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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.160, #6)

Oat (Avena sativa) Seed Extract as an Antifungal Food Preservative Through the Catalytic Activity of a Highly Abundant Class I Chitinase by Hans Peter Sørensen; Lone Søvad Madsen; Jørgen Petersen; Jesper Tapdrup Andersen; Anne Maria Hansen; Hans Christian Beck (pp. 1573-1584).

Extracts from different higher plants were screened for the ability to inhibit the growth of Penicillium roqueforti, a major contaminating species in industrial food processing. Oat (Avena sativa) seed extracts exhibited a high degree of antifungal activity and could be used directly on rye bread to prevent the formation of P. roqueforti colonies. Proteins in the oat seed extracts were fractionated by column chromatography and proteins in fractions containing antifungal activity were identified by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and database searches. Identified antifungal candidates included thaumatin-like proteins, 1,3-beta-glucanase, permatin precursor, pathogenesis-related protein type 1, and chitinases of class I and II. Class I chitinase could be specifically removed from the extracts and was found to be indispensable for 50% of the P. roqueforti inhibiting activity. The purified class I chitinase has a molecular weight of approximately 34 kDa, optimal chitinase activity at pH 7, and exists as at least two basic isoforms (pI values of 7.6 and 8.0). Partial sequencing of the class I chitinase isoforms by LC-MS/MS revealed a primary structure with high similarity to class I chitinases of wheat (Triticum aestivum), barley (Hordeum vulgare), and rye (Secale cereale). Oat, wheat, barley, and rye seed extracts were compared with respect to the abundance of the class I chitinase and decrease in antifungal activity when class I chitinase is removed. We found that the oat seed class I chitinase is at least ten times more abundant than the wheat, barley, and rye homologs and that oat seed extracts are highly active toward P. roqueforti as opposed to extracts of other cereal seeds.

Keywords: Oat; Chitinase; Protein purification; Antifungal; Mass spectrometry

Biochemical Properties and PCR Performance of a Family B DNA Polymerase from Hyperthermophilic Euryarchaeon Thermococcus peptonophilus by Jong Il Lee; Yun Jae Kim; Heejin Bae; Sung Suk Cho; Jung-Hyun Lee; Suk-Tae Kwon (pp. 1585-1599).

The Thermococcus peptonophilus (Tpe) DNA polymerase gene was expressed under the control of the T7lac promoter on pET-22b(+) in Escherichia coli BL21-CodonPlus(DE3)-RIL in order to fully elucidate its biochemical properties and evaluate its feasibility in polymerase chain reaction (PCR) application. The expressed enzyme was then purified by heat treatment followed by two steps of column chromatography after which optimum pH and temperature of the enzyme were evaluated to be 7.0 and 75 °C, respectively. The optimal buffer for PCR with Tpe DNA polymerase consisted of 50 mM Tris–HCl (pH 8.0), 2 mM MgCl₂, 80 mM KCl, and 0.02% Triton X-100. Tpe DNA polymerase revealed a 3.6-fold higher fidelity (3.37 × 10⁻⁶) than Taq DNA polymerase (12.13 × 10⁻⁶) and performed significantly more efficiently in PCR amplification than both Taq and Pfu DNA polymerases. Ratios of 31:1 of Taq to Tpe DNA polymerases allowed PCR amplification of targets up to 15 kb in length with a 2.2-fold higher fidelity than Taq DNA polymerase. The results of the PCR experiments indicate that Tpe DNA polymerase may provide a higher fidelity DNA amplification in a shorter reaction time.

Keywords: DNA polymerase; Thermococcus peptonophilus ; Polymerase chain reaction; PCR efficiency; PCR fidelity; Archaea

Rapid Screening of Serum-Free Media for the Growth of Adherent Vero Cells by Using a Small-Scale and Non-invasive Tool by Emma Petiot; Frantz Fournier; Cécile Gény; Hervé Pinton; Annie Marc (pp. 1600-1615).

