Thesis (MSc. (Biochemistry)) -- University of Limpopo, 2013 / Hydroxynitrile lyases (HNLs) are enzymes that catalyse enantioselective cleavage of the substrate in a reaction and are also used as important industrial biocatalysts for the synthesis of chiral cyanohydrins. The aim of the study was to screen indigenous South African plants for potential hydroxynitrile lyase activity, purify and biochemically characterise the active hydroxynitrile lyase(s) from the selected plants. Several indigenous plants were randomly collected, identified and screened for HNL activity. The plant parts (leaves, seeds or fruits) were processed using established experimental protocols in order to obtain the crude enzyme extracts. The enzymatic conversion of benzaldehyde and potassium cyanide to mandelonitrile was optimised and consequently used for the screening of HNL activity. Enzyme activity was detected in the crude enzyme extracts of Kalanchoe spp and Senecio spp and these were then designated as Ks and Sb, respectively. Ammonium sulphate fractionation, DEAE Toyopearl 650M and Concanavalin A chromatography techniques were then used in the purification process of the active crude enzyme extracts. Subsequently, two purified active fractions were isolated from each plant species with molecular masses estimated at 64.64 kDa and 64.06 kDa for the KsHNL enzymes and 70.60 kDa and 74.04 kDa for SbHNL enzymes. The optimum temperature and pH of all the isolated enzymes were determined as 50°C and pH 5, respectively. The experimental Km and Vmax values of the enzymes were respectively determined to be 0.33 and 0.73 mM and 1.238 and 1.948 μM/min for KsHNL; while that for SbHNL enzymes were 5.86 and 0.22 mM and 9.741 and 1.905 μM/min. The effect of additives and metal ions (viz., DTT, DEP, mercury chloride, magnesium chloride and zinc chloride) was determined. The experimental data obtained alluded to the notion that both KsHNL and SbHNL enzymes may contain the cysteine and serine residues next to their active sites and that a histidine residue may be involved in the catalytic activities of both the isolated KsHNL enzymes and one of the SbHNL enzymes. All the isolated enzymes from the two plant species did not seem to contain an FAD group. These findings compared favourably to the theoretical type II HNLs, although with a slight difference in that they displayed high molecular weights. Kalanchoe spp and Senecio spp are the two indigenous South African plants that were found to contain active HNLs. The isolated HNLs from the two plants have a potential to be xv purified to homogeneity, cloned and overexpressed into robust recombinant enzymes that can be used for large scale industrial applications.

Plants inherently produce polyphenols (i.e., antioxidants) as a response to reduce oxidative stress caused by abusive environmental pre- and postharvest conditions. These antioxidants, as well as vitamin C, are present in considerable levels in strawberries; however, excessive oxidative stress brought on by improper postharvest handling conditions can reduce the levels of antioxidants in the fruit and shorten the shelf-life of strawberries. Nevertheless, it may be possible to utilize strawberry’s naturally occurring polyphenols to reduce postharvest stress and extend their shelf life. The polyphenolic profile has been previously investigated in several strawberry cultivars, however no studies have determined the unique polyphenolic profiles of three important Florida strawberry cultivars (‘Florida Radiance’, Sweet Sensation® ‘Florida 127’and ‘Florida Beauty’) at harvest and during cold storage. Therefore, in order to better understand the distribution of individual polyphenols within these cultivars and their impact on postharvest shelf-life, this study examined the polyphenolic profiles throughout 7 days of cold storage (1 °C) using an HPLC-DAD. The activity of phenylalanine ammonia lyase (PAL), a key enzyme in the biosynthesis of polyphenols, and polyphenol oxidase (PPO), the enzyme responsible for polyphenol degradation, were also examined during cold storage to understand their possible influences on postharvest synthesis or degradation of polyphenols. This study revealed that the polyphenolic profile of strawberry fruit was genotype dependent; however, pelargonidin 3-glucoside was consistently the anthocyanin found in higher concentrations in the fruit regardless of the cultivar. Apart from the anthocyanins, the flavonols showed the most variation among the three cultivars. PAL was slightly induced during strawberry postharvest storage suggesting that a stress response occurred during cold storage while PPO showed variable induction patterns across all three cultivars most likely due to their different polyphenol profiles. Analysis of the distribution of polyphenols in the cortex and pith of strawberries showed that polyphenols were mostly concentrated in the cortex of the fruit and that the concentration of individual polyphenol in each fruit tissue varied by cultivar. These results indicate that the oxidative stress response varies in each of the strawberry cultivars studied contributing to their unique polyphenolic profile. Results from this study can ultimately help to identify the polyphenols and enzymes related to superior postharvest quality in future studies.

