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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.169, #1)

Purification Studies on a Thermo-active Amidase of Geobacillus pallidus BTP-5x MTCC 9225 Isolated from Thermal Springs of Tatapani (Himachal Pradesh) by Monica Sharma; Nitya Nand Sharma; Tek Chand Bhalla (pp. 1-14).

An intracellular aliphatic amide degrading inducible thermo-active amidase produced by Geobacillus pallidus BTP-5x MTCC 9225 was purified to apparent homogeneity using anion exchange and gel filtration chromatography, giving a yield of 6.7 % and a specific activity of 30.49 units mg⁻¹. The purified protein migrated as a single band of estimated molecular mass of 158 kDa (homo-tetramer) in 8 % polyacrylamide gel electrophoresis and ∼38.5 kDa in 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Optima of pH and temperature varied widely in broad pH range (pH 6–9) and temperature range (45–70 °C). The purified amidase was stable up to 6 h at 50 °C, with a t _1/2 of 7 h at 55 °C. The multimeric nature of the holozyme (tetramer) contributed to protection of the enzyme against thermal denaturation. The enzyme showed resistance to metal chelating agents (EDTA, 8-hydroxyquinoline, and sodium azide), explaining its non-metallic nature, and is strongly inhibited by thiol reagents that means cysteine is involved in catalysis. The amidase of G. pallidus BTP-5x preferentially hydrolyzed only small aliphatic amides and has a narrow substrate spectrum. The K _M value for acrylamide is 10.54 mM, V _max 45.19 μmol⁻¹ min⁻¹ mg⁻¹ protein, and k _cat 4.29 min⁻¹. The sequence of amino acids of the purified enzyme MRHGDISSSHDTVGI appears similar to thermophilic amidases. Sequence analysis of the amidase gene showed that the enzyme is 347 amino-acid-long with a molecular weight of 38.4 kDa (as observed in SDS-PAGE), theoretical pI 5.38, and show strong similarity to thermostable amidases, possessing unique restriction sites.

Keywords: Geobacillus pallidus BTP-5x MTCC 9225; Aliphatic amidase; Thermostable amidase; Cloning

Isolation and Biochemical Characterization of Two Novel Metagenome-Derived Esterases by Li-Ming Ouyang; Jia-Ying Liu; Ming Qiao; Jian-He Xu (pp. 15-28).

Environmental DNA from soil and water samples was extracted to construct a plasmid library and a fosmid library containing 19,500 and 20,400 clones, respectively. Two esterases (EstP2K and EstF4K) were finally isolated from each library based on activity screening, and both of them were characterized in this study. The esterase EstF4K consists of 396 amino acids with an SMTK motif which belongs to family VIII esterase/lipase. The amino acid sequence of EstF4K showed 83 % identity with that of EstA3, a reported esterase isolated from uncultured organisms of soil. EstP2K is composed of 224 amino acids in size and shows only 37 % identity with a putative lipase of Neisseria elongata subsp. The purified EstF4K was optimally active at pH 8.0 and 50 °C. It was remarkably active and very stable in the presence of 30 % dimethyl sulfoxide. Activity fingerprint of EstF4K displayed a higher level of activity toward short-chain fatty acid p-nitrophenyl (pNP) esters, while EstP2K preferred bias for pNP caprylate ester. The optimum reaction temperature and pH for EstP2K are 45 °C and 7.5, respectively, and the enzyme exhibited strong tolerance in the presence of 30 % methanol. EstF4K and EstP2K showed opposite enantioselectivity for methyl 3-phenylglycidate, a chiral synthon for the synthesis of Taxol® side chain.

Keywords: Esterase; Metagenomic library; Plasmid; Fosmid; p-Nitrophenyl ester

Cloning, Expression, and Characterization of a Recombinant Esterase from Cold-Adapted Pseudomonas mandelii by ChangWoo Lee; Junyoung Kim; Seunghee Hong; Bonlon Goo; Seungyeon Lee; Sei-Heon Jang (pp. 29-40).

A gene coding for the extracellular esterase (EstK) was cloned from the psychrotrophic bacterium Pseudomonas mandelii based on its partial amino acid sequence as determined by mass spectrometry. The entire open reading frame consisting of 1,011 bp was expressed in Escherichia coli as a soluble protein and purified by nickel-chelated affinity chromatography and Capto Q column chromatography. Here, we show that the 33-kDa recombinant EstK protein (rEstKsp) had a substrate preference for esters of short-chain fatty acids, especially, p-nitrophenyl acetate. Optimum activity of rEstKsp was at pH 8.5 and 40 °C. The esterase activity remained similar from a range of 4∼20 °C, but the maximum activity varied depending upon pH. With p-nitrophenyl acetate as the substrate, K _M was 210 μM and k _cat was 3.4 s⁻¹. Circular dichroism and fluorescence spectroscopy results revealed that rEstKsp had a predominantly α-helical structure and maintained its folded state at 4∼40 °C. Interestingly, the tertiary structure of rEstKsp was predicted based on the structures of other hyperthermophilic esterases. Our results demonstrated that both native and rEstKsp are active at low temperatures and have a unique substrate preference for p-nitrophenyl acetate.

Keywords: Esterase; Lipase; Psychrotrophic bacterium; Pseudomonas mandelii

Signal Peptide-Independent Secretory Expression and Characterization of Pullulanase from a Newly Isolated Klebsiella variicola SHN-1 in Escherichia coli by Wen-Bo Chen; Yao Nie; Yan Xu (pp. 41-54).