The paper proposes a rapid screening method for a first step improvement of an animal component-free medium dedicated to the growth of the anchorage-dependent Vero cell line. A new, rapid, and non-invasive technique is presented to specifically monitor cultures of adherent cells in 96-well plates. The operating conditions of an image analyzer are adapted to take into account the decrease of cell size when the attached cell density increases. An experimental design is carried out to assess the influence of ten component groups in the original medium. Two groups including protein extracts, growth factor, insulin, glucose, and pyruvate show significant positive effects. The groups with vitamins and molecules related to nitrogenous bases display a less pronounced influence. The mixture of amino acids, B₁ vitamin, magnesium sulfate, and sodium phosphate as well as the couple sodium citrate and ferric chloride lead to a downward trend. The screening results are proved to be scalable in stirred cultures with cells on microcarriers. An improved serum-free medium, with some component groups being removed or added, can be rapidly formulated to reach respectively similar or 1.6 times higher cell density than in the original medium. The results from this global approach could be helpful to further focus experiments on identified medium components.

Keywords: Adherent Vero cells; Serum-free medium; Small-scale screening; Non-invasive cell counting; Microcarriers

Biosynthesis of Indigo Dye by Newly Isolated Naphthalene-Degrading Strain Pseudomonas sp. HOB1 and its Application in Dyeing Cotton Fabric by Hilor Pathak; Datta Madamwar (pp. 1616-1626).

Indigo is one of the oldest dyes manufactured chemically and is mostly used in textile, food, and pharmaceutical industries. However, owing to the environmental hazards posed by the chemical production, the present scenario in the field stipulates a biosynthesis alternative for indigo production. The present study describes an indigenously isolated naphthalene-degrading strain Pseudomonas sp. HOB1 producing a blue pigment when indole was added in the growth medium. This blue pigment was analyzed by high-pressure thin-layer chromatography and other spectroscopic techniques which revealed it to be the indigo dye. Pseudomonas sp. HOB1 showed ability to produce 246 mg indigo liter⁻¹ of the medium. The K _m for the enzyme naphthalene dioxygenase which is involved in indigo formation is 0.3 mM, and V _max was as high as 50 nmol min⁻¹ mg dry biomass⁻¹. The bacterial indigo dye was further successfully applied for dyeing cotton fabrics. The high indigo productivity of Pseudomonas sp. HOB1 using naphthalene as growth substrate and its applicability on cotton fabrics, therefore, stems the probability of using this culture for commercial indigo production.

Keywords: Dyeing; Indole; Indigo production; Naphthalene degradation; Naphthalene dioxygenase (NDO); Pseudomonas sp.

A Novel Endoglucanase (Cel9P) From a Marine Bacterium Paenibacillus sp. BME-14 by Xiaoyu Fu; Pengfu Liu; Ling Lin; Yuzhi Hong; Xiaoluo Huang; Xin Meng; Ziduo Liu (pp. 1627-1636).

By constructing a genomic library, an endoglucanase gene (cel9P) was cloned from Paenibacillus sp. BME-14 which was isolated from the sea. It had an open-reading frame of 1,629 bp, encoding a peptide of 542-amino acid residue with a calculated molecular mass of 60 kDa. The enzyme showed the highest amino acid identity of 52% with other known endoglucanases and had a C-terminal catalytic domain belonging to the glycosyl hydrolases family 9. The optimum pH and temperature for enzymatic activity was pH 6.5 and 35 °C. The metal ions of Ca²⁺, Mg²⁺, and Mn²⁺ had a positive effect on the activity while Hg²⁺, Cu²⁺, and EDTA had a negative effect. Notably, Cel9P had 65% of the maximal activity at 5 °C. Based on the special characteristic of Cel9P, it had a potential significance for study of cold-active mechanism and industry applications.

Keywords: Paenibacillus sp.; Sea bacteria; Endoglucanase; Glycoside hydrolase family 9; Alkali tolerant

Increased Saccharification Yields from Aspen Biomass Upon Treatment with Enzymatically Generated Peracetic Acid by Shona Duncan; Qing Jing; Adrian Katona; Romas J. Kazlauskas; Jonathan Schilling; Ulrike Tschirner; Waleed Wafa AlDajani (pp. 1637-1652).

