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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.168, #7)

Elicitation of Andrographolide in the Suspension Cultures of Andrographis paniculata by Suryakala Gandi; Kiranmayee Rao; Bhuvaneswari Chodisetti; Archana Giri (pp. 1729-1738).

Andrographis paniculata belonging to the family Acanthaceae produces a group of diterpene lactones, one of which is the pharmaceutically important—andrographolide. It is known to possess various important biological properties like anticancer, anti-HIV, anti-inflammatory, etc. This is the first report on the production of andrographolide in the cell suspension cultures of Andrographis paniculata by ‘elicitation’. Elicitation was attempted to enhance the andrographolide content in the suspension cultures of Andrographis paniculata and also to ascertain its stimulation under stress conditions or in response to pathogen attack. The maximum andrographolide production was found to be 1.53 mg/g dry cell weight (DCW) at the end of stationary phase during the growth curve. The biotic elicitors (yeast, Escherichia coli, Bacillus subtilis, Agrobacterium rhizogenes 532 and Agrobacterium tumefaciens C 58) were more effective in eliciting the response when compared to the abiotic elicitors (CdCl₂, AgNO₃, CuCl₂ and HgCl₂). Yeast has shown to stimulate maximum accumulation of 13.5 mg/g DCW andrographolide, which was found to be 8.82-fold higher than the untreated cultures.

Keywords: Andrographis paniculata ; Andrographolide; Suspension cultures; Elicitation; Biotic; Abiotic

An Efficient and Reproducible Method for in vitro Clonal Multiplication of Rauvolfia tetraphylla L. and Evaluation of Genetic Stability using DNA-Based Markers by Mohammad Faisal; Abdulrahman A. Alatar; Naseem Ahmad; Mohammad Anis; Ahmad K. Hegazy (pp. 1739-1752).

An efficient protocol is described for the rapid in vitro clonal propagation of an endangered medicinal plant, Rauvolfia tetraphylla L., through high frequency shoot induction from nodal explants collected from young shoots of a field grown plant. Effects of growth regulators [6-benzyladenine (BA), kinetin (Kin) 2iP, or α-naphthalene acetic acid (NAA)], carbohydrates, different medium [Murashige and Skoog (MS), Woody Plant Medium (WPM), Gamborg medium (B5), Linsmier and Skoog medium (LS)], and various pH levels on in vitro morphogenesis were investigated. The highest frequency of shoot regeneration (90 %) and maximum number of shoot (35.4 ± 2.3) per explant were observed on WPM medium supplemented with 7.5 μM BA, 2.5 μM NAA, and 30 g/l sucrose at pH 5.8. Well-developed shoots, 4–5 cm in length, were successfully rooted ex vitro at 90 % by a 30-min pulse treatment with 150 μM IBA prior to their transfer in planting substrates. The survival rate of transplantation reached 90 % when transferred to field condition. Genetic stability of micropropagated plantlets was assessed and compared with mother plant using Random Amplified Polymorphic DNA and Inter Simple Sequence Repeats markers. No variation was observed in DNA fingerprinting patterns among the micropropagated plants, which were similar to that of the donor plant illustrating their genetic uniformity and clonal fidelity. This confirms that clonal propagation of this plant using axillary shoot buds can be used for commercial exploitation of the selected genotype where a high degree of fidelity is an essential prerequisite. The work contributed to a better in vitro regeneration and clonal mass multiplication of R. tetraphylla and to develop a strategy for the germplasm conservation of this endangered medicinal plant.

Keywords: Acclimatization; Conservation; Ex vitro rooting; Micropropagation; Genetic fidelity; RAPD; ISSR

Strain Screening, Fermentation, Separation, and Encapsulation for Production of Nattokinase Functional Food by Xuetuan Wei; Mingfang Luo; Yuchun Xie; Liangrong Yang; Haojian Li; Lin Xu; Huizhou Liu (pp. 1753-1764).

This study presents a novel and integrated preparation technology for nattokinase functional food, including strain screening, fermentation, separation, and encapsulation. To rapidly screen a nattokinase-productive strain, PCR-based screening method was combined with fibrinolytic activity-based method, and a high productive strain, Bacillus subtilis LSSE-22, was isolated from Chinese soybean paste. Reduction of poly-γ-glutamic acid (γ-PGA) concentration may contribute to separation of nattokinase and reduction of late-onset anaphylaxis risk. Chickpeas were confirmed as the favorable substrate for enhancement of nattokinase production and reduction of γ-PGA yield. Using cracked chickpeas, the nattokinase activity reached 356.25 ± 17.18 FU/g (dry weight), which is much higher than previous reports. To further reduce γ-PGA concentration, ethanol fractional extraction and precipitation were applied for separation of nattokinase. By extraction with 50 % and precipitation with 75 % ethanol solution, 4,000.58 ± 192.98 FU/g of nattokinase powders were obtained, and the activity recovery reached 89 ± 1 %, while γ-PGA recovery was reduced to 21 ± 2 %. To improve the nattokinase stability at acidic pH condition, the nattokinase powders were encapsulated, and then coated with methacrylic acid–ethyl acrylate copolymer. After encapsulation, the nattokinase was protected from being denatured under various acid conditions, and pH-responsible controlled release at simulated intestinal fluid was realized.