A strain with the power to produce extracellular pullulanase was obtained from the sample taken from a flour mill. By sequencing its 16S rDNA, the isolate was identified as Klebsiella variicola SHN-1. When the gene encoding pullulanase, containing the N-terminal signal sequence, was cloned into Escherichia coli BL21 (DE3), extracellular activity was detected up to 10 U/ml, a higher level compared with the results in published literature. Subsequently, the recombinant pullulanase was purified and characterized. The main end product from pullulan hydrolyzed by recombinant pullulanase was determined as maltotriose with HPLC, and hence, the recombinant pullulanase was identified as type I pullulanase, which could be efficiently employed in starch processing to produce maltotriose with higher purity and even to evaluate the purity of pullulan. To investigate the effect of signal peptide on secretion of the recombinant enzyme, the signal sequence was removed from the constructed vector. However, secretion of pullulanase in E. coli was not influenced, which was seldom reported previously. By localizing the distribution of pullulanase on subcellular fractions, the secretion of recombinant pullulanase in E. coli BL21 (DE3) was confirmed, even from the expression system of nonsecretory type without the assistance of signal peptide.

Keywords: Pullulanase; Heterologous expression; Secretion; Klebsiella variicola ; Signal peptide

Perspectives in Metabolic Engineering: Understanding Cellular Regulation Towards the Control of Metabolic Routes by Sohila Zadran; Raphael D. Levine (pp. 55-65).

Metabolic engineering seeks to redirect metabolic pathways through the modification of specific biochemical reactions or the introduction of new ones with the use of recombinant technology. Many of the chemicals synthesized via introduction of product-specific enzymes or the reconstruction of entire metabolic pathways into engineered hosts that can sustain production and can synthesize high yields of the desired product as yields of natural product-derived compounds are frequently low, and chemical processes can be both energy and material expensive; current endeavors have focused on using biologically derived processes as alternatives to chemical synthesis. Such economically favorable manufacturing processes pursue goals related to sustainable development and “green chemistry”. Metabolic engineering is a multidisciplinary approach, involving chemical engineering, molecular biology, biochemistry, and analytical chemistry. Recent advances in molecular biology, genome-scale models, theoretical understanding, and kinetic modeling has increased interest in using metabolic engineering to redirect metabolic fluxes for industrial and therapeutic purposes. The use of metabolic engineering has increased the productivity of industrially pertinent small molecules, alcohol-based biofuels, and biodiesel. Here, we highlight developments in the practical and theoretical strategies and technologies available for the metabolic engineering of simple systems and address current limitations.

Keywords: Metabolomics; Bioengineering; Bioenergetics; Gene networks; Metabolic flux analysis

Chemometrics-Based Process Analytical Technology (PAT) Tools: Applications and Adaptation in Pharmaceutical and Biopharmaceutical Industries by Shruthi Challa; Ravichandra Potumarthi (pp. 66-76).

Process analytical technology (PAT) is used to monitor and control critical process parameters in raw materials and in-process products to maintain the critical quality attributes and build quality into the product. Process analytical technology can be successfully implemented in pharmaceutical and biopharmaceutical industries not only to impart quality into the products but also to prevent out-of-specifications and improve the productivity. PAT implementation eliminates the drawbacks of traditional methods which involves excessive sampling and facilitates rapid testing through direct sampling without any destruction of sample. However, to successfully adapt PAT tools into pharmaceutical and biopharmaceutical environment, thorough understanding of the process is needed along with mathematical and statistical tools to analyze large multidimensional spectral data generated by PAT tools. Chemometrics is a chemical discipline which incorporates both statistical and mathematical methods to obtain and analyze relevant information from PAT spectral tools. Applications of commonly used PAT tools in combination with appropriate chemometric method along with their advantages and working principle are discussed. Finally, systematic application of PAT tools in biopharmaceutical environment to control critical process parameters for achieving product quality is diagrammatically represented.

Keywords: Process analytical technology (PAT); Chemometrics; Pharmaceutical; Biopharmaceutical; PAT tools; Applications

Enzymatic Characterization of Human Immunodeficiency Virus Type 1 Reverse Transcriptase for Use in cDNA Synthesis by Atsushi Konishi; Mayu Shinomura; Kiyoshi Yasukawa (pp. 77-87).

The aim of this study is to explore the advantages of using human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in cDNA synthesis. Recombinant HIV-1 group M (HIV-1 M) RT and HIV-1 group O (HIV-1 O) RT were produced in an Escherichia coli expression system. In the incorporation of dTTP into poly(rA)-p(dT)₁₅ (T/P), the K _m values for dTTP of HIV-1 M RT and HIV-1 O RT were 8 and 12 % of that of Moloney murine leukemia virus (MMLV) RT, respectively, and the K _m values for T/P were 25 and 23 % of that of MMLV RT, respectively. Compared with MMLV RT, HIV-1 M RT and HIV-1 O RT were less susceptible to formamide, which is frequently used for cDNA synthesis with a G + C-rich RNA to improve specificity. The high substrate affinity and low susceptibility to formamide of HIV-1 RT might be advantageous for its use in cDNA synthesis.

Keywords: cDNA; Formamide; HIV-1; MMLV; Reverse transcriptase

Control of the Harmful Alga Microcystis aeruginosa and Absorption of Nitrogen and Phosphorus by Candida utilis by Yun Kong; Xiangyang Xu; Liang Zhu; Lihong Miao (pp. 88-99).

This study is aimed at controlling eutrophication through converting the nutrients such as nitrogen and phosphorus into microbial protein and simultaneously inhibiting the growth of Microcystis aeruginosa by Candida utilis. C. utilis and M. aeruginosa (initial cell density was 2.25 × 10⁷ and 4.15 × 10⁷ cells·mL⁻¹) were cultured together in the absence or presence of a carbon source (glucose) during a 10-day experiment. In the absence of carbon source, the measured removal efficiencies of NH₄ ⁺–N and PO₄ ³⁻–P were 41.39 ± 2.19 % and 82.93 ± 3.95 %, respectively, at the second day, with the removal efficiency of 67.82 ± 2.29 % for M. aeruginosa at the fourth day. In contrast, the removal efficiencies of NH₄ ⁺–N and PO₄ ³⁻–P were increased to 87.45 ± 4.25 % and 83.73 ± 3.55 %, respectively, while the removal efficiency of M. aeruginosa decreased to 37.89 ± 8.41 % in the presence of the carbon source (C/N = 2:1). These results showed that the growth of M. aeruginosa was inhibited by C. utilis. Our finding sheds light on a novel potential approach for yeast to consume nutrients and control harmful algal during bloom events.