The recalcitrance of lignocellulosic biomass to enzymatic release of sugars (saccharification) currently limits its use as feedstock for biofuels. Enzymatic hydrolysis of untreated aspen wood releases only 21.8% of the available sugars due primarily to the lignin barrier. Nature uses oxidative enzymes to selectively degrade lignin in lignocellulosic biomass, but thus far, natural enzymes have been too slow for industrial use. In this study, oxidative pretreatment with commercial peracetic acid (470 mM) removed 40% of the lignin (from 19.9 to 12.0 wt.% lignin) from aspen and enhanced the sugar yields in subsequent enzymatic hydrolysis to about 90%. Increasing the amount of lignin removed correlated with increasing yields of sugar release. Unfortunately, peracetic acid is expensive, and concentrated forms can be hazardous. To reduce costs and hazards associated with using commercial peracetic acid, we used a hydrolase to catalyze the perhydrolysis of ethyl acetate generating 60–70 mM peracetic acid in situ as a pretreatment to remove lignin from aspen wood. A single pretreatment was insufficient, but multiple cycles (up to eight) removed up to 61.7% of the lignin enabling release of >90% of the sugars during saccharification. This value corresponds to a predicted 581 g of fermentable sugars from 1 kg of aspen wood. Improvements in the enzyme stability are needed before the enzymatically generated peracetic acid is a commercially viable alternative.

Keywords: Perhydrolysis; Pseudomonas fluorescens esterase; Peracetic acid; Lignocellulosic biomass; Aspen; Pretreatment; Saccharification; Enzyme

Fermentation Performance and Structure Characteristics of Xanthan Produced by Xanthomonas campestris with a Glucose/Xylose Mixture by Zhiguo Zhang; Hongzhang Chen (pp. 1653-1663).

The ability of Xanthomonas campestris to convert glucose and xylose to xanthan and the structure of xanthan derived from the glucose/xylose mixture media are important when the lignocelluloses hydrolysate was used in xanthan production. In this paper, the features related to xanthan fermentation in the glucose/xylose mixture media and the structures of xanthan derived from the mixture media were studied. Glucose was the preferred carbon source to produce xanthan while xylose was also utilized with a very low consumption rate. When the fraction of glucose decreased from 100% to 25%, the glucose consumption rate and xanthan production rate reduced from 0.44 g L⁻¹ h⁻¹ to 0.25 g L⁻¹ h⁻¹ and 0.21 g L⁻¹ h⁻¹ to 0.04 g L⁻¹ h⁻¹ respectively while xylose was consumed at a very stable rate (0.053–0.060 g L⁻¹ h⁻¹). On the other hand, when the xylose fraction increased from 0% to 50%, pyruvate and acetate content of xanthan increased from 2.43% to 3.78% and 2.55% to 7.05%. The existence of xylose also led to higher average molecular weight. Therefore, it could be concluded that xylose was not efficiently utilized by X. campestris to produce xanthan. The concentration of glucose rather than the total sugar was the main factor to determine the xanthan production. But xylose was helpful to improve the quality of xanthan.

Keywords: Xanthan gum; Glucose; Xylose; Xanthomonas campestris ; Molecular structure; Lignocellulose

Sequential Injection Kinetic Flow Assay for Monitoring Glycerol in a Sugar Fermentation Process by Saccharomyces cerevisiae by Karina B. Hueso Domínguez; Ildikó V. Tóth; M. Renata S. Souto; Filipa Mendes; Cándido García De María; Isabel Vasconcelos; António O. S. S. Rangel (pp. 1664-1673).

A sequential injection system to monitor glycerol in a Saccharomyces cerevisiae fermentation process was developed. The method relies on the rate of formation of nicotinamide adenine dinucleotide in its reduced form (NADH, measured spectrophotometrically at 340 nm) from the reaction of glycerol with NAD⁺ cofactor, catalysed by the enzyme glycerol dehydrogenase present in solution. This procedure enables the determination of glycerol between 0.046 and 0.46 g/l, (corresponding to yeast fermentation samples with concentrations up to 50 g/l) with good repeatability (relative standard deviation for n = 10 lower than 2.2% for three different samples) at a sampling frequency of 25/h. The detection and quantification limits using a miniaturised spectrophotometer were 0.13 and 0.44 mM, respectively. Reagent consumption was of 0.45 μmol NAD⁺ and 1.8 μg enzyme per assay, and the waste production was 2.8 ml per determination. Results obtained for samples were in agreement with those obtained with a high-performance liquid chromatography method.

