Beef tallow injection matrix for serial crystallography

Serial crystallography (SX) enables the visualization of the time-resolved molecular dynamics of macromolecular structures at room temperature while minimizing radiation damage. In SX experiments, the delivery of a large number of crystals into an X-ray interaction point in a serial and stable manner is key. Sample delivery using viscous medium maintains the stable injection stream at low flow rates, markedly reducing sample consumption compared with that of a liquid jet injector and is widely applied in SX experiments with low repetition rates. As the sample properties and experimental environment can affect the stability of the injection stream of a viscous medium, it is important to develop sample delivery media with various characteristics to optimize the experimental environment. In this study, a beef tallow injection matrix possessing a higher melting temperature than previously reported fat-based shortening and lard media was introduced as a sample delivery medium and applied to SX. Beef tallow was prepared by heat treating fats from cattle, followed by the removal of soluble impurities from the extract by phase separation. Beef tallow exhibited a very stable injection stream at room temperature and a flow rate of < 10 nL/min. The room-temperature structures of lysozyme and glucose isomerase embedded in beef tallow were successfully determined at 1.55 and 1.60 Å, respectively. The background scattering of beef tallow was higher than that of previously reported fat-based shortening and lard media but negligible for data processing. In conclusion, the beef tallow matrix can be employed for sample delivery in SX experiments conducted at temperatures exceeding room temperature.

Glucose Isomerase: Functions, Structures, and Applications

Glucose isomerase (GI, also known as xylose isomerase) reversibly isomerizes D-glucose and D-xylose to D-fructose and D-xylulose, respectively. GI plays an important role in sugar metabolism, fulfilling nutritional requirements in bacteria. In addition, GI is an important industrial enzyme for the production of high-fructose corn syrup and bioethanol. This review introduces the functions, structure, and applications of GI, in addition to presenting updated information on the characteristics of newly discovered GIs and structural information regarding the metal-binding active site of GI and its interaction with the inhibitor xylitol. This review provides an overview of recent advancements in the characterization and engineering of GI, as well as its industrial applications, and will help to guide future research in this field.

Valorisation of whey for lactose recycling products production = Sūkalu valorizācija laktozes pārstrādes produktu ieguvei

The doctoral thesis “Valorisation of whey for lactose recycling products production” was developed from 2016 to 2021. Experiments were carried out in the research laboratories of the Faculty of Food Technology, Latvia University of Life Sciences and Technologies; Dairy Innovation Institute, California Polytechnic State University (USA); Institute of Microbiology and Biotechnology, Latvia University; Faculty of Chemistry, Latvia University; Institute of Solid State Physics, Latvia University and J.S. Hamilton Baltic Ltd. The aim of the doctoral thesis was to improve the lactose hydrolysis process for obtaining glucose-galactose and oligosaccharide syrups. The hypothesis of the doctoral thesis – the two-stage fermentation increases the sweetness of glucose-galactose syrup. The hypothesis of the doctoral thesis has been confirmed by the defended thesis: 1. The presence of cations affects the β-galactosidase activity in the sweet and acid whey permeate. 2. The chemical composition and quality of whey affect the physical properties of lactose. 3. Enzymatic reactions affect the functional and sensory properties of syrups. The research objects – sweet and acid whey permeates, glucose isomerase, commercial β-galactosidases and glucose-galactose syrup. The following tasks were set to achieve the aim of the doctoral thesis: 1. To evaluate the effect of cation concentration to ensure the β-galactosidase activity in substrate. 2. To investigate the physical properties of whey lactose in order to better understand its behaviour. 3. To study the changes of monosaccharide concentration in the lactose hydrolysis, varying with the solids concentration of the substrates and enzyme units. 4. To assess the possibilities of glucose isomerase to increase the sweetness of glucose-galactose syrup. 5. To evaluate the sensory properties of the developed syrups. The novelty of the doctoral thesis: 1. The study of the relationship of lactose hydrolysis process in the formation of galacto-oligosaccharides and lactulose. 2. The combination of β-galactosidase and glucose isomerase increases the sweetness of glucose-galactose syrup. The economic significance of the doctoral thesis: 1. The studies have shown the possibility to obtain syrup that can be used as sugar and sweeteners replacer in the food industry and to produce functional products. 2. A technological solution for hydrolysis of lactose is proposed, comprehensively evaluating the physical properties of lactose, fermentation parameters and whey composition. The doctoral thesis consists of three chapters: Chapter 1 describes the composition of whey and the possibilities of using it. An overview of the chemical and physical properties of lactose, lactose hydrolysis methods, the application of β-galactosidases and the properties of glucose-galactose syrup are provided. Chapter 2 summarises the materials and methods used in the thesis. Chapter 3 provides a summary of the results obtained in the study, the properties of commercial enzymes in different cation concentrations, the stability of enzymes in the gastrointestinal tract model, methods for the determination of lactose, the properties of dehydrated permeates are evaluated. The influence of factors on the hydrolysis of permeates and the profile of the obtained sugars was analysed. Possibilities for lactulose synthesis are considered. Sensory analysis of glucose-galactose syrups and syrups obtained in the two-stage fermentation are given. During the PhD studies the author had an internship at the Dairy Innovation Institute California Polytechnic State University (USA), where the experimental work was done. Internship was provided by the Baltic – American Freedom Foundation (BAFF) and the Council on International Education Exchange (CIEE). The study was partly financed by the LLU programme “Strengthening Research Capacity at the Latvia University of Agriculture” grant (Contract No. 3.2.-10/2017/LLU/27) “The optimization of bioprocesses for lactose recycling products”. The study was partly financed by the doctoral studies grant “Transition to the new doctoral funding model at the Latvia University of Life Sciences and Technologies” (Contract No. 3.2.-10/90). The thesis is written in English, it consists of 111 pages, 32 tables, 41 figures, 3 appendixes, and 233 bibliographic sources.