Keywords: Eutrophication; Biological control; Candida utilis ; Microcystis aeruginosa ; Competition

Extracellular Enzymes of the White-Rot Fungus Fomes fomentarius and Purification of 1,4-β-Glucosidase by Tomáš Větrovský; Petr Baldrian; Jiří Gabriel (pp. 100-109).

Production of the lignocellulose-degrading enzymes endo-1,4-β-glucanase, 1,4-β-glucosidase, cellobiohydrolase, endo-1,4-β-xylanase, 1,4-β-xylosidase, Mn peroxidase, and laccase was characterized in a common wood-rotting fungus Fomes fomentarius, a species able to efficiently decompose dead wood, and compared to the production in eight other fungal species. The main aim of this study was to characterize the 1,4-β-glucosidase produced by F. fomentarius that was produced in high quantities in liquid stationary culture (25.9 U ml⁻¹), at least threefold compared to other saprotrophic basidiomycetes, such as Rhodocollybia butyracea, Hypholoma fasciculare, Irpex lacteus, Fomitopsis pinicola, Pleurotus ostreatus, Piptoporus betulinus, and Gymnopus sp. (between 0.7 and 7.9 U ml⁻¹). The 1,4-β-glucosidase enzyme was purified to electrophoretic homogeneity by both anion-exchange and size-exclusion chromatography. A single 1,4-β-glucosidase was found to have an apparent molecular mass of 58 kDa and a pI of 6.7. The enzyme exhibited high thermotolerance with an optimum temperature of 60 °C. Maximal activity was found in the pH range of 4.5–5.0, and K _M and V _max values were 62 μM and 15.8 μmol min⁻¹ l⁻¹, respectively, when p-nitrophenylglucoside was used as a substrate. The enzyme was competitively inhibited by glucose with a K _i of 3.37 mM. The enzyme also acted on p-nitrophenylxyloside, p-nitrophenylcellobioside, p-nitrophenylgalactoside, and p-nitrophenylmannoside with optimal pH values of 6.0, 3.5, 5.0, and 4.0–6.0, respectively. The combination of relatively low molecular mass and low K _M value make the 1,4-β-glucosidase a promising enzyme for biotechnological applications.

Keywords: Cellulose; 1,4-β-glucosidase; Glycosyl hydrolase; Saprotrophic basidiomycetes; Wood-rotting fungi; Fomes fomentarius

Industrial Waste Utilization for Low-Cost Production of Raw Material Oil Through Microbial Fermentation by Yasmi Louhasakul; Benjamas Cheirsilp (pp. 110-122).

In view of ever-growing demand of biodiesel, there is an urgent need to look for inexpensive and promising renewable raw material oils for its production. In this context, the aim of this study was to evaluate the potential use of industrial wastes for low-cost production of oils through microbial fermentation. Among the strains tested, Yarrowia lipolytica grew best and produced highest lipid when grown on decanter effluent from palm oil mill. When crude glycerol by-product from a biodiesel plant was added into the effluent as a co-substrate, Y. lipolytica produced a higher biomass of 3.21 g/L and a higher amount of lipid of 2.21 g/L which was 68 % of the dry weight. The scale up and process improvement in a 5-L bioreactor increased the biomass and lipid up to 5.53 and 2.81 g/L, respectively. A semi-continuous mode of operation was an effective mode for biomass enhancement while a fed-batch mode was effective for lipid enhancement. These yeast lipids have potential to be used as biodiesel feedstocks because of their similar fatty acid composition to that of plant oil.

Keywords: Biodiesel; Palm oil mill effluent; Crude glycerol; Lipid; Yarrowia lipolytica

Evaluation for Rock Phosphate Solubilization in Fermentation and Soil–Plant System Using A Stress-Tolerant Phosphate-Solubilizing Aspergillus niger WHAK1 by Chunqiao Xiao; Huaxiang Zhang; Yujuan Fang; Ruan Chi (pp. 123-133).

A strain WHAK1, identified as Aspergillus niger, was isolated from Yichang phosphate mines in Hubei province of China. The fungus developed a phosphate solubilization zone on modified National Botanical Research Institute’s phosphate growth (NBRIP) agar medium, supplemented with tricalcium phosphate. The fungus was applied in a repeated-batch fermentation process in order to test its effect on solubilization of rock phosphate (RP). The results showed that A. niger WHAK1 could effectively solubilize RP in NBRIP liquid medium and released soluble phosphate in the broth, which can be illustrated by the observation of scanning electron microscope, energy-dispersive X-ray microanalysis, and Fourier transform infrared spectroscopy. Acidification of the broth seemed to be the major mechanism for RP solubilization by the fungus. Indeed, multiple organic acids (mainly gluconic acid) were detected in the broth by high-performance liquid chromatography analysis. These organic acids caused a significant drop of pH and an obvious rise of titratable acidity in the broth. The fungus also exhibited high levels of tolerance against temperature, pH, salinity, and desiccation stresses, although a significant decline in the fungal growth and release of soluble phosphate was marked under increasing intensity of stress parameters. Further, the fungus was introduced into the soil supplemented with RP to analyze its effect on plant growth and phosphate uptake of wheat plants. The result revealed that inoculation of A. niger WHAK1 significantly increased the growth and phosphate uptake of wheat plants in the RP-amended soil compared to the control soil.

Keywords: Rock phosphate; Solubilization; Aspergillus niger ; Stress; Soluble phosphate; Wheat

Molecular Cloning, Tissue Expression, and Analysis with Genome DNA Methylation of Porcine LSD1 Gene by Jin Chai; Lina Liu; Qi Xiong; Chen Chen; Yongdong Peng; Wei Jin; Rong Zheng; Jian Peng; Siwen Jiang (pp. 134-144).

