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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.160, #3)

The Operable Modeling of Simultaneous Saccharification and Fermentation of Ethanol Production from Cellulose by Jiacheng Shen; Foster A. Agblevor (pp. 665-681).

An operable batch model of simultaneous saccharification and fermentation (SSF) for ethanol production from cellulose has been developed. The model includes four ordinary differential equations that describe the changes of cellobiose, glucose, yeast, and ethanol concentrations with respect to time. These equations were used to simulate the experimental data of the four main components in the SSF process of ethanol production from microcrystalline cellulose (Avicel PH101). The model parameters at 95% confidence intervals were determined by a MATLAB program based on the batch experimental data of the SSF. Both experimental data and model simulations showed that the cell growth was the rate-controlling step at the initial period in a series of reactions of cellulose to ethanol, and later, the conversion of cellulose to cellobiose controlled the process. The batch model was extended to the continuous and fed-batch operating models. For the continuous operation in the SSF, the ethanol productivities increased with increasing dilution rate, until a maximum value was attained, and rapidly decreased as the dilution rate approached the washout point. The model also predicted a relatively high ethanol mass for the fed-batch operation than the batch operation.

Keywords: Simultaneous saccharification and fermentation; Ethanol; Cellulose; Model; Operating mode

Partial Characterization of Inulinases Obtained by Submerged and Solid-State Fermentation Using Agroindustrial Residues as Substrates: A Comparative Study by Marcio Antonio Mazutti; Aline Skrowonski; Gabriela Boni; Giovani Leone Zabot; Marceli Fernandes Silva; Débora de Oliveira; Marco Di Luccio; Francisco Maugeri Filho; Maria Isabel Rodrigues; Helen Treichel (pp. 682-693).

Inulinase belongs to an important class of enzymes as it can be used to produce high-fructose syrups by enzymatic hydrolysis of inulin and fructooligosaccharides, which has been used as functional food. This work aimed to carry out a partial characterization of the crude enzymatic extract of two different inulinases, obtained by solid-state fermentation (SSF) and submerged fermentation (SmF), using agroindustrial residues as substrates. The crude enzymatic extract obtained by SmF showed an optimal pH and temperature for hydrolytic activity of 4.5 and 55 °C, respectively; and that obtained by SSF conducted to optimal pH and temperature of 5.0 and 55 °C, respectively. Both enzymes presented high thermostability, with a D value of 230.4 h and 123.1 h for SmF and SSF, respectively. The inulinase produced by SmF showed highest stability at pH 4.4, while inulinase obtained by SSF was more stable at pH 4.8. The results showed that inulinase obtained by SmF is less susceptible to pH effect and the inulinase obtained by SSF is more resistant to higher temperatures.

Keywords: Inulinase; Agroindustrial residues; Solid-state fermentation; Submerged fermentation; Partial characterization

The Catalytic Property of 3-Hydroxyisobutyrate Dehydrogenase from Bacillus cereus on 3-Hydroxypropionate by Tianran Yao; Lin Xu; Hanjie Ying; He Huang; Ming Yan (pp. 694-703).

The MmsB gene product from Bacillus cereus ATCC14579 exhibits 3-hydroxypropionate dehydrogenase activity. It encodes the 32-kDa enzyme protein composed of 292 amino acids. Recombinant 3-hydroxyisobutyrate dehydrogenase (3-HIBADH) was purified 100-fold from cell extract by ammonium sulfate fractionation and column chromatography. The enzyme catalyzed oxidation of 3-hydroxypropionate (3-HP) between pH 7.0 and 10.0 with optimal activity between 8.8 and 9.0. A Km of 16.8 mM for 3-HP was calculated from a Lineweaver–Burk plot. The semialdehyde as products has been proven by spectrophotometric determination. The dehydrogenase apparently has no metal ion requirement. Kinetic determinations established that 3-HIBADH was more active with NADP⁺ than NAD⁺, which did not show similarity with previously reported 3-HIBADH except that from Thermus thermophilus.

Keywords: 3-Hydroxyisobutyrate dehydrogenase; 3-Hydroxypropionate; Bacillus cereus

Salicylic-Acid-Mediated Enhanced Biological Treatment of Wastewater by Anshuman A. Khardenavis; Atya Kapley; Hemant J. Purohit (pp. 704-718).

