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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.169, #6)

Crystalline Xylitol Production by a Novel Yeast, Pichia caribbica (HQ222812), and Its Application for Quorum Sensing Inhibition in Gram-Negative Marker Strain Chromobacterium violaceum CV026 by Ruchira Mukherji; Kasturi Joshi-Navare; Asmita Prabhune (pp. 1753-1763).

Xylitol, a sugar alcohol, is fast gaining ground over other artificial sugar substitutes owing to its advantageous properties. Xylitol is a safer alternative for diabetics because of insulin-independent metabolism. It has beneficial properties suitable to form an important part of odontological formulations. Conventional commercial production of xylitol involves harsh chemical method operating at high temperature and pressure. Thus, microbial production of xylitol is preferred over chemical method, and yeasts have been extensively exploited for this purpose. In the present manuscript, quantitative production of xylitol from d-xylose with the yield of 0.852 gm/gm and volumetric productivity of 1.83 gm/l/h in crystalline form, using novel yeast Pichia caribbica is reported. Also, a mild, safe procedure for product extraction is described. The ability of xylitol to act as a quorum sensing antagonist in gram-negative marker strain Chromobacterium violaceum CV026 has been demonstrated for the first time.

Keywords: Pichia caribbica ; Xylitol; Crystallization; Quorum sensing antagonist; CV026

Characterization of Novel EGs Reconstructed from Bacillus subtilis Endoglucanase by Tang Zi-Zhong; Wu Zhen-Fang; Chen Hui; Lai Xin; Han Xue-yi; Wu Qi (pp. 1764-1773).

Bacterial cellulases have taken on satisfactory application performance and economic value in detergent industry. Neutral endoglucanase (EG1) gene was cloned from Bacillus subtilis and expressed Pichia pastoris in our previous study. Redesigned endoglucanases enhanced cellulase domain, added and deleted carbohydrate-binding module (CBM), named EG2, EG3, and EG4, respectively, were constructed in this study. The redesigned EG genes were expressed in P. pastoris, and their characters were also discussed. The optimal temperature and pH value of the all EGs was 65 °C and 6.0, respectively, where their enzymatic activities in P. pastoris cultivation supernatant reached 867, 651, 966, and 881 U/mL. EG2 showed 24.9 % enzymatic activity loss compared to natural endoglucanase. EG4 showed specific activity 30 % loss and thermostability decrease compared to EG1, which suggested CBM played an important role in improving the catalytic power and heat stability of cellulase family which attached. The specific activity of EG2 and EG3 showed similar to EG1, which suggested neither enhancement of CD nor CBM to endoglucanase can improve its catalytic power, which might rest with its intact topologic structure.

Keywords: Bacillus subtilis ; Neutral endoglucanase; Cellulase domain; Carbohydrate-binding module; Reconstruction; Pichia pastoris

RNA Interference—A Silent but an Efficient Therapeutic Tool by Puthucode Venkatakrishnan Ramachandran; Savarimuthu Ignacimuthu (pp. 1774-1789).

RNA interference (RNAi) is an evolutionary conserved gene regulation pathway that has emerged as an important discovery in the field of molecular biology. One of the important advantages of RNAi in therapy is that it brings about efficient downregulation of gene expression by targeting complementary transcripts in comparison with other antisense-based techniques. RNAi can be can be achieved by introducing chemically synthesized small interfering RNAs (siRNAs) into a cell system. A more stable knockdown effect can be brought about by the use of plasmid or viral vectors encoding the siRNA. RNAi has been used in reverse genetics to understand the function of specific genes and also as a therapeutic tool in treating human diseases. This review provides a brief insight into the therapeutic applications of RNAi against debilitating diseases.

Keywords: RNAi; siRNA; shRNA; miRNA; Cancer; Ocular diseases; Cardiovascular diseases; Diabetes mellitus; Viral diseases; Gene silencing; Therapeutic tool

Structural Insights on Mycobacterium tuberculosis Thiazole Synthase—A Molecular Dynamics/Docking Approach by K. Rohini; P. S. Srikumar (pp. 1790-1798).

