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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.162, #4)

Enzymatic Synthesis of Semiconductor Polymers by Chloroperoxidase of Caldariomyces fumago by Adriana M. Longoria; Hailin Hu; Rafael Vazquez-Duhalt (pp. 927-934).

Among intrinsically conducting polymers, polyaniline is traditionally synthesized by chemical or electrochemical methods. Recently enzymatic synthesis of conducting polymers has been explored. In this work, polymers were synthesized using chloroperoxidase from Caldariomyces fumago and substituted anilines such as 2,6-dimethylaniline, 2,6-dichloroaniline, and 2,3,5,6-tetrachloroaniline (TCA), in order to promote a linear polymerization. These polymers were doped with (1S)-(+)-10-camphorsulfonic acid, dodecylbenzenesulfonic acid, and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPSA) with molar ratios of 1:0.25 and 1:0.5. Doped polymers showed conductivity corresponding to the semiconductors. TCA polymer doped with AMPSA showed the higher conductivity values. Different AMPSA concentrations have been tested, and the highest conductivity value of 1.6 × 10⁻² S•m⁻¹ was obtained for the complex with molar ratio of 1:0.5. This value is similar to those found with other substituted anilines with sulfonic groups. In addition, the enzymatically synthesized polymeric film showed combined transparency and semiconducting properties.

Keywords: Chloroperoxidase; Intrinsically conducting polymers; Substituted anilines; Transparent semiconductor

Increase of Docosahexaenoic Acid Production by Schizochytrium sp. Through Mutagenesis and Enzyme Assay by Min Lian; He Huang; Lujing Ren; Xiaojun Ji; Jingyao Zhu; Lijing Jin (pp. 935-941).

The present study focused on improving docosahexaenoic acid (DHA) production by Schizochytrium sp. through N-methyl-N-nitro-N-nitrisiguanidine treatment coupled with ultraviolet radiation based on the metabolic pathway analysis. The activity of glucose-6-phosphate dehydrogenase of the mutant was higher than the parent strain, which indicated that the hexose monophosphate pathway of the mutant was strengthened, and more NADPH was thus produced. Also, the activities of malic enzyme and ATP–citrate lyase in the cell extract of the mutant were higher than the parent strain, which indicated that the screening method increased NADPH and acetyl–CoA supply in vivo effectively. Finally, in the batch culturing of the mutant, 34.84% higher lipid was accumulated with the cell dry weight at the same level compared with the parent strain. Moreover, the DHA percentage of the total fatty acids up to 56.22% was achieved using the mutant, which was 38.88% higher than the parent strain. When the cultures were maintained under appropriate conditions, the final DHA yield was 0.20 and 0.11 g/g dry biomass, for the mutant and parent, respectively.

Keywords: Docosahexaenoic acid; Schizochytrium sp.; Mutagenesis; Enzyme activity

Snail Hepatopancreatic Lipase: A New Member of Invertebrates Lipases' Group by Sawsan Amara; Ahmed Fendri; Nadia Ben Salem; Youssef Gargouri; Nabil Miled (pp. 942-952).

Higher animal's lipases are well characterized; however, much less is known about lipases from mollusks. A lipolytic activity was located in the land snail (Eobania vermiculata) digestive glands (hepatopancreas), from which a snail digestive lipase (SnDL) was purified. Pure SnDL has a molecular mass of 60 kDa; it does not present the interfacial activation phenomenon. It was found to be more active on short-chain triacylglycerols than on long-chain triacylglycerols. The NH₂-terminal sequence of the SnDL shows 66% of identity with the 17 NH₂-terminal amino acids of a putative lipase from sea urchin (Strongylocentrotus purpuratus). No sequence identity was found with known lipases. Interestingly, neither colipase nor bile salts were detected in the snail hepatopancreas. This suggests that colipase evolved in vertebrates simultaneously with the appearance of an exocrine pancreas and a true liver which produces bile salts. Altogether, these results suggest that SnDL is a member of a new group of digestive lipases belonging to invertebrates.

Keywords: Hepatopancreas; Snail; Invertebrate; Digestive lipase; Purification; Characterization

Gene Cloning, Expression, and Characterization of a Thermostable Xylanase from Nesterenkonia xinjiangensis CCTCC AA001025 by Hong Kui; Huiying Luo; Pengjun Shi; Yingguo Bai; Tiezheng Yuan; Yaru Wang; Peilong Yang; Shouliang Dong; Bin Yao (pp. 953-965).

An endo-β-1,4-xylanase-encoding gene, xyn11NX, was cloned from Nesterenkonia xinjiangensis CCTCC AA001025 and expressed in Escherichia coli. The gene encoded a 192-amino acid polypeptide and a putative 50-amino acid signal peptide. The deduced amino acid sequence exhibited a high degree of similarity with the xylanases from Streptomyces thermocyaneoviolaceus (68%) and Thermobifida fusca (66%) belonging to glycoside hydrolase family 11. After purification to homogeneity, the recombinant Xyn11NX exhibited optimal activity at pH 7.0 and 55 °C and remained stable at weakly acidic to alkaline pH (pH 5.0–11.0). The enzyme was thermostable, retaining more than 80% of the initial activity after incubation at 60 °C for 1 h and more than 40% of the activity at 90 °C for 15 min. The K _m and V _max values for oat spelt xylan and birchwood xylan were 16.08 mg ml⁻¹ and 45.66 μmol min⁻¹ mg⁻¹ and 9.22 mg ml⁻¹ and 16.05 μmol min⁻¹ mg⁻¹, respectively. The predominant hydrolysis products were xylobiose and xylotriose when using oat spelt xylan or birchwood xylan as substrate.