Keywords: Sequential injection analysis; Glycerol; Glucose fermentation; Saccharomyces cerevisiae ; Kinetic method; Enzymatic assay

Heterotrophic Culture of Chlorella protothecoides in Various Nitrogen Sources for Lipid Production by Y. Shen; W. Yuan; Z. Pei; E. Mao (pp. 1674-1684).

The influences of urea, yeast extract, and nitrate as the nitrogen source on heterotrophic growth of four strains of Chlorella protothecoides were investigated in 9-day feed-batch cultures. Biomass dry weight concentration (DWC) and lipid yield (LY) of the four strains in all media were compared. The highest LY in 9 days was 654 mg/L/day by UTEX 255 in 2.4 g/L KNO₃ medium with a biomass DWC of 11.7 g/L and lipid content of 50.5%. Using green autotrophic seeds instead of yellow heterotrophic seeds improved the biomass DWC (13.1 vs. 11.7 g/L), LY (850 vs. 654 mg/L/day), and lipid to glucose consumption ratio (0.607 vs. 0.162). Moreover, 17.0 g/L DWC and 489 mg/L/day LY were obtained from the sequentially mixed-nitrogen medium, and the lipid to glucose consumption ratio was improved to 0.197 from 0.162 in 2.4 g/L nitrate medium and from 0.108 in 4.2 g/L yeast extract medium in the first batch.

Keywords: Algae; Biofuel; Nitrogen; Chlorella protothecoides ; Lipid

Toxicity of Methanol and Formaldehyde Towards Saccharomyces cerevisiae as Assessed by DNA Microarray Analysis by Daisuke Yasokawa; Satomi Murata; Yumiko Iwahashi; Emiko Kitagawa; Ryoji Nakagawa; Tazusa Hashido; Hitoshi Iwahashi (pp. 1685-1698).

To assess the toxicity of the C1 compounds methanol and formaldehyde, gene expression profiles of treated baker’s yeast were analyzed using DNA microarrays. Among approximately 6,000 open reading frames (ORFs), 314 were repressed and 375 were induced in response to methanol. The gene process category “energy” comprised the greatest number of induced genes while “protein synthesis” comprised the greatest number of repressed genes. Products of genes induced by methanol were mainly integral membrane proteins or were localized to the plasma membrane. A total of 622 and 610 ORFs were induced or repressed by formaldehyde, respectively. More than one-third of the genes found to be strongly repressed by formaldehyde belonged to the “protein synthesis” functional category. Conversely, genes in the subcategory of “nitrogen, sulfur, and selenium metabolism” within “metabolism” and in the category of “cell rescue, defense, and virulence” were up-regulated by exposure to formaldehyde. Our data suggest that membrane structure is a major target of methanol toxicity, while proteins were major targets of formaldehyde toxicity.

Keywords: Microarray; Methanol; Formaldehyde; Toxicity; Saccharomyces cerevisiae

Role of Human Liver Microsomes in In Vitro Metabolism of Drugs—A Review by Sepuri Asha; Maravajhala Vidyavathi (pp. 1699-1722).

Drug metabolism studies are essential and necessary during the evaluation of drugs. This review discusses the in vitro human liver models to estimate the drug metabolic fates in vivo. Different approaches are provided and emphasis is placed on the potential of human liver microsomes for drug metabolism and inhibition studies. The methodology for these studies using human liver microsomes, applications of human liver microsomes, and the drugs studied by human liver microsomes are listed. Human liver microsomes represent a critical experimental model for the evaluation of drug metabolites with a high probability of clinical success.

Keywords: Biotransformation; CYP450; Drug; Human liver microsomes; In vitro metabolism study; Metabolism; Metabolites

In Silico Identification of Significant Detrimental Missense Mutations of EGFR and Their Effect with 4-Anilinoquinazoline-Based Drugs by R. Rajasekaran; Rao Sethumadhavan (pp. 1723-1733).

In this work, we identified the detrimental missense mutations (point mutations) in epidermal growth factor receptor (EGFR) and its binding efficiency with the inhibitors namely Erlotinib, Gefitinib, and Lapatinib. Out of 26 point mutations on EGFR, 12 point mutations were commonly less stable, deleterious, and damaged as shown by all the three servers, I-Mutant2.0, SIFT, and PolyPhen. Further, we modeled 12 mutants and superimposed with the native EGFR to get RMSD values. Docking studies showed that Erlotinib had lesser binding affinity against both native and all the 12 mutants. Gefitinib had maximum binding affinity only with two mutants, viz., R748P and L858R. Lapatinib had maximum binding affinity with both native and other 10 mutants. Based on our computational analysis, we recommend that the combined administration of Gefitinib and Lapatinib could give a better effect in combating the disease.