Keywords: Nattokinase; Screening; Solid-state fermentation; Separation; Enteric coating

Isolation and Characterization of Pseudomonas sp. STM 997 from Soil Sample having Potentiality to Degrade 3,6-Dimethyl-1-keto-1,2,3,4-tetrahydrocarbazole: A Novel Approach by Biswanath Chakraborty; Suchandra Chakraborty; Anjan Kumar Basu; Bhrigu Aditya; T. P. Sinha; Tanima Modak Dhar; Chandan Saha (pp. 1765-1777).

A pure colony of a bacterium from contaminated soil was isolated by exploiting 3,6-dimethyl-1-keto-1,2,3,4–tetrahydrocarbazole, a novel carbazole derivative, having indole moiety as well as 3-methyl functionality both in aromatic and hydro-aromatic moiety, as a sole source of carbon and energy. Taxonomical studies, biochemical analysis, and 16S rDNA sequence analysis indicated that the isolated strain has close similarity with Pseudomonas sp. Thin-layer chromatography followed by HPLC and mass spectroscopic study indicates that the isolated Pseudomonas sp. STM 997 degrades 3,6-dimethyl-1-keto-1,2,3,4–tetrahydrocarbazole, and this strain may be useful in the bioremediation of environments contaminated by the compounds containing carbazole moiety with methyl substituents at various reactive sites. This study also provides an evidence in favor of the suggested biodegradation of 3-methylcarbazole to carbazole in plants.

Keywords: 3,6-Dimethyl-1-keto-1,2,3,4-tetrahydrocarbazole; Biodegradation; 16S rDNA analysis; Pseudomonas sp. STM 997

New Carbosilane Polymers with Interacting Ferrocenes as Support and Bioelectrocatalysts of Oxidases to Develop Versatile and Specific Amperometric Biodevices by M. Pilar García Armada; Almudena Jiménez; José Losada; Beatriz Alonso; Carmen M. Casado (pp. 1778-1791).

In this work, the bioelectrocatalytical properties and kinetic characteristics of new oxidase amperometric biosensors based on two different ferrocene functionalized carbosilane polymers, polydiallylmethylsilane (PDAMS) and polymethyldiundecenylsilane (PMDUS) are described. In the development of these biodevices, glucose oxidase has been used as example of oxidase enzyme, and two different immobilization procedures have been studied. The polymer-modified electrodes act as efficient transducers for glucose sensing in anodic and cathodic aerobic conditions and also in anodic anaerobic conditions, and this fact turns them into useful devices for a wide field of applications. PMDUS has shown to be the bioelectrocatalyst with best kinetic and analytical properties in aerobic media while PDAMS was better in anaerobic conditions. The best aerobic biosensor developed displayed a strictly linear range from 0 to 3.0 mM, a detection limit of 7.8 μM and a response time less than 2 s in an ascorbate interference free work potential interval. The apparent Michaelis–Menten constant was calculated to be 1.36 mM according to the Lineweaver–Burk equation.

Keywords: Bioelectrocatalysis; Glucose oxidase; Biosensors; Carbosilane polymers; Interacting ferrocenes; Electron transfer

In Silico Study and Validation of Phosphotransacetylase (PTA) as a Putative Drug Target for Staphylococcus aureus by Homology-Based Modelling and Virtual Screening by V. K. Morya; Varun Dewaker; Eun-Ki Kim (pp. 1792-1805).

Staphylococcus aureus, a Gram-positive bacterium, can cause a range of illnesses from minor skin infections to life-threatening diseases, such as bacteraemia, endocarditis, meningitis, osteomyelitis, pneumonia, toxic shock syndrome and sepsis. Due to the emergence of antibiotic resistance strains, there is a need to develop of new class of antibiotics or drug for this pathogen. The phosphotransacetylase enzyme plays an important role in the acetate metabolism and found to be essential for the survival of the S. aureus. This enzyme was evaluated as a putative drug target for S. aureus by in silico analysis. The 3D structure of the phosphotransacetylase from S. aureus was modelled, using the 1TD9 chain ‘A’ from Bacillus subtilis as a template at the resolution of 2.75 Å. The generated model has been validated by PROCHECK, WHAT IF and SuperPose. The docking was performed by the Molegro virtual docker using the ZINC database generated ligand library. The ligand library was generated within the limitation of the Lipinski rule of five. Based on the dock-score, five molecules have been subjected to ADME/TOX analysis and subjected for pharmacophore model generation. The zinc IDs of the potential inhibitors are ZINC08442078, ZINC8442200, ZINC 8442087 and ZINC 8442184 and found to be pharmacologically active antagonist of phosphotransacetylase. The molecules were evaluated as no-carcinogenic and persistent molecule by START programme.