Lysine-specific demethylase 1 (LSD1) functioned as a demethyl methylase gene, underlying a wide range of biological processes, including cancer, cell apoptosis, differentiation, and development. To further understand the functions of the porcine LSD1 gene, we first obtained cDNA sequence of porcine LSD1 gene, using in silico cloning method. We further found that the porcine LSD1 gene has two transcripts, in which cDNA sequences are 2,716 and 2,656 bp, ORF are 2,622 and 2,562 bp, respectively. Then, RT-PCR analysis showed that the LSD1 gene is expressed in various tissues and relatively higher in the tissues of ovary, kidney, and spleen. Besides, the LSD1 gene was expressed higher in the growth nonage and peaked at 3 days in muscle tissue. Meanwhile, the expression of two transcript variants of LSD1 gene presented the same change trend. Besides, the level of DNA methylation was approximately fourfold higher in a 3-day muscle than in an old pig (180 days), significantly positive related to the gene expression of LSD1 (R = 0.9362, P < 0.05), and declined with growing age. Cloning, expression pattern, and analysis with genome DNA methylation of porcine LSD1 gene laid a foundation to clarify the molecular mechanisms of porcine growth and development and also for further work on animal breeding.

Keywords: LSD1 gene; Cloning; Expression; DNA methylation; Porcine

Kinetics and Computational Docking Studies on the Inhibition of Tyrosinase Induced by Oxymatrine by Xiao-Xia Liu; Shi-Qing Sun; Yu-Jie Wang; Wei Xu; Yi-Fang Wang; Daeui Park; Hai-Meng Zhou; Hong-Yan Han (pp. 145-158).

A combination of enzymatic inhibition kinetics and computational prediction was employed to search for an effective inhibitor of tyrosinase. We found that oxymatrine significantly inhibited tyrosinase, and that this reaction was not accompanied by detectable conformational changes. Kinetic analysis showed that oxymatrine reversibly inhibited tyrosinase in a mixed-type manner. Measurements of intrinsic and ANS-binding fluorescences showed that oxymatrine did not induce any conspicuous changes in the tertiary structure. We also conducted a docking simulation between tyrosinase and oxymatrine using two docking programs, Dock6.3 and AutoDock4.2 (binding energy was −118.81 kcal/mol for Dock6 and −8.04 kcal/mol for AutoDock4). The results also suggested that oxymatrine interacts mostly with the residues of CYS83 and HIS263 in the active site of tyrosinase. This strategy of predicting tyrosinase inhibition by simulation of docking coupling with kinetics may prove useful in screening for potential tyrosinase inhibitors. Knowledge of tyrosinase inhibition can provide medical, cosmetic, and agricultural applications. Our study suggests that oxymatrine is an important agent for various applications related to pigment formation.

Keywords: Oxymatrine; Tyrosinase; Inhibitor; Kinetics; Computational docking

Recombinant Single-Chain Antibody with the Trojan Peptide Penetratin Positioned in the Linker Region Enables Cargo Transfer Across the Blood–Brain Barrier by Nives Škrlj; Gorazd Drevenšek; Samo Hudoklin; Rok Romih; Vladka Čurin Šerbec; Marko Dolinar (pp. 159-169).

Delivery of therapeutic proteins into tissues and across the blood–brain barrier (BBB) is limited by the size and biochemical properties of the proteins. Efficient delivery across BBB is generally restricted to small, highly lipophilic molecules. However, in the last decades, several peptides that can pass cell membranes have been identified. It has been shown that these peptides are also capable of delivering large hydrophilic cargoes into cells and are therefore a powerful biological tool for transporting drugs across cell membranes and even into the brain. We designed and prepared a single-chain antibody fragment (scFvs), specific for the pathological form of the prion protein (PrP^Sc), where a cell-penetrating peptide (CPP) was used as a linker between the two variable domains of the scFv. The intravenously administered recombinant scFv-CPP was successfully targeted to and delivered into mouse brain cells. Our single-chain antibody fragments are of special interest in view of possible therapeutic reagents design not only for prion diseases but also for other neurodegenerative diseases.

Keywords: Antibody fragment; Cell-penetrating peptides; Brain delivery; Prion; Penetratin

Overexpression of the Formaldehyde Dehydrogenase Gene from Brevibacillus brevis to Enhance Formaldehyde Tolerance and Detoxification of Tobacco by Hongjuan Nian; Qingchao Meng; Wei Zhang; Limei Chen (pp. 170-180).

The faldh gene coding for a putative Brevibacillus brevis formaldehyde dehydrogenase (FALDH) was isolated and then transformed into tobacco. A total of three lines of transgenic plants were generated, with each showing 2- to 3-fold higher specific formaldehyde dehydrogenase activities than wild-type tobacco, a result that demonstrates the functional activity of the enzyme in formaldehyde (HCHO) oxidation. Overexpression of faldh in tobacco confers a high tolerance to exogenous HCHO and an increased ability to take up HCHO. A ¹³C-nuclear magnetic resonance technique revealed that the transgenic plants were able to oxidize more aqueous HCHO to formate than the wild-type (WT) plants. When treated with gaseous HCHO, the transgenic tobacco exhibited an enhanced ability to transform more HCHO into formate, citrate acid, and malate but less glycine than the WT plants. These results indicate that the increased capacity of the transgenic tobacco to take up, tolerate, and metabolize higher concentrations of HCHO was due to the overexpression of B. brevis FALDH, revealing the essential function of this enzyme in HCHO detoxification. Our results provide a potential genetic engineering strategy for improving the phytoremediation of HCHO pollution.

Keywords: Brevibacillus brevis ; Formaldehyde dehydrogenase; Transgenic tobacco; HCHO metabolism; HCHO detoxification

Effect of Dissolved Oxygen Tension and Agitation Rates on Sulfur-Utilizing Autotrophic Denitrification: Batch Tests by Naveed Ahmed Qambrani; Sang-Eun Oh (pp. 181-191).

