Nanoporous Gold as a Solid Support for Protein Immobilization for the Development of Immunoassays, and for Biomolecular Interaction Studies

Nanoporous gold (NPG) is a versatile material of high surface area to volume ratio that can be readily modified with self-assembled monolayers of alkanethiols to which biomolecules can be linked. NPG presents new opportunities for the development of immunoassays, and for the development of carbohydrate based assays. This thesis explores the use of NPG as a support for self-assembled monolayers, their linkage to antibody-enzyme conjugates for immunoassay development, and for the study and application of carbohydrate-protein interactions. Direct kinetic electrochemical immunoassays were developed on NPG for prostate specific antigen (PSA) and carcinoembryonic antigen (CEA). The decrease in enzymatic conversion of p-aminophenylphosphate to p-aminophenol, by alkaline phosphatase conjugated to an antibody, due to steric hindrance caused by the presence of antigen on antibody, was observed as a drop in peak current in square-wave voltammetry. Detection limit of these assays was 0.075 ng mL-1 and 0.015 ng mL-1 for PSA and CEA, respectively. Similarly, the linear range of determination of these biomarkers extended up to 30 ng mL-1 and 10 ng mL-1 for PSA and CEA, respectively. Minimal interference was observed using newborn calf serum as a substitute for the human serum matrix. A rapid and sensitive enzyme linked lectinsorbant assay was also developed for the study of glycoprotein-lectin interactions on the NPG surface. Self-assembled monolayers of alkanethiols on NPG were characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Similarly, the applicability of this surface for the formation of carbohydrate monolayers and its application for lectin carbohydrate interactions was also studied. Pure and mixed SAMs of 8-mercaptooctyl -D-mannopyranoside (Man-C8-SH) and α-D-Gal-(1→4)-β-D-Gal-(1→4)-D-Glc1-O-mercaptooctane (Gb3-C8-SH) with alkanethiols having varying tail groups were prepared. Binding affinity and binding kinetics of concanavalin A to mannoside and soybean agglutinin to galactose in these SAMs were found to be different on NPG than on flat polycrystalline gold, and was also sensitive to the chemical composition of the modified surfaces

Solid-state Fermentation for the Production of Poly(hydroxyalkanoates)

Poly(hydroxyalkanoates) (PHAs) can be synthesized by adopting different microbial fermentation strategies, of which submerged fermentation has been exploited largely. In the past few years, solid-state fermentation (SSF) has been reassessed as an alternative to submerged fermentation, and could be a possible strategy for the cost-effective production of PHAs. The capital investment for SSF is usually lower than that of submerged fermentation and the cost of raw materials for SSF would be cheap, since it uses waste agricultural residues. These positive factors make SSF a potential technique for PHAs production. However, this method is still too immature for commercialization. The major drawback to address is the proper maintenance of the culture conditions under SSF. The present review discusses the current developments in solid-state fermentation for the production of PHAs and addresses the various issues for its commercialization

Aspergillus spp., a versatile cell factory for enzymes and metabolites: Interventions through genome editing

Aspergillus sp. is widely distributed in nature and plays significant roles in the degradation of lignocellulose biomass and extensively used in bioprocess and fermentation technology and many species are also a generally regarded safe. Many of the Aspergillus species are established cell factories due to their inherent capacity in secreting large number of hydrolytic enzymes. With the advent of next generation genomic technologies and metabolic engineering technologies, the production potential of Aspergillus cell factory has improved over the years. Various genome editing tools has been developed for Aspergillus like engineered nucleases, zinc finger nucleases, TALEN and CRISPR-Cas9 system. Currently, the CRISPR/Cas9-based technique is extensively used to enhance the effectiveness of gene manipulation in model system Aspergillus nidulans and other strains like Aspergillus oryzae, Aspergillus niger and Aspergillus fumigatus. This review describes the recent developments of genome editing technologies in Aspergillus the synthesis of heterologous proteins and secondary metabolites in the Aspergillus species

Optimization of Process Parameters for the Production of γ-Linolenic Acid by Cunninghamella elegans CFR C07 in Submerged Fermentation