Keywords: Phosphotransacetylase (PTA); S. aureus ; Homology modelling; Molecular docking; Pharmacophore

Effect of Alkali Pretreatment on the Structural Properties and Enzymatic Hydrolysis of Corn Cob by Padmavati Sahare; Rajkumar Singh; R. Seeta Laxman; Mala Rao (pp. 1806-1819).

An effective alkali pretreatment which affects the structural properties of cellulose (corn cob) has been studied. The pretreatment of corn cob was carried out with different combinations of alkali at varying temperatures. The most effective pretreatment of corn cob was achieved with 1 % alkali at 50 °C in 4 h. The crystallinity index (CrI) and specific surface area (SSA) of untreated corn cob was 39 % and 0.52 m²/g wherein after alkali pretreatment CrI decreased to 15 % and SSA increased to 3.32 m²/g. The fungal organism was identified as Penicillium pinophilum on the basis of ITS sequence. At 5 % substrate concentration using a complete cellulase from Penicillium pinophilum the hydrolysis of untreated corn cob with 5, 10 and 20 FPU/g enzyme loadings were 11 %, 13 % and 16 %, whereas after alkali treatment the hydrolysis increased to 78 %, 90 % and 100 %, respectively. Further hydrolytic potential of commercial cellulases viz. Accellerase™ 1,000, Palkofeel-30 and Palkocel-40 were investigated under similar conditions.

Keywords: Corn cob; Crystallinity index; Specific surface area; Penicillium pinophilum ; Enzymatic hydrolysis

Expression and Characterization of a Novel Enantioselective Lipase from Aspergillus fumigatus by Jiao-Jiao Shangguan; Li-qiang Fan; Xin Ju; Qing-qing Zhu; Fu-Jun Wang; Jian Zhao; Jian-He Xu (pp. 1820-1833).

A 1,080-bp cDNA (CGMCC 2873) encoding of a cold-active lipase of Aspergillus fumigatus (AFL67) was cloned and expressed in Escherichia coli for the first time. The new lipase, AFL67, was one-step purified by 8.30 folds through Ni–NTA affinity chromatography with a recovery of 86.8 %. The specific activity of purified AFL67 was 449 U mg⁻¹ on p-NP hexanoate. AFL67 preferentially hydrolyzed p-nitrophenyl esters of short- and medium-chain fatty acids, with p-nitrophenyl hexanoate the maximum. The optimum temperature and pH was 15 °C and 7.5, respectively. The purified AFL67 was stable at 10–25 °C for 30 min, and in the pH range of 6.0–9.0 for 16 h (at 4 °C). Its activity was increased by 47 and 50 %, in the presence of 10 % (v/v) ethanol and isopropanol, respectively. The new lipase AFL67 highly enantioselectively deacylated (S)-α-acetoxyphenylacetic acid (APA) and o-Cl-APA, m-Cl-APA, and p-Cl-APA to (S)-mandelic acid and its derivates. These features render this cold-active novel lipase AFL67 attractive for biotechnological applications in the field of enantioselective synthesis of chiral mandelic acids, o-acylated mandelic acids, and their derivates and detergent additives.

Keywords: Aspergillus fumigatus ; Recombinant lipase; Characterization; Enantioselective deacylation; α-Acetoxyphenylacetic acid; Enantiopure mandelic acid

Evidence Showing Duplication and Recombination of cel Genes in Tandem from Hyperthermophilic Thermotoga sp. by Min Keun Kim; Tae Ho Kang; Jungho Kim; Hoon Kim; Han Dae Yun (pp. 1834-1848).

This study was conducted to assess the gene duplication and diversification of tandem cellulase genes in thermophilic bacteria. The tandem cellulase genes cel5C and cel5D were cloned from Thermotoga maritima MSB8, and a survey of the thermophilic bacterial genome for tandem cel genes from the databases was carried out. A clone having 2.3 kb fragment from T. maritima MSB8 showed cellulase activity, which had two open reading frames in tandem (cel5C and cel5D). The cel5C gene has 954 bp, which encodes a protein of 317 amino acid residues with a signal peptide of 23 amino acids, and the other gene cel5D consisting of 990 bp encoding a protein of 329 amino acid residues. These two proteins have similarity with the enzymes of glycosyl hydrolase family 5. From the enzyme assay, it was observed that Cel5C was extracellular and Cel5D was intracellular cellulase. Phylogenetic and homology matrix analyses of DNA and protein sequences revealed that family 12 cellulase enzymes Cel12A and Cel12B displayed higher homology (>50 %), but Cel5C and Cel5D enzymes belong to family 5 displayed lower homology (<30 %). In addition, repeated and mirror sequences in tandem genes are supposed to show the existence of gene duplication and recombination.

Keywords: Thermotoga maritima MSB8; el5C; cel5D; Tandem genes; Gene duplication

Characterization of Thermostable Serine Alkaline Protease from an Alkaliphilic Strain Bacillus pumilus MCAS8 and Its Applications by Renganathan Jayakumar; Shanmugam Jayashree; Balumuri Annapurna; Sundaram Seshadri (pp. 1849-1866).

