Applied Biochemistry and Biotechnology (v.157, #2)

Gene Cloning, Expression, and Characterization of a Novel Phytase from Dickeya paradisiaca by Weina Gu; Huoqing Huang; Kun Meng; Peilong Yang; Dawei Fu; Huiying Luo; Yaru Wang; Bin Yao; Zhichun Zhan (113-123).
A novel phytase gene, appA, was isolated by degenerate polymerase chain reaction (PCR) and thermal asymmetric interlaced PCR from Dickeya paradisiaca. The full-length appA comprises 1278 bp and encodes 425 amino acid residues, including a 23-residue putative N-terminal signal peptide. The deduced amino acid sequence of appA reveals the conserved motifs RHGXRXP and HD, which are typical of histidine acid phosphatases; significantly, APPA shows maximum identity (49%) to a phytase from Klebsiella pneumoniae. To characterize the properties of APPA, appA was expressed in Escherichia coli and purified. The purified recombinant APPA has two pH optima at pH 4.5 and 5.5, optimum temperature at 55 °C, specific activity of 769 U/mg, and good pH stability. The K m value for the substrate sodium phytate is 0.399 mM with a V max of 666 U/mg. To our knowledge, this is the first report of a phytase or phytase gene isolated from Dickeya.
Keywords: Degenerate PCR; Dickeya paradisiaca ; Histidine acid phosphatase; Phytase; TAIL-PCR

Serine Hydroxamate and the Transcriptome of High Cell Density Recombinant Escherichia coli MG1655 by Fu’ad T. Haddadin; Harry Kurtz; Sarah W. Harcum (124-139).
For more than 30 years, serine hydroxamate has been used to chemically stimulate a stringent response in Escherichia coli and other bacteria. These studies have elucidated numerous characteristics of the classical stringent response beyond the simple cellular response to an amino acid shortage, including phospholipid synthesis and protease upregulation. In this study, the effects of a serine hydroxamate addition on high-cell-density recombinant E. coli were examined and compared to the effects of recombinant protein production to determine overlaps, as recombinant protein production stress has often been attributed to amino acid shortages. Both the transcriptome and growth characteristics were evaluated and compared. The serine hydroxamate addition profoundly decreased the culture growth rate, whereas recombinant protein production did not. Conversely, the transcriptome profile of the recombinant E. coli cultures were relatively unaffected by the serine hydroxamate addition, yet recombinant protein production dramatically changed the transcriptome profile. A subset of the classical stringent response genes were effected by the serine hydroxamate addition, whereas recombinant protein production regulated numerous classical stringent response genes but not all. The genes that were regulated by the serine hydroxamate addition include numerous fatty acid synthesis genes, in agreement with altered phospholipids synthesis reports. These results indicate that recombinant protein production and the stringent response have many overlapping responses but are far from identical.
Keywords: Gene expression; Heat shock; Stringent response; Proteases; Chloramphenicol acetyltransferase

Effects of Feed Time, Organic Loading and Shock Loads in Anaerobic Whey Treatment by an AnSBBR with Circulation by Roberto A. Bezerra Jr.; José A. D. Rodrigues; Suzana M. Ratusznei; Marcelo Zaiat; Eugenio Foresti (140-158).
The aim of this work was to investigate the effect of different feeding times (2, 4, and 6 h) and organic loading rates (3, 6 and 12 gCOD l−1 day−1) on the performance of an anaerobic sequencing batch reactor containing immobilized biomass, as well as to verify the minimum amount of alkalinity that can be added to the influent. The reactor, in which mixing was achieved by recirculation of the liquid phase, was maintained at 30 ± 1°C, possessed 2.5 l reactional volume and treated 1.5 l cheese whey in 8-h cycles. Results showed that the effect of feeding time on reactor performance was more pronounced at higher values of organic loading rates (OLR). During operation at an OLR of 3 gCOD l−1 day−1, change in feeding time did not affect efficiency of organic matter removal from the reactor. At an OLR of 6 gCOD l−1 day−1, reactor efficiency improved in relation to the lower loading rate and tended to drop at longer feeding times. At an OLR of 12 gCOD l−1 day−1 the reactor showed to depend more on feeding time; higher feeding times resulted in a decrease in reactor efficiency. Under all conditions shock loads of 24 gCOD l−1 day−1 caused an increase in acids concentration in the effluent. However, despite this increase, the reactor regained stability readily and alkalinity supplied to the influent showed to be sufficient to maintain pH close to neutral during operation. Regardless of applied OLR, operation with feeding time of 2 h was which provided improved stability and rendered the process less susceptible to shock loads.
Keywords: AnSBBR; Organic loading rate; Shock load; Feeding time; Cheese whey

