Biochemical Characterization of Kluyveromyces lactis Adenine Deaminase and Guanine Deaminase and Their Potential Application in Lowering Purine Content in Beer

Excess amounts of uric acid in humans leads to hyperuricemia, which is a biochemical precursor of gout and is also associated with various other disorders. Gout is termed as crystallization of uric acid, predominantly within joints. The burden of hyperuricemia and gout has increased worldwide due to lifestyle changes, obesity, and consumption of purine-rich foods, fructose-containing drinks, and alcoholic beverages. Some of the therapies available to cure gout are associated with unwanted side-effects and antigenicity. We propose an attractive and safe strategy to reduce purine content in beverages using enzymatic application of purine degrading enzymes such as adenine deaminase (ADA) and guanine deaminase (GDA) that convert adenine and guanine into hypoxanthine and xanthine, respectively. We cloned, expressed, purified, and biochemically characterized both adenine deaminase (ADA) and guanine deaminase (GDA) enzymes that play important roles in the purine degradation pathway of Kluyveromyces lactis, and demonstrate their application in lowering purine content in a beverage. The popular beverage beer has been selected as an experimental sample as it confers higher risks of hyperuricemia and gout. Quantification of purine content in 16 different beers from the Indian market showed varying concentrations of different purines. Enzymatic treatment of beer samples with ADA and GDA showed a reduction of adenine and guanine content, respectively. These enzymes in combination with other purine degrading enzymes showed marked reduction in purine content in beer samples. Both enzymes can work at 5.0–8.0 pH range and retain >50% activity at 40°C, making them good candidates for industrial applications

The role of culture collections as an interface between providers and users: the example of yeasts

The importance and species diversity of yeasts in food production are described, including a listing of agricultural applications. Two yeast species were selected for case studies on distribution patterns from microbial culture collections: the high representation of Saccharomyces cerevisiae in culture collections enabled global comparison, while Dekkera bruxellensis deposits and distributions were analyzed from the perspective of a single culture collection. In conclusion, culture collections need to cover temporal gaps between deposit and application of strains. The further development of culture collections in countries of high but underexplored species diversity should facilitate the conservation and management of microbial resources. (C) 2010 Elsevier Masson SAS. All rights reserved

Development of suitable solvent system for downstream processing of biopolymer pullulan using response surface methodology.

Downstream processing is an important aspect of all biotechnological processes and has significant implications on quality and yield of the final product. Several solvents were examined for their efficacy on pullulan precipitation from fermentation broth. Interactions among four selected solvents and their effect on pullulan yield were studied using response surface methodology. A polynomial model was developed using D-optimal design and three contour plots were generated by performing 20 different experiments and the model was validated by performing optimization experiments. The results indicated that lower concentration of ethanol in combination with the other three solvents has resulted in higher yield of polymer from fermentation broth and the optimized solvent system was able to recover 1.44 times more pullulan as compared to the conventional ethanolic precipitation method. These observations may help in enhancing efficiency of pullulan recovery from fermentation broth and also result in reduced cost of production for the final product

Functional and Structural Characterization of Purine Nucleoside Phosphorylase from Kluyveromyces lactis and Its Potential Applications in Reducing Purine Content in Food.

Consumption of foods and beverages with high purine content increases the risk of hyperuricemia, which causes gout and can lead to cardiovascular, renal, and other metabolic disorders. As patients often find dietary restrictions challenging, enzymatically lowering purine content in popular foods and beverages offers a safe and attractive strategy to control hyperuricemia. Here, we report structurally and functionally characterized purine nucleoside phosphorylase (PNP) from Kluyveromyces lactis (KlacPNP), a key enzyme involved in the purine degradation pathway. We report a 1.97 Å resolution crystal structure of homotrimeric KlacPNP with an intrinsically bound hypoxanthine in the active site. KlacPNP belongs to the nucleoside phosphorylase-I (NP-I) family, and it specifically utilizes 6-oxopurine substrates in the following order: inosine > guanosine > xanthosine, but is inactive towards adenosine. To engineer enzymes with broad substrate specificity, we created two point variants, KlacPNPN256D and KlacPNPN256E, by replacing the catalytically active Asn256 with Asp and Glu, respectively, based on structural and comparative sequence analysis. KlacPNPN256D not only displayed broad substrate specificity by utilizing both 6-oxopurines and 6-aminopurines in the order adenosine > inosine > xanthosine > guanosine, but also displayed reversal of substrate specificity. In contrast, KlacPNPN256E was highly specific to inosine and could not utilize other tested substrates. Beer consumption is associated with increased risk of developing gout, owing to its high purine content. Here, we demonstrate that KlacPNP and KlacPNPN256D could be used to catalyze a key reaction involved in lowering beer purine content. Biochemical properties of these enzymes such as activity across a wide pH range, optimum activity at about 25°C, and stability for months at about 8°C, make them suitable candidates for food and beverage industries. Since KlacPNPN256D has broad substrate specificity, a combination of engineered KlacPNP and other enzymes involved in purine degradation could effectively lower the purine content in foods and beverages

Parity plot: showing the relation between actual and predicted values for pullulan recovery.