Activated sludge represents a microbial community which is responsible for reduction in pollution load from wastewaters and whose performance depends upon the composition and the expression of degradative capacity. In the present study, the role of salicylic acid (SA) has been evaluated for acclimatization of activated sludge collected from a combined effluent treatment plant followed by analysis of the physiological performance and microbial community of the sludge. The biodegradative capacity of the acclimatized activated sludge was further evaluated for improvement in efficiency of chemical oxygen demand (COD) removal from wastewater samples collected from industries manufacturing bulk drugs and dyes and dye intermediates (wastewater 1) and from dye industry (wastewater 2). An increase in COD removal efficiency from 50% to 58% and from 78% to 82% was observed for wastewater 1 and wastewater 2, respectively. Microbial community analysis data showed selective enrichment and change in composition due to acclimatization by SA, with 50% of the clones showing sequence homology to unidentified and uncultured bacteria. This was demonstrated by analysis of partial 16S rDNA sequence data generated from dominating clones representing the metagenome which also showed the appearance of a unique population of clones after acclimatization, which was distinct from those obtained before acclimatization and clustered away from the dominating population.

Keywords: Acclimatization; Activated sludge; Salicylic acid; Microbial diversity; Wastewater treatment

Response of an Algal Consortium to Diesel under Varying Culture Conditions by Anal Chavan; Suparna Mukherji (pp. 719-729).

A diesel-tolerant sessile freshwater algal consortium obtained from the vicinity of Powai Lake (Mumbai, India) was cultured in the laboratory. The presence of diesel in batch cultures enhanced the maximum specific growth rate of the algal consortium. With decrease in light–dark (L:D) cycle from 20:4 to 4:20 h, the chlorophyll-a levels decreased; however, the removal of diesel was found to be maximum at L:D of 18:6 h with 37.6% degradation over and above controls. In addition to growth in the form of green clumps, white floating biomass was found surrounding the diesel droplets on the surface. This culture predominated at the least L:D ratio of 4:20 h. Studies confirmed the ability of the floating organisms to grow heterotrophically in the dark utilizing diesel as carbon source and also in the presence of light in a medium devoid of organic carbon sources.

Keywords: Biodegradation; Cyanobacteria; Diesel; Heterotrophic growth; Hydrocarbons

A New Aminopeptidase from the Keratin-Degrading Strain Streptomyces fradiae var. k11 by Bo Wu; Pengjun Shi; Jiang Li; Yaru Wang; Kun Meng; Yingguo Bai; Huiying Luo; Peilong Yang; Zhigang Zhou; Bin Yao (pp. 730-739).

An aminopeptidase gene fragment was isolated from a keratin-degrading strain, Streptomyces fradiae var. k11, by PCR amplification using a degenerate primer set designed based on the partial amino acid sequence of the native enzyme. The gene, designated sfap, encoded a polypeptide of 461 amino acids comprised of three domains: a signal peptide, a mature region, and a C-terminal propeptide. The aminopeptidase, SFAP, had highest amino acid sequence identity (79%) with a putative aminopeptidase from Streptomyces griseus subsp. griseus NBRC 13350. The gene with and without C-terminal propeptide was successfully overexpressed in Escherichia coli BL21 (DE3), and the gene without C-terminal propeptide encoded a functional enzyme. Purified recombinant SFAP exhibited optimal activity at pH 8.0 and 60 °C, and retained >60% peak activity over a broad range of temperature. The enzyme was thermal and pH stable, and showed metalloprotease characteristics, which was inhibited by EDTA but activated by Ca²⁺ and Co²⁺. This is the first study to report the gene cloning and expression of a leucine aminopeptidase from S. fradiae.

Keywords: Aminopeptidase; Streptomyces fradiae var. k11; C-terminal propeptide

Enhancing Production of l-Serine by Increasing the glyA Gene Expression in Methylobacterium sp. MB200 by Peihong Shen; Hongjun Chao; Chengjian Jiang; Zhangde Long; Changhao Wang; Bo Wu (pp. 740-750).

Microbial fermentation using methylotrophic bacteria is one of the most promising methods for l-serine production. Here we describe the metabolic engineering of a Methylobacterium strain to increase the production of l-serine. The glyA gene, encoding serine hydroxymethyltransferase (SHMT), was isolated from the genomic DNA of Methylobacterium sp. MB200, using a DNA fragment encoding Methylobacterium extorquens AM1 SHMT as a probe, and inserted into the vector pLAFR3. The resulting construct was transformed into Methylobacterium sp. MB200 using triparental mating. The genetic-engineered strain, designated as Methylobacterium sp. MB202, was shown to produce 11.4 ± 0.6 mg/ml serine in resting cell reactions from 30 mg/ml wet cells, 20 mg/ml glycine, and 70 mg/ml methanol in 2 days, representing a 4.4-fold increase from that of the wild strain. The results demonstrated the potential for improving l-serine production by manipulating the glyA in bacteria and should facilitate the production of l-serine using Methylobacterium sp. strains.