Tuberculosis (TB), an epidemic disease, affects the world with death rate of two million people every year. The bacterium Mycobacterium tuberculosis was found to be a more potent and disease-prolonged bacterium among the world due to multi-drug resistance. Emergence of new drug targets is needed to overcome the bacterial resistance that leads to control epidemic tuberculosis. The pathway thiamine biosynthesis was targeting M. tuberculosis due to its role in intracellular growth of the bacterium. The screening of enzymes involved in thiamin biosynthesis showed novel target thiazole synthase (ThiG) involved in catalysis of rearrangement of 1-deoxy-d-xylulose 5-phosphate (DXP) to produce the thiazole phosphate moiety of thiamine. We carried out homology modeling for ThiG to understand the structure–function relationship, and the model was refined with MD simulations. The results showed that the model predicted with (α + β)₈-fold of synthase family proteins. Molecular docking of ThiG model with substrate DXP showed binding mode and key residues ARG46, ASN69, THR41, and LYS96 involved in the catalysis. First-line anti-tuberculosis drugs were docked with ThiG to identify the inhibition. The report showed the anti-tuberculosis drugs interact well with ThiG which may lead to block thiamin biosynthesis pathway.

Keywords: Tuberculosis; Mycobacterium tuberculosis ; Homology modeling; MD simulations; DXP

Plant Regeneration Through Callus Organogenesis and True-to-Type Conformity of Plants by RAPD Analysis in Desmodium gangeticum (Linn.) DC. by Meena K. Cheruvathur; Jyothi Abraham; T. Dennis Thomas (pp. 1799-1810).

An efficient plant regeneration protocol was established for an endangered ethnomedicinal plant Desmodium gangeticum (Linn.) DC. Morphogenic calli were produced from 96 % of the cultures comprising the immature leaf explants on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (4.0 mg l⁻¹) in combination with 6-benzylaminopurine (BA; 0.8 mg l⁻¹). For callus regeneration, various concentrations of BA (1.0–5.0 mg l⁻¹) or thidiazuron (TDZ; 1.0–5.0 mg l⁻¹) alone or in combination with indole-3-acetic acid (IAA; 0.2–1.0 mg l⁻¹) were used. Highest response of shoot regeneration was observed on MS medium fortified with TDZ (4.0 mg l⁻¹) and IAA (0.5 mg l⁻¹) combination. Here, 100 % cultures responded with an average number of 22.3 shoots per gram calli. Inclusion of indole-3-butyric acid in half MS medium favored rooting of recovered shoots. Out of 45 rooted plants transferred to soil, 40 survived. Total DNA was extracted from the leaves of the acclimatized plants of D. gangeticum. Analysis of random amplified polymorphic DNA using 13 arbitrary decanucleotide primers showed the genetic homogeneity in all the ten plants regenerated from callus with parental plant, suggesting that shoot regeneration from callus could be used for the true-to-type multiplication of this plant.

Keywords: Callus; Desmodium gangeticum ; Immature leaf; RAPD analysis; Regeneration

Preparation, Characterization, and Bacteriostasis of AgNP-Coated β-CD Grafting Cellulose Beads by Ting Wang; Bin Li; Li Lin (pp. 1811-1821).

A novel functional material of β-cyclodextrin (β-CD) grafting cellulose beads containing immobilized silver nanoparticles (AgNPs) is presented in this paper. The morphology was characterized by scanning electron microscopy, energy-dispersive X-ray, and X-ray photoelectron spectroscopy. Phenolphthalein probe molecule technique was used to detect the activity of the grafting β-CD, and the results demonstrated that the deposition of AgNPs had no influence on its encapsulation ability. Acid resistance of the AgNPs on the bead material was studied by atomic absorption spectrometry. The stability of the AgNPs was enhanced due to the grafting of β-CD. Tube dilution method was applied to study the bacteriostatic effect, and the minimal inhibitory doses of the novel material against Escherichia coli and Staphylococcus aureus were 12.5 and 25 mg, respectively. The minimal bactericidal doses for the two bacteria were 25 and 25 mg, respectively.

Keywords: Cellulose; Beads; β-Cyclodextrin; Silver nanoparticles

Linking Performance with Microbial Community Characteristics in an Anaerobic Baffled Reactor by Qiaoying Ban; Jianzheng Li; Liguo Zhang; Ajay Kumar Jha; Loring Nies (pp. 1822-1836).