Keywords: Point mutation; EGFR; Erlotinib; Gefitinib; Lapatinib

Scale-up of Thermally Dried Kefir Production as Starter Culture for Hard-Type Cheese Making: An Economic Evaluation by Athanasios A. Koutinas; Argyro Bekatorou; Eleftheria Katechaki; Dimitra Dimitrellou; Nikolaos Kopsahelis; Harris Papapostolou; Panayiotis Panas; Kostas Sideris; Mihalis Kallis; Loulouda A. Bosnea; Dionisis Koliopoulos; Panayiotis Sotiropoulos; Ageliki Panteli; Yiannis Kourkoutas; Maria Kanellaki; Magdalini Soupioni (pp. 1734-1743).

This paper concerns the effect of thermal-drying methodology on the investment cost for dried kefir cells production in order to be used as starter culture in cheese manufacturing. Kefir cells were produced at pilot plant scale using a 250-L bioreactor and whey as the main substrate. Kefir cells were subsequently dried in a thermal dryer at 38 °C and used as a starter culture in industrial-scale production of hard-type cheeses. The use of thermally dried kefir as starter culture accelerated ripening of cheeses by increasing both lipolysis and fermentation rate as indicated by the ethanol, lactic acid, and glycerol formation. Additionally, it reduced coliforms and enterobacteria as ripening proceeded. This constituted the basis of developing an economic study in which industrial-scale production of thermally dried kefir starter culture is discussed. The industrial design involved a three-step process using three bioreactors of 100, 3,000, and 30,000 L for a plant capacity of 300 kg of thermally dried kefir culture per day. The cost of investment was estimated at 238,000 €, which is the 46% of the corresponding cost using freeze-drying methodology. Production cost was estimated at 4.9 €/kg of kefir biomass for a 300-kg/day plant capacity, which is the same as with the corresponding cost of freeze-dried cells. However, the estimated added value is up to 10.8 × 10⁹ € within the European Union.

Keywords: Kefir; Freeze-dried; Thermal-dried; Industrial scale-up; Whey; Hard-type cheese

3-Isopropyloxy-6-morpholino-2-phenylphenalen-1-one as Lipophilic Fluorescent Probe for Lymphocyte Investigations by Elena M. Kirilova; Inta Kalnina (pp. 1744-1751).

Fluorescent properties of two naphthalimides and a phenalenone derivative in organic solvents and when they bind to human peripheral blood lymphocytes were investigated. Different spectral characteristics were observed using lymphocytes of healthy donors and patients with nonmalignant (chronic myeloid leukemia) and malignant (B-cell lymphoid leukemia) diseases. It was found that spectral properties of the used fluorophores in cell suspension qualitatively characterize its structural and functional alterations during pathological phenomena. The intensity of fluorescence increased in samples from patients with B-cell lymphoid leukemia, and the fluorescence maximum shifted to the long-wavelength region by 20 nm compared with normal lymphocytes. It is concluded that 3-isopropyloxy-6-morpholino-2-phenylphenalen-1-one as most promising probe may be applied to the study of malignant diseases.

Keywords: Lipophilic fluorescent probe; Phenalenone derivative; Naphthalimide; Lymphocytes; Malignant diseases

Inhibition of Beta-Lactamase by 1,4-Naphthalenedione from the Plant Holoptelea integrifolia by N. V. Vinod; R. Shijina; K. V. Dileep; C. Sadasivan (pp. 1752-1759).

The most important mechanism of the beta-lactam antibiotic resistance is the destruction of the antibiotics by the enzyme beta-lactamase. Use of beta-lactamase inhibitors in combination with antibiotics is one of the successful antibacterial strategies. The inhibitory effect of a phytochemical, 1,4-naphthalenedione, isolated from the plant Holoptelea integrifolia on beta-lactamase is reported here. This compound was found to have a synergistic effect with the antibiotic amoxicillin against a resistant strain of Staphylococcus aureus. The enzyme was purified from the organism and incubated with the compound. An assay showed that the compound can inhibit the enzymatic activity of beta-lactamase. Modeling and molecular docking studies indicated that the compound can fit into the active site of beta-lactamase. Hence, the compound can serve as a potential lead compound for the development of effective beta-lactamase inhibitor that can be used against beta-lactam-resistant microbial strains.