This study describes the characterization and optimization of medium components for an extracellular detergent, surfactant, organic solvent and thermostable serine alkaline protease produced by alkaliphilic Bacillus pumilus MCAS8 strain isolated from Pulicat lake sediments, Tamil Nadu, India. The strain yielded maximum protease (2,214 U/ml) under optimized conditions: carbon source, citric acid—1.5 % (w/w); inducer, soyabean meal—2 % (w/w); pH 11.0; shaking condition 37 °C for 48 h. The enzyme had pH and temperature optima of 9.0 and 60 °C, respectively. The enzyme displayed the molecular mass of 36 kDa in sodium dodecyl sulphate–polyacrylamide gel electrophoresis study and exhibited activity at a wide range of pH (6.0–11.0) and thermostability (20–70 °C). More than 70 % residual activity was observed when the enzyme was incubated with dithiothreitol, ethylenediaminetetraacetic acid, ethylene glycol tetraacetic acid and H₂O₂ for 30 min. The protease activity was also enhanced by divalent cations such as Ba²⁺, Ca²⁺ and Mg²⁺ and was strongly inhibited by Fe²⁺, Zn²⁺, Sr²⁺, Hg²⁺ and urea. The enzyme retained more than 50 % of its initial activity after pre-incubation for 1 h in the presence of 5 % (v/v) organic solvents such as dimethyl sulphoxide and acetone. The protease could hydrolyse various native proteinaceous substrates (1 % w/v) such as bovine serum albumin, casein, skim milk, gelatine, azocasein and haemoglobin. Wash performance analysis of enzyme revealed that it could effectively remove blood stains from the cotton fabric, thus making it suitable to use as an effective detergent additive. The protease enzyme also exhibited promising result in the dehairing of goat skin. The potency of the eco-friendly enzyme without using any chemicals against washing and dehairing showed that the enzyme could be used for various industrial applications.

Keywords: Serine alkaline protease; Dehairing; Pulicat Lake; Soyabean meal; Stain removal

Two-Stage Cultivation of Pseudomonas sp. F12 for the Production of Enzymes Converting dl-2-Amino-Δ²-thiazoline-4-carboxylic Acid to l-Cysteine by Cuili Fan; Zhimin Li; Qin Ye (pp. 1867-1879).

Pseudomonas sp. F12 isolated from soil could transform dl-2-amino-Δ²-thiazoline-4-carboxylic acid (DL-ATC) to l-cysteine. It could grow in minimal medium containing DL-ATC as the sole carbon and nitrogen source, and the apparent activity of l-cysteine synthesis (CS) achieved 122 U/mL in a 5-L bioreactor. Pseudomonas sp. F12 could utilize glucose as carbon source and ammonia as nitrogen source for growth, but no CS activity was formed. To reduce the cost of DL-ATC, the cultivation process was divided into a growth stage on glucose and ammonia and a production stage induced by DL-ATC. The excessive glucose led to the production of byproduct(s) which seriously inhibited cell growth and CS production. Ammonium was accumulated when DL-ATC was consumed, and ammonium did not inhibit CS activity formation until 60 mM. Based on the above features, fed-batch cultivation of the growth stage was developed by supplying glucose restrictively. The volumetric CS activity was enhanced more than two times that obtained under the initial conditions.

Keywords: l-Cysteine; Pseudomonas ; dl-2-Amino-Δ²-thiazoline-4-carboxylic acid; Two-stage cultivation; Glucose; Ammonium

Probiotics—Interactions with Bile Acids and Impact on Cholesterol Metabolism by Nebojša Pavlović; Karmen Stankov; Momir Mikov (pp. 1880-1895).

The use of probiotics, alone or in interaction with bile acids, is a modern strategy in the prevention and treatment of hypercholesterolemia. Numerous mechanisms for hypocholesterolemic effect of probiotics have been hypothesized, based mostly on in vitro evidence. Interaction with bile acids through reaction of deconjugation catalyzed by bile salt hydrolase enzymes (BSH) is considered as the main mechanism of cholesterol-lowering effects of probiotic bacteria, but it has been reported that microbial BSH activity could be potentially detrimental to the human host. There are several approaches for prevention of possible side effects associated with BSH activity, which at the same time increase the viability of probiotics in the intestines and also in food matrices. The aim of our study was to summarize present knowledge of probiotics—bile acids interactions, with special reference to cholesterol-lowering mechanisms of probiotics, and to report novel biotechnological approaches for increasing the pharmacological benefits of probiotics.

Keywords: Probiotics; Bile acids; Cholesterol; Bile salt hydrolase; Interactions

Effects of External Enzymes on the Fermentation of Soybean Hulls to Generate Lipids by Mortierella isabellina by Jianguo Zhang; Bo Hu (pp. 1896-1906).