The performance and microbial community characteristics of a laboratory scale anaerobic baffled reactor (ABR) with four compartments (C1–C4) treating sugar refinery wastewater were investigated. The COD removal was 94.8 % with a CH₄ yield of 0.21 L g⁻¹ COD_removed at total organic loading rate (OLR) of 5.33 kg COD/m⁻³ day⁻¹. Fermentative bacteria were dominant in C1 and C2, while syntrophic acetogens and methanogens were dominant in C3 and C4. Some acid-tolerant methanogens were enriched in acidogenic phase. The present of the acid-tolerant methanogens could improve the efficiency and stability of the ABR as the most of the methanogens are vulnerable to low pH. In addition, high functional redundancy of the fermentative bacteria implicated that the microbial communities in acidogenic phase were stable functionally and allowed the ABR to balance perturbation. It was also found that syntrophic acetogenesis might be a weakness in the ABR as syntrophic acetogens were poor as compared with fermentative bacteria and methanogens.

Keywords: Anaerobic baffled reactor; Performance; Bacteria; Methanogens; PCR-DGGE; Real-time PCR

Characterization of a Heavy Metal Translocating P-Type ATPase Gene from an Environmental Heavy Metal Resistance Enterobacter sp. Isolate by Chih-Ching Chien; Chia-Hsuan Huang; Yi-Wei Lin (pp. 1837-1846).

Heavy metals are common contaminants found in polluted areas. We have identified a heavy metal translocating P-type ATPase gene (hmtp) via fosmid library and in vitro transposon mutagenesis from an Enterobacter sp. isolate. This gene is believed to participate in the bacterium’s heavy metal resistance traits. The complete gene was identified, cloned, and expressed in a suitable Escherichia coli host cell. E. coli W3110, RW3110 (zntA::Km), GG48 (ΔzitB::Cm zntA::Km), and GG51 (ΔzitB::Cm) were used to study the possible effects of this gene for heavy metal (cadmium and zinc in particular) resistance. Among the E. coli strains tested, RW3110 and GG48 showed more sensitivity to cadmium and zinc compared to the wild-type E. coli W3110 and strain GG51. Therefore, strains RW3110 and GG48 were chosen for the reference hosts for further evaluation of the gene’s effect. The results showed that expression of this heavy metal translocating P-type ATPase gene could increase the ability for zinc and cadmium resistance in the tested microorganisms.

Keywords: Heavy metal resistance; Environmental isolate; Cadmium; Enterobacter sp.; P-type ATPase gene

Recombinant Production of Crab Antimicrobial Protein Scygonadin Expressed as Thioredoxin and SUMO Fusions in Escherichia coli by Yifeng Li (pp. 1847-1857).

Scygonadin is an antimicrobial protein isolated from the mud crab, Scylla serrate. The mature protein comprises 102 amino acids and has a theoretical molecular weight of 11,272 Da. The protein’s specific expression pattern strongly suggests that it plays a role in reproductive immunity. In this study, I developed a protocol for producing recombinant scygonadin in Escherichia coli. The target protein was expressed as both thioredoxin and SUMO fusions, and released by TEV and SUMO protease-mediated cleavages, respectively. In either case, the liberated scygonadin was separated from its carrier using a HisTrap HP column. From thioredoxin and SUMO fusion constructs, 32.7 and 29.2 mg target protein per liter of culture was obtained, respectively. The described protocol provides an effective means for producing scygonadin in relatively large quantities, which facilities its further characterization.

Keywords: Antimicrobial protein; Fusion expression; Maltose-binding protein; Scygonadin; SUMO; SUMO protease; TEV protease; Thioredoxin

Secretory Phospholipase A₂ in Dromedary Tears: a Host Defense Against Staphylococci and Other Gram-Positive Bacteria by Abir Ben Bacha; Islem Abid (pp. 1858-1869).