Keywords: Beta-lactamase; Staphylococcus aureus ; Amoxicillin; Holoptelea integrifolia ; Phytochemicals; 1,4-Naphthalenedione

Ligninolytic Fungal Laccases and Their Biotechnological Applications by Daljit Singh Arora; Rakesh Kumar Sharma (pp. 1760-1788).

Lignin is amorphous in nature, lacks stereoregularity, and is not susceptible to hydrolytic attack. Despite its resistant nature, it is however degraded by various microorganisms, particularly, white-rot fungi. Such fungi are capable of extracellular production of lignin peroxidase, manganese peroxidase, and laccase, the three major enzymes associated with ligninolysis. Though all white-rot fungi do not produce all the three enzymes, laccase occupies an important place in ligninolysis. Laccase belongs to a diverse group of enzymes called oxidoreductases and is also known as benzenediol: oxygen oxidoreductase. They have low substrate specificity. The copper-containing enzyme laccase has been detected in a variety of organisms such as bacteria, fungi, plants, and insects. Mostly, these are extracellular proteins, although intracellular laccases have also been detected in some fungi and insects. Fungal laccases are believed to play a variety of roles, such as, morphogenesis, pathogenesis, and lignin degradation. As an oxidase, laccase is used in many agricultural, industrial, and medicinal applications. Current investigations are focused on laccase-based biooxidation, biotransformation, biosensor, and enzymatic synthesis of organic compounds. By enhancing laccase production using different physiochemical parameters, better understanding of the mechanism for the reactions of interest, and optimizing the catalytic activity of laccase, it can be used in a better way in diverse fields of biotechnology.

Keywords: Bioremediation; Laccase; Lignin; Lignocellulosics; White-rot fungi

Low-Pressure Lipase-Catalyzed Production of Mono- and Diglycerides with and Without N-Butane and AOT Surfactant by Alexsandra Valério; Karina G. Fiametti; Suzimara Rovani; Helen Treichel; Débora de Oliveira; J. Vladimir Oliveira (pp. 1789-1796).

The aim of this work is to report the production of mono- and diglycerides from olive oil at ambient condition and in pressurized n-butane as solvent medium. For this purpose, a commercial immobilized lipase (Novozym 435) was employed as catalyst and sodium (bis-2-ethyl-hexyl) sulfosuccinate (Aerosol-OT or AOT) as surfactant. The experiments were conducted in batch mode varying the temperature, pressure, and AOT concentration. Results showed that lipase-catalyzed glycerolysis either with compressed n-butane or in solvent-free system with AOT as surfactant might be a potential alternative route to conventional methods, as high contents of reaction products, especially monoglycerides (∼ 60 wt.%), were achieved at mild temperature and pressure with a relatively low solvent to substrates mass ratio (4:1) in short reaction times (2 h).

Keywords: Glycerolysis; Olive oil; Lipase; AOT; n-Butane; Solvent-free system

Screening of Variables Influencing the Clavulanic Acid Production by Streptomyces DAUFPE 3060 Strain by D. A. Viana; M. N. Carneiro-Cunha; J. M. Araújo; B. Barros-Neto; J. L. Lima-Filho; A. Converti; A. Pessoa-Júnior; A. L. F. Porto (pp. 1797-1807).

Clavulanic acid (CA) is a β-lactam antibiotic, which has a potent β-lactamase inhibiting activity. The influence of five variables, namely pH (6.0, 6.4, and 6.8), temperature (28°C, 30°C, and 32°C), agitation intensity (150, 200, and 250 rpm), glycerol concentration (5.0, 7.5, and 10 g/L) and soybean flour concentration (5.0, 12.5, and 20 g/L), on CA production by a new isolate of Streptomyces (DAUFPE 3060) was investigated in 250-mL Erlenmeyer flasks using a fractional factorial design. Temperature and soybean flour concentration were shown to be the two variables that exerted the most important effects on the production of CA at 95% confidence level. The highest CA concentration (494 mg/L) was obtained after 48 h at 150 rpm, 32°C, pH 6.0, 5.0 g/L glycerol, and 20 g/L soybean flour concentrations. Under these conditions, the yields of biomass and product on consumed substrate were 0.26 g_X/g_S and 64.3 mg_P/g_S, respectively. Fermentations performed in 3.0-L bench-scale fermenter allowed increasing the CA production by about 60%.