Keywords: Methylobacterium sp.; glyA ; Serine hydroxymethyltransferase (SHMT); l-Serine; Genetic engineering

Cytotoxicity of Bacterial-Derived Toxins to Immortal Lung Epithelial and Macrophage Cells by Dianne E. Peterson; Jayne M. Collier; Matthew E. Katterman; Rachael A. Turner; Mark R. Riley (pp. 751-763).

Health risks associated with inhalation and deposition of biological materials have been a topic of great concern due to highly publicized cases of inhalation anthrax, of new regulations on the release of particulate matter, and to increased concerns on the hazards of indoor air pollution. Here, we present an evaluation of the sensitivity of two immortal cell lines (A549, human lung carcinoma epithelia) and NR8383 (rat alveolar macrophages) to a variety of bacterial-derived inhalation hazards and simulants including etoposide, gliotoxin, streptolysin O, and warfarin. The cell response is evaluated through quantification of changes in mitochondrial succinate dehydrogenase activity, release of lactate dehydrogenase, initiation of apoptosis, and through changes in morphology as determined by visible light microscopy and scanning electron microscopy. These cells display dose–response relations to each toxin, except for triton which has a step change response. The first observable responses of the epithelial cells to these compounds are changes in metabolism for one toxin (warfarin) and alterations in membrane permeability for another (gliotoxin). The other four toxins display a similar time course in response as gauged by changes in metabolism and loss of membrane integrity. Macrophages are more sensitive to most toxins; however, they display a lower level of stability. This information can be used in the design of cell-based sensors responding to these and similar hazards.

Keywords: Human lung cells; Cell culture; Toxicity monitoring

Semi-continuous Cultivation of Haematococcus pluvialis for Commercial Production by Esra Imamoglu; Meltem Conk Dalay; Fazilet Vardar Sukan (pp. 764-772).

The objectives of the present study on the growth of Haematococcus pluvialis were to indicate the effects of a long-term semi-continuous cultivation, sterilization, carbon dioxide, and different culture media by using artesian well water. This investigation was an enterprise in order to commercialize the production economically. When the effect of CO₂ was investigated in basal culture medium, the influence of sterilization was also researched in Rudic’s culture medium in vertical panel-type photobioreactors for 31 days of semi-continuous cultivation. The maximum cell concentration of 10.55 × 10⁵ cells ml⁻¹, which corresponds to the growth rate of 0.271 day⁻¹ with the areal productivity of 3.531 g m⁻² day⁻¹, was found in non-sterilized RM medium on the 24th day of the third run of semi-continuous cultivation at a renewal rate of 50% in a vertical panel-type photobioreactor.

Keywords: Carbon dioxide; Haematococcus pluvialis ; Nutrient medium; Photobioreactor; Semi-continuous cultivation; Sterilization

Mechanisms of Analgesic Action of Gln49-PLA₂ from Gloydius ussurensis Snake Venom by Yu Zhang; Bo Jiang; Wenli Li; Cheng Zhou; Fangling Ji; Qun Xie; Xiaojiao Sun; Lijia An; Yongming Bao (pp. 773-779).

Gln49-PLA₂ is a phospholipase A₂ isolated from the Gloydius ussurensis snake venom. In this paper, we studied its effect on the function of neural conduction. Electrophysiological studies demonstrated that Gln49-PLA₂ reduced the amplitude of the action potential and the velocity of nerve conduction on isolated mouse sciatic nerve. Patch clamp recordings confirmed that Gln49-PLA₂ significantly decreased neural excitability by the potentiation of sodium channels and the blockade of potassium channels in nerve terminal. In freshly isolated hippocampal pyramidal neurons, 54.25% of potassium current was inhibited by 20 μg/ml Gln49-PLA₂. However, sodium current was potentiated by 158.99% under the same condition. These findings demonstrate that the effect of Gln49-PLA₂ on ion channels is the main mechanism of analgesic action.

Keywords: Analgesic action; Gln49 phospholipase A₂ ; Neurotoxin; Patch clamp; Ion channel; Gloydius ussurensis

Enhanced Biodegradation of Hydrocarbons in Soil by Microbial Biosurfactant, Sophorolipid by Seok-Whan Kang; Young-Bum Kim; Jae-Dong Shin; Eun-Ki Kim (pp. 780-790).