<p>Parity plot: showing the relation between actual and predicted values for pullulan recovery.</p

Effect of Lasiodiplodia pseudotheobromae Isolates, a Potential Bioherbicide for Amaranthus hybridus L. in Maize Culture

The use of phytopathogenic fungi in biological control of weeds may represent a promising alternative to the use of chemicals and may lead to the sustainability of agro-ecosystems. In the current study, strain C1136 was developed into various formulations and their activities were compared with a chemical herbicides using glyphosate in a screen house, applying standard procedure. All the bioherbicide treatments gave significantly higher yield component on the following parameters: tasselling, number of cobs, day of silking, number of ears, number of grain/cob, weight of 100 grains, number of cob, weight of cob, and length of cob from maize when compared to the chemical-treated herbicides and weedy cheek. Moreover, the bioherbicidal formulation coded BH4 (32 g of semolina + 6 g kaolin + 20 mL of glycerol + mutant strain of Lasiodiplodia pseudotheobromae + glucose+ sucrose + fructose + dextrose + lactose sugar + peptone) showed the highest activities when compared to other formulations. Conclusively, this study revealed that Lasiodiplodia pseudotheobromae C1136 strain has bioherbicidal activity and could therefore be exploited for large scale production of bioherbicides for weed control in conventional farming, to improve yield and enhance food security

Identification of yeasts by polymerase-chain-reaction-mediated denaturing gradient gel electrophoresis in marcha, an ethnic amylolytic starter of India

Background: Marcha is an ethnic amylolytic starter that is used to ferment boiled cereals to produce alcoholic drinks, commonly called jaanr, in the Himalayan Regions of Sikkim and Darjeeling of India. Methods: The aim of this study was to investigate yeast flora of marcha collected from Sikkim in India by phenotypic characterization and polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE). Results: The average load of yeast in marcha was 6.0 × 108 colony-forming units/g. The phenotypic characterization of yeast isolates from marcha showed the presence of Candida, Pichia, Torulospora, Schizosaccharomyces, Kluveromyces, Issatchenki, and Saccharomycopsis. The PCR-DGGE bands showed the dominance of Wickerhamomyces anomalus (72%) and Pichia anomalus (28%) in marcha. W. anomalus was reported for the first time from marcha using PCR-mediated DGGE. Conclusion: This is the first report on the yeast community associated with marcha analyzed by PCR-mediated DGGE

Active site architecture of <i>Klac</i>PNP with bound hypoxanthine in the active site.

<p>(A) ESI-MS analysis of intrinsically bound ligand (upper panel) and expected mass of the possible ligands (lower panel) is shown. Mass spectrometry analysis unambiguously suggests that hypoxanthine is bound to the active site. (B) Possible conventional hydrogen bond and weak C–H⋯O interactions are represented by broken black and broken red lines, respectively. The Phe213 side chain is oriented almost perpendicular to the substrate and mediates the π-π interactions. Hypoxanthine, amino acids, and water molecules are represented as ball and stick, sticks, and red spheres, respectively. The atoms are colored according to the following color code: carbon, grey; nitrogen, blue; oxygen, red.</p

Multiple sequence alignment of <i>Klac</i>PNP homologs.

<p>Sequence comparison of <i>Klac</i>PNP with homotrimeric PNPs (specific for 6-oxopurines: <i>S</i>. <i>cerevisiae</i>, human, and calf spleen PNPs), homohexameric PNPs (specific for 6-aminopurines: <i>B</i>. <i>cereus</i>, <i>B</i>. <i>subtilis</i>, and <i>E</i>. <i>coli</i> PNPs). Highly conserved regions are highlighted with red boxes; conservative substitutions are also boxed. The figure was drawn by using the ESPript 3 server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164279#pone.0164279.ref059" target="_blank">59</a>]. Active site residues involved in the interaction with hypoxanthine are shown with an asterisk and the catalytically active residue that is known to play an important role in substrate specificity is shown with a red filled circle [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164279#pone.0164279.ref045" target="_blank">45</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164279#pone.0164279.ref049" target="_blank">49</a>].</p

Comparative structural analysis of the active sites of PNPs.

<p>Crystal structures of <i>K</i>. <i>lactis</i> (green, hypoxanthine bound structure), calf spleen (magenta, PDB ID 1VFN, hypoxanthine bound structure) and human (cyan, PDB ID 1RCT, inosine bound structure) PNPs were used for the structural comparison [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164279#pone.0164279.ref064" target="_blank">64</a>]. The active site is located in the vicinity of the intersubunit interface, and a structurally equivalent Phe172 is contributed from the neighboring monomer. Water molecules at probable phosphate and ribose binding sites in the <i>Klac</i>PNP and the calf spleen PNP are shown as blue and orange spheres, respectively. The two conserved water molecules involved in the water-mediated interactions are encircled (red broken circle). Ligands and amino acids in the active site are shown in the stick representation. Oxygen, nitrogen, and sulfur atoms are shown in red, blue, and yellow colors, respectively. A sulfate ion occupies the potential phosphate binding site. Most of the residues forming the active site superpose well, except for variations in the turn connecting β1 and 3₁₀ helix. For clarity, some residues, which are not a part of the active site, have been removed.</p