The best known physiologic function of secreted phospholipase A₂ (sPLA₂) group IIA (sPLA₂-IIA) is defense against bacterial infection through hydrolytic degradation of bacterial membrane phospholipids. In fact, sPLA₂-IIA effectively kills Gram-positive bacteria and to a lesser extent Gram-negative bacteria and is considered a major component of the eye’s innate immune defense system. The antibacterial properties of sPLA₂ have been demonstrated in rabbit and human tears. In this report, we have analyzed the bactericidal activity of dromedary tears and the subsequently purified sPLA₂ on several Gram-positive bacteria. Our results showed that the sPLA₂ displays a potent bactericidal activity against all the tested bacteria particularly against the Staphylococcus strains when tested in the ionic environment of tears. There is a synergic action of the sPLA₂ with lysozyme when added to the bacteria culture prior to sPLA₂. Interestingly, lysozyme purified from dromedary tears showed a significant bactericidal activity against Listeria monocytogene and Staphylococcus epidermidis, whereas the one purified from human tears displayed no activity against these two strains. We have also demonstrated that Ca²⁺ is crucial for the activity of dromedary tear sPLA₂ and to a less extent Mg²⁺ ions. Given the presence of sPLA₂ in tears and intestinal secretions, this enzyme may play a substantial role in innate mucosal and systemic bactericidal defenses against Gram-positive bacteria.

Keywords: Dromedary tears; sPLA₂-IIA; Lysozyme; Purification; Bactericidal effect

Recombinant Expression and Characterization of an Organic-Solvent-Tolerant α-Amylase from Exiguobacterium sp. DAU5 by Jie Chang; Yong-Suk Lee; Shu-Jun Fang; In-Hye Park; Yong-Lark Choi (pp. 1870-1883).

The enzyme from halophilic microorganisms often has unique properties such as organic-solvent-tolerance. In this study, a novel organic-solvent-tolerant α-amylase gene was cloned from the mild halophile Exiguobacterium sp. DAU5. The open reading frame (ORF) of the enzyme consisted of 1,545 bp and encoded 514 amino acids, the primary sequence revealed that it belongs to the glycoside hydrolase (GH) family 13. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed an AmyH monomer of 57 kDa. The enzyme exhibited maximal activity at 40 °C in pH 8.5 glycine–NaOH buffer, and the activity was strongly inhibited by Zn²⁺, Cu²⁺, and Fe²⁺. The α-amylase AmyH exhibited high hydrolysis activity toward soluble starch, and the major hydrolysis products were maltose, maltotriose, and maltopentaose; the AmyH could not efficiently hydrolyze oligosaccharides smaller than maltoheptaose, nor could it act on the β-1,4 or α-1,6 glucosidic bonds in xylan or pullulan, respectively. In addition, the α-amylase exhibited better tolerance to organic solvents, as it was stable in the presence of dimethylsulfoxide (DMSO), methanol, ethanol, and acetone. Base on all of these results, the enzyme could be useful for practical application in the bakery industry and in biotechnological processes that occur in the presence of organic solvents.

Keywords: Purification; Characterization; α-Amylase; Organic solvent tolerant; Exiguobacterium

Prediction of Optimal pH in Hydrolytic Reaction of Beta-glucosidase by Shaomin Yan; Guang Wu (pp. 1884-1894).

This is the continuation of our studies to use very basic information on enzyme to predict optimal reaction parameters in enzymatic reactions because the gap between available enzyme sequences and their available reaction parameters is widening. In this study, 23 features selected from 540 plus features of individual amino acid as well as a feature combined whole protein information were screened as independents in a 20-1 feedforward backpropagation neural network for predicting optimal pH in beta-glucosidase’s hydrolytic reaction because this enzyme drew attention recently due to its role in biofuel industry. The results show that 11 features can be used as independents for the prediction, while the feature of amino acid distribution probability works better than the rest independents for the prediction. Our study paves a way to predict the optimal reaction parameters of enzymes based on the amino acid features of enzyme sequences.

Keywords: Amino acid feature; Beta-glucosidase; Distribution probability; Optimal pH; Prediction

Optimized Fed-Batch Fermentation of Scheffersomyces stipitis for Efficient Production of Ethanol from Hexoses and Pentoses by Pornkamol Unrean; Nhung H.A. Nguyen (pp. 1895-1909).