Biosorption of copper (II) and cadmium (II) by live Phanerochaete chrysosporium immobilized by growing onto polyurethane foam material in individual packed bed columns over two successive cycles of sorption–desorption were investigated in this study. Initial pH and concentrations of the metals in their respective solutions were set optimum to each of those: 4.6 and 35 mg·l−1 in case of copper and 5.3 and 11 mg·l−1 for cadmium. The breakthrough curves obtained for the two metals during sorption in both the cycles exhibited a constant pattern at various bed depths in the columns. The maximum yield of the columns in removing these metals were found to be, respectively, 57% and 43% for copper and cadmium indicating that copper biosorption by the immobilized fungus in its column was better than for cadmium. Recovery values of the sorbed copper and cadmium metals from the respective loaded columns by using 0.1 N HCl as eluant was observed to be quite high at more than 65% and 75%, respectively, at the end of desorption in both the cycles. Breakthrough models of bed-depth service time, Adams–Bohart, Wolborska, and Clark were fitted to the experimental data on sorption of copper and cadmium in the columns, and only the Clark model could fit the sorption performance of the columns well over the entire range of ratios of concentrations of effluent to influent, i.e., C/C 0 for both copper and cadmium biosorption. The kinetic coefficients of mass transfer and other suitable parameters in the system were determined by applying the experimental data at C/C 0 ratios lower than 0.5 to the other three models.
Keywords: Heavy metals; Copper; Cadmium; Phanerochaete chrysosporium ; Packed bed column; Immobilized fungus; Biosorption; Desorption; Breakthrough models

Lignin is the most abundant renewable source of aromatic polymer in nature, and its decomposition is indispensable for carbon recycling. It is chemically recalcitrant to breakdown by most organisms because of the complex, heterogeneous structure. The white-rot fungi produce an array of extracellular oxidative enzymes that synergistically and efficiently degrade lignin. The major groups of ligninolytic enzymes include lignin peroxidases, manganese peroxidases, versatile peroxidases, and laccases. The peroxidases are heme-containing enzymes with catalytic cycles that involve the activation by H2O2 and substrate reduction of compound I and compound II intermediates. Lignin peroxidases have the unique ability to catalyze oxidative cleavage of C–C bonds and ether (C–O–C) bonds in non-phenolic aromatic substrates of high redox potential. Manganese peroxidases oxidize Mn(II) to Mn(III), which facilitates the degradation of phenolic compounds or, in turn, oxidizes a second mediator for the breakdown of non-phenolic compounds. Versatile peroxidases are hybrids of lignin peroxidase and manganese peroxidase with a bifunctional characteristic. Laccases are multi-copper-containing proteins that catalyze the oxidation of phenolic substrates with concomitant reduction of molecular oxygen to water. This review covers the chemical nature of lignin substrates and focuses on the biochemical properties, molecular structures, reaction mechanisms, and related structures/functions of these enzymes.
Keywords: Lignin peroxidase; Manganese peroxidase; Versatile peroxidase; Laccase; Lignin degradation

Hyoscyamine biosynthesis in Datura stramonium hairy roots with different ploidy levels was investigated. For the first time, we report that hairy roots undergo endoreduplication and therefore consist mainly of cells with doupled sets of chromosomes of primary plant tissues, used for Agrobacterium transformation. The alkaloid profiles of hairy roots obtained from diploid and tetraploid plants were similar in terms of the major compounds, but they differed significantly with respect to the minor compounds (here defined as those that accounted for <1% of the total ion current of the alkaloid mixture in gas chromatography–mass spectrometric analyses). Significant differences in the effects of the main nutrients on the growth of the hairy roots obtained from diploid and tetraploid plants and their hyoscyamine contents were observed. The maximal yield of hyoscyamine (177 mg/L) was obtained when hairy roots from tetraploid plants were cultivated in Murashige–Skoog nutrient medium supplemented with 6% sucrose. Time courses of utilization of the main nutrients in the medium during cultivation of D. stramonium hairy root cultures are also presented.
Keywords: Alkaloids; Datura stramonium ; Endoreduplication; Flow Cytometry; Gas chromatography–mass spectroscopy; Hairy roots; Ploidy level