Although enzymes show great potential for use in industrial applications, their implementation from a practical perspective is still somewhat limited by various shortcomings in the area of enzyme immobilization. The use of silica sol-gels for protein entrapment has been studied extensively over the past 15 years or so. However, our understanding of the interactions between the immobilization matrix and the entrapped biomolecules is still relatively poor. Non-invasive in situ spectroscopic characterization is a promising approach to gain a better understanding of the fundamentals governing sol-gel immobilization. This thesis describes the application of Fourier transform infrared (FTIR) microscopy, two dimensional (2D) FTIR and fluorescence spectroscopy to characterize the immobilization matrix, entrapped model proteins and their interactions. Hydroperoxide lyase (HPL [E.C. 4.1.2.]) was chosen as a potential model protein for sol-gel entrapment. HPL activity was evaluated by use of factorial experimental design investigating the effects of KCl and Triton X-100 on HPL activity with 13-hydroperoxy-octadecadienoic acid (LA-OOH) and the novel water soluble 13-hydroperoxy-octadienoyl sulfate (LS-OOH) as substrates. The highest HPL activity was achieved under aqueous conditions with high salt and low surfactant concentrations and LA-OOH as the substrate. A significant interaction between salt and surfactant as well as salt and substrate was identified and a hypothesis to explain the basis of the interaction phenomena is presented. To analyze sol-gels with spectroscopic techniques, a sample format amenable to these techniques was needed. Therefore, a spin-coating technique for the preparation of aluminum or glass supported sol-gel thin films containing immobilized protein and a varying degree of the organically modified precursor propyltrimethoxysilane (PTMS) was developed. This approach produced samples that were suitable for chemical mapping using FTIR microscopy or fluorescence spectroscopic investigations. A data analysis method was developed to extract information on chemical speciation and distribution from FTIR data matrices obtained through FTIR microscopy. Results indicate that sol-gel thin films are not homogeneous on the microscopic level. Instead, they are heterogeneous with a clustering in the distribution of the model proteins studied (lysozyme [E.C. 3.2.1.17], lipase [E.C. 3.1.1.3] and bovine serum albumin (BSA)) at the scale investigated. The appearance of these clusters was found to depend on the type of protein entrapped, as well in some cases on the composition of the sol-gel. Moreover, the PTMS distribution was positively correlated with the protein distribution in the case of lipase and negatively correlated in the case of lysozyme and BSA. Additionally, sol-gels with a higher PTMS content appeared to conserve protein structure in areas where lipase clustered. Lysozyme and BSA, on the other hand, seemed to retain their structures in high concentration clusters better at lower PTMS content. A hypothesis taking into account the surface hydrophobicity of the proteins and the sol-gel composition as the basis for these phenomena is proposed. Fluorescence spectroscopy revealed that the PTMS content of the sol-gels had a direct effect on the physical properties of the immobilized proteins as evidenced by a blue shift of the intrinsic tryptophan (TRP) fluorescence. Temperature-dependent fluorescence spectroscopy revealed that the amount of TRP quenching was inversely proportional to the PTMS content of the sol-gel, suggesting that there were varying amounts of water available for quenching for the different immobilized enzyme systems. Analysis of the sol-gels by 2D FTIR spectroscopy with a focus on the amide A region using Gaussian peak deconvolution revealed two different species of water for the 50 % PTMS thin film sol-gels with BSA that could be described as fully and not fully H-bonded. It was also found that these species of water showed different removal profiles during thermal treatment. 2D FTIR of the amide I region followed by absorbance difference spectrum evaluation revealed that the temperature stability of the three model proteins was also sol-gel composition dependent. A hypothesis that the surface characteristics of the proteins determine the nature of the composition dependence is presented.