Effectiveness of a microbial biosurfactant, sophorolipid, was evaluated in washing and biodegradation of model hydrocarbons and crude oil in soil. Thirty percent of 2-methylnaphthalene was effectively washed and solubilized with 10 g/L of sophorolipid with similar or higher efficiency than that of commercial surfactants. Addition of sophorolipid in soil increased biodegradation of model compounds: 2-methylnaphthalene (95% degradation in 2 days), hexadecane (97%, 6 days), and pristane (85%, 6 days). Also, effective biodegradation method of crude oil in soil was observed by the addition of sophorolipid, resulting in 80% biodegradation of saturates and 72% aromatics in 8 weeks. These results showed the potentials of the microbial biosurfactant, sophorolipid, as an effective surfactant for soil washing and as an in situ biodegradation enhancer.

Keywords: Sophorolipid; Petroleum hydrocarbon; Bioremediation; Microbial surfactant

Isolation and Characterization of Low Molecular Weight Glycosaminoglycans from Marine Mollusc Amussium pleuronectus (Linne) using Chromatography by R. Saravanan; A. Shanmugam (pp. 791-799).

The glycosaminoglycan (GAG) heparin is a polyanionic sulfated polysaccharide most recognized for its anticoagulant activity. In the present study, the GAGs were extracted from bivalve mollusc Amussium pleuronectus. The crude GAGs were fractionated by ion-exchange (DEAE-cellulose and Amberlite IRA-900 & 120) chromatography. The recovered active fractions (as determined by metachromatic assay) were confirmed by agarose gel electrophoresis and the active fractions were purified in Sephadex G-100 column. Fractionated and purified GAG molecular weight was determined through gradient polyacrylamide gel electrophoresis. The structural characterization of low molecular weight GAG was analyzed by Fourier transform infrared spectroscopy. The activated partial thromboplastin time of purified GAG is 95 IU/mg and has molecular weight 6,500–7,500 Da. The disaccharide compositional analysis on the GAG sample was sulfated like porcine intestinal mucosal heparan sulfate, and it contains equivalent amount of uronic acid and hexosamine. The results of this study suggest that the GAG from A. pleuronectus could be an alternative source of heparin.

Keywords: Glycosaminoglycans; DEAE-cellulose; Amberlite IRA-900&120; Sephadex G-100; APTT; Amussium pleuronectus

Effect of Resistant Starch on Hydrolysis and Fermentation of Corn Starch for Ethanol by Vivek Sharma; Kent D. Rausch; James V. Graeber; Shelly J. Schmidt; Philip Buriak; M. E. Tumbleson; Vijay Singh (pp. 800-811).

Starch samples with 0% or 30% amylose were subjected to four different liquefaction enzyme treatments (at various temperature and pH conditions) followed by simultaneous saccharification and fermentation (SSF). Resistant starch (RS) measurements were conducted for the initial starch sample, after liquefaction and after SSF. Initial RS was higher for 30% amylose starch samples (16.53 g/100 g sample) compared with 0% amylose (0.76 g/100 g sample). Higher initial RS resulted in lower conversion of starch into sugars and lower final ethanol yields. The four enzymes hydrolyzed RS, but in varying amounts. Higher temperature liquefaction hydrolyzed a larger portion of RS, resulting in higher ethanol concentrations and lower final residual solids (non-fermentables), whereas lower temperature liquefaction hydrolyzed a smaller portion of RS and resulted in lower ethanol concentrations and higher final residual solids. Decreases in resistant starch after high temperature liquefaction were 55% to 74%, whereas low temperature liquefaction decreases were 11% to 43%. For all enzyme treatments, RS content of starch samples decreased further after SSF.

Keywords: Amylose; Dry grind corn process; Enzymes; Ethanol; Fermentation; Liquefaction; Resistant starch

Structural Characterization of Lipopeptides from Brevibacillus brevis HOB1 by Ji Wang; Namir I. A. Haddad; Shi-Zhong Yang; Bo-Zhong Mu (pp. 812-821).

Brevibacillus brevis HOB1 was isolated from the formation water of an oil field and found to produce lipopeptides. The separation of lipopeptides was successfully achieved by reversed-phase high-performance liquid chromatography (HPLC) leading to nine separated peaks. The chemical structures of these lipopeptides were studied by means of electrospray ionization mass spectrometry (ESI-MS), gas chromatography-mass spectrometry (GC/MS), HPLC and electrospray ionization tandem mass spectrometry (ESI-MS/MS). As the results, all the lipopeptides had peptide parts with the same amino acid composition of Asp, Glu, Val, and Leu in the molar ratio 1:1:1:4, while the lipid part was composed of C₁₃–C₁₅ β-hydroxy fatty acids. As the sequence of fraction 1 was determined to be N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C, the same as surfactin, they were proposed to be surfactin isoforms. Fraction 4 (C₁₅ surfactin) exhibited a good surface activity of 26.8 mN/m with CMC of 9 × 10⁻⁶M. Surfactin is a powerful biosurfactant possessing biological activities. As far as we know, Br. brevis is a new surfactin-producing species.