Isolation and Functional Characterization of Single Domain Antibody Modulators of Caspase-3 and Apoptosis by Katrina McGonigal; Jamshid Tanha; Elitza Palazov; Shenghua Li; Deyzi Gueorguieva-Owens; Siyaram Pandey (226-236).
Apoptosis, or programmed cell death, is an essential process affecting homeostasis of cell growth, development, and the elimination of damaged or dangerous cells. Inappropriate cell death caused by oxidative stress has been implicated in the development of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and stroke. On the other hand, a defect in the cell death process leads to the development of cancer. For example, the main player of apoptosis, p53, is defective in many of the human cancers. Apoptosis is regulated by the interplay of pro-apoptotic and anti-apoptotic proteins from the Bcl-2 family and caspases. In particular, specific modulators of the activity of Caspase 3 could be very important for the development of therapies for diseases such as neurodegeneration and cancer. In this study, two VHHs specific to Caspase 3 (VhhCasp31 and VhhCasp32) were isolated from a heavy chain antibody variable domain (VHH) phage display library and tested for their apoptosis-modulating effects. While VhhCasp31 was found to be antagonistic towards Caspase 3, VhhCasp32 was agonistic. Furthermore, when expressed as intrabodies in SHSY-5Y neuroblastoma cells, VhhCasp31 rendered cells resistant to oxidative-stress-induced apoptosis, whereas VhhCasp32 resulted in apoptosis. These VHH antagonist and agonist of apoptosis could have potential for the development of therapeutics for neurodegenerative diseases and cancer, respectively.
Keywords: Caspase 3; Apoptosis; VHH; Intrabodies; Oxidative stress; Neurodegenerative Diseases; Cancer

Culture fluorescence measurement technique has the potential for on-line characterization of metabolic status of fermentation processes. Many fluorophores present inside the living cells such as NADH + H+, tryptophan, pyridoxine, and riboflavin fluoresce at specific excitation and emission wavelength combinations. Since these key intracellular metabolites are involved in cell growth and metabolism, their concentration change at any time inside the cell could reflect the changes in cell metabolic activity. NADH + H+ spectrofluorometry was used for on-line characterization of physiological state during batch cultivation of poly-β-hydroxybutyric acid (PHB) production by Wautersia eutropha. The culture fluorescence increased with an increase in the biomass concentration with time. A linear correlation between cell mass concentration and net NADH + H+ fluorescence was established during active growth phase (13 to 38 h) of batch cultivation. The rate of change of culture fluorescence (dF/dt) exhibited a gradual increase during the predominantly growth phase of batch cultivation (till 20 h). Thereafter, a sudden drop in the dF/dt rate and its leveling was recorded indicating major changes in culture metabolism status which synchronized with the start-up of accumulation of PHB. After 48 h, yet another decrease in the rate of change of fluorescence (dF/dt) was observed primarily due to severe substrate limitation in the reactor. On-line NADH + H+ fluorescence signal and its rate (dF/dt) could therefore be used to distinguish the growth, product formation, and nutrient depletion stage (the metabolic state marker) during the batch cultivation of W. eutropha.
Keywords: PHB; Wautersia eutropha ; NADH + H+ ; Fluorescence; Batch cultivation

Plant growth and productivity are adversely affected by various abiotic and biotic stress factors. Despite the wealth of information on abiotic stress and stress tolerance in plants, many aspects still remain unclear. Prosopis juliflora is a hardy plant reported to be tolerant to drought, salinity, extremes of soil pH, and heavy metal stress. In this paper, we report the isolation and characterization of the complementary DNA clone for an atypical late embryogenesis abundant (LEA) protein (Pj LEA3) and its putative promoter sequence from P. juliflora. Unlike typical LEA proteins, rich in glycine, Pj LEA3 has alanine as the most abundant amino acid followed by serine and shows an average negative hydropathy. Pj LEA3 is significantly different from other LEA proteins in the NCBI database and shows high similarity to indole-3 acetic-acid-induced protein ARG2 from Vigna radiata. Northern analysis for Pj LEA3 in P. juliflora leaves under 90 mM H2O2 stress revealed up-regulation of transcript at 24 and 48 h. A 1.5-kb fragment upstream the 5′ UTR of this gene (putative promoter) was isolated and analyzed in silico. The possible reasons for changes in gene expression during stress in relation to the host plant’s stress tolerance mechanisms are discussed.
Keywords: Prosopis juliflora ; Abiotic stress; Atypical LEA proteins; Promoter; Phylogenetic analysis