The effect of dissolved oxygen (DO) and agitation rate in open and closed reactors was examined for sulfur-utilizing autotrophic denitrification. The reaction rate constants were determined based on a half-order kinetic model. Declining denitrification rate constants obtained for open reactors those of 8.46, 8.03, and 2.18 for 50 mg NO₃ ⁻-N/L, while 11.12, 9.14, and 0.12 mg^1/2/L^1/2 h were for 100 mg NO₃ ⁻-N/L at agitation speeds of 0, 100, and 200 rpm. In closed reactors, the ever-increasing denitrification rates were 10.13, 22.56, and 37.03, whereas for the same nitrate concentrations and speeds the rates were 13.17, 15.63, and 26.67 mg^1/2/L^1/2 h. The rate constants correlated well (r 2 = 0.89–0.99) with a half-order kinetic model. In open reactors, high SO₄ ²⁻/N ratios (8.02–75.10) while in closed reactors comparatively low SO₄ ²⁻/N ratios (6.10–13.39) were obtained. Sulfur oxidation occurred continuously in the presence of DO, resulting in mixed cultures acclimated to sulfur and nitrate. SO₄ ²⁻ was produced as an end product, which reduced alkalinity and lowered pH over time. Furthermore, DO inhibited sulfur denitrification in open reactors, while agitation in closed reactors increased the rate of denitrification.

Keywords: Dissolved oxygen; Sulfur-utilizing autotrophic denitrification; Nitrate removal; pH control; Sulfur reactor

Lipid Biomarkers of Lens Aging by Bimal Prasanna Mohanty; Soma Bhattacharjee; Prasenjit Paria; Arabinda Mahanty; Anil Prakash Sharma (pp. 192-200).

Lipids are important structural components of cell membranes and have profound effect on membrane fluidity. Lipid profiling and lipidomics have captured increased attention due to the well-recognized roles of lipids in numerous human diseases. Investigating lipid profiles not only provides insights into the specific roles of lipid molecular species in health and diseases, but can also help in identifying potential preventive or therapeutic biomarkers. Cataract, the loss of transparency of eye lens, is a disease of protein aggregation. There are several factors contributing to the stability in protein conformation. Age-related changes in lipid composition could be a contributing factor for altered protein–lipid interaction leading to protein aggregation and cataract. Keeping this in view, in the present study, fatty acid profiling from different age groups of lenses was carried out, using a freshwater catfish as the model. Total lipids were extracted from lenses of three different age groups of fishes (young, adult, and aged) and fatty acid methyl esters (FAME) were prepared and FAME analysis was carried out using gas chromatography–mass spectrometry. The results showed that three fatty acids viz. heneicosylic acid (C21), docosahexaenoic acid (C22:6), nervonic acid (C24:1) which were not present in the adult lens, appeared in the aged lens. On the other hand, eicosenoic acid (C20:1) present in the adult lens was found to be absent in the aged lens. The appearance or disappearance of these fatty acids can possibly serve as biomarkers of aging lens which is the most vulnerable stage for cataract development.

Keywords: Lipid biomarkers; Eye lens; Aging; GC–MS; Lipid profiling; Lipidomics

Purification, Characterization, and Specificity Determination of a New Serine Protease Secreted by Penicillium waksmanii by Eduardo Rezende Graminho; Ronivaldo Rodrigues da Silva; Tatiana Pereira de Freitas Cabral; Eliane Candiani Arantes; Nathalia Gonsales da Rosa; Luiz Juliano; Debora Noma Okamoto; Lilian Caroline Gonçalves de Oliveira; Marcia Yuri Kondo; Maria Aparecida Juliano; Hamilton Cabral (pp. 201-214).

The purpose of this work was to purify a protease from Penicillium waksmanii and to determine its biochemical characteristics and specificity. The extracellular protease isolated that was produced by P. waksmanii is a serine protease that is essential for the reproduction and growth of the fungus. The protease isolated showed 32 kDa, and has optimal activity at pH 8.0 and 35 °C towards the substrate Abz-KLRSSKQ-EDDnp. The protease is active in the presence of CaCl₂, KCl, and BaCl, and partially inhibited by CuCl₂, CoCl₂ and totally inhibited by AlCl₃ and LiCl. In the presence of 1 M urea, the protease remains 50 % active. The activity of the protease increases 60 % when it is exposed to 0.4 % nonionic surfactant-Triton X-100 and loses 10 % activity in the presence of 0.4 % Tween-80. Using fluorescence resonance energy transfer analysis, the protease showed the most specificity for the peptide Abz-KIRSSKQ-EDDnp with k _cat/K _m of 10,666 mM⁻¹ s⁻¹, followed by the peptide Abz-GLRSSKQ-EDDnp with a k _cat/K _m of 7,500 mM⁻¹ s⁻¹. Basic and acidic side chain-containing amino acids performed best at subsite S₁. Subsites S₂, S₃, S^′ ₂, and S^′ ₁, S^′ ₃ showed a preference for binding for amino acids with hydrophobic and basic amino acid side chain, respectively. High values of k _cat/K _m were observed for the subsites S₂, S₃, and S^′ _2. The sequence of the N-terminus (ANVVQSNVPSWGLARLSSKKTGTTDYTYD) showed high similarity to the fungi Penicillium citrinum and Penicillium chrysogenum, with 89 % of identity at the amino acid level.

Keywords: Protease; Fungal enzymes; Purification; Specificity; N-terminal Penicillium

The Constitutive Production of Pectinase by the CT1 Mutant of Penicillium Occitainis is Modulated by pH by Zamen Ben Romdhane; Hajer Tounsi; Azza Hadj-Sassi; Noomen Hadj-Taieb; Ali Gargouri (pp. 215-227).

