23^rd World Congress on Biotechnology November 14-15, 2019 | Park Plaza Amsterdam Airport | Melbournestraat 1, 1175 RM Lijnden, Netherlands

Biography:

Dr. Duraid Al-Midfai is currently a member of Fuwai Central China Cardiovascular Hospital as a post-doctoral research in the cardio surgery department-in patient building as well as his work also collaborated with Henan Hospital in the Research Centre building.

Abstract:

The interleukin 1 family plays an important role in the immune and inflammatory responses. Coronary artery disease (CAD) is a chronic inflammatory disease. However, the genetic association between IL-37, the seventh member of the IL-1 family, and CAD is unknown. Here we show that a single nucleotide polymorphism in the IL-37 gene (rs3811047) confers a significant risk of CAD. We have performed an association analysis between rs3811047 and CAD in two independent populations with 2,501 patients and 3,116 controls from China. Quantitative RT-PCR analysis has been performed to determine if the IL-37 expression level is influenced by rs3811047. We show that the minor allele A of rs3811047 is significantly associated with CAD in two independent populations under a recessive model (Padj=5.51×10-3/OR=1.56 in the GeneID Northernern population and Padj=1.23×10-3/OR=1.45 in the GeneID Central population). The association became more significant in the combined population (Padj=9.70×10-6/OR=1.47). Moreover, the association remains significant in a CAD case control population matched for age and sex. Allele A of rs3811047 shows significant association with a decreased mRNA expression level of IL-37 (n=168, P=3.78×10-4). These data suggest that IL37 is a new susceptibility gene for CAD, which provides a potential target for the prevention and treatment of CAD.

Biography:

Hauke Dieken has completed his Bachelor of Science in bioprocess engineering at the University of Applied Sciences Flensburg and received his Master of Science in the same field from the Technical University of Braunschweig (Germany). Currently he is working on his PhD Thesis focusing on the chromatographic purification of oncolytic measles virus at the University of Applied Sciences Mittelhessen.

Abstract:

Cancer still remains a major global health burden with 8.7 million related deaths in 2015. A novel therapy concept, which achieved full remission of even advanced-stage cancer, is engineered oncolytic measles virus (OMV). To ensure an effective cancer lysis, extremely high doses of at least 10⁸-10¹¹ TCID₅₀ (50% tissue culture infectious dose) of infective OMV particles are necessary. Furthermore, the OMV must be extremely pure. Impurities like host cell proteins (HCP), host cell DNA (hcDNA) or non-infective viral fragments must be reduced below the regulatory limits.

An upstream process for high titer OMV production has already been established in our lab and we are currently focusing on the development of an efficient chromatographic downstream process.

We first characterized the OMV’s size distribution, isoelectric point and physicochemical stability. Based on these characteristics, we tested different cationic and hydrophobic interaction resins. We found that a two-step purification process which combined cation exchange chromatography flowed by hydrophobic interaction chromatography leads to high yield of infectious OMV (~70%) and strong reduction of HCP (~98%) as well as hcDNA (~80%). The tested resin-stationary phases were beneficial over monolithic stationary phases which only led to average yields for OMV. Our findings are contrary to the common scientific opinion which assesses resin-based phases as unsuitable for virus purification processes. Nonetheless, we showed that it is worth to investigate resin-based stationary phases also for larger particle such as OMV and that resin-based stationary phases are a versatile option to reach highest OMV yield, purity and potency.

Biography:

Sanusi Magaji holds a bachelor of technology from ATB University Bauchi, PGD public health ATB Bauchi, Msc Molecular Biology with Bioinformatics University of Wolverhampton. Sanusi is a lecturer at the department of science lab. Tech. ATAPOLY, Nigeria.

Abstract:

Two experiments on alkaline lysis method of plasmid DNA isolation was carried out utilizing strains of E.coli JM109, a non- pathogenic bacteria that has been deliberately disabled. Restriction digests and gel electrophoresis carried out. The result of the first gel electrophoresis reveals only traces of RNA, hence no DNA fragments on the samples loaded labeled A to D this could be attributed to possible contamination from the procedures. For the second gel electrophoresis samples, A is plasmid free, B and C contains small fragments approximately 2.3 to 5.6 and D contains the fertility plasmids.

Biography:

Hiroto Saeki is a student of master course at Ritsumeikan University.

