysed on pure cellulose in a solution of MSW wastewater. Results showed no effect of surfactant addition to the hydrolysis media as measured by viscosity and particle size distribution. MSW treatment wastewater was found to contain a high amount of calcium, potassium, sodium, chloride and others that may affect cellulolytic enzymes. Cellulase performance showed no effect of adding the metal ion-chelating agent EDTA to the solution. The cellulases were stable, tolerated and functioned in the presence of several contaminants.

Keywords: MSW; Enzymatic hydrolysis; Cellulase; Surfactants; PEG; EDTA

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.165, #7-8)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.165, #7-8)