Keywords: Nesterenkonia xinjiangensis ; Xylanase; Thermostability; Glycoside hydrolase (GH) family 11

Purification and Characterization of 3-Ketovalidoxylamine A C-N Lyase Produced by Stenotrophomonas maltrophilia by Jian-Fen Zhang; Yu-Guo Zheng; Yin-Chu Shen (pp. 966-974).

A soluble 3-ketovalidoxylamine A C-N lyase from Stenotrophomonas maltrophilia was purified to 367.5-fold from the crude enzyme, with a yield of 16.4% by column chromatography on High S IEX, Methyl HIC, High Q IEX, and Sephadex G 100. The molecular mass of the enzyme was estimated to be 34 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the enzyme was a neutral protein having an isoelectric point value at pH 7.0. The optimal pH of 3-ketovalidoxylamine A C-N lyase was around 7.0. The enzyme was stable within a pH range of 7.0–10.5. The optimal temperature was found to be near 40 °C, and the enzyme was sensitive to heat. The enzyme was completely inhibited by ethylenediaminetetraacetic acid, and it was reversed by Ca²⁺. The product, p-nitroaniline, inhibited the enzyme activity significantly at low concentration. The enzyme has C-N lyase activity and C-O lyase activity, and need 3-keto groups. The apparent K _m value for p-nitrophenyl-3-ketovalidamine was 0.14 mM.

Keywords: 3-Ketovalidoxylamine A C-N lyase; Valienamine; p-Nitrophenyl-3-ketovalidamine; Stenotrophomonas maltrophilia

A Simplified Method for the Measurement of Insoluble Solids in Pretreated Biomass Slurries by Noah D. Weiss; Jonathan J. Stickel; Jeffrey L. Wolfe; Quang A. Nguyen (pp. 975-987).

The biochemical conversion of cellulosic biomass to liquid transportation fuels includes the breakdown of biomass into its soluble, fermentable components. Pretreatment, the initial step in the conversion process, results in heterogeneous slurry comprised of both soluble and insoluble biomass components. For the purpose of tracking the progress of the conversion process, it is important to be able to accurately measure the fraction of insoluble biomass solids in the slurry. The current standard method involves separating the solids from the free liquor and then repeatedly washing the solids to remove the soluble fraction, a laborious and tedious process susceptible to operator variations. In this paper, we propose an alternative method for calculating the fraction of insoluble solids which does not require a washing step. The proposed method involves measuring the dry matter content of the whole slurry as well as the dry matter content in the isolated liquor fraction. We compared the two methods using three different pretreated biomass slurry samples and two oven-drying techniques for determining dry matter content, an important measurement for both methods. We also evaluated a large set of fraction insoluble solids data collected from previously analyzed pretreated samples. The proposed new method provided statistically equivalent results to the standard washing method when an infrared balance was used for determining dry matter content in the controlled measurement experiment. Similarly, in the large historical data set, there was no statistical difference shown between the wash and no-wash methods. The new method is offered as an alternative method for determining the fraction of insoluble solids.

Keywords: Pretreatment; Fraction insoluble solids; Biomass; Lignocellulosic ethanol; Corn stover

Expression and Functional Characterization of Tumor-Targeted Fusion Protein Composed of NGR Peptide and 15-kDa Actin Fragment by Huanzong Lei; Peng Cao; Guoping Miao; Zhihua Lin; Zhenyu Diao (pp. 988-995).

To induce tumor cell apoptosis, a modified 15 kDa actin linked with a peptide NGR “homing” into tumor or tumor vessels was expressed in Escherichia coli. After refolding and purification, this fusion protein NGR-15actin was labeled with FITC to testify whether NGR-15actin could integrate into the cytoskeleton. It was found that this targeted peptide could induce HepG2 and HeLa cells apoptosis through its effect on the cytoskeleton function by binding to cytoskeleton protein. Thus, targeted NGR-15actin could be a candidate molecule for the therapy of cancer.

Keywords: Apoptosis; NGR; Actin; Cytoskeleton

A Statistical Approach for Optimization of Polyhydroxybutyrate Production by Bacillus sphaericus NCIM 5149 under Submerged Fermentation Using Central Composite Design by Nisha V. Ramadas; Carlos R. Soccol; Ashok Pandey (pp. 996-1007).