Keywords: Biosurfactant; Lipopeptide; Structure; Brevibacillus brevis ; Surfactin

d-glucose Enhanced 5-Aminolevulinic Acid Production in Recombinant Escherichia coli Culture by Xiao Xia Liu; Lan Wang; Yu Jie Wang; Li Ling Cai (pp. 822-830).

In this study, we introduced a new strategy, feeding d-glucose, to overproduce extracellular 5-aminolevulinic acid (ALA) in the recombinant Escherichia coli. We investigated that the d-glucose concentration is dependent on extracellular ALA production. The results indicated that increasing d-glucose concentration in bacteria culture enhanced final cell density and ALA yield and simultaneously decreased the activities of ALA synthase (ALAS) and ALA dehydratase (ALAD); then, the inhibitory effect of d-glucose on ALAS activity was relieved with the metabolism of d-glucose. when 4.0 g/L d-glucose was added at late exponential phase; 1.46 g/L ALA was achieved in shaking culture, which is 47% or 109% higher than the ALA yields with 30 mM levulinic acid of ALAD inhibitor or no inhibitor. In jar fermenter, final extracellular ALA concentration reached 3.1 g/L by feeding with d-glucose.

Keywords: 5-Aminolevulinic acid (ALA); Recombinant Escherichia coli (recombinant E. coli); d-glucose; ALA dehydratase (ALAD); ALA synthase (ALAS)

Structural Analysis and Inhibitory Kinetics of Brain Type Creatine Kinase by Sodium Dodecyl Sulfate by Zhi-Rong Lü; Sang Ho Oh; Shan-Shan Zhou; He-Chang Zou; Daeui Park; Seong Jin Park; Hong-Wei Zhou; Jong Bhak; Yong-Doo Park; Fei Zou (pp. 831-842).

The studies regarding the effect of sodium dodecyl sulfate (SDS) on enzyme activities and structures can provide a valuable insight into public health. We have predicted the 3D structure of the brain creatine kinase (CK-BB) with a high resolution and simulated the docking between CK-BB and SDS. The predicted structure had a root mean square deviation of 0.51 Å. The docking between CK-BB and SDS was successful with significant scores (−4.67 kcal/mol, AutoDock4 and −48.32 kcal/mol, DOCK6). We have also investigated the inactivation by using SDS to study CK-BB’s folding behaviors. The two-phase rate constants as a first-order reaction were measured during inactivation. SDS strongly inhibited the CK-BB activity in a noncompetitive inhibition manner (K _i = 1.22 mM). The tertiary structural change was induced by SDS binding with the exposure of hydrophobic surface. The methyl-β-cyclodextrin was used to strip SDS from the enzyme molecule to reactivate. The changes of thermodynamic parameters for the SDS ligand binding such as enthalpy, Gibbs free energy, and entropy were obtained as −13 ± 7.0 MJ/mol, 8.39 kJ/mol, and −42.754 kJ/(K mol), respectively. Our study provides important structural information for CK-BB and its interaction with SDS with an insight on its folding and inhibition kinetics.

Keywords: Brain creatine kinase; Sodium dodecyl sulfate; Structure prediction; Folding; Thermodynamics

Colloidal Nanogold-Based Immunochromatographic Strip Test for the Detection of Digoxin Toxicity by Kobra Omidfar; Solmaz Kia; Soheila Kashanian; Malihe Paknejad; Afsaneh Besharatie; Susan Kashanian; Bagher Larijani (pp. 843-855).

Digoxin is widely used as a cardiac glycoside drug in the treatment of various heart conditions. Because it is a toxic drug, it should be regularly monitored in the serum of patients under treatment. In this study, colloidal nanogold is synthesized and the preparation of nanogold-labeled monoclonal antibody probe to digoxin is described under optimal conditions. In addition, an immunochromatographic (IC) method for digoxin analysis employing nanogold-labeled probe is developed. With this technique, it requires only 5 min to complete the quantitative detection of digoxin. The detection time is decreased 20–30 times in comparison to radioimmunoassay (RIA). The sensitivity to digoxin was about 2 ng/ml by naked eye, which is within the therapeutic and toxic ranges of digoxin. The results of serum samples obtained by IC strip were in agreement with those obtained by RIA. The IC strip was sufficiently sensitive and accurate to be used for the rapid detection of digoxin in serum samples.