U radu je ispitana proizvodnja γ-linolenske kiseline submerznom fermentacijom s pomoću gljivice Cunninghamella elegans CFR C07, te je proces optimiran odabirom najprikladnijeg izvora ugljika i optimalnog vremena inkubacije. Radi poboljšanja ekstrakcije lipida iz biomase nakon fermentacije ispitane su četiri različite metode: ekstrakcija pomoću otapala i pijeska tretiranog kiselinom, ekstrakcija pomoću otapala i staklenih kuglica, liofilizacija ili ekstrakcija u Soxhlet uređaju. Proizvodnja je γ-linolenske kiseline prvo optimirana u tikvici zapremnine 250 mL na tresilici, a zatim u fermentoru od 3 L. Postignut je prinos γ-linolenske kiseline od 882 mg/L na tresilici, te 733 mg/L u fermentoru. Rezultati istraživanja potvrđuju da je C. elegans CFR C07 odličan mikroorganizam za proizvodnju γ-linolenske kiseline u submerznim uvjetima.The production of γ-linolenic acid (GLA) by the fungus Cunninghamella elegans CFR C07 in submerged fermentation was studied. Culture parameters such as carbon source and incubation time were optimized. Four different extraction methods using solvents with acid washed sand, glass beads, lyophilization and Soxhlet extraction were evaluated for improved extraction of lipids from the fungal biomass after fermentation. The GLA production was initially optimized in 250-mL flask and the process was demonstrated in a 3-litre fermentor. The maximum GLA production was 882 mg/L in shake flask culture and 733 mg/L in the fermentor. The study shows that Cunninghamella elegans CFR C07 is a potent organism for the production of GLA under submerged conditions

Analysis of Yield Attributing Characters of Different Genotypes of Wheat in Rupandehi, Nepal

Field experiment was conducted at National Wheat Research Program, Bhairahawa, Rupandehi with the objective to identify high yielding superior wheat genotypes for Rupandehi district of Nepalduring 2014. Experiment was laid out in one factorial Randomized completely block design with ten wheat genotypes including both released and promising; Annapurna 1, Annapurna 3, Pasang Lahmu, Bijaya, BL 3623, Bhirkuti, NL 297, BL 4316, BL 3978 and BL 4347with three replications. The results showed that the grain yield of BL 3978 was found higher (4.03 t ha-1) than other genotypes followed by BL 4347 (3.93t ha-1). BL 3978 have also higher number of effective tillers m-2 and test weight. Among release varieties, NL 297 show higher yield (4 t ha-1) followed by Bhirkuti (3.43 t ha-1)and Bijaya (3.37 t ha-1). From this experiment it can be concluded that BL 3978 was found promising among all genotypes however should be tested at on-farms before promoted for general cultivation in Rupandehi district of Nepal

Production of Pectinase from Bacillus sonorensis MPTD1

U radu je ispitanaproizvodnja pektinaze na podlozi s agarom s pomoću sedam sojeva bakterija izoliranih iz pokvarenog voća i povrća. Najučinkovitiji soj, MPTD1, identificiran je kao soj bakterije Bacillus sonorensis. Primjenom Plackett-Burman i Box-Behnken statističkih planova optimirani su različiti parametri, te je utvrđeno da udjeliekstrakta kvasca, K2HPO4,NaNO3i KCl te vrijeme inkubacije negativno utječu na proizvodnju pektinaze. Najveća postignuta aktivnost enzima bila je 2,43 (μM/mL)/min. U ovom je radu po prvi put opisana proizvodnja pektinaze s pomoću bakterije Bacillus sonorensis.Seven isolates from spoiled fruits and vegetables were screened for pectinase produc¬tion using pectin agar plates and the most efficient bacterial strain, MPTD1, was identified as Bacillus sonorensis. Optimisation of various process parameters was done using Plack¬ett-Burman and Box-Behnken designs and it was found that parameters like yeast extract, K2HPO4, incubation time, NaNO3 and KCl have a negative impact on pectinase production. Parameters like pH and MgSO4 and pectin mass fractions have a positive impact on pecti¬nase production. The maximum obtained enzyme activity was 2.43 (μM/mL)/min. This is the first report on pectinase production by Bacillus sonorensis