The aim of the present study was to investigate pectinases production by CT1 mutant of Penicillium occitanis on glucose based media. Two main groups of pectinases were followed: lyases (pectin and pectate lyases) and hydrolases (polygalacturonases and polymethylgalacturonases). When cultivated in different liquid media, where either the starting glucose concentration or the nature of nitrogen sources used was varied, the CT1 mutant secreted either lyases or hydrolases. In fact, the pH of these various media seemed to correlate with the activity produced: The lyases were highly and exclusively produced at neutral or alkaline ambient pH, whereas hydrolases were highly produced on acidic ambient pH. Such conclusion was confirmed by following pectinase production in the same culture medium (with the same glucose concentration and the same nitrogen source) set at two initial pH of 4 and 7. Altogether, these results suggest that the pectinases control by PacC signaling pathway of P. occitanis should resemble to that of Aspergillus and its ability to “activate the expression of alkaline-expressed genes and repress acid-expressed genes” remains intact in the CT1 over-producing and constitutive strain. Enzymes produced at acidic pH (hydrolases) and at neutral pH (lyases) were applied in the hydrolysis of orange peel and gave results comparable to commercial enzymes.

Keywords: Pectinase; Lyase; Hydrolase; Constitutive mutant; pH; Orange peel hydrolysis

Mining of EST-SSR Markers of Musa and Their Transferability Studies Among the Members of Order the Zingiberales by S. Backiyarani; S. Uma; P. Varatharj; M. S. Saraswathi (pp. 228-238).

Expressed sequence tags (ESTs) databases of 11 Musa complementary DNA libraries were retrieved from National Center of Biotechnology Information and used for mining simple sequence repeats (SSRs). Out of 21,056 unique ESTs, SSR regions were found only in 5,158 ESTs. Among these SSR containing ESTs, the occurrence of trinucleotide repeats are the most abundant followed by mono-, di-, tetra-, hexa-, and pentanucleotides. Moreover, this study showed that the rate of class II SSRs (<20 nucleotides) was higher than the class I SSRs (<20 nucleotides), and proportion of class I and II SSRs as abundant for tri-repeats. As a representative sample, primers were synthesized for 24 ESTs, carrying >12 nucleotides of SSR region, and tested among the various genomic group of Musa accessions. The result showed that 88 % of primers were functional primers, and 43 % are showing polymorphism among the Musa accessions. Transferability studies of Musa EST-SSRs among the genera of the order Zingiberales exhibited 100 and 58 % transferability in Musaceae and Zingiberaceae, respectively. The sequence comparison of SSR regions among the different Musa accessions confirmed that polymorphism is mainly due to the variation in repeat length. High percentage of cross-species, cross-genera, and cross-family transferability also suggested that these Musa EST-SSR markers will be a valuable resource for the comparative mapping by developing COS markers, in evolutionary studies and in improvement of the members of Zingiberaceae and Musaceae.

Keywords: Musa sp.; EST-SSR markers; Transferability; Zingiberaceae

Preparation of Hyaluronic Acid Micro-Hydrogel by Biotin–Avidin-Specific Bonding for Doxorubicin-Targeted Delivery by Yuan Cui; Yanhui Li; Qian Duan; Toyoji Kakuchi (pp. 239-249).

Hyaluronic acid is a naturally ionic polysaccharide with cancer cell selectivity. It is an ideal candidate material for delivery of anticancer agents. In this study, hyaluronic acid (HA) micro-hydrogel loaded with anticancer drugs was prepared by the biotin–avidin system approach. Firstly, carboxyl groups on HA were changed into amino groups with adipic acid dihydrazide (ADH) to graft with biotin by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride named as HA–biotin. When HA–biotin solution mixed with doxorubicin hydrochloride (DOX·HCl) was blended with neutravidin, the micro-hydrogels would be formed with DOX loading. If excess biotin was added into the microgel, it would be disjointed, and DOX will be released quickly. The results of the synthesis procedure were characterized by ¹H-NMR and FTIR; ADH and biotin have been demonstrated to graft on the HA molecule. A field emission scanning electron microscope was used to observe morphologies of HA micro-hydrogels. Furthermore, the in vitro DOX release results revealed that the release behaviors can be adjusted by adding biotin. Therefore, the HA micro-hydrogel can deliver anticancer drugs efficiently, and the rate of release can be controlled by biotin-specific bonding with the neutravidin. Consequently, the micro-hydrogel will perform the promising property of switching in the specific site in cancer therapy.

Keywords: Hyaluronic acid; Biotin–avidin; Micro-hydrogel; Drug delivery; DOX

Selection of DNA Aptamers with Affinity for Pro-gastrin-Releasing Peptide (proGRP), a Tumor Marker for Small Cell Lung Cancer by Masayasu Mie; Tetsuro Kai; Thao Le; Anthony E. G. Cass; Eiry Kobatake (pp. 250-255).

Aptamers are single-strand oligonucleotides that are generated by the systemic evolution of ligands by exponential enrichment (SELEX) technique and that can bind to target molecules specifically. However, only a few aptamers have been developed to date against tumor markers. To utilize aptamers for tumor diagnosis, a variety of aptamers are required. Here, a single-stranded DNA aptamer specific for pro-gastrin-releasing peptide (proGRP), a marker for small cell lung cancer, was selected using SELEX. After selection, identical sequences were found in the DNA library. This sequence was selected and its binding affinity to proGRP was evaluated using surface plasmon resonance.

Keywords: Aptamer; SELEX; proGRP; Small cell lung cancer

Biochemical Basis of Mercury Remediation and Bioaccumulation by Enterobacter sp. EMB21 by Arvind Sinha; Sumit Kumar; Sunil Kumar Khare (pp. 256-267).