Keywords: Digoxin; Immunochromatography; Colloidal nanogold; Monoclonal antibody

Amperometric Phenol Biosensor Based on Horseradish Peroxidase Entrapped PVF and PPy Composite Film Coated GC Electrode by Meral Topcu Sulak; Elif Erhan; Bülent Keskinler (pp. 856-867).

Polyvinylferrocene (PVF) was used as a mediator for the fabrication of a horseradish peroxidase (HRP)-modified electrode to detect phenol derivatives via a composite polymeric matrix of conducting polypyrrole (PPy). Through an electropolymerization process, enzyme HRP was entrapped with PPy in a three-electrode system onto a glassy carbon electrode previously covered with PVF, resulting in a composite polymeric matrix. Steady-state amperometric measurements were performed at −200 mV vs. Ag/AgCl in aqueous phosphate buffer containing NaCl 0.1 M (pH 6.8) in the presence of hydrogen peroxide. The response of the HRP-modified PVF electrode was investigated for various phenol derivatives, which were 4-chlorophenol, phenol, catechol, hydroquinone, 2-aminophenol, pyrogallol, m-cresol, and 4-methoxyphenol. Analytical parameters for the fabricated PVF electrode were obtained from the calibration curves. The highest sensitivity was obtained from the calibration of 4-chlorophenol as 29.91 nA/μM. The lowest detection limit was found to be 0.22 μM (S/N = 3) for catechol, and the highest detection limit was found to be 0.79 μM (S/N = 3) for 4-methoxyphenol among the tested derivatives. The biosensor can reach 95% of steady-state current in about 5 min. The electrode is stable for 2 months at 4 °C.

Keywords: Phenol biosensor; Conducting polymer; HRP; PVF; Polypyrrole

Improved Production of (R)-1-phenyl-1,2-ethanediol by a Codon-optimized R-specific Carbonyl Reductase from Candida parapsilosis in Escherichia coli by Rongzhen Zhang; Yan Xu; Yawei Geng; Shanshan Wang; Ying Sun; Rong Xiao (pp. 868-878).

An R-specific carbonyl reductase from Candida parapsilosis (CprCR) catalyzes the transformation of (R)-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone. The gene rcr coding CprCR contains a few codons rarely used by Escherichia coli. In order to improve chiral alcohol production, three codon variants Δ24, aRCR, and mRCR of CprCR were designed through truncation of 4–27 bp disorder sequence at the 5′-terminus or/and adaption of nine rare codons. The effects of codon optimization on enzyme activity, protein production, and biotransformation were studied. Among these three types, the disorder sequence-truncated and rare codon-adapted variant mRCR presents the highest enzyme activity. When compared with CprCR, mRCR showed an increase of 35.6% in the total activity of cell-free extracts. The specific activity of mRCR presented similar increase in the cell-free extract with purified protein, which suggested that the codon optimization caused positive effect on protein productivity of variant enzyme. When microbial cells concentration was 30% (w/v), the molar conversion yield and enantiomeric excess of the mRCR variant reached 86.4% and 93.6%, which were increased 36.5% and 15.8% than those of wild-type at a high substrate concentration of 5 g/L. The work will supply a new method for improving chiral alcohol preparation with codon engineered microorganisms.

Keywords: (R)-1-phenyl-1,2-ethanediol; 2-ethanediol; Carbonyl reductase; Codon optimization; Candida parapsilosis

Characterization of a Neutral and Thermostable Glucoamylase from the Thermophilic Mold Thermomucor indicae-seudaticae: Activity, Stability, and Structural Correlation by Pardeep Kumar; Asimul Islam; Faizan Ahmad; T. Satyanarayana (pp. 879-890).