Applications of Microbial Enzymes in Food Industry

Uporaba enzima i mikroorganizama za pripremu hrane poznata je od davnina. S napretkom tehnologije razvijeni su novi enzimi specifičnih svojstava i širokog raspona primjene, te se neprestano traga za novim mogućnostima njihove uporabe. Bakterije, kvasci i gljivice te njihovi enzimi često se upotrebljavaju za pripremu hrane poboljšanog okusa i teksture, a ekonomski su isplativi. Mikrobni enzimi se koriste u većoj mjeri nego biljni i životinjski enzimi, i to zbog jednostavnije i jeftinije proizvodnje te njihove postojane kvalitete. U ovom se revijalnom prikazu raspravlja o najnovijim postignućima u tehnologiji proizvodnje enzima u prehrambenoj industriji. Naveden je opsežan popis enzima koji se koriste za obradu hrane, mikroorganizama iz kojih su proizvedeni, te je dan pregled njihove raznovrsne primjene.The use of enzymes or microorganisms in food preparations is an age-old process. With the advancement of technology, novel enzymes with wide range of applications and specificity have been developed and new application areas are still being explored. Microorganisms such as bacteria, yeast and fungi and their enzymes are widely used in several food preparations for improving the taste and texture and they offer huge economic benefits to industries. Microbial enzymes are the preferred source to plants or animals due to several advantages such as easy, cost-effective and consistent production. The present review discusses the recent advancement in enzyme technology for food industries. A comprehensive list of enzymes used in food processing, the microbial source of these enzymes and the wide range of their application are discussed

Napredak u istraživanju polisaharida izoliranih iz gljive vrste Cordyceps

Cordyceps sinensis (Berk.) Sacc. is one of the well-described fungi that has been used in traditional Chinese medicine for over 700 years. Fungal mycelia contain some polysaccharides that are responsible for their biological activity. C. sinensis has traditionally been cultivated on the high Tibetan plateau as a parasitic fungus growing on caterpillars. However, currently it is being cultivated on some insects and in artificial media. This article deals with the advances in the production, isolation and purification of Cordyceps polysaccharide (CP) in recent years, as well as the structure elucidation and pharmacological action. The article also aims to provide some references for further application and exploitation in the future.Cordyceps sinensis (Berk.) Sacc. jedna je od dobro poznatih gljiva što se više od 700 godina koristi u tradicionalnoj kineskoj medicini. Micelij gljive sadrži polisaharide koji utječu na njezinu biološku aktivnost. C. sinensis se tradicionalno uzgaja na Tibetanskom platou kao parazitska gljiva na gusjenicama leptira, a može se uzgajati i na ličinkama drugih insekata ili na umjetnoj podlozi. U radu se istražuje napredak postignut posljednjih godina u proizvodnji, izolaciji, pročišćavanju, strukturnoj analizi i procjeni farmakološkog djelovanja polisaharida iz gljive Cordyceps. Također je prikazan pregled referencija potrebnih za buduća istraživanja i primjenu tih polisaharida

Recent advances in microbial biosynthesis of C3 – C5 diols: Genetics and process engineering approaches

Diols derived from renewable feedstocks have significant commercial interest in polymer, pharmaceutical, cosmetics, flavors and fragrances, food and feed industries. In C3-C5 diols biological processes of 1,3-propanediol, 1,2-propanediol and 2,3-butanediol have been commercialized as other isomers are non-natural metabolites and lack natural biosynthetic pathways. However, the developments in the field of systems and synthetic biology paved a new path to learn, build, construct, and test for efficient chassis strains. The current review addresses the recent advancements in metabolic engineering, construction of novel pathways, process developments aimed at enhancing in production of C3-C5 diols. The requisites on developing an efficient and sustainable commercial bioprocess for C3-C5 diols were also discusse