A thermophilic microorganism growing within the temperature range of 40–65 °C (optimum at 55 °C) was isolated from hot water springs near Konkan, Maharashtra, India. Based on 16S rDNA sequence analysis, it was concluded that the isolate belongs to the genus Brevibacillus. The present paper reports the isolation, identification, and standardization of fermentation conditions for the production of enzyme, bile salt hydrolase (EC 3.5.1.24) which is produced intracellularly at high temperatures. This is the first report regarding the production of bile salt hydrolase from a thermophilic source. Optimization of fermentation conditions resulted in a 2.9-fold enhancement in enzyme production.
Keywords: Brevibacillus sp ; Thermophile; Bile salt hydrolase; Glycodeoxycholic acid; Hot springs; Fermentation

Enzymatic Polymerization of Natural Anacardic Acid and Antibiofouling Effects of Polyanacardic Acid Coatings by Rahul Chelikani; Yong Hwan Kim; Do-Young Yoon; Dong-Shik Kim (263-277).
Anacardic acid, separated from cashew nut shell liquid, is well known for its strong antibiotic and antioxidant activities. Recent findings indicate that phenolic compounds from plant sources have an effect on Gram-negative bacteria biofilm formation. In this work, a polyphenolic coating was prepared from anacardic acid using enzymatic synthesis and tested for its effects on biofilm formation of both Gram-negative and Gram-positive bacteria. Natural anacardic acid was enzymatically polymerized using soybean peroxidase. Hydrogen peroxide and phenothiazine-10-propionic acid were used as an oxidizing agent and redox mediator, respectively. Nuclear magnetic resonance and Fourier transform infrared (FTIR) analyses showed the formation of oxyphenylene and phenylene units through the phenol rings. No linkage through the alkyl chain was observed, which proved a high chemo-selectivity of the enzyme. Aqueous solvents turned out to play an important role in the polymer production yield and molecular weight. With 2-propanol, the highest production yield (61%) of polymer (molecular weight = 3,900) was observed, and with methanol, higher-molecular-weight polymers (5,000) were produced with lower production yields (43%). The resulting polyanacardic acid was cross-linked on a solid surface to form a permanent natural polymer coating. The FTIR analysis indicates that the cross-linking between the polymers took place through the unsaturated alkyl side chains. The polyanacardic acid coating was then tested for its antibiofouling effect against Gram-negative and Gram-positive bacteria and compared with the antibiofouling effects of polycardanol coatings reported in the literature. The polyanacardic acid coating showed more reduction in biofilm formation on its surface than polycardanol coatings in the case of Gram-positive bacteria, while in the case of Gram-negative bacteria, it showed a similar reduction in biofilm formation as polycardanol.
Keywords: Enzymatic polymerization; Anacardic acid; CNSL; Antibiofouling; Soybean peroxidase; Redox mediator

Production of Calcium-Stearate by Lipase Using Hydrogenated Beef Tallow by Hyang-Bok Lee; Jin-Soo Kwon; Young-Bum Kim; Eun-Ki Kim (278-284).
Calcium-stearate has been traditionally produced by chemical methods, producing wastes and requiring high energy because of high temperature operation. To achieve enzymatic production of calcium-stearate at unfavorable conditions, i.e., pH 10 and 60 °C, suitable lipase was selected and reaction conditions were optimized using calcium hydroxide and hydrogenated beef tallow as substrates. Under optimum conditions, 95% of beef tallow, in 2.5 h, was converted into calcium-stearate by using commercial lipase SDL 451. Investigation of the time-course reaction revealed that fatty acid was initially produced by lipase, followed by conversion into calcium-stearate. The fatty acid production rate was faster than that of the conversion into calcium-stearate at the beginning of the reaction. Alkaline pH, originating from the addition of calcium hydroxide, increased the converting reaction. This is the first report demonstrating that chemical production of calcium-stearate can be replaced by enzymatic reaction, thereby creating a cleaner process.
Keywords: Calcium-stearate; Lipase SDL 451; Hydrogenated beef tallow