The aim of this work was to statistically optimize the cultural and nutritional parameters for the production of polyhydroxybutyrate (PHB) under submerged fermentation using jackfruit seed hydrolysate as the sole carbon source. On the basis of results obtained from “one variable at a time” experiment, inoculum age, jackfruit seed hydrolysate concentration, and pH were selected for response surface methodology studies. A central composite design (CCD) was employed to get the optimum level of these three factors to maximize the PHB production. The CCD results predicted that jackfruit seed hydrolysates containing 2.5% reducing sugar, inoculum age of 18 h, and initial medium pH 6 could enhance the production of PHB to reach 49% of the biomass (biomass 4.5 g/l and PHB concentration 2.2 g/l). Analysis of variance exhibited a high coefficient of determination (R ²) value of 0.910 and 0.928 for biomass and PHB concentration, respectively, and ensured that the quadratic model with the experimental data was a satisfactory one. This is the first report on PHB production by Bacillus sphaericus using statistical experimental design and RSM in submerged fermentation with jackfruit seed hydrolysate as the sole source of carbon.

Keywords: Central composite design; Response surface methodology; Bacillus sphaericus ; Polyhydroxybutyrate; Jackfruit seed hydrolysate; Submerged fermentation

Hemolytic and Antimicrobial Activities Differ Among Saponin-rich Extracts From Guar, Quillaja, Yucca, and Soybean by Sherif M. Hassan; James A. Byrd; Aubry L. Cartwright; Chris A. Bailey (pp. 1008-1017).

Hemolytic and antibacterial activities of eight serial concentrations ranged from 5-666 µg/mL of saponin-rich extracts from guar meal (GM), quillaja, yucca, and soybean were tested in 96-well plates and read by enzyme-linked immunosorbent assay plate-well as 650 nm. Hemolytic assay used a 1% suspension of chicken red blood cells with water and phosphate buffered saline as positive and negative controls, respectively. Antibacterial activity against Staphylococcus aureus, Salmonella typhimurium, and Escherichia coli were evaluated using ampicillin and bacteria without saponin-rich extract as positive and negative controls, respectively. The 100% MeOH GM and commercial quillaja saponin-rich extracts were significantly the highest in both hemolytic and antibacterial activities against all bacteria at the same concentration tested. Soybean saponin-rich extract had no antibacterial activity against any of the bacteria at the concentrations tested while yucca saponin-rich extract had no antibacterial activity against the gram-negative bacteria at the concentrations tested. GM and quillaja saponin-rich extracts were hemolytic, while yucca and soybean saponin-rich extracts were not hemolytic at the concentrations tested. No saponin-rich extract source had antibacterial activity against S. typhimurium or E. coli at the concentrations tested. Both GM and quillaja saponin-rich extracts exhibited antibacterial activity against S. aureus. Saponin-rich extracts from different plant sources have different hemolytic and antibacterial activities.

Keywords: Bacteria; Guar meal; Hemolytic; Quillaja; Saponin; Soybean; Yucca

A Novel α-Amylase from Bacillus mojavensis A21: Purification and Biochemical Characterization by Noomen Hmidet; Hana Maalej; Anissa Haddar; Moncef Nasri (pp. 1018-1030).

α-Amylase from Bacillus mojavensis A21 (BMA.2) was purified to homogeneity by ultrafiltration, Sephadex G-75 gel filtration and Sepharose mono Q anion exchange chromatography, with a 15.3-fold increase in specific activity and 11% recovery. The molecular weight of the BMA.2 enzyme was estimated to be 58 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration. The optimum temperature and pH were 80 °C and 6.5, respectively. BMA.2 belonged to the EDTA-sensitive α-amylase, but its activity was not stimulated by the presence of Ca²⁺ ions. The major end-products of starch hydrolysis were maltohexaose, maltopentaose and maltotriose. The N-terminal amino acid sequence of the first ten amino acids of the purified α-amylase was ASVNGTLMQY. Compared to sequences of other amylases, the ten amino acid sequence contains Val at position 3, while amylases from Bacillus licheniformis NH1 and Bacillus sp. SG-1 have Leu and Thr at position 3, respectively.

Keywords: α-Amylase; B. mojavensis A21; Purification; Biochemical characterization

Enhancement of Fumaric Acid Production by Rhizopus oryzae Using a Two-stage Dissolved Oxygen Control Strategy by Yong-Qian Fu; Shuang Li; Yao Chen; Qing Xu; He Huang; Xiao-Yan Sheng (pp. 1031-1038).

Batch fermentative production of fumaric acid by Rhizopus oryzae ME-F12 was investigated in a 7-l stirred tank fermentor under different dissolved oxygen (DO) concentrations. High fumaric acid yield on glucose (0.56 g/g) was achieved under high DO concentration (80%), but the glucose consumption rate and fumaric acid productivity were rather low (0.91 and 0.51 g/l/h). Fumaric acid productivity was enhanced under low DO concentration (30%), but the fuamric acid yield on glucose decreased to 0.52 g/g. In order to achieve the high fumaric acid yield and productivity simultaneously, a two-stage dissolved oxygen control strategy was proposed, in which the DO concentration was controlled at 80% in the first 18 h and then switched to 30%. This was experimentally proven to be successful. Relatively high fumaric acid production (56.2 g/l), high fumaric acid yield on glucose (0.54 g/g), and high glucose consumption rate (1.3 g/l/h) were achieved by applying this strategy. The productivity (0.7 g/l/h) was improved by 37%, 21%, and 9%, respectively, compared with fermentations in which DO concentrations were kept constant at 80%, 60%, and 30%.