Glucoamylase from the thermophilic mold Thermomucor indicae-seudaticae was purified by anion exchange and gel filtration chromatographic techniques using a fast protein liquid chromatographic system. The structure and thermal stability of this unique ‘thermostable and neutral glucoamylase’ were analyzed by circular dichroism (CD). T. indicae-seudaticae glucoamylase (TGA) contained typical aromatic amino acid (tryptophan/tyrosine) fingerprints in its tertiary structure. Analysis of the far-UV CD spectrum at pH 7.0 and 25 °C revealed the presence of 45% α-helix, 43% β-sheet, and 12% remaining structures. The α-helix content was highest at pH 7.0, where glucoamylase is optimally active. This observation points towards the possible (α/α)₆ barrel catalytic domain in TGA, as reported in microbial glucoamylases. Thermal denaturation curves of the pure protein at different pH values revealed maximum stability at pH 7.0, where no change in the secondary structure was observed upon heating in the temperature range between 20 °C and 60 °C. The observed midpoint of thermal denaturation (T _m) of glucoamylase at pH 7.0 was 67.1 °C, which decreased on either sides of this pH. Thermostability of TGA enhanced in the presence of starch (0.1%) as no transition curve was obtained in the temperature range between 20 °C and 85 °C. The only product of TGA action on starch was glucose, and it did not exhibit transglycosylation activity even at 40% glucose that can also be considered as an advantage during starch saccharification.

Keywords: Glucoamylase; Thermomucor indicae-seudaticae ; Circular dichroism; Heat denaturation; Transglycosylation

In Vitro Osteoblast Differentiation is Negatively Regulated by Hoxc8 by Yan-Jun Zheng; Hyun Joo Chung; Hyehyun Min; Myengmo Kang; Seong Hwan Kim; Jogeswar Gadi; Myoung Hee Kim (pp. 891-900).

Hoxc8 has multiple roles in normal skeletal development. In this paper, a MC3T3-E1 subclone 4 osteogenic cell differentiation model was used to examine expression of Hoxc8 at multiple stages of osteogenesis. We found that Hoxc8 expression levels do not change in the early stage but increase in the middle stage and decrease in the late stage of osteogenesis. A knockdown of Hoxc8 by small-interfering RNA transfection in C2C12 cells indicated that Hoxc8 is a negative regulator of osteogenesis. Similarly, expression of Hoxc8 in C2C12 cells decreases alkaline phosphatase levels induced by bone morphogenetic protein-2 (BMP-2). The results of this study showed that Hoxc8 is involved in BMP-2-induced osteogenesis, and osteoblast differentiation in vitro is negatively regulated by Hoxc8, suggesting that Hoxc8 regulation is essential for osteoblast differentiation.

Keywords: Hoxc8 ; Osteoblast differentiation; C2C12; MC3T3-E1; BMP signaling

Activation and Stabilization of The Hydroperoxide Lyase Enzymatic Extract from Mint Leaves (Mentha spicata) Using Selected Chemical Additives by Najla B. Akacha; Salwa Karboune; Mohamed Gargouri; Selim Kermasha (pp. 901-911).

The effects of selected lyoprotecting excipients and chemical additives on the specific activity and the thermal stability of the hydroperoxide lyase (HPL) enzymatic extract from mint leaves were investigated. The addition of KCl (5%, w/w) and dextran (2.5%, w/w) to the enzymatic extract, prior to lyophilization, increased the HPL specific activity by 2.0- and 1.2-fold, respectively, compared to the control lyophilized extract. From half-life time (t _1/2), it can be seen that KCl has enhanced the HPL stability by 1.3- to 2.3-fold, during long-period storage at −20 °C and 4 °C. Among the selected additives used throughout this study, glycine appeared to be the most effective one. In addition to the activation effect conferred by glycine, it also enhanced the HPL thermal stability. In contrast, polyhydroxyl-containing additives were not effective for stabilizing the HPL enzymatic extract. On the other hand, there was no signification increase in HPL activity and its thermal stability with the presence of Triton X-100. The results also showed that in the presence of glycine (10%), the catalytic efficiency of HPL was increased by 2.45-fold than that without additive.

Keywords: Hydroperoxide lyase; Chemical additives; Lyoprotectants; Stabilization; Kinetics

Biogas Plasticization Coupled Anaerobic Digestion: Continuous Flow Anaerobic Pump Test Results by Keith A. Schimel; David R. Boone (pp. 912-926).