Scheffersomyces stipitis was cultivated in an optimized, controlled fed-batch fermentation for production of ethanol from glucose–xylose mixture. Effect of feed medium composition was investigated on sugar utilization and ethanol production. Studying influence of specific cell growth rate on ethanol fermentation performance showed the carbon flow towards ethanol synthesis decreased with increasing cell growth rate. The optimum specific growth rate to achieve efficient ethanol production performance from a glucose-xylose mixture existed at 0.1 h⁻¹. With these optimized feed medium and cell growth rate, a kinetic model has been utilized to avoid overflow metabolism as well as to ensure a balanced feeding of nutrient substrate in fed-batch system. Fed-batch culture with feeding profile designed based on the model resulted in high titer, yield, and productivity of ethanol compared with batch cultures. The maximal ethanol concentration was 40.7 g/L. The yield and productivity of ethanol production in the optimized fed-batch culture was 1.3 and 2 times higher than those in batch culture. Thus, higher efficiency ethanol production was achieved in this study through fed-batch process optimization. This strategy may contribute to an improvement of ethanol fermentation from lignocellulosic biomass by S. stipitis on the industrial scale.

Keywords: Ethanol fermentation; Scheffersomyces stipitis ; Fed-batch; Kinetic model; Feed medium composition; Controlled specific growth rate

Heterologous Expression and Characterization of a Glycoside Hydrolase Family 45 endo-β-1,4-Glucanase from a Symbiotic Protist of the Lower Termite, Reticulitermes speratus by Masato Otagiri; Crisanto M. Lopez; Katsuhiko Kitamoto; Manabu Arioka; Toshiaki Kudo; Shigeharu Moriya (pp. 1910-1918).

The termite symbiotic system is one of the efficient lignocellulose degradation systems. We tried to express and characterize a novel cellulolytic enzyme from this system. Here, we report the isolation of an endo-β-1,4-glucanase gene homolog of glycoside hydrolase family 45 from a symbiotic protistan community of Reticulitermes speratus. Heterologous expression of this gene was performed using the expression system of Aspergillus oryzae. Analysis of enzymatic properties revealed 786 μmol/min/mg protein in specific activity, a V _max of 833.0 units/mg protein, and a K _m value of 2.58 mg/ml with carboxymethyl cellulose as the substrate. Thin-layer chromatography analysis showed that RsSymEG2 produces cellobiose from cellodextrins larger than cellohexaose. This enzyme showed high specific activity like other endo-β-1,4-glucanases from the symbiotic system of termites. It means that the termite symbiotic system is a good resource for highly active endo-β-1,4-glucanases.

Keywords: Cellulase; Biomass; Termite; Protist

Endopolysaccharides from Ganoderma resinaceum, Phlebia rufa, and Trametes versicolor Affect Differently the Proliferation Rate of HepG2 Cells by Amélia M. Silva; Andreia Miranda; Elisabete Fernandes; Susana Santos; Irene Fraga; Dario L. Santos; Albino A. Dias; Rui M. Bezerra (pp. 1919-1926).

Fungi have been used for medicinal purposes for long time by Asian countries, being a putative source of powerful new phytopharmaceuticals such as polysaccharides. The aim of this study was to extract endopolysaccharides (IPS) from Ganoderma resinaceum, Phlebia rufa, and Trametes versicolor, grown under submerged culture, to compare crude IPS production, total carbohydrate, and protein yield, and to study the effect of these IPS on HepG2 cells proliferation rate. Total biomass produced by G. resinaceum, P. rufa, and T. versicolor was (in gram per liter) 3.32 ± 0.80, 5.42 ± 0.58, and 4.2 ± 1.29 and the IPS yield (as the biomass percent) was 9.9 ± 0.05, 29.0 ± 6.3, and 9.1 ± 3.1 %, respectively. Characterization of IPS has shown different proportion between total sugar and protein being, on average 6.04, 10.74, and 22.62, for G. resinaceum, T. versicolor, and P. rufa, respectively. The IPS effect, at 50, 100, and 200 μg mL⁻¹ on HepG2 cell growth and viability was negligible for G. resinaceum and P. rufa but, in the case of T. versicolor, 200 μg mL⁻¹ of IPS evoked 40 % reduction on cell growth. The results suggest that the intracellular polysaccharides from T. versicolor are a potential source for bioactive molecules with anti-proliferative properties.