Hydrolytic enzymes were evaluated on the lipid accumulation via an oleaginous fungal species, Mortierella isabellina, cultivated on sugars released from soybean hulls. The weight loss of soybean hull, fungal growth, and lipid production were tested under different loads of hydrolytic enzymes. M. isabellina could not directly utilize cellulose and adding cellulase and β-glucosidase significantly increased the cell growth and oil accumulation of M. isabellina on soybean hulls. The highest weight loss of soybean hulls was 47.80 % and the lipid production reached 0.14 g from 1 g of soybean hull when 12 U cellulase, 27.2 U β-glucosidase, 2,278.56 U pectinase, and 15 U hemicellulase were added. Fatty acids (76.82 %) accumulated in M. isabellina were C16 and C18, which are suitable for biodiesel production. These results provide a new application for soybean hulls to be applied as the raw material for the production of biodiesel fuel, besides its traditional role as animal feed supplements.

Keywords: Soybean hull; Mortierella isabellina ; Cellulose hydrolysis; Lipid accumulation

Structural and Docking Studies of a Nucleoside Diphosphate Kinase 1 (CsNDPK1) from Tea [Camellia sinensis (L.) O. Kuntze] by Gajjeraman Prabu; Krishnaraj Thirugnanasambantham; Abul Kalam Azad Mandal (pp. 1907-1916).

Nucleoside diphosphate kinase (NDPK, EC 2.7.4.6) is a housekeeping gene, which functions in the general homeostasis of cellular nucleoside triphosphate (NTP) pools. Among the various NDPK isoforms, cytosolic NDPK1 has been shown to be the main NDPK isoform in plants, accounting for more than 70 % of total NDPK activity in plants. For the first time, a full-length cDNA (697 bp), designated as CsNDPK1 was cloned from tea leaves and consisted of a 448-bp open reading frame (ORF) encoding a 147-amino-acid polypeptide with calculated molecular mass of 16.1 kDa and a pI of 6.3. Homology modeling of CsNDPK1 shows that the presented tea NDPK1 also contains several motifs, binding and catalytic sites which are highly conserved among other NDPKs. Docking studies of CsNDPK1 with its substrates (NTPs) are discussed in detail. In summary, we describe a reliable model of CsNDPK1 that can be used in structure-based protein–protein interaction studies for identifying its potential role in intracellular communication and its physiological significance in tea.

Keywords: Cytosolic; Docking; Modeling; Nucleoside diphosphate kinase; Camellia sinensis

Suppressive Subtractive Hybridization Approach Revealed Differential Expression of Hypersensitive Response and Reactive Oxygen Species Production Genes in Tea (Camellia sinensis (L.) O. Kuntze) Leaves during Pestalotiopsis thea Infection by Palanisamy Senthilkumar; Krishnaraj Thirugnanasambantham; Abul Kalam Azad Mandal (pp. 1917-1927).

Tea (Camellia sinensis (L.) O. Kuntze) is an economically important plant cultivated for its leaves. Infection of Pestalotiopsis theae in leaves causes gray blight disease and enormous loss to the tea industry. We used suppressive subtractive hybridization (SSH) technique to unravel the differential gene expression pattern during gray blight disease development in tea. Complementary DNA from P. theae-infected and uninfected leaves of disease tolerant cultivar UPASI-10 was used as tester and driver populations respectively. Subtraction efficiency was confirmed by comparing abundance of β-actin gene. A total of 377 and 720 clones with insert size >250 bp from forward and reverse library respectively were sequenced and analyzed. Basic Local Alignment Search Tool analysis revealed 17 sequences in forward SSH library have high degree of similarity with disease and hypersensitive response related genes and 20 sequences with hypothetical proteins while in reverse SSH library, 23 sequences have high degree of similarity with disease and stress response-related genes and 15 sequences with hypothetical proteins. Functional analysis indicated unknown (61 and 59 %) or hypothetical functions (23 and 18 %) for most of the differentially regulated genes in forward and reverse SSH library, respectively, while others have important role in different cellular activities. Majority of the upregulated genes are related to hypersensitive response and reactive oxygen species production. Based on these expressed sequence tag data, putative role of differentially expressed genes were discussed in relation to disease. We also demonstrated the efficiency of SSH as a tool in enriching gray blight disease related up- and downregulated genes in tea. The present study revealed that many genes related to disease resistance were suppressed during P. theae infection and enhancing these genes by the application of inducers may impart better disease tolerance to the plants.

Keywords: cDNA; EST; Pestalotiopsis thea ; Suppressive subtractive hybridization

Rotating Biological Contactor Reactor with Biofilm Promoting Mats for Treatment of Benzene and Xylene Containing Wastewater by K. Sarayu; S. Sandhya (pp. 1928-1937).

A novel rotating biological contactor (RBC) bioreactor immobilized with microorganisms was designed to remove volatile organic compounds (VOC), such as benzene and xylene from emissions, and its performance was investigated. Gas-phase VOCs stripped by air injection were 98 % removed in the RBC when the superficial air flow rate was 375 ml/h (1,193 and 1,226 mg/l of benzene and xylene, respectively). The maximum removal rate was observed to be 1,007 and 1,872 mg/m³/day for benzene and xylene, respectively. The concentration profile of benzene and xylene along the RBC was dependent on the air flow rate and the degree of microbial adaptation. Air flow rate and residence time were found to be the most important operational parameters for the RBC reactor. By manipulating these operational parameters, the removal efficiency and capacity of the bioreactor could be enhanced. The kinetic constant K _s demonstrated a linear relationship that indicated the maximum removal of benzene and xylene in RBC reactor. The phylogenic profile shows the presence of bacterium like Pseudomonas sp., Bacillus sp., and Enterococcus sp., which belonged to the phylum Firmicutes, and Proteobacteria that were responsible for the 98 % organic removal in the RBC.