Keywords: Streptomyces ; Soybean flour; Glycerol; Clavulanic acid; Screening of variables; Fermentation

Single-Chain Fv Antibody Fragments Retain Binding Properties of the Monoclonal Antibody Raised Against Peptide P1 of the Human Prion Protein by Nives Škrlj; Vladka Čurin Šerbec; Marko Dolinar (pp. 1808-1821).

Prion diseases are incurable neurodegenerative diseases that affect both humans and animals. The infectious agent is a pathogenic form of the prion protein that accumulates in brain as amyloids. Currently, there is neither cure nor reliable preclinical diagnostics on the market available. The growing number of reports shows that passive immunisation is one of the most promising strategies for prion disease therapy, where antibodies against prions may prevent and even cure the infection. Since antibodies are large molecules and, thus, might not be suitable for the therapy, different antibody fragments are a good alternative. Therefore, we have designed and prepared single-chain antibody fragments (scFvs) derived from the PrP^Sc-specific murine monoclonal antibody V5B2. Using a new expression vector pMD204, we produced scFvs in two opposing chain orientations in the periplasm of Escherichia coli. Both recombinant antibody fragments retained the specificity of the parent antibody and one of these exhibited binding properties comparable to the corresponding murine Fab fragments with the affinity in nM range. Our monovalent antibody fragments are of special interest in view of possible therapeutic reagents for prion diseases as well as for development of a new generation of diagnostics.

Keywords: Prion protein; Recombinant antibody; scFv; Escherichia coli

Treatment of Sanitary Landfill Leachates in a Lab-Scale Gradual Concentric Chamber (GCC) Reactor by Lourdes Mendoza; Willy Verstraete; Marta Carballa (pp. 1822-1832).

Sanitary landfill leachates are a major environmental problem in South American countries where sanitary landfills are still constructed and appropriate designs for the treatment of these leachates remain problematic. The performance of a lab-scale Gradual Concentric Chamber (GCC) reactor for leachates treatment is presented in this study. Two types of sanitary landfill residuals were evaluated, one directly collected from the garbage trucks (JGL), with high organic strength (84 g COD/l) and the second one, a 6-month-generated leachate (YL) collected from the lagoon of the sanitary landfill in Quito, Ecuador, with an organic strength of 66 g COD/l. Different operational parameters, such as organic loading rate (OLR), temperature, recycling and aeration, were tested. The GCC reactor was found to be a robust technology to treat these high-strength streams with organic matter removal efficiencies higher than 65%. The best performance of the reactors (COD removal efficiencies of 75–80%) was obtained at a Hydraulic Retention Time (HRT) of around 20 h and at 35 °C, with an applied OLR up to 70 and 100 g COD/l per day. Overall, the GCC reactor concept appears worth to be further developed for the treatment of leachates in low-income countries.

Keywords: Anaerobic treatment; Developing countries; Food wastes; UASB reactor

Anticancer Properties of Highly Purified l-Asparaginase from Withania somnifera L. against Acute Lymphoblastic Leukemia by Vishal P. Oza; Pritesh P. Parmar; Sushil Kumar; R. B. Subramanian (pp. 1833-1840).

Withania somnifera L. has been traditionally used as a sedative and hypnotic. The present study was carried out for the purification, characterization, and in vitro cytotoxicity of l-asparaginase from W. somnifera L. l-Asparaginase was purified from the fruits of W. somnifera L. up to 95% through chromatography. The purified l-asparaginase was characterized by size exclusion chromatography, polyacrylamide gel electrophoresis (PAGE), and 2D PAGE. The antitumor and growth inhibition effect of the l-asparaginase was assessed using [3-(4, 5-dimethyl-thiazol-2yl)-2, 5-diphenyl-tetrazolium bromide] (MTT) colorimetric dye reduction method. The purified enzyme is a homodimer, with a molecular mass of 72 ± 0.5 kDa, and the pI value of the enzyme was around 5.1. This is the first report of the plant containing l-asparaginase with antitumor activity. Data obtained from the MTT assay showed a LD₅₀ value of 1.45 ± 0.05 IU/ml. W. somnifera L. proved to be an effective and a novel source of l-asparaginase. Furthermore, it shows a lot of similarity with bacterial l-asparaginases EC-2.

