Browsing by Subject "β-glucosidase"
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(2018)Production of biofuels from non-food-based materials, such as lignocellulose, provides a good alternative for the traditional burning of fossil fuels. Some of the researched and existing biofuel applications are based on the utilization of enzymes. There are multiple cellulolytic enzymes required in the efficient hydrolysis of lignocellulose, and one of the key enzyme group is β-glucosidases. These enzymatic systems are mainly adopted from wood-decaying fungi. The overall enzymatic system consists of different types of cellulases that first degrade the crystalline cellulose to oligosaccharides and cellobiose. In the final step, β-glucosidases hydrolyse the oligosaccharides to glucose (a fermentable sugar). In fact, β-glucosidases are one of the limiting enzyme classes in this process, due to phenomena such as end-product inhibition. β-Glucosidases belong to Glycoside hydrolases (GH), that can be classified into different protein families. In an industrial perspective, the main interest resides in GH1 and GH3 family enzymes. Many industrially relevant extracellular β-glucosidases belong to GH3 family. However, intracellular GH1 β-glucosidases often exhibit higher tolerance to harsh conditions such as high substrate and product concentrations, high temperatures and low pH. The goal of this MSc thesis work was to purify and characterize a novel GH1 β-glucosidase, named NBG. Both GH1 and GH3 family enzymes were used as references for the characterization work. The GH3 reference enzyme was a β-glucosidase from Aspergillus niger (An Cel3A), derived from the commercial enzyme preparation Novozym 188. The used GH1 reference was a β-glucosidase from termite Nasutitermes takasagoensis (Nt GH1). The applicability of NBG β-glucosidase in biomass hydrolysis was also examined, together with possible considerations for applicability by other type of applications. The purification of An Cel3 reference enzyme was performed as described previously in literature. A novel protocol combining thermal treatment and low resolution IEX purification was developed for the NBG enzyme in this study. The enzyme’s activity on various pNP-substrates was determined, followed by pH stability, thermostability and inhibition studies. According to the result, NBG is a potential candidate for industrial use. The enzyme was found to be thermostable and active in a wide pH range when compared to the reference enzymes (stable up to 20 h at +60 ˚C and in pH 3.5 – 6.0). NBG also exhibited wider activity on pNP-substrates than the reference enzymes, highest specific activity being on pNPG, followed by moderate activity on pNPFuc and low activities on pNPGal and pNPXyl. Furthermore, NBG exhibited higher tolerance to inhibitors such as glucose and ethanol. Glucose inhibition was not observed until concentration of 200 mM for NBG, while in the same concentration the reference enzymes were almost completely inhibited. A Clear activation (of +16 %) by 100 mM glucose was observed with NBG. This enzyme also outperformed the An Cel3A-reference in ethanol tolerance, retaining activity better in 15 and 20 % ethanol. Activation by ethanol was also observed for both of the fungal enzymes, the most pronounced effect being observed for NBG in 15 % ethanol (+21 % of initial activity). The hydrolysis of insoluble cellulosic substrate (Avicel) was investigated using a commercial cellulase mixture (Celluclast 1.5L), where a semi-pure β-glucosidase preparation was added: novel β-glucosidase preparation (NBG (2-S2)) or the reference preparation An Cel3A (Nz188). According to the results, the NBG (2S-2) was outperformed by An Cel3A (Nz188) in Avicel 4 – 72 h hydrolysis experiments. The amount of reducing sugars released from Avicel was approximately 18–19 % higher with the commercial Nz188 preparation when compared to the 2S-2 preparation. Further analyses of samples revealed accumulation of cello-oligosacchardes, which may accumulate due to two possible reasons: Either the NBG enzyme does not possess high enough cellobiase activity (needed in biomass hydrolysis to glucose), or accumulation of cellobiose is due to transglycosylation activity of NBG. According to activity (and 3D modelling) data, NBG may not be a true β-glucosidase belonging to the EC 3.2.1.21 (and having cellobiase activity). Further investigation of the possible substrate specificity and transglycosylation activity of the NBG will be needed in assessing its applicability in other types of biotechnical applications.
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(2014)Divicine and isouramil are the causative agents of favism. The stability of divicine is of vital importance in faba bean detoxification. The literature review of this thesis focused on the hydrolysis of vicine to divicine, the oxidation processes of divicine and its stability studies. The main aims of this thesis were to produce divicine using the hydrolysis of vicine with ?-glucosidase, and study the effects of nitrogen/air atmosphere, reducing agent, pH and temperature on the stability of divicine. The identities of other compounds observed in vicine hydrolysis were also to be investigated. In addition, convicine was also hydrolyzed in the extracts and pure convicine fractions. Vicine and convicine were co-extracted from dehulled faba bean flour and were separated with preparative HPLC-MS. The extracts and the pure vicine and convicine fractions were hydrolyzed with ?-glucosidase to yield divicine and isouramil. The identities of the compounds formed during vicine fraction hydrolysis were studied by MS. In the following stability studies, the pure vicine fractions were hydrolyzed with ?-glucosidase under nitrogen and in the presence of (+)-sodium L-ascorbate. Moreover, the fractions were firstly hydrolyzed under air, next, the formed divicine was incubated at pH 3.0 or 5.0 at 20 or 37 ºC. An analytical HPLC method was used to study the changes during hydrolysis and stability tests. It was found that higher ?-glucosidase concentration and longer incubation period resulted in higher hydrolysis degrees of vicine and convicine. Further, vicine was hydrolyzed more rapidly at pH 3.0 than 5.0. Vicine was hydrolyzed to divicine. Divicine further generated two compounds, named compound 1 and compound 2 in this thesis. Their corresponding retention times and absorption maxima were: 2.15 min, 282 nm; 1.79 min, 262 nm; and 1.94 min, 210 nm. Compound 1 was directly generated from divicine. It was possibly oxidized divicine, but its characterization with MS failed in this study. Compound 1 decomposed to compound 2 at pH 5.0 at 20 ºC, but at pH 3.0 at 20 ºC, divicine might directly decompose to compound 2. Only one compound (named compound 3) was formed during convicine hydrolysis, and its retention time and absorption maximum were 2.50 min and 280 nm. Divicine and compounds 1, 2, and 3 were not stable, they finally decomposed to non-UV absorbing substances. Divicine was more stable under nitrogen than under air, and in the presence of (+)-sodium L-ascorbate than without its presence. Divicine decomposed similarly at pH 3.0 and 5.0 at 37 ºC, but at 20 ºC, divicine was more stable at pH 5.0. At both pH values, the stability of divicine was increased at 20 ºC compared with 37 ºC.
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