Production of Glucose Isomerase from Streptomyces roseiscleroticus

This study was aimed at the isolation and characterization of a microbial strain capable of producing glucose isomerase. Microbial strain was isolated from soil using starch casein agar as a differential media. The isolated microbial strain was capable of producing glucose isomerase which was tested using 2, 3, 5 - triphenyltetrazolium solution as a chromogenic substrate. The microbial strain was identified as Streptomyces species based on its morphological and microscopic characteristics. It was further subjected to molecular characterization using 16S rRNA sequencing and was subsequently confirmed as Streptomyces roseiscleroticus. Glucose isomerase was produced from Streptomyces roseiscleroticus after 120 hr of submerged fermentation at pH 6.8 and at 37°C utilizing xylose as the sole carbon source and a compendium of peptone, beef and yeast extracts as nitrogen sources. These findings suggest that the microbial strain, Streptomyces roseiscleroticus can be a useful bacterial source for the production of glucose isomerase needed for commercial and industrial utilization.

Catalyst Replacement Policy on Multienzymatic Systems: Theoretical Study in the One-Pot Sequential Batch Production of Lactofructose Syrup

One-pot systems are an interesting proposal to carry out multi-enzymatic reactions, though this strategy implies establishing an optimal balance between the activity and operability of the involved enzymes. This is crucial for enzymes with marked differences in their operational stability, such as one-pot production of lactofructose syrup from cheese whey permeate, which involves two enzymes—β-galactosidase (β-gal) and glucose isomerase (GI). The aim of this work was to study the behavior of one-pot sequential batch production of lactofructose syrup considering both enzymes immobilized individually, in order to evaluate and design a strategy of replacement of the catalysts according to their stabilities. To this end, the modelling and simulation of the process was carried out, considering simultaneously the kinetics of both reactions and the kinetics of inactivation of both enzymes. For the latter, it was also considered the modulating effect that sugars present in the medium may have on the stability of β-gal, which is the less stable enzyme. At the simulated reaction conditions of 40 °C, pH 7, and 0.46 (IUGI/IUβ-gal), the results showed that considering the stability of β-gal under non-reactive conditions, meaning in absence of the effect of modulation, it is necessary to carry out four replacements of β-gal for each cycle of use of GI. On the other hand, when considering the modulation caused by the sugars on the β-gal stability, the productivity increases up to 23% in the case of the highest modulation factor studied (η = 0.8). This work shows the feasibility of conducting a one-pot operation with immobilized enzymes of quite different operational stability, and that a proper strategy of biocatalyst replacement increases the productivity of the process.