Abstract:

L-Trptophanyl-L-histidine (Trp-His) is the anti-atherosclerotic dipeptide and acts as a vasorelaxant by binding to an extracellular site of Ca²⁺ channels and regulating intracellular Ca²⁺ concentration. In addition, Trp-His reduces plaque formation in blood vessels and prevents arteriosclerosis. The dipeptides are generally produced by chemical synthetic methods, but such methods often require the complicated steps and the use of organic solvents which have a negative impact on the environment. L-Amino acid esterase (LAE) can synthesize dipeptides from aminoacyl methyl esters and free amino acids without ATP in one step under mild conditions. In this study, we characterized LAE from Pseudomonas aeruginosa PAO1 (PaLAE) for the synthesis of Trp-His.

The PaLAE gene was inserted into the pCold TEV vector and expressed in Escherichia coli BL21 (DE3). PaLAE was purified by Ni-affinity and gel filtration chromatography with a yield of 35.6% and a purification of 2.37 fold. The apparent molecular mass of the purified enzyme was calculated to be 64 kDa by gel filtration, and the molecular mass of the denatured enzyme was determined to be 66 kDa by SDS-PAGE, indicating that PaLAE was a monomer. The reaction products were measured by HPLC for enzyme activity. PaLAE could synthesize Trp-His using Trp-OMe and L-histidineas. However, PaLAE hydrolyzed Phe-OMe, Tyr-OMe, and Leu-OMe more than Trp-OMe as substrates. These results suggested that PaLAE preferred bulky or hydrophobic amino acid residues.

Biography:

Taizo. Y has completed his B. Engineering. He is 22 years old and a master 1st grade student at  Ritsumeikan University.

Abstract:

Biography:

Hiroki Ikezoe has completed his BE at the top of his class from Ritsumeikan University in March 2019, and studies life science in master’s course of Ritsumeikan University Graduate School. 

Abstract:

γ-Glutamyltranspeptidase (GGT) is a ubiquitous enzyme that catalyzes the transfer of the γ-glutamyl moiety from γ-glutamyl compounds to acceptor substrates or the hydrolysis of γ-glutamyl compounds. GGT from Pseudomonas nitroreducens (PnGGT) is used for the industrial synthesis of γ-glutamyl-ethylamide (theanine), which contributes the fifth taste that is known as umami. However, the detailed properties of PnGGT are still unclear. Hence, it is necessary to reveal the structural basis which is involved in hydrolysis and transpeptidation reactions and to identify the acceptor site of PnGGT for more efficient synthesis of theanine. Structural studies of PnGGT have shown that the three amino acid residues (Trp385, Phe417, and Trp525) might be involved in the recognition of acceptor substrates. Here we further investigated the role of Trp385 in PnGGT through site-directed mutagenesis and biochemical analyses.

We substituted Trp385 by the other four amino acids (Ala, Ser, Asp and Lys) and got PnGGT mutants (W385A, W385S, W385D and W385K). W385A was expressed as a precursor form in insoluble fraction, and W385D was not expressed in any fractions, whereas W385S and W385K were expressed as a mature form in soluble fraction.  The activity assays with p-nitroaniline or γ-l-glutamyl hydroxamate showed that substitution of Trp385 resulted in drastic decrease in hydrolysis activity and significant increase in transpeptidase activity. W385S and W385K had a lower substrate specificity towards ethylamine compared to wild-type PnGGT. These results suggested that Trp385 was essential residue for auto processing, catalytic reaction and substrate recognition mechanism of PnGGT.

Biography:

Naoki Yamahata is a student of doctoral course at Ritsumeikan University.

Abstract:

Whey is a main by-product of the cheese production and it contains lactose, protein, ash, fat, and other bioactive substances. Many researchers developed whey-based beverages; however, these beverages were low-alcohol content drinks, distilled liquors or mixtures of fruit. Here, we consider the raw materials and the optimal concentration of substance for the development of new fermented whey beverages conducting the sensory evaluation. Whey powder and demineralized whey (DMW) powder were dissolved in mineral water and fermented by a lactose fermenting yeast, Kluyveromyces marxianus, at 30°C. After fermentation, the 33 participants in the preference test scored the fermented beverages made from whey and DMW. The score on the overall acceptability of the fermented beverages made from whey and DMW was 2.3 and 4.3 on a scale of 1 (Dislike extremely) to 7 (Like extremely), respectively. The result showed that the fermented beverage made from DMW had a better acceptance to drink. Subsequently, we determined the optimal concentration of DMW as fermented beverages comparing three different concentration of the substance, that is 10%, 15% and 20%. The fermented beverage from 20% of DMW was the best preferred by sensory evaluation and the amount of ethanol of this beverage was 9.5 v/v%. The results indicated that higher concentration of ethanol was better to develop whey-based fermented beverages. We propose that this new fermented beverage made from DMW containing high amount of alcohol could be acceptable and that other approaches to improve preference of whey-based beverages were remained.