The aims of this study were to isolate metal bioaccumulating bacterial strains and to study their applications in removal of environmental problematic heavy metals like mercury. Five bacterial strains belonging to genera Enterobacter, Bacillus, and Pseudomonas were isolated from oil-spilled soil. Among these, one of the strains Enterobacter sp. EMB21 showed mercury bioaccumulation inside the cells simultaneous to its bioremediation. The bioaccumulation of remediated mercury was confirmed by transmission electron microscopy and energy dispersive X-ray. The mercury-resistant loci in the Enterobacter sp. EMB21 cells were plasmid-mediated as confirmed by transformation of mercury-sensitive Escherichia coli DH5α by Enterobacter sp. EMB21 plasmid. Effect of different culture parameters viz-a-viz inoculum size, pH, carbon, and nitrogen source revealed that alkaline pH and presence of dextrose and yeast extract favored better remediation. The results indicated the usefulness of Enterobacter sp. EMB21 for the effective remediation of mercury in bioaccumulated form. The Enterobacter sp. EMB21 seems promising for heavy metal remediation wherein the remediated metal can be trapped inside the cells. The process can further be developed for the synthesis of valuable high-end functional alloy, nanoparticles, or metal conjugates from the metal being remediated.

Keywords: Enterobacter sp.; Mercury bioremediation; Mercury reductase

Inactivation of Recombinant Human Brain-Type Creatine Kinase During Denaturation by Guanidine Hydrochloride in a Macromolecular Crowding System by Yong-Qiang Fan; Hong-Jian Liu; Chang Li; Yu-Shi Luan; Jun-Mo Yang; Yu-Long Wang (pp. 268-280).

In this study, we quantitatively examined the effects of the macromolecular crowding agents, polyethylene glycol 2000 (PEG 2000) and dextran 70, on guanidine hydrochloride (GdnHCl)-induced denaturation of recombinant human brain-type creatine kinase (rHBCK). Our results showed that both PEG 2000 and dextran 70 had a protective effect on the inactivation of rHBCK induced by 0.5 M GdnHCl at 25 °C. The presence of 200 g/L PEG 2000 resulted in the retention of 35.33 % of rHBCK activity after 4 h of inactivation, while no rHBCK activity was observed after denaturation in the absence of macromolecular crowding agents. The presence of PEG 2000 and dextran 70 at a concentration of 100 g/L could decelerate the k ₂ value of the slow track to 21 and 33 %, respectively, in comparison to values obtained in the absence of crowding agents. Interestingly, inactivation of rHBCK in the presence of 200 g/L PEG 2000 followed first-order monophasic kinetics, with an apparent rate constant of 8 × 10⁻⁵ s⁻¹. The intrinsic fluorescence results showed that PEG 2000 was better than dextran 70 at stabilizing rHBCK conformation. In addition, the results of the phase diagram indicate that more intermediates may be captured when rHBCK is denatured in a macromolecular crowding system. Mixed crowding agents did not produce better results than single crowding agents, but the protective effects of PEG 2000 on the inactivation and unfolding of rHBCK tended to increase as the ratio of PEG 2000 increased in the mixed crowding agent solution. Though it is not clear which crowding agents more accurately simulated the intracellular environment, this study could lead to a better understanding of protein unfolding in the intracellular environment.

Keywords: Human brain-type creatine kinase; Macromolecular crowding; Guanidine; Hydrochloride denature; Inactivation; Kinetics

Nonspecific Effect of Double-Stranded (ds) RNA on Prophenoloxidase (proPO) Expression in Penaeus monodo n by Anutosh Paria; S. S. Greeshma; Aparna Chaudhari; M. Makesh; C. S. Purushothaman; K. V. Rajendran (pp. 281-289).

RNA interference-mediated silencing is an effective way of controlling white spot syndrome virus (WSSV). However, the effect of RNAi on the innate immune mechanism is not well understood. Prophenoloxidase (proPO) is an important component of the shrimp innate immunity. In the present study, nonspecific effect of two double-stranded (ds)RNA-expressing constructs, one targeting vp28 gene of WSSV (pCMV-VP28-LH) and another targeting green fluorescent protein (GFP) (pCMV-GFP-LH) on proPO2 gene expression, is investigated. mRNA expression levels of proPO2 in hemocytes of DNA construct-injected shrimp were estimated using real-time PCR with elongation factor 1-α as internal control. Empty vector (pcDNA)-injected shrimp were used as experimental control. In pCMV-VP28-LH-injected shrimp, proPO2 showed significant upregulation until 48 h post-injection (p.i.). Similarly, pCMV-GFP-LH-injected animals showed high levels of expression until 72 h p.i. WSSV-challenged animals, compared to pcDNA-injected control group, showed no significant change in expression of the gene until 24 h. However, an increased expression was noticed at 48 h p.i. Our results suggest that neither the plasmids nor the long hairpin RNA expressed by the constructs has any nonspecific silencing effect on the proPO2 expression. On the contrary, the consistent upregulation of proPO2 observed in shrimp injected with dsRNA at early time-points indicates the possibility of nonspecific protection against WSSV infection.

Keywords: Double-stranded RNA; RNAi; dsRNA construct; Prophenoloxidase; Penaeus monodon ; White spot syndrome virus (WSSV); Shrimp

Ionic Liquid-Induced Structural and Activity Changes in Hen Egg White Lysozyme by Le-Ping Dang; Wen-Zhi Fang; Yan Li; Qian Wang; Hua-Zhi Xiao; Zhan-Zhong Wang (pp. 290-300).

Lysozyme crystals in the presence of 1-butyl-3-methylimidazolium tetrafluoroborate ([C₄mim]BF₄), 1-butyl-3-methylimidazolium chloride ([C₄mim]Cl), 1-butyl-3-methylimidazolium bromide([C₄mim]Br), and 1,3-dimethylimidazolium iodine([dmim]I) were prepared, and the influence of ionic liquids (ILs) on the structure and activity change of lysozyme was investigated. Fourier transform infrared spectroscopy revealed the major secondary structures of α-helix and β-sheet for lysozyme. It was interesting to note that increases of the band near 2,935 and 1,656 cm⁻¹ from Raman spectroscopy are attributed to the unfolding of lysozyme molecules. A shift in amide III from 1,230 to 1,270 cm⁻¹ in adding [dmim]I occurs, indicating a transformation from β-sheet to random coil. With regard to adding [C₄mim]BF₄, [C₄mim]Cl, and [C₄mim]Br, α-helix and β-sheet are the predominant structures for lysozyme. The activity study showed that the ILs used brought a positive effect. Especially, [dmim]I leads to a drastic increase in relative activity, and its value reaches 50 %.