Keywords: Batch fermentation; Fumaric acid; Kinetics; Rhizopus oryzae ; Two-stage DO control strategy

Antimicrobial Lipids from the Hemolymph of Brachyuran Crabs by Samuthirapandian Ravichandran; Solimabi Wahidulla; Lisette D’Souza; Ganapathy Rameshkumar (pp. 1039-1051).

The potential of marine crabs as a source of biologically active products is largely unexplored. In the present study, antimicrobial activity of the hemolymph (plasma) and hemocytes (plasma cells) of six brachyuran crabs was investigated against 16 pathogenic strains. Among the 16 strains tested maximum zone of inhibition was recorded in the hemolymph of Hyas araneus against Shigella flexineri. Interestingly Staphylococcus aureus and Salmonella typhi were susceptible to all the hemolymph and hemocytes samples. Likewise, the highest zone of inhibition was exhibited by both hemolymph and hemocytes samples against Vibrio cholerae. On the basis of TLC, ¹HNMR, and ¹³CNMR it may be concluded that the antimicrobial activity in the hemolymph extract is due to the presence of lipids. This observation is further supported by the ESI-MS of the methanolic extract of hemolymph of H. araneus. ESI-MS shows cluster of peaks in the region m/z 445 to m/z 491 due to lysoglycerolipids/glycerides and cluster of signals between m/z 216 and 246, due to fatty acids/esters present in the sample.

Keywords: Crab; Hemolymph; Lipid; Antimicrobial; TLC; ESI-MS

Enzymatic Hydrolysis of Sodium Dodecyl Sulphate (SDS)—Pretreated Newspaper for Cellulosic Ethanol Production by Saccharomyces cerevisiae and Pichia stipitis by Fengxue Xin; Anli Geng; Ming Li Chen; Ming Jun Marcus Gum (pp. 1052-1064).

Fermentation of enzymatic hydrolysate of waste newspaper was investigated for cellulosic ethanol production in this study. Various nonionic and ionic surfactants were applied for waste newspaper pretreatment to increase the enzymatic digestibility. The surfactant-pretreated newspaper was enzymatically digested in 0.05 M sodium citrate buffer (pH 4.8) with varying solid content, filter paper unit loading (FPU/g newspaper), and ratio of filter paper unit/β-glucosidase unit (FPU/CBU). Newspaper pretreated with the anionic surfactant sodium dodecyl sulphate (SDS) demonstrated the highest sugar yield. The addition of Tween-80 in the enzymatic hydrolysis process enhanced the enzymatic digestibility of newspaper pretreated with all of the surfactants. Enzymatic hydrolysis of SDS-pretreated newspaper with 15% solid content, 15 FPU/g newspaper, and FPU/CBU of 1:4 resulted in a newspaper hydrolysate conditioning 29.07 g/L glucose and 4.08 g/L xylose after 72 h of incubation at 50 °C. The fermentation of the enzymatic hydrolysate with Saccharomyces cerevisiae, Pichia stipitis, and their co-culture produced 14.29, 13.45, and 14.03 g/L of ethanol, respectively. Their corresponding ethanol yields were 0.43, 0.41, and 0.42 g/g.

Keywords: Newspaper; Surfactant; Enzymatic hydrolysis; Saccharomyces cerevisiae ; Pichia stipitis ; Co-culture; Fermentation; Ethanol

Influence of Ionic Strength, pH, and SDS Concentration on Subunit Analysis of Phycoerythrins by SDS-PAGE by Mingri Zhao; Li Sun; Xuejun Fu; Xueqin Gong (pp. 1065-1079).

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is often used for subunit analysis of proteins, but it is not efficient to make the α- and β-subunits of phycoerythrins separated by normal SDS-PAGE. In this research, subunit components and subunit molecular weights of four purified phycoerythrins were analyzed by SDS-PAGE. Four factors including Tris concentration, pH, ammonium persulfate (APS), and SDS concentration were studied for their effects on SDS-PAGE of phycoerythrins. It showed that these factors can influence the separation of α- and β-subunits, electrophoresis effect of γ-subunits, apparent molecular weights of subunits, and mobility of marker proteins. The α- and β-subunits separated better in the case of lower SDS concentration, lower Tris concentration, higher pH, and/or lower APS addition in separating gels. The molecular weights of α- and β-subunits increased when Tris concentration increased in a certain range. It can be concluded that factors critical to subunit analysis by SDS-PAGE are SDS concentration and ionic strength, both of which are related to critical micelle concentration of SDS and ratio of SDS monomer to micelle in SDS-PAGE system. The ratio is postulated to influence SDS-PAGE by influencing the amount of SDS bound to polypeptides and shapes of polypeptide–SDS complexes.