Keywords: Volatile organic compounds (VOCs); Treatment; Benzene and xylene degradation; Biofilm; RBC reactor

Metabolic Flux and Nodes Control Analysis of Brewer’s Yeasts Under Different Fermentation Temperature During Beer Brewing by Zhimin Yu; Haifeng Zhao; Mouming Zhao; Hongjie Lei; Huiping Li (pp. 1938-1952).

The aim of this work was to further investigate the glycolysis performance of lager and ale brewer’s yeasts under different fermentation temperature using a combined analysis of metabolic flux, glycolytic enzyme activities, and flux control. The results indicated that the fluxes through glycolytic pathway decreased with the change of the fermentation temperature from 15 °C to 10 °C, which resulted in the prolonged fermentation times. The maximum activities (V _max) of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK) at key nodes of glycolytic pathway decreased with decreasing fermentation temperature, which was estimated to have different control extent (22–84 %) on the glycolytic fluxes in exponential or flocculent phase. Moreover, the decrease of V _max of PFK or PK displayed the crucial role in down-regulation of flux in flocculent phase. In addition, the metabolic state of ale strain was more sensitive to the variation of temperature than that of lager strain. The results of the metabolic flux and nodes control analysis in brewer’s yeasts under different fermentation temperature may provide an alternative approach to regulate glycolytic flux by changing V _max and improve the production efficiency and beer quality.

Keywords: Metabolic flux analysis; Nodes control analysis; Lager yeast; Ale yeast; Glycolytic key enzyme; Fermentation temperature

Agrobacterium-Mediated Gene Transfer in Plants and Biosafety Considerations by Shweta Mehrotra; Vinod Goyal (pp. 1953-1975).

Agrobacterium, the natures’ genetic engineer, has been used as a vector to create transgenic plants. Agrobacterium-mediated gene transfer in plants is a highly efficient transformation process which is governed by various factors including genotype of the host plant, explant, vector, plasmid, bacterial strain, composition of culture medium, tissue damage, and temperature of co-cultivation. Agrobacterium has been successfully used to transform various economically and horticulturally important monocot and dicot species by standard tissue culture and in planta transformation techniques like floral or seedling infilteration, apical meristem transformation, and the pistil drip methods. Monocots have been comparatively difficult to transform by Agrobacterium. However, successful transformations have been reported in the last few years based on the adjustment of the parameters that govern the responses of monocots to Agrobacterium. A novel Agrobacterium transferred DNA-derived nanocomplex method has been developed which will be highly valuable for plant biology and biotechnology. Agrobacterium-mediated genetic transformation is known to be the preferred method of creating transgenic plants from a commercial and biosafety perspective. Agrobacterium-mediated gene transfer predominantly results in the integration of foreign genes at a single locus in the host plant, without associated vector backbone and is also known to produce marker free plants, which are the prerequisites for commercialization of transgenic crops. Research in Agrobacterium-mediated transformation can provide new and novel insights into the understanding of the regulatory process controlling molecular, cellular, biochemical, physiological, and developmental processes occurring during Agrobacterium-mediated transformation and also into a wide range of aspects on biological safety of transgenic crops to improve crop production to meet the demands of ever-growing world’s population.

Keywords: Agrobacterium ; T-DNA; Inplanta transformation; Biosafety

Pathways for Degrading TNT by Thu-Z: a Pantoea sp. Strain by Liangdong Zou; Diannan Lu; Zheng Liu (pp. 1976-1988).

2,4,6-Trinitrotoluene (TNT), an extensively used and versatile explosive, is harmful in soil and water. In the present study, four bacterial strains capable of degrading TNT have been isolated from contaminated sites and named as Thu-A, Thu-B, Thu-C, and Thu-Z. Thu-Z, which gave the highest degradation efficiency compared to the others, was assigned to the genus Pantoea according to its 16S rRNA gene. Similarities in both biochemical properties and morphology suggested that Thu-Z was a Pantoea sp. strain. Thu-Z was proved to be capable of using TNT as a sole nitrogen source by cleaving NO₂ from the nitroaromatic ring by direct aromatic ring reduction. Under nitrogen-limited conditions, 96.6 % N of TNT was consumed by Thu-Z for growth, which was determined in terms of NaNO₂. Trace nitro reduction metabolites such as 2,4-diamino-6-nitrotoluene (24Dam) and 2,6-diamino-4-nitrotoluene (26Dam) were identified in the presence of (NH₄)₂SO₄. On the other hand, 4,4′,6,6′-tetranitro-2,2′-azoxytoluene (22Azo) and 2,2′,6,6′-tetranitro-4,4′-azoxytoluene (44Azo) were detected in the absence of (NH₄)₂SO₄. These indicated the existence of a dual pathway for Thu-Z, while the direct aromatic ring reduction was predominant. Addition of a nitrogen source ((NH₄)₂SO₄) after inoculation stimulated the growth of Thu-Z and accelerated TNT degradation.