Although enzymes show great potential for use in industrial applications, their implementation from a practical perspective is still somewhat limited by various shortcomings in the area of enzyme immobilization. The use of silica sol-gels for protein entrapment has been studied extensively over the past 15 years or so. However, our understanding of the interactions between the immobilization matrix and the entrapped biomolecules is still relatively poor. Non-invasive in situ spectroscopic characterization is a promising approach to gain a better understanding of the fundamentals governing sol-gel immobilization. This thesis describes the application of Fourier transform infrared (FTIR) microscopy, two dimensional (2D) FTIR and fluorescence spectroscopy to characterize the immobilization matrix, entrapped model proteins and their interactions. Hydroperoxide lyase (HPL [E.C. 4.1.2.]) was chosen as a potential model protein for sol-gel entrapment. HPL activity was evaluated by use of factorial experimental design investigating the effects of KCl and Triton X-100 on HPL activity with 13-hydroperoxy-octadecadienoic acid (LA-OOH) and the novel water soluble 13-hydroperoxy-octadienoyl sulfate (LS-OOH) as substrates. The highest HPL activity was achieved under aqueous conditions with high salt and low surfactant concentrations and LA-OOH as the substrate. A significant interaction between salt and surfactant as well as salt and substrate was identified and a hypothesis to explain the basis of the interaction phenomena is presented. To analyze sol-gels with spectroscopic techniques, a sample format amenable to these techniques was needed. Therefore, a spin-coating technique for the preparation of aluminum or glass supported sol-gel thin films containing immobilized protein and a varying degree of the organically modified precursor propyltrimethoxysilane (PTMS) was developed. This approach produced samples that were suitable for chemical mapping using FTIR microscopy or fluorescence spectroscopic investigations. A data analysis method was developed to extract information on chemical speciation and distribution from FTIR data matrices obtained through FTIR microscopy. Results indicate that sol-gel thin films are not homogeneous on the microscopic level. Instead, they are heterogeneous with a clustering in the distribution of the model proteins studied (lysozyme [E.C. 3.2.1.17], lipase [E.C. 3.1.1.3] and bovine serum albumin (BSA)) at the scale investigated. The appearance of these clusters was found to depend on the type of protein entrapped, as well in some cases on the composition of the sol-gel. Moreover, the PTMS distribution was positively correlated with the protein distribution in the case of lipase and negatively correlated in the case of lysozyme and BSA. Additionally, sol-gels with a higher PTMS content appeared to conserve protein structure in areas where lipase clustered. Lysozyme and BSA, on the other hand, seemed to retain their structures in high concentration clusters better at lower PTMS content. A hypothesis taking into account the surface hydrophobicity of the proteins and the sol-gel composition as the basis for these phenomena is proposed. Fluorescence spectroscopy revealed that the PTMS content of the sol-gels had a direct effect on the physical properties of the immobilized proteins as evidenced by a blue shift of the intrinsic tryptophan (TRP) fluorescence. Temperature-dependent fluorescence spectroscopy revealed that the amount of TRP quenching was inversely proportional to the PTMS content of the sol-gel, suggesting that there were varying amounts of water available for quenching for the different immobilized enzyme systems. Analysis of the sol-gels by 2D FTIR spectroscopy with a focus on the amide A region using Gaussian peak deconvolution revealed two different species of water for the 50 % PTMS thin film sol-gels with BSA that could be described as fully and not fully H-bonded. It was also found that these species of water showed different removal profiles during thermal treatment. 2D FTIR of the amide I region followed by absorbance difference spectrum evaluation revealed that the temperature stability of the three model proteins was also sol-gel composition dependent. A hypothesis that the surface characteristics of the proteins determine the nature of the composition dependence is presented.