Autoacetylation of Purified Calreticulin Transacetylase Utilizing Acetoxycoumarin as the Acetyl Group Donor by Seema Bansal; Prija Ponnan; Hanumantharao G. Raj; Susan T. Weintraub; Madhu Chopra; Ranju Kumari; Daman Saluja; Ajit Kumar; Tapesh K. Tyagi; Prabhjot Singh; Ashok K. Prasad; Luciano Saso; Ramesh C. Rastogi; Virinder S. Parmar (285-298).
Our earlier reports documented that calreticulin, a multifunctional Ca2+-binding protein in endoplasmic reticulum lumen, possessed protein acetyltransferase function termed Calreticulin Transacetylase (CRTAase). The autoacetylation of purified human placental CRTAase concomitant with the acetylation of receptor proteins by a model acetoxycoumarin, 7,8-Diacetoxy-4-methylcoumarin, was observed. Here, we have examined the autoacetylation property of CRTAase by immunoblotting and mass spectrometry. Ca2+ was found to inhibit CRTAase activity. The inhibition of both autoacetylation of CRTAase as well as acetylation of the receptor protein was apparent when Ca2+ was included in the reaction mixture as visualized by interaction with anti-acetyl lysine antibody. The acetylation of lysines residues: −48, −62, −64, −153, and −159 in N-domain and −206, −207, −209, and −238 in P-domain of CRTAase were located by high-performance liquid chromatography-electronspray ionization tandem mass spectrometry. Further, computer assisted protein structure modeling studies were undertaken to probe the effect of autoacetylation of CRTAase. Accordingly, the predicted CRTAase 3D model showed that all the loop regions of both N- and P-domain bear the acetylated lysines. Energy minimization of the acetylated residues revealed charge neutralization of lysines due to the N-ε-acetylation which may facilitate the interaction of CRTAase with the protein substrate and the subsequent transacetylase action.
Keywords: Protein acetyltransferase; Calreticulin; Acetoxycoumarin; Protein acetylation; Mass spectrometry

Enhancement of Recombinant Human ADAM15 Disintegrin Domain Expression Level by Releasing the Rare Codons and Amino Acids Restriction by Jing Wu; Lianfen Zhang; Jianyong Lei; Gangming Cai; Wei Zhu; Daru Lu; Jian Jin (299-310).
This study was aimed at increasing the production of the recombinant human ADAM15 disintegrin domain (RADD) in Escherichia coli by releasing the rare codons and restricting amino acid residues. Three different strategies for increasing RADD production were examined: to select the suitable host strain, to optimize the rare codons, and to delete the amino acids residues. The total fusion protein glutathione-S-transferase (GST)-RADD concentration of 298 mg/l and 326 mg/l were achieved by selecting E. coli Rosetta (DE3) as the host strain and by changing GGA to GGC at the GST-RADD cassette, respectively. The RADD concentration was increased by 35.7% by eliminating the “Pro-Glu-Phe” residues at the GST–RADD junction. By combinational utilizing the preferred codon introduction and amino acid sequence optimization in E. coli Rosetta (DE3), the highest RADD concentration of 68 mg/l was achieved. The proposed strategy may provide an alternative approach for other enhanced recombinant protein production by E. coli.
Keywords: Recombinant ADAM15 disintegrin domain (RADD); Rare codon; Amino acids residues

Purification and Characterization of Laccase Secreted by L. lividus by R. Sahay; R. S. S. Yadav; K. D. S. Yadav (311-320).
The culture conditions for maximum secretion of laccase by Loweporus lividus MTCC-1178 have been optimized. The laccase from the culture filtrate of L. lividus MTCC-1178 has been purified to homogeneity. The molecular weight of the purified laccase is 64.8 kDa. The enzymatic characteristics like K m, pH, and temperature optimum using 2,6-dimethoxyphenol have been determined and found to be 480 μM, 5.0, and 60 °C, respectively. The K m values for other substrates like catechol, m-cresol, pyrogallol, and syringaldazine have also been determined and found to be 230, 210, 320, and 350 μM, respectively.
Keywords: Laccases; L. lividus ; Lignolytic enzymes; Metalloenzymes; Cu containing enzymes; Lignolytic fungi

Bioremediation Potential of Formaldehyde by the Marine Microalga Nannochloropsis oculata ST-3 Strain by Kosuke Yoshida; Hiroshi Ishii; Yoshimi Ishihara; Hiroshi Saito; Yoshihiro Okada (321-328).
The present work is intended to investigate biodegradation of formaldehyde by the marine microalga Nannochloropsis oculata ST-3 strain. Formaldehyde concentration in the medium decreased with the growth of the ST-3 strain. It is observed that the degradation of formaldehyde concentration depends on the increased cell number of the ST-3 strain. The ST-3 strain which was adapted to formaldehyde stepwise was able to tolerate to 19.9 ppm formaldehyde and degrade 99.3% of it in the medium for 22 days. Tolerance and degradation ability of formaldehyde by the ST-3 strain was improved by stepwise increasing of the formaldehyde concentration. Transformation of [13C]formaldehyde in the medium with the passage of incubation was monitored by using a nuclear magnetic resonance (NMR) spectrometer. Formaldehyde was transformed into formate, and these two substances degraded in the medium with the passage of incubation as clearly shown by the NMR spectrum.
Keywords: Formaldehyde; Marine microalga; Growth inhibition; Adaptation; Biodegradation

Characterization of Rhamnolipid Produced by Pseudomonas aeruginosa Isolate Bs20 by Ahmad Mohammad Abdel-Mawgoud; Mohammad Mabrouk Aboulwafa; Nadia Abdel-Haleem Hassouna (329-345).
Rhamnolipid produced by Pseudomonas aeruginosa isolate Bs20 is viscous sticky oily yellowish brown liquid with a fruity odor. It showed solubility at aqueous pH > 4 with optimum solubility at pH 7–7.5 and freely soluble in ethyl acetate. This biosurfactant has a very high surface activity as it could lower the surface tension of water to 30 mN/m at about 13.4 mg/L, and it exhibited excellent stabilities at high temperatures (heating at 100°C for 1 h and autoclaving at 121°C for 10 min), salinities (up to 6% NaCl), and pH values (up to pH 13). The produced biosurfactant can be used in the crude form either as cell-free or cell-containing culture broth of the grown bacteria, since both preparations showed high emulsification indices ranged between 59% and 66% against kerosene, diesel, and motor oil. These characters make the test rhamnolipid a potential candidate for use in bioremediation of hydrocarbon-contaminated sites or in the petroleum industry. High-performance thin-layer chromatography densitometry revealed that the extracted rhamnolipid contained the two most active rhamnolipid homologues dirhamno dilipidic rhamnolipid and monorhamno dilipidic rhamnolipid at 44% and 56%, respectively, as compared to 51% and 29.5%, respectively, in a standard rhamnolipid preparation. The nature and ratio of these two rhamnolipid homologues showed to be strain dependent rather than medium-component dependent.
Keywords: Rhamnolipid; Pseudomonas aeruginosa ; Characterization

Slug, a member of the Snail family of zinc-finger transcription factors, is involved in regulating embryonic development and tumorigenesis. The aim of this study was to investigate the expression of Slug in mouse endometrium during early pregnancy and its possible role during embryo implantation. Fluorescence quantitative polymerase chain reaction and immunohistochemistry were applied to detect Slug mRNA and Slug protein expression in endometrium of nonpregnant and early pregnant mice, respectively. The expressions of Slug mRNA and its protein in pregnant group were higher than that in nonpregnant group and gradually increased from pregnancy day 1, reaching its maximum level on day 4 and then declining on days 5, 6, and 7. Immunohistochemistry showed that Slug protein was mainly present in luminal epithelium from pregnancy days 2 to 5 and in glandular epithelium from days 2 to 6 and enhanced significantly in stromal cells on days 4, 5, and 6. The number of embryos implanted was greatly decreased after Slug function in mouse endometrium was blocked by the intrauterine injection with anti-Slug polyclonal antibody on day 3 of pregnancy before implantation. These results suggested that up-regulation of Slug expression may play a key role in the embryo implantation in mice.
Keywords: Embryo implantation; Expression study; Mouse endometrium; Slug