Keywords: Ganoderma resinaceum ; Phlebia rufa ; Trametes versicolor ; Intracelular polysaccharides; Phytopharmaceuticals; Anti-proliferative agents

A New Trend on Biosensor for Neurotransmitter Choline/Acetylcholine—an Overview by Anish Khan; Aftab Aslam Parwaz Khan; Abdullah M. Asiri; Malik Abdul Rub; Naved Azum; Mohammed M. Rahman; Sher Bahadar Khan; Sulaiman Ab Ghani (pp. 1927-1939).

Technology always has been an indispensible part in the development of biosensors. The performance of biosensors is being tremendously improved using new materials as transducer as well as binding material in their construction. The use of new materials allowed innovation on transduction technology in biosensor preparations. Because of the submicron dimensions of these sensors, simple and rapid analyses in vitro as well as in vivo are now possible. Portable instruments capable of analysing multiple components are becoming available, too. Sensors that provide excellent temporal and spatial resolution for in vivo monitoring such as for measurement of neurotransmitters have become prominent. The interest to improve the stability, sensitivity and selectivity of the sensors is paramount. This study tries to give an overview of the present status of the material-based biosensor design and new generation of choline/acetylcholine neurotransmitter biosensors.

Keywords: Neurotransmitter; Biosensor; Acetylcholine; Choline; Amperometry

Expression and Large-Scale Production of Human Tissue Plasminogen Activator (t-PA) in Transgenic Tobacco Plants Using Different Signal Peptides by Hojjat Ghasemi Goojani; Mokhtar Jalali Javaran; Jaber Nasiri; Esmaeel Ghasemi Goojani; Houshang Alizadeh (pp. 1940-1951).

An attempt was made to assess the expression level and targeting of a human protein entitled recombinant tissue plasminogen activator (rt-PA) through accumulation in three cellular compartments including the endoplasmic reticulum and cytosolic and apoplastic spaces in transgenic tobacco plants. In this context, three chimeric constructs pBI-SP-tPA, pBI-tPA-KDEL, and pBI-Ext-tPA were employed and transferred into the tobacco plants through a popular transformation-based system called Agrobacterium tumefaciens. As an initial screening system, the incorporation of the rt-PA gene in the genomic DNA of tobacco transgenic plants and the possible existence of the rt-PA-specific transcript in the total RNAs of transgenic plant leaves were confirmed via PCR and reverse transcription (RT)-PCR, respectively. Southern blot analysis, in addition, was used to determine the copy number of the corresponding gene (i.e., t-PA) transformed into the each transgenic plant; one or more copies were detected regarding transformants derived from all three abovementioned constructs. According to the enzyme-linked immunosorbent assay, the mean values of t-PA expression were calculated as 0.50, 0.68, and 0.69 μg/mg of the total soluble protein when a collection containing 30 transgenic plants transformed with pBI-SP-tPA, pBI-tPA-KDEL, and pBI-Ext-tPA was taken into account, respectively. The zymography assay was lastly performed and concluded the expression of the properly folded rt-PA in this expression system. Our results, altogether, revealed that tobacco plants could be utilized as a bioreactor system for the large-scale production of enzymatically active t-PA and presumably other therapeutic recombinant proteins in large quantities.

Keywords: Agrobacterium tumefaciens ; Bioreactor system; Transgenic tobacco; Human tissue plasminogen activator

Biodiesel Residual Glycerol Metabolism by Klebsiella pneumoniae: Pool of Metabolites Under Anaerobiosis and Oxygen Limitation as a Function of Feeding Rates by Daniele Misturini Rossi; Elisangela Aquino de Souza; Marco Antônio Záchia Ayub (pp. 1952-1964).