The enzyme L-methionine γ-lyase (MGL) utilizes a pyridoxal-5’-phosphate-cofactor in order to convert L-methionine to α-ketobutyrate, ammonia and methyl mercaptan. MGL is proposed to be a potential drug target since it is expressed in the human pathogens, Trichomonas vaginalis and Entamoeba histolytica, but not in humans. There is currently a need to find alternative drug targets for these pathogens, because the misuse and overuse of the currently prescribed drugs of choice, metronidazole and tinidazole, have lead to drug resistance. The overall goal of this thesis was to examine the chemistry of MGL 1 from T. vaginalis (TvMGL1) by probing the active site by site-directed mutagenesis and with fluorinated methionine analogs. The mutation of the active site residue Cys113 to Ser led to a 5-fold decrease in turnover rate for L-methionine relative to the wild-type enzyme. The results suggest that the active site C113 residue plays an important role in catalysis and is consistent with literature reports for MGL homologs from Pseudomonas putida and E. histolytica. Probing the active site of TvMGL1 with the fluorinated methionine analogs, L-difluoromethionine (DFM) and L-trifluoromethionine (TFM), were found to increase the turnover rate of the enzyme with an increase in fluorine substitution. The results suggest that the bulky fluorine atoms do not interfere with the Michaelis-Menten kinetics of the enzyme, and the γ-elimination step is rate determining. The second goal of this thesis was to identify the reactive intermediates generated by the processing of TFM and the uninvestigated DFM by TvMGL1, and to investigate the theoretical and experimental chemistry and biochemistry of these fluorinated groups (CF3S- and CF2HS-). The reactivity of the intermediates, generated from the processing of DFM by TvMGL1 was correlated to the cytotoxicity observed in model organisms expressing TvMGL1, and consistent with the hypothesis that the intermediates will result in the thioformylation of primary amines. The results suggest that cytotoxicity requires thioacylation of a single primary amine, while sequential cross-linking of primary amines is not an absolute requirement. The relationship between the chemical structure of the reactive intermediates produced from the enzymatic processing of these analogs and their cellular toxicity is discussed. Attempts at the synthesis of 3,3-difluoro-O-methyl-L-homoserine were undertaken in order to examine the catalytic mechanism of TvMGL1, since the compound is expected to inhibit the enzyme. To ensure that the oxo moiety does not impede the chemistry of the enzyme, the analog, O-methyl-L-homoserine was examined as a potential substrate for TvMGL1. Several synthetic routes to 3,3-difluoro-O-methyl-L-homoserine were examined; however, attempts to fluorinate the β-carbon atom of the starting material were unsuccessful.

In this study, a methodology for separation of chiral molecules, by using enhanced ultrafiltration system was developed. Benzoin was the model chiral molecule studied. In the scope of developing this methodology, some parameters were investigated in the preliminary ultrafiltration experiments in order to set the operation conditions for enhanced ultrafiltration experiments. Due to the slight solubility of benzoin in pure water, 15% (v/v) Polyethylene glycol (PEG 400) and 30 % (v/v) Dimethyl sulfoxide (DMSO) were selected as cosolvents. Because of the high retention capacity of RC-10000 Da membranes for benzoin, a membrane saturation strategy was developed. In polymer enhanced ultrafiltration (PEUF) experiments bovine serum albumin (BSA) was used as ligand. Effects of ligand concentration and pH on total benzoin retention and on enantiomeric excess (ee %) were investigated. Benzoin concentration was almost kept constant at ~10 ppm and ~50 ppm for 15% (v/v) PEG 400 and 30 % (v/v) DMSO cosolvents, respectively. It was observed that the increase either in pH or in BSA concentration yielded an increase in total benzoin retention. In 15% (v/v) PEG 400-water, with BSA concentration of 10000 ppm, at pH 10, total benzoin retention reached to 48.7%. For this cosolvent, at different pH values and at different BSA concentrations, all ee % values were about or less than 10%. When 50000 ppm BSA was dissolved in 30 % (v/v) DMSO-water, total benzoin retention increased to 41.3% at