Biography:

Risa Yamashiro has completed her B. Engineering. She is 22 years old and a master 1st grade student at the Ritsumeikan University. She majors in life science.

Abstract:

l-Asparaginase hydrolyzes l-asparagine into l-aspartate and ammonia, and is widely distributed in microorganisms, plants, and animals. l-Asparaginase has been applied in the food industry for reducing acrylamide, a carcinogenic compound in foods, which is mainly formed by a reaction between reducing sugars and asparagine at high temperature more than 120ºC. Although l-asparaginases from Escherichia coli and Erwinia chrysanthemi are commercially available, approved l-asparaginases for the application are few because of their lack of appropriate properties. Therefore, new l-asparaginases with better performance have been investigated worldwide.

In the present study, we focused on l-asparaginases from lactic acid bacteria, Lactobacillus delbrueckii and Lactobacillus sakei, designated as LdASNase and LsASNase, respectively. The expression plasmids were constructed by inserting LdASNase or LsASNase gene into pET-28a. Escherichia coli BL21 (DE3) transformed with the plasmid was cultured in LB medium in the presence of isopropyl β-D-thiogalactoside at 37ºC. The SDS-PAGE analysis showed that LdASNase was only expressed in insoluble form, whereas LsASNase was expressed in soluble and insoluble form. The soluble LsASNase was purified by Ni-affinity chromatography to electrophoretic homogeneity. The enzyme activity was measured by determining the amount of ammonia formed with glutamate dehydrogenase. The specific activity of purified LsASNase was calculated to be 1.32 U/mg. The optimum pH and reaction temperature were found to be 7-9 and 40ºC, respectively. LsASNase showed high substrate specificity towards l-asparagine compared with l-glutamine. In addition, LsASNase activity was inhibited by Zn²⁺ and Fe³⁺.

Biography:

Momoko Tachibana is a student of master course at Ritsumeikan University.

Abstract:

Whey is a by-product of cheeses and rich in nutrients. Although, whey had been thrown away into the river untill the 1980’s, it has been banned to stop eutrophication and to preserve environment. Therefore, various methods for effectively utilizing whey waste have been proposed. In our laboratory, we have been developing alcoholic drink made from whey for the better use of it. It is known that amino acids contained in alcoholic drink have an influence on its taste. We have been interested in an ability of amino acid assimilation of yeast from the view point of alcohol fermentation.

In the present study, we have constructed the deletion mutant of autophagy- related gene in Saccharomyces cerevisiae to compare the capability of amino acid assimilation between wild- and mutant-yeast. Autophagy- related genes are involved in the intracellular recycling of amino acids. Among them, we have focused on ATG1 gene because it is the trigger for autophagy in starvation. Because autophagy-defective mutants are considered to be lack of recycling ability of amino acids, external amino acids would much more positively be taken up by mutant-yeast for life support than wild-yeast. ATG1-defective mutant of S. cerevisiae BY4742 was constructed by homologous recombination method. The wild- and mutant-yeasts fermented and compared between them the total amino acids, ethanol concentration, and glucose. As a result, little differences of medium constituents between the wild- and mutant-yeasts have been observed in the fermentation under nutrient rich condition like YPD medium. Experiments under various fermentation conditions are in progress.

Biography:

Ayako Tanaka is a student of master course at Ritsumeikan University.

Abstract:

All amino acids except for glycine can exist in either of two enantiomers, called l- or d-amino acids. l-Amino acids are predominant in nature, but d-amino acids also play an important role as physiologically active substances. d-Valine and d-Phenylalanine are components of some peptide antibiotics, and d-Aspartic acid affects the production of collagens in the skin. The increasing demand of d-amino acids by pharmaceutical and biotechnological industries and the difficulty of mass production of d-amino acids by enzymatic synthesis motivate us to establish a novel method which can produce d-amino acids on large-scale. In this study, we developed and evaluated the fermentation system for the production of N-acetyl-d-amino acids using the recombinant E. coli.