Keywords: Red alga; Phycoerythrin; SDS-PAGE; Subunit; Ionic strength; pH; SDS concentration

Potential of Xanthones from Tropical Fruit Mangosteen as Anti-cancer Agents: Caspase-Dependent Apoptosis Induction In Vitro and in Mice by Ramida Watanapokasin; Faongchat Jarinthanan; Alan Jerusalmi; Sunit Suksamrarn; Yukio Nakamura; Supawadee Sukseree; Wanlaya Uthaisang-Tanethpongtamb; Piniti Ratananukul; Takeshi Sano (pp. 1080-1094).

The pericarp of mangosteen (Garcinia mangostana L.) is rich in various xanthones that are known to possess unique biological activities. In this work, we characterized the anti-proliferative and cytotoxic activities of mangosteen xanthones both in vitro and in mice. In vitro analysis with a human colorectal adenocarcinoma cell line, COLO 205, showed that mangosteen xanthones not only inhibit the proliferation of target cells but also induce their death by apoptosis that involves the activation of the caspase cascade. In vivo analysis using a mouse subcutaneous tumor model with COLO 205 cells showed that, at relatively low doses, the growth of tumors was repressed upon intratumoral administration of mangosteen xanthones. When a higher dose of mangosteen xanthones was administered, the size of tumors was reduced gradually, and, in some mice, the disappearance of tumors was seen. Histopathological evaluation and biochemical analysis of tumors that received mangosteen xanthones indicate the induction of apoptosis in tumors, which resulted in the repression of their growth and the reduction of their sizes. These results demonstrate the potential of mangosteen xanthones to serve as anti-cancer agents for the chemotherapy of cancer.

Keywords: Garcinia mangostana L (mangosteen); Apoptosis; Caspase; Mangostin; Cancer chemotherapy

Bioflocculant Exopolysaccharide Production by Azotobacter indicus Using Flower Extract of Madhuca latifolia L by Satish V. Patil; Rahul B. Salunkhe; Chandrashekhar D. Patil; Deepak M. Patil; Bipinchandra K. Salunke (pp. 1095-1108).

Efficacy of Azotobacter indicus ATCC 9540 strain for production exopolysaccharide (EPS) bioflocculant was investigated. Mahua flower extract (Madhuca latifolia L), a natural substrate at the concentration of 20 g L⁻¹, gave maximum recovery of EPS followed by sucrose and mannitol as compared to other carbon sources after 172 h. Yeast extract was found to be the most effective nitrogen source as compared to beef extract, sodium nitrate, ammonium sulfate, casein hydrolysate, and urea for the production of EPS. EPS production was increased in presence of nitrogen (5.51 g L⁻¹) as compared to nitrogen-free medium (3.51 g L⁻¹), and fermentation time was also reduced by 28 h. Maximum EPS production (6.10 g L⁻¹) was found in the presence of 20 g L⁻¹ flower extract and 0.5 g L⁻¹ yeast extract containing Ashby’s media with 180 rpm at 30 °C at 144 h, under controlled conditions in 2.5 L fermenter using optimized medium. The isolated EPS showed cation-dependent flocculating activity. Concentration of EPS played an important role in bioflocculating activity which increased in a concentration-dependent manner up to a certain limit, with the maximum flocculation of 72% at 500 mg L⁻¹ concentration but remained almost static after this concentration. Extracted polymer was characterized by different chemical tests, FT-IR spectroscopy, and TLC which showed presence of uronic acids, O-acetyl groups, and Orcinol with suggestive indication of alginate like polymer. This study suggests that use of M. latifolia L. flowers can be a potential alternative bioresource for production of exopolysaccharide.

Keywords: Bioflocculant; Azotobacter indicus ; Polysaccharide; EPS; Cations

A New Process for Wine Production by Penetration of Yeast in Uncrushed Frozen Grapes by Argyris Tsakiris; Athanasios A. Koutinas; Costas Psarianos; Yiannis Kourkoutas; Argyro Bekatorou (pp. 1109-1121).

Different types of red and white wines were prepared by fermentation of the juice which was naturally separated from uncrushed frozen grapes during thawing (A) and from the residual juice by fermentation inside the berries (B). Yeast penetrated the skin of uncrushed grapes and fermented the content completely. The new types of wines were compared with wines prepared conventionally from the whole material of frozen grapes. Chemical and chromatographic analysis (gas chromatography (GC) and solid-phase microextraction–GC/mass spectrometry) showed similar profiles of the aroma volatiles but with significant quantitative differences among the new types of wines, which reflected to the differences observed during the sensory evaluations. The majority of identified compounds were esters, with higher amounts found in (A) wines due to the higher concentration of the must which was separated during thawing. The proposed process is new and of industrial interest for the production of different types of wines from the same raw material in one fermentation batch.

Keywords: Wine; Frozen grapes; Yeast; Fermentation; Volatiles; Phenolics; Sensory evaluation

Silk Mat as Bio-matrix for the Immobilization of Cholesterol Oxidase by Urmila Saxena; Pranab Goswami (pp. 1122-1131).