Keywords: 2,4,6-Trinitrotoluene (TNT); Explosive; Biodegradation; Pathways; Pantoea sp

Chemical Modifications of Laccase from White-Rot Basidiomycete Cerrena unicolor by K. H. Kucharzyk; G. Janusz; I. Karczmarczyk; J. Rogalski (pp. 1989-2003).

Laccases belong to the group of phenol oxidizes and constitute one of the most promising classes of enzymes for future use in various fields. For industrial and biotechnological purposes, laccases were among the first enzymes providing larger-scale applications such as removal of polyphenols or conversion of toxic compounds. The wood-degrading basidiomycete Cerrena unicolor C-139, reported in this study, is one of the high-laccase producers. In order to facilitate novel and more efficient biocatalytic process applications, there is a need for laccases with improved biochemical properties, such as thermostability or stability in broad ranges of pH. In this work, modifications of laccase isoforms by hydrophobization, hydrophilization, and polymerization were performed. The hydrophobized and hydrophilized enzyme showed enhanced surface activity and higher ranges of pH and temperatures in comparison to its native form. However, performed modifications did not appear to noticeably alter enzyme’s native structure possibly due to the formation of coating by particles of saccharides around the molecule. Additionally, surface charge of modified laccase shifted towards the negative charge for the hydrophobized laccase forms. In all tested modifications, the size exclusion method led to average 80 % inhibition removal for hydrophilized samples after an hour of incubation with fluoride ions. Samples that were hydrophilized with lactose and cellobiose showed an additional 90 % reversibility of inhibition by fluoride ions after an hour of concluding the reaction and 40 % after 24 h. The hydrophobized laccase showed higher level of the reversibility after 1 h (above 80 %) and 24 h (above 70 %) incubation with fluoride ions. The addition of ascorbate to laccase solution before a fluoride spike resulted in more efficient reversibility of fluoride inhibitory effect in comparison to the treatments with reagents used in the reversed sequence.

Keywords: Laccase; Cerrena unicolor ; Hydrophobization; Hydrophilization

Enhanced Exoglucanase Production by Brown Rot Fungus Fomitopsis sp. RCK2010 and its Application for Cellulose Saccharification by Deepa Deswal; Rishi Gupta; Ramesh Chander Kuhad (pp. 2004-2016).

Exoglucanase production by brown rot fungus Fomitopsis sp. RCK2010 was optimized under solid-state fermentation using Plackett–Burman design (PBD) and response surface methodology (RSM). Four fermentation variables (moisture, inoculum level, casein, and Triton X-100) were identified to effect cellulase production significantly by PBD, which were further optimized using RSM of central composite design. An overall 130 % increase in enzyme production was achieved by the optimization of variables using statistical approaches. Moreover, crude cellulase from Fomitopsis sp. RCK2010 was applied to saccharify pretreated Prosopis juliflora (cellulosic fraction), which resulted in the release of 327.35 mg/g of reducing sugars that could further be utilized for bioethanol production.

Keywords: Cellulase; Exoglucanase; Filamentous fungus; Optimization; Solid-state fermentation

An Extremely Alkaline Novel Xylanase from a Newly Isolated Streptomyces Strain Cultivated in Corncob Medium by Jaya Ram Simkhada; Hah-Young Yoo; Yun Hee Choi; Seung Wook Kim; Jin Cheol Yoo (pp. 2017-2027).

Streptomyces sp. CS802, recently isolated from Korean soil, produced xylanase in corncob medium. An extracellular xylanase (Xyn802) was purified by a single-step gel filtration and biochemical properties were studied. It showed high activity in extremely alkaline condition with optimum pH at 12.0 and exhibited stability between pH 7.5 and 13.0. It produced xylobiose and xylotriose as the major products from xylan, suggesting its endoxylanase nature. N-terminal amino acid sequences of Xyn802 were ADRNANRD which are significantly different from the reported xylanase. The activity was enhanced by various detergents and a reducing agent and stable in various organic solvents. Xyn802 produced by utilizing corncob, an agro-waste material, might be a novel xylanase based on its peculiar biochemical characteristics, and it can be a suitable candidate for the production of xylooligosaccharides including other useful products.

Keywords: Agro-waste; Corncob; Hyperalkaline xylanase; Streptomyces ; Xylooligosaccharides production

Infrared Spectroscopy as Alternative to Wet Chemical Analysis to Characterize Eucalyptus globulus Pulps and Predict Their Ethanol Yield for a Simultaneous Saccharification and Fermentation Process by Rosario del P. Castillo; Jaime Baeza; Joselyn Rubilar; Álvaro Rivera; Juanita Freer (pp. 2028-2042).