The co-expression vector of alanine racemase (Alr) from Pseudomonas putida KT2440 and d-amino-acid N-acetyltransferase (DNT) from Saccharomyces cerevisiae was constructed using a pETDuet-1 plasmid. E. coli Rosetta-Gami B (DE3) transformed with the vector was grown in LB medium, harvested and disrupted by sonication. We first checked the activity of Alr and DNT using the resulting cell lysate in vitro. The specific activity of Alr and DNT was found to be 20.2 U/mg and 3.42 U/mg, respectively. Then, we monitered the fermentation performance of the recombinant E. coli grown in LB medium containing l-Methionine (l-Met) as a substrate. After 10 hours incubation, only d-Met, but not N-acetyl-d-Met, was detected in the culture supernatant. These results suggested that Alr could convert l-Met into d-Met in the periplasmic space, and vast majority of d-Met was leaked outside the cells.

Biography:

Keita Nishio is a student of master course at Ritsumeikan University.

Abstract:

Sesame oil cake is a by-product in a production process of sesame oil. It is rich in protein, phosphoric acid and nitrogen. It is currently used for fertilizers and animal feed. Although sesame oil cake is rich in dietary fiber and has high antioxidant activity, it has not been used as food. Fermented seasoning with high antioxidant activity and high amino acid content has been obtained by using sesame oil cake as a raw material for koji. In my laboratory, the fermented seasoning using milk ingredients as a raw material have been performed in my laboratory. It is necessary to improve the quality of fermented seasoning such as taste and physiological function. We have tried to improve the function of milk-fermented seasoning by using sesame oil cake as a raw material for koji. In the present study, we aims to develop a new fermented seasoning made from sesame oil koji. Fermentation conditions, sensory evaluation, amino acid analysis, and antioxidant activity were examined. As a result, the optimal water content of the sesame oil cake koji was determined at 60% and the fermentation was performed. The fermented sample prepared by adding skimmed milk powder and saline water to sesame oil cake has a higher amino acid content and higher antioxidant activity than the other brewed samples. In addition, it got a higher evaluation than other fermented samples in sensory test.

Biography:

Alica Chroňáková has completed her PhD in ecology in 2009 from South Bohemia University in ÄŒeské BudÄ›jovice and postdoctoral studies from Institute of Soil Biology (ISB), BC CAS. She is the head of the Laboratory for environmental microbiology, head of Culture Collection of Soil Actinomycetes at ISB and member of the Scientific board of the ISB.

Abstract:

Pharmaceuticals enter agricultural soils mainly with wastewater, manure and sewage sludge and potentially cause unknown biological effects on soil microbiota. We explored the structural and functional potential of the soil microbiome to respond on six pharmaceuticals and their mixture in seven agricultural soils in 1, 13 and 61 incubation days. Pharmaceuticals from different therapeutic classes highly abundant in effluents in the Czech Republic - Atenolol, Carbamazepine, Citalopram, Clarithromycin, Clindamycin, Fexofenadine, Irbesartan And Sulfamethoxazole, were used. Basal respiration was used to indicate the microbial activity, phospholipid fatty acids were used to determine microbial biomass, and  HTP sequencing of bacterial 16S rRNA gene to assess bacterial community structure. Four responses of pharmaceuticals on soil microbial community were observed: (i) stimulatory, (ii) inhibitory, (iii) stress and (iv) dormancy, which highly dependend on soil type. Antibiotics affected the bacterial alpha diversity, which increased with the amendment of sulfamethoxazole and decreased with clindamycin. However, we provide an evidence that non-antibiotic pharmaceuticals are capable of shifting microbial community along with their antibiotic counterparts. Simultaneously, we studied the parmaceutical biodegradation of microbial cultures to identify their metabolites and follow them in soil matrix using LC-HRMS. Single strain culture experiments did not mimic complex soil incubation experiments, as we identified completely different transformation products. Our findings indicated that microbial responses were highly dependent on the type of the soil, amended pharmaceuticals and incubation time, highlighting the importance of these parameters to be considered for long-term consequences of agricultural soil management and resilience of soil microbial communities to contaminants.