Keywords: Spectral studies; Lysozyme; Structural changes; Activity; Ionic liquids

Production of Metabolites with Antioxidant and Emulsifying Properties by Antarctic Strain Sporobolomyces salmonicolor AL₁ by Stela Dimitrova; Kostantsa Pavlova; Ludmil Lukanov; Elena Korotkova; Ekaterina Petrova; Plamen Zagorchev; Margarita Kuncheva (pp. 301-311).

The Sporobolomyces salmonicolor AL₁ Antarctic strain was cultivated and two bioproducts were obtained: exopolysaccharide and biomass. The biologically active substances ergosterol, torularhodin, torulene, β-carotene and CoQ₁₀ were extracted from the biomass and were quantified as follows: ergosterol 5.2 ± 0.2 mg/g, torularhodin 458.3 ± 24.5 μg/g, torulene 273.7 ± 14.5 μg/g, β-carotene 129.2 ± 7.3 μg/g and coenzyme Q₁₀ (CoQ₁₀) 236.1 ± 12.1 μg/g. Their antioxidant activity was estimated according to the cathode voltammetry method. The most pronounced antioxidant activity (according to trolox) was exhibited by β-carotene 3.78, followed by CoQ₁₀ 3.60, both of them being the main contributors to the total extract activity of 3.19. The biologically active metabolites in combination with exoglucomannan as emulsifier were used for the creation of model emulsion systems characterised by great stability. The absorption of UVA rays by the model emulsions was studied.

Keywords: S. salmonicolor AL₁ ; Carotenoids; Coenzyme Q₁₀ ; Ergosterol; Antioxidant activity; Exopolysaccharide

Kinetic Modeling of Fermentative Production of 1, 3-Propanediol by Klebsiella pneumoniae HR526 with Consideration of Multiple Product Inhibitions by Lu He; Xuebing Zhao; Keke Cheng; Yan Sun; Dehua Liu (pp. 312-326).

During the fermentative production of 1, 3-propanediol (1,3-PD), the multiple product inhibitions cannot be negligible to accurately describe the kinetics of fermentation process. A kinetic model for fermentative production of 1,3-PD by Klebsiella pneumoniae HR526 with glycerol as carbon source under aerobic condition was proposed. The inhibitions of multiple products including 1,3-PD, 2, 3-butanediol (2,3-BD), acetate, and succinate were considered in the model. It was found that 1,3-PD, 2,3-BD, and acetate showed strong inhibitions to cell growth depending on their concentrations. The kinetic model was relatively accurate to predict the experimental data of batch, fed-batch, and continuous fermentations. The model thus can serve as a tool for further controlling and optimizing the fermentation process.

Keywords: 1, 3-propanediol; Kinetic modeling; Fermentation; Product inhibition; Klebsiella pneumoniae

Kinetic Modeling of Esterification Reaction of Surfactin-C₁₅ in Methanol Solution by Yue Zhao; Shi-Zhong Yang; Bo-Zhong Mu (pp. 327-337).

Surfactin in methanol solution with acid would be spontaneously esterified into the mono- or dimethyl ester surfactin even at a temperature as low as 4 °C because there were two free carboxyl groups in the peptide loop of surfactin. Using trifluoroacetic acid as the catalyst, the esterification and the contents change in surfactin-C₁₅, mono- and dimethyl ester surfactin-C₁₅ with time were investigated at 4, 25, and 45 °C, respectively. The kinetic model was established for prediction of the esterification degree under experimental conditions. At 4, 25, and 45 °C, more than 10 % of the surfactin-C₁₅ in methanol solution in the presence of 0.05 % trifluoroacetic acid was changed into the esterified surfactin-C₁₅ after 37.6, 14.1, and 7.4 h, respectively. The maximum of intermediate of the mono-methyl ester surfactin-C₁₅ was observed at 4, 25, and 45 °C after 25, 10, and 5 days, respectively. Our results indicated that the time for preparation should be strictly controlled to avoid an unexpected esterification of surfactin during its storage and experimental treatment, and the kinetic results could be adopted as the reference condition for preparation of monomethyl ester surfactin-C₁₅.

Keywords: Surfactin; Esterification; Mono-methyl ester; Kinetic; Lipopeptide

Combining Genome Shuffling and Interspecific Hybridization Among Streptomyces Improved ε-Poly-l-Lysine Production by Shu Li; Xusheng Chen; Chuanliang Dong; Fulin Zhao; Lei Tang; Zhonggui Mao (pp. 338-350).

Here we first improved the ε-PL productivity in five species of wild-type strains in Streptomyces (Streptomyces padanus, Streptomyces griseofuscus, Streptomyces graminearus, Streptomyces hygroscopicus, and Streptomyces albulus) by genome shuffling. Then all the shuffled strains were suffered from an interspecific hybridization through stochastic protoplast fusion. One hybrid designated FEEL-1 was selected by morphology and spore color with ε-PL production of 1.12 g/L in shake flask, about 2.75-fold higher than that in wild types. The ε-PL production of FEEL-1 was then obtained as 24.5 g/L in fed-batch fermentation, which was 63–81 % higher than those in shuffled strains. Random amplified polymorphic DNA revealed that FEEL-1 was probably hybridized from S. padanus, S. griseofuscus, and S. albulus. Activities of several enzymes in FEEL-1 (hexokinase, phosphoenolpyruvate carboxylase, aspartokinase, and citrate synthase) were more active than those in shuffled strains, which was a possible reason for the enhancement of ε-PL production. This research highlights the importance of genome shuffling along with interspecific hybridization as a new breeding strategy for improving phenotype of industrial strains.

Keywords: Genome shuffling; ε-poly-l-lysine; Hybridization; Fermentation; RAPD

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.169, #1)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.169, #1)