The metabolism of residual glycerol from biodiesel synthesis by Klebsiella pneumoniae BLh-1 was investigated in this study. Batch and fed-batch cultivations were performed in bioreactors under anaerobic and oxygen limitation conditions. Results of batch cultivations showed that the main product was 1,3-propanediol (1,3-PD) in both conditions, although the higher yields and productivities (0.46 mol mol⁻¹ glycerol and 1.22 g L⁻¹ h⁻¹, respectively) were obtained under anaerobic condition. Large amounts of ethanol were also produced under batch anaerobic condition, peaking at 12.30 g L⁻¹. Batch cultivations under oxygen limitation were characterized by faster growth kinetics, with higher biomass production but lower conversions of glycerol into 1,3-PD, with yields and productivities of 0.33 mol mol⁻¹ glycerol and 0.99 g L⁻¹ h⁻¹, respectively. The fed-batch cultivations were carried out in order to investigate the effects of feeding of raw glycerol on cells. Fed-batch under anaerobiosis showed that 1,3-PD and ethanol concentrations increased with the feeding rate, with maximal productions of 26.12 and 19.2 g L⁻¹, respectively. The oxygen limitation conditions diverted the bacterium metabolism to an elevated lactic acid formation, reaching 59 g L⁻¹ in higher feeding rates of glycerol, but lowering the production of ethanol.

Keywords: Biodiesel residual glycerol; Klebsiella pneumoniae ; 1,3-Propanediol; Bioethanol; Bioreactor cultivations

Overproduction of Polygalacturonase by Penicillium griseoroseum Recombinant Strains and Functional Analysis by Targeted Disruption of the pgg2 Gene by Janaina Aparecida Teixeira; João Batista Ribeiro; Daniel Bonoto Gonçalves; Marisa Vieira de Queiroz; Elza Fernandes de Araújo (pp. 1965-1977).

Inactivation of the pgg2 gene, a polygalacturonase-encoding gene from Penicillium griseoroseum, reduced the total activity of polygalacturonase (PG) by 90 % in wild-type P. griseoroseum, which indicates that the pgg2 gene is the major gene responsible for PG production in this species. To increase PG production, the coding region of the pgg2 gene was cloned under the control of the glyceraldehyde 3-phosphate dehydrogenase (gpd) promoter and the terminator region of the tryptophan synthase (trpC) gene from Aspergillus nidulans (pAN52pgg2 vector). This vector was then used to transform P. griseoroseum. The transformed strains were characterized according to PG production using glucose, sucrose, or sugar cane juice as the carbon sources. The recombinant P. griseoroseum T146 strain contained an additional copy of the pgg2 gene, which resulted in a 12-fold increase in PG activity when compared with that detected in the supernatant of the control PG63 strain. The proteins secreted by the recombinant strain T146 showed a strong band at 38 kDa, which corresponds to the molecular weight of PG of the P. griseoroseum. The results demonstrate the significant biotechnological potential of recombinant P. griseoroseum T146 for use in PG production.

Keywords: Polygalacturonase; Penicillium griseoroseum ; Gene inactivation; gpd promoter; Overproduction

Heterotrophic Nitrifying and Oxygen Tolerant Denitrifying Bacteria from Greenwater System of Coastal Aquaculture by Kathiravan Velusamy; Kishore Kumar Krishnani (pp. 1978-1992).

In this work, herbivorous fish Mugil cephalus has been cultured to secrete protein rich green slime, which helps nitrifying and oxygen tolerant denitrifying bacteria to grow and colonize. Four strains representing Alcaligenaceae family have been isolated from greenwater system and characterized using biochemical test, fatty acid methyl ester (GC-FAME) analysis, 16S rRNA and functional gene approaches. They were tested for an ability to nitrify ammonia and nitrite aerobically. Two strains showed notable nitrification activity, when grown in a mineral salts medium containing ammonium sulfate and potassium nitrite. Functional gene analysis confirmed the presence of nitrous oxide reductase (nosZ) gene showing that they have an oxygen-tolerant denitrification system. It has been proposed that Alcaligenes faecalis strains heterotrophically nitrify ammonia into nitrite via formation of hydroxyl amine, which is oxidized to nitrous oxide using oxygen or nitrite as electron acceptor. These results provide a possible advantage of having nitrification and denitrification capabilities in the same organism, which plays an important role in biological wastewater system.

Keywords: Heterotrophic nitrification; Oxygen tolerant denitrification; Greenwater; Coastal aquaculture

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.169, #6)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.169, #6)