Cholesterol oxidase (ChOx) was covalently immobilized onto the woven silk fiber (silk mat) produced by Antheraea assamensis. The immobilization was done using N-ethyl-N’-(3-dimethylaminopropyl) carbodimide and N-hydroxysuccinimide ligand chemistry. The attachment of ChOx to the silk mat was demonstrated by scanning electron microscopy and activity study. The kinetic studies of the immobilized ChOx were performed by using a biological oxygen monitor. The enzyme loading was found to be 0.046 U cm⁻² of silk mat and the enzyme loading efficiency of the silk mat was estimated to be 70%. Remarkably high storage and operational stability (t_1/2 of initial activities) corresponding to 13 months and 25 numbers of assay (for a period of 6 h), respectively, of the fabricated ChOx electrode were demonstrated.

Keywords: Cholesterol; Cholesterol oxidase; Silk mat; Immobilization

Inhibition of Fungi and Gram-Negative Bacteria by Bacteriocin BacTN635 Produced by Lactobacillus plantarum sp. TN635 by Slim Smaoui; Lobna Elleuch; Wacim Bejar; Ines Karray-Rebai; Imen Ayadi; Bassem Jaouadi; Florence Mathieu; Hichem Chouayekh; Samir Bejar; Lotfi Mellouli (pp. 1132-1146).

The aim of this study was to evaluate 54 lactic acid bacteria (LAB) strains isolated from meat, fermented vegetables and dairy products for their capacity to produce antimicrobial activities against several bacteria and fungi. The strain designed TN635 has been selected for advanced studies. The supernatant culture of this strain inhibits the growth of all tested pathogenic including the four Gram-negative bacteria (Salmonella enterica ATCC43972, Pseudomonas aeruginosa ATCC 49189, Hafnia sp. and Serratia sp.) and the pathogenic fungus Candida tropicalis R2 CIP203. Based on the nucleotide sequence of the 16S rRNA gene of the strain TN635 (1,540 pb accession no FN252881) and the phylogenetic analysis, we propose the assignment of our new isolate bacterium as Lactobacillus plantarum sp. TN635 strain. Its antimicrobial compound was determined as a proteinaceous substance, stable to heat and to treatment with surfactants and organic solvents. Highest antimicrobial activity was found between pH 3 and 11 with an optimum at pH = 7. The BacTN635 was purified to homogeneity by a four-step protocol involving ammonium sulfate precipitation, centrifugal microconcentrators with a 10-kDa membrane cutoff, gel filtration Sephadex G-25, and C18 reverse-phase HPLC. SDS-PAGE analysis of the purified BacTN635, revealed a single band with an estimated molecular mass of approximately 4 kDa. The maximum bacteriocin production (5,000 AU/ml) was recorded after a 16-h incubation in Man, Rogosa, and Sharpe (MRS) medium at 30 °C. The mode of action of the partial purified BacTN635 was identified as bactericidal against Listeria ivanovii BUG 496 and as fungistatic against C. tropicalis R2 CIP203.

Keywords: Lactobacillus plantarum sp. TN635; Identification; BacTN635; Purification; Broad inhibitory spectrum

A Proof for Substitution of Endogenous Iron (II) in Lipoxygenase by Exogenous Cu²⁺ by Yan Cai; Hong Xu; Yongmei Xia; Yafen Su; Yun Fang (pp. 1147-1152).

Soybean lipoxygenase (LOX) contains endogenous iron (II) at the active site, which is important for the enzyme activity. The activity of LOX can be accelerated by some exogenous metal ions including Cu²⁺. However, the mechanism of the activity improvement caused by exogenous metal ions remains unclear, not only for LOX but for most other metalloenzymes. Meanwhile, the possibility that exogenous metal ions can displace endogenous iron (II) is still in discussion for a lack of a direct and quantitative proof. In this paper, a quantitative proof of replacing iron (II) inside LOX by exogenous Cu²⁺ was provided, simply using UV-Vis spectrometry with two indicators p-carboxylantipyrylazo and 9-(4-carboxyphenyl)-2,3,7-trihydroxyl-6-fluorine. A 0.56 μM free iron (II) was observed in the bulk solution after incubating 9.45 μM Cu²⁺ with 16.10 μM LOX at 20°C for 5 min, which is in coincidence with the decrement of Cu²⁺ in the bulk solution (0.53 μM), implying that iron (II) was replaced by Cu²⁺.

Keywords: Soybean lipoxygenase; Iron; Copper; Substitution; Trace determination; Metalloenzyme

Antidiabetic Activity of Ethanolic Extract of Zaleya decandra in Alloxan-Induced Diabetic Rats by Periasamy Meenakshi; Rajendran Bhuvaneshwari; Muthaiyan Ahalliya Rathi; Lakshmanan Thirumoorthi; Dugganaboyana Chinna Guravaiah; Muthedath Janardhanan Jiji; Velliyur Kanniappan Gopalakrishnan (pp. 1153-1159).