Bioethanol can be obtained from wood by simultaneous enzymatic saccharification and fermentation step (SSF). However, for enzymatic process to be effective, a pretreatment is needed to break the wood structure and to remove lignin to expose the carbohydrates components. Evaluation of these processes requires characterization of the materials generated in the different stages. The traditional analytical methods of wood, pretreated materials (pulps), monosaccharides in the hydrolyzated pulps, and ethanol involve laborious and destructive methodologies. This, together with the high cost of enzymes and the possibility to obtain low ethanol yields from some pulps, makes it suitable to have rapid, nondestructive, less expensive, and quantitative methods to monitoring the processes to obtain ethanol from wood. In this work, infrared spectroscopy (IR) accompanied with multivariate analysis is used to characterize chemically organosolv pretreated Eucalyptus globulus pulps (glucans, lignin, and hemicellulosic sugars), as well as to predict the ethanol yield after a SSF process. Mid (4,000–400 cm⁻¹) and near-infrared (12,500–4,000 cm⁻¹) spectra of pulps were used in order to obtain calibration models through of partial least squares regression (PLS). The obtained multivariate models were validated by cross validation and by external validation. Mid-infrared (mid-IR)/NIR PLS models to quantify ethanol concentration were also compared with a mathematical approach to predict ethanol yield estimated from the chemical composition of the pulps determined by wet chemical methods (discrete chemical data). Results show the high ability of the infrared spectra in both regions, mid-IR and NIR, to calibrate and predict the ethanol yield and the chemical components of pulps, with low values of standard calibration and validation errors (root mean square error of calibration, root mean square error of validation (RMSEV), and root mean square error of prediction), high correlation between predicted and measured by the reference methods values (R ² between 0.789 and 0.997), and adequate values of the ratio between the standard deviation of the reference methods and the standard errors of infrared PLS models relative performance determinant (RPD) (greater than 3 for majority of the models). Use of IR for ethanol quantification showed similar and even better results to the obtained with the discrete chemical data, especially in the case of mid-IR models, where ethanol concentration can be estimated with a RMSEV equal to 1.9 g L⁻¹. These results could facilitate the analysis of high number of samples required in the evaluation and optimization of the processes.

Keywords: Bioethanol; Infrared spectroscopy; SSF; Eucalyptus globulus ; Organosolv

Study of the Release Mechanism of Terminalia chebula Extract from Nanoporous Silica Gel by Suparna Chakraborty; Manoj Kumar Mitra; Mahua Ghosh Chaudhuri; Biswanath Sa; Satadal Das; Rajib Dey (pp. 2043-2056).

Sol/gel-derived silica gel was prepared at room temperature from tetraethyl orthosilicate precursor. The extracts of Terminalia chebula (Haritoki) were entrapped into the porous silica gel. Fourier transform infrared analysis revealed the proper adsorption of herbal values in the nanopores of the silica gel. Porosity was estimated by transmission electron microscope studies. The release kinetics of the extract in both 0.1 N HCl, pH 1.2, and Phosphate-buffer saline (PBS), pH 7.2, were determined using UV–Vis spectroscopy. Different dissolution models were applied to release data in order to evaluate the release mechanisms and kinetics. Biphasic release patterns were found in every formulation for both the buffer systems. The kinetics followed a zero-order equation for first 4 h and a Higuchi expression in a subsequent timeline in the case of 0.1 N HCl. In the case of PBS, the formulations showed best linearity with a first-order equation followed by Higuchi’s model. The sustained release of the extract predominantly followed diffusion and super case II transport mechanism. The release value was always above the minimum inhibitory concentration.

Keywords: Nano porous silica gel; Terminalia chebula ; Kinetics; Sustained release; MIC

Gibberellic Acid Increases Secondary Metabolite Production in Echinacea purpurea Hairy Roots by Bilal H. Abbasi; Amanda R. Stiles; Praveen K. Saxena; Chun-Zhao Liu (pp. 2057-2066).

Gibberellic acid (GA₃) is reported to have diverse effects on hairy root cultures of many plant species; therefore, the effects of GA₃ on the growth, secondary metabolite production (caffeic acid derivatives and lignin), phenylalanine ammonia lyase (PAL) activity, and free radical scavenging activity of light-grown Echinacea purpurea L. hairy roots were investigated. Eight concentrations of GA₃, ranging from 0.005 to 1.0 μM, were added to shake flask cultures. The moderate GA₃ concentration, 0.025 μM, resulted in the highest concentrations of cichoric acid, caftaric acid, and chlorogenic acid, as well as increased PAL activity, cell viability, and free radical scavenging activity, while higher and lower GA₃ concentrations resulted in reduced levels compared to the control (lacking GA₃). The moderate GA₃ concentration also affected root morphogenesis; supplementation with 0.025 μM GA₃ resulted in the development of thick, dense, purple-colored roots, while roots exposed to the higher and lower concentrations of GA₃ were thin and off-white. This study demonstrates that supplementation with GA₃ may be an excellent strategy to optimize the production of secondary metabolites from E. purpurea hairy root cultures; however, the GA₃ concentration is a critical factor.

Keywords: Echinacea purpurea ; Hairy roots; Cichoric acid; Anthocyanins; GA₃ ; Light

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.168, #7)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.168, #7)