Keywords: Antitumor activity; l-Asparaginase; MTT; Purification; Withania somnifera L.

Optimization of Growth Medium and Enzyme Assay Conditions for Crude Cellulases Produced by a Novel Thermophilic and Cellulolytic Bacterium, Anoxybacillus sp. 527 by Yanna Liang; Zisong Feng; Jemil Yesuf; James W. Blackburn (pp. 1841-1852).

A newly isolated Anoxybacillus sp. 527 was found to grow on crystalline cellulose as sole carbon and energy sources. Cellulases secreted by strain 527 were better induced by cellobiose, followed by glucose, lactose, sucrose, and cellulose. Cellulase secretion was enhanced by an optimized medium. Cellulase activity was increased by the addition of Ca²⁺ and NH ₄ ⁺ and achieved maximum as 7.0 FPU ml⁻¹ at 70 °C and pH 6.0. Even at 100 °C, the enzymes were still active, which implies their potential application in large-scale cellulose conversion process.

Keywords: Cellulose; Thermophilic; Cellulase; Anoxybacillus sp. 527

Conjugal Transferring of Resistance Gene ptr for Improvement of Pristinamycin-Producing Streptomyces pristinaespiralis by Zhihua Jin; Xin Jin; Qingchao Jin (pp. 1853-1864).

Improving pristinamycin production from Streptomyces pristinaespiralis was performed by introducing the resistance gene ptr followed by selection for enhanced tolerance to pristinamycin and fermentation test. To transfer ptr into S. pristinaespiralis, an effective method was established for the first time by using the intergeneric conjugation of DNA from Escherichia coli to S. pristinaespiralis. The procedure was optimized with heat treatment, spore concentration, optimum medium used in conjugation, concentration of MgCl₂, etc. With the optimized conditions, the conjugation frequency was up to 1.36 × 10⁻³ exconjugants per recipient. The procedure was used to transfer the ptr gene into S. pristinaespiralis, resulting in 146 exconjugants. These exconjugants were screened on the pristinamycin-resistant plates, and then the fermentation test subsequently. Finally, two strains (SPR1 and SPR2) were obtained with a high yield of 0.11 and 0.15 g/l, respectively, which is about six to eight times more than that of wild-strain ATCC25486. The subculture experiments indicated that the hereditary character of the high-producing S. pristinaespiralis SPR1 and SPR2 was stable. Our work suggests that introducing resistance gene ptr into S. pristinaespiralis could be the way to improve the production of pristinamycin through the enhancement of antibiotic tolerance.

Keywords: Strain improvement; ptr ; Intergeneric conjugation; Pristinamycin; Streptomyces pristinaespiralis

A Closed Concept of Extractive Whole Cell Microbial Transformation of Benzaldehyde into l-Phenylacetylcarbinol by Saccharomyces cerevisiae in Novel Polyethylene-Glycol-Induced Cloud-Point System by Zhilong Wang; Rui Liang; Jian-He Xu; Yubo Liu; Hanshi Qi (pp. 1865-1877).

Extractive microbial transformation of benzaldehyde into l-phenylacetylcarbinol (l-PAC) by Saccharomyces cerevisiae (Baker’s yeast) has been carried out in a novel polyethylene-glycol-induced cloud-point system (PEG-CPS). The extractive microbial transformation in the PEG-CPS and a downstream process for stripping of the product from the microbial transformation broth with microemulsion extraction are demonstrated. The results indicate that the PEG-CPS maintains the advantage of CPS for in situ extraction of polar product in the microbial transformation. At the same time, the utilization of hydrophilic nonionic surfactant in the PEG-CPS is favorable for stripping of product from the nonionic surfactant in the microbial transformation broth by Winsor I microemulsion extraction. Thus, a closed concept of in situ extraction of polar product in microbial transformation and its downstream process of product recovery are fulfilled at the same time.

Keywords: Microbial transformation; Nonionic surfactant; Extraction; Cloud-point system; Microemulsion

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.160, #6)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.160, #6)