Diabetes mellitus is a complex disorder that disturbs the metabolism of carbohydrates, fats, and proteins. Medicinal plants play an important role in the management of diabetes mellitus. The present study was aimed to evaluate the antidiabetic potential of Zaleya decandra roots on alloxan-induced diabetes in rats. Oral administration of ethanolic extract of the root (200 mg/kg body weight/day) for 15 days restored the levels of glucose, cholesterol, triglycerides, total proteins, urea, creatinine, lipid peroxidation level, and antioxidant enzymes significantly in diabetic rats. Histopathological studies showed significant changes like necrosis and degeneration in the liver and pancreas of alloxan-induced diabetic rats. Also these histopathological abnormalities were found to be normalized after treatment with Z. decandra extract. The efficacy of the root extract was found to be equivalent when compared to the standard hypoglycemic drug glibenclamide (1.25 mg/kg body weight/day, orally) in diabetic rats.

Keywords: Zaleya decandra ; Antidiabetic activity; Alloxan-induced diabetes; Ethanolic extract

Self-immobilization of Recombinant Caulobacter crescentus and Its Application in Removal of Cadmium from Water by Jigar Patel; Gregory Wilson; R. Michael L. McKay; Robert Vincent; Zhaohui Xu (pp. 1160-1173).

Microbial biofilms can be valuable in many biotechnological applications, including bioremediation. We have previously constructed a recombinant strain of Caulobacter crescentus JS4022/p723-6H by inserting a hexahistidine peptide to a permissive site of the host surface layer (S-layer) protein RsaA. This engineered strain is highly effective in removal of cadmium from water as free cells. In this communication, we examined the biofilms formed by self-immobilized JS4022/p723-6H and evaluated their ability to retrieve cadmium from contaminated water samples. According to light and electron microscopic observations, JS4022/p723-6H cells developed a uniform monolayer biofilm on borosilicate surfaces through their intrinsic appendage, a stalk with an adhesive holdfast. The density of the biofilms reached a maximum after 48 h of incubation and was not affected by exposure to at least 1 ppm cadmium. When 0.4 ppm Cd(II) was added to the growth medium, immobilized JS4022/p723-6H removed 76.9% of the total metal, whereas the control strain only removed 13.5%. When a water sample collected from Lake Erie was spiked with various amounts of CdCl₂, immobilized JS4022/p723-6H was able to sequester 44∼51% of the total metal, compared to 37∼42% accumulated by the control strain. By combining two powerful techniques, cell surface display and self-immobilization, we achieved complete separation of dissolved heavy metals from contaminated water in a single step. This study laid down the foundation to cost-effectively construct large-scale bioreactors with high efficiency and specificity to retrieve environmental contaminants from water.

Keywords: Caulobacter crescentus ; Immobilization; Biofilm; Cadmium; Heavy metals; Bioremediation; S-layer

Cultivation of Green Algae Chlorella sp. in Different Wastewaters from Municipal Wastewater Treatment Plant by Liang Wang; Min Min; Yecong Li; Paul Chen; Yifeng Chen; Yuhuan Liu; Yingkuan Wang; Roger Ruan (pp. 1174-1186).

The objective of this study was to evaluate the growth of green algae Chlorella sp. on wastewaters sampled from four different points of the treatment process flow of a local municipal wastewater treatment plant (MWTP) and how well the algal growth removed nitrogen, phosphorus, chemical oxygen demand (COD), and metal ions from the wastewaters. The four wastewaters were wastewater before primary settling (#1 wastewater), wastewater after primary settling (#2 wastewater), wastewater after activated sludge tank (#3 wastewater), and centrate (#4 wastewater), which is the wastewater generated in sludge centrifuge. The average specific growth rates in the exponential period were 0.412, 0.429, 0.343, and 0.948 day⁻¹ for wastewaters #1, #2, #3, and #4, respectively. The removal rates of NH₄–N were 82.4%, 74.7%, and 78.3% for wastewaters #1, #2, and #4, respectively. For #3 wastewater, 62.5% of NO₃–N, the major inorganic nitrogen form, was removed with 6.3-fold of NO₂–N generated. From wastewaters #1, #2, and #4, 83.2%, 90.6%, and 85.6% phosphorus and 50.9%, 56.5%, and 83.0% COD were removed, respectively. Only 4.7% was removed in #3 wastewater and the COD in #3 wastewater increased slightly after algal growth, probably due to the excretion of small photosynthetic organic molecules by algae. Metal ions, especially Al, Ca, Fe, Mg, and Mn in centrate, were found to be removed very efficiently. The results of this study suggest that growing algae in nutrient-rich centrate offers a new option of applying algal process in MWTP to manage the nutrient load for the aeration tank to which the centrate is returned, serving the dual roles of nutrient reduction and valuable biofuel feedstock production.

Keywords: Municipal wastewater; Centrate; Algae; Nutrients removal; Metal; Chlorella

Polymerase Chain Reaction-based Detection of Total and Specific Vibrio Species by Byeong Hee Hwang; Jae Won Lee; Hyung Joon Cha (pp. 1187-1194).