KINETIC STUDY COMPARISON OF IMMOBILIZED GLUCOSE ISOMERASE (GENSWEET AND SWEETZYME IT) IN STIRRED TANK REACTOR AND PACKED BED REACTOR

D- Glucose/xylose isomerase catalysis the reversible isomerization of aldoses to ketoses such as D-glucose and D-xylose to D-fructose and D-xylose respectively. High fructose corn syrup (HFCS), a low calorie sugar substitute for cane sugar, utilizes Glucose isomerase enzyme for conversion of glucose to fructose. The conversion of glucose to fructose favours more at high temperature, providing an incentive to utilize thermostable and thermoactive glucose isomerase in High fructose corn syrup (HFCS) production. Present studies emphasize on enzymatic conversion and optimization using Sweetzyme IT extra & Gensweet, commercially available glucose isomerases. The experiments were carried out for enzymatic conversion of glucose to fructose using Gensweet and Sweetzyme in Packed bed reactor (PBR) and Stirred tank reactor (STR). Maximum conversion was seen in Stirred tank reactor (STR) using both of these enzymes, approx 10 % more Fructose conversion comparing it to packed bed reactor (PBR). Also, Stirred tank reactor (STR) reaction conditions such as pH, buffers, cofactor (MgSO4) requirement were optimized to achieve optimum enzyme activity. Analysis of enzymatic conversion samples was done using HPLC-RID (using Zorbax Column). The importance of the divalent cation MgCl2 for optimal enzyme activity was investigated. The enzyme performed best at pH 7.5 and 60°C, using 10mM MgSO4 as a cofactor. Utilizing Gensweet in Stirred tank reactor (STR), the maximum fructose transformation was 44 %. The most activity was detected with Sodium phosphate buffers, and EPPS buffers at pH 7 and 8, accordingly, whereas the least activity was reported with TRIS HCl buffer.

Production of D-Allose From D-Allulose Using Commercial Immobilized Glucose Isomerase

Rare sugars are regarded as functional biological materials due to their potential applications as low-calorie sweeteners, antioxidants, nucleoside analogs, and immunosuppressants. D-Allose is a rare sugar that has attracted substantial attention in recent years, owing to its pharmaceutical activities, but it is still not widely available. To address this limitation, we continuously produced D-allose from D-allulose using a packed bed reactor with commercial glucose isomerase (Sweetzyme IT). The optimal conditions for D-allose production were determined to be pH 8.0 and 60°C, with 500 g/L D-allulose as a substrate at a dilution rate of 0.24/h. Using these optimum conditions, the commercial glucose isomerase produced an average of 150 g/L D-allose over 20 days, with a productivity of 36 g/L/h and a conversion yield of 30%. This is the first report of the successful continuous production of D-allose from D-allulose by commercial glucose isomerase using a packed bed reactor, which can potentially provide a continuous production system for industrial applications of D-allose.

Control of strain in subgrains of protein crystals by the introduction of grown-in dislocations

It is important to reveal the exact cause of poor diffractivity in protein crystals in order to determine the accurate structure of protein molecules. It is shown that there is a large amount of local strain in subgrains of glucose isomerase crystals even though the overall crystal quality is rather high, as shown by clear equal-thickness fringes in X-ray topography. Thus, a large stress is exerted on the subgrains of protein crystals, which could significantly lower the resistance of the crystals to radiation damage. It is also demonstrated that this local strain can be reduced through the introduction of dislocations in the crystal. This suggests that the introduction of dislocations in protein crystals can be effective in enhancing the crystal quality of subgrains of protein crystals. By exploiting this effect, the radiation damage in subgrains could be decreased, leading to the collection of X-ray diffraction data sets with high diffractivity.