In this investigation, the Anaerobic Pump (®TAP) and a conventional continuous flow stirred tank reactor (CFSTR) were tested side by side to compare performance. TAP integrates anaerobic digestion (AD) with biogas plasticization–disruption cycle to improve mass conversion to methane. Both prototypes were fed a “real world” 50:50 mixture of waste-activated sludge (WAS) and primary sludge and operated at room temperature (20°C). The quantitative results from three steady states show TAP peaked at 97% conversion of the particulate COD in a system hydraulic residence time (HRT) of only 6 days. It achieved a methane production of 0.32 STP cubic meter CH₄ per kilogram COD fed and specific methane yield of 0.78 m³ CH₄ per cubic meter per day. This was more than three times the CFSTR specific methane yield (0.22 m³ CH₄ per cubic meter per day) and more than double the CFSTR methane production (0.15 m³ CH₄ per kilogram COD fed). A comparative kinetics analysis showed the TAP peak substrate COD removal rate (R _o) was 2.24 kg COD per cubic meter per day, more than three times the CFSTR substrate removal rate of 0.67 kg COD per cubic meter per day. The three important factors contributing to the superior TAP performance were (1) effective solids capture (96%) with (2) mass recycle and (3) stage II plasticization–disruption during active AD. The Anaerobic Pump (®TAP) is a high rate, high efficiency–low temperature microbial energy engine that could be used to improve renewable energy yields from classic AD waste substrates like refuse-derived fuels, treatment plant sludges, food wastes, livestock residues, green wastes and crop residuals.

Keywords: Advanced anaerobic bioconversion; Biogas; Biomass; Continuous flow; Pressure cycle; Pressure swing; Renewable energy; Waste

Development of a Sensitive Deaminated Single-Strand Conformation Polymorphism (DSSCP) by Sadeq Vallian; Isar Nassiri (pp. 927-931).

The single-strand conformation polymorphism (SSCP), accompanied by sequencing, is a useful methods for identifying mutations in a DNA fragment. In this study, we have developed a modified SSCP with the aid of sodium bisulfite treatment. The corresponding PCR products for exon 3 of Hb gene were sequenced and samples with homozygote and heterozygote single nucleotide substitutions were identified. The PCR products were treated with sodium bisulfite, which deaminates all the cytosine residues. The reaction mixture was then analyzed on non-denaturing polyacrylamide gels. The modified method, which is called deaminated SSCP (DSSCP), was applied successfully in analysis of mutations in the beta-globin gene at positions relevant to codon 6. DSSCP is a very effective and reproducible method providing clear results that are easy to interpret without the involvement of radioactivity.

Keywords: Deaminated SSCP; DNA polymorphisms; SSCP; PCR

Proteomics Approach to Identify Unique Xylem Sap Proteins in Pierce’s Disease-Tolerant Vitis Species by Sheikh M. Basha; Hifza Mazhar; Hemanth K. N. Vasanthaiah (pp. 932-944).

Pierce’s disease (PD) is a destructive bacterial disease of grapes caused by Xylella fastidiosa which is xylem-confined. The tolerance level to this disease varies among Vitis species. Our research was aimed at identifying unique xylem sap proteins present in PD-tolerant Vitis species. The results showed wide variation in the xylem sap protein composition, where a set of polypeptides with pI between 4.5 and 4.7 and M _r of 31 kDa were present in abundant amount in muscadine (Vitis rotundifolia, PD-tolerant), in reduced levels in Florida hybrid bunch (Vitis spp., PD-tolerant) and absent in bunch grapes (Vitis vinifera, PD-susceptible). Liquid chromatography/mass spectrometry/mass spectrometry analysis of these proteins revealed their similarity to β-1, 3-glucanase, peroxidase, and a subunit of oxygen-evolving enhancer protein 1, which are known to play role in defense and oxygen generation. In addition, the amount of free amino acids and soluble sugars was found to be significantly lower in xylem sap of muscadine genotypes compared to V. vinifera genotypes, indicating that the higher nutritional value of bunch grape sap may be more suitable for Xylella growth. These data suggest that the presence of these unique proteins in xylem sap is vital for PD tolerance in muscadine and Florida hybrid bunch grapes.

Keywords: Grape; Pierce’s disease; Differential protein expression; Xylem sap

Phytochelatins: Peptides Involved in Heavy Metal Detoxification by Rama Pal; J. P. N. Rai (pp. 945-963).

Phytochelatins (PCs) are enzymatically synthesized peptides known to involve in heavy metal detoxification and accumulation, which have been measured in plants grown at high heavy metal concentrations, but few studies have examined the response of plants even at lower environmentally relevant metal concentrations. Recently, genes encoding the enzyme PC synthase have been identified in plants and other species enabling molecular biological studies to untangle the mechanisms underlying PC synthesis and its regulation. The present paper embodies review on recent advances in structure of PCs, their biosynthetic regulation, roles in heavy metal detoxification and/or accumulation, and PC synthase gene expression for better understanding of mechanism involved and to improve phytoremediation efficiency of plants for wider application.

Keywords: Heavy metals; Phytochelatins; Vacuolar sequestration; Sulfide ions

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.160, #3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.160, #3)