The polymerase chain reaction (PCR) technique is widely used for efficient detection of food-borne pathogens because of speed and specificity. However, PCR methods have focused mostly on species-specific detection. In the present work, we describe a PCR-based method for the simultaneous detection of all Vibrio species because lots of them are notorious food-borne human pathogens. We then combined this total detection method with specific detection of Vibrio cholerae pathogen. Using a degenerate primer set based on the sequence of the potassium uptake gene, trkA, we were able to successfully detect all Vibrio species. Specific detection of V. cholerae was also possible using primer sets based on putative flagellin sequence. Importantly, simultaneous total and species-specific Vibrio detection was possible using all two primer sets in a multiplexed PCR strategy. Thus, the PCR method we have developed is applicable to both simultaneous and two-step detection of total and specific Vibrio species.

Keywords: Polymerase chain reaction; Multiplex PCR; Total Vibrio detection; Species-specific detection; Vibrio cholerae

Xylooligosaccharides Production from Alkali-Pretreated Sugarcane Bagasse Using Xylanases from Thermoascus aurantiacus by Michel Brienzo; Walter Carvalho; Adriane M. F. Milagres (pp. 1195-1205).

Sugarcane bagasse hemicellulose was isolated in a one-step chemical extraction using hydrogen peroxide in alkaline media. The polysaccharide containing 80.9% xylose and small amounts of l-arabinose, 4-O-methyl-d-glucuronic acid and glucose, was hydrolyzed by crude enzymatic extracts from Thermoascus aurantiacus at 50 °C. Conditions of enzymatic hydrolysis leading to the best yields of xylose and xylooligosaccharides (DP 2-5) were investigated using substrate concentration in the range 0.5–3.5% (w/v), enzyme load 40–80 U/g of the substrate, and reaction time from 3 to 96 h, applying a 2² factorial design. The maximum conversion to xylooligosaccharides (37.1%) was obtained with 2.6% of substrate and xylanase load of 60 U/g. The predicted maximum yield of xylobiose by a polynomial model was 41.6%. Crude enzymatic extract of T. aurantiacus generate from sugarcane bagasse hemicellulose 39% of xylose, 59% of xylobiose, and 2% of other xylooligosaccharides.

Keywords: Xylooligosaccharides; Thermoascus aurantiacus ; Lignocellulose degradation; Biomass; Optimization; Enzyme

Magnetic Screening of the Potential Targeted Protein of Salvianolic Acid B Using T7 Phage Display Library by Shupeng Liu; Ye Sun; Heng Chen; Suxian Song; Yuhong Xu (pp. 1206-1213).

Salvianolic acid B is one of the effective components from the Chinese traditional drug Salvia miltiorrhiza (Danshen), which is widely used as a usual clinic drug for atherosclerosis-related disorder patients in China. But the targeting protein of salvianolic acid B is still not known. The possible targeting proteins of salvianolic acid B were explored by high throughput screening in this paper. Attached to the magnetic nanoparticles, salvianolic acid B was used for screening the high-affinity protein from the displaying cDNA peptide library phage. After biopanning, the selected protein or peptide sequences were used to explore the whole proteins containing the selected sequences in the National Center for Biotechnology Information website using blast. One of the selected phages was carried out by affinity analysis with salvianolic acid B using capillary electrophoresis (CE). The CE results indicated that the protein or peptide on the surface of the selected phages could bind the drug salvianolic acid B. The results are helpful to preliminarily explain the pharmacology of salvianolic acid B.

Keywords: Phage display library; Magnetic nanoparticles; Targeted protein; Salvianolic acid B

Disclosure of the Tuberous Lectin Composed of Homogeneous Tetramers in Pinellia pedatisecda Schott by Xiaochuan Liu; Xiaoping Tian; Tong Liu; Jiangli Liang (pp. 1214-1223).

Pinellia pedatisecta (Schott) and Pinellia ternata (Thumb) Breit, whose tuberous stems are an important Chinese medicine, taxonomically belong to Pinellia, Araceae species. Pinellia contains various lectins in their tubers, leading to distinct roles in Chinese medicine. Difference of the lectins, however, is little known between P. pedatisecta and P. ternata tubers. For addressing to this purpose, lectins were isolated from their tuberous stems, purified through porcine thyroglobulin chromatography, analyzed with 2D-gel and Q-Trap mass spectrometry, and evaluated with hemagglutinating assays. The results showed that they possess completely different components of lectins though the lectins could specifically bind to mannose. P. ternata had the tuberous lectin composed of heterogeneous tetramer (L1)₂(L2)₂ with the similar molecular weight but distinct pI 5.8 and pI 6.2. Comparatively, P. pedatisecta mainly contained the tuberous lectin composed of homogeneous tetramer with the same molecular weight and pI 5.8. As a result of the lectin difference between P. pedatisecta and P. ternata, it probably leads to distinct pharmacologic variability. From this perspective, P. pedatisecta could be useful for anticancer research in some ways.

Keywords: Pinellia pedatisecta ; Pinellia ternata ; Tuberous lectins; Homogeneous tetramer; Heterogeneous tetramer

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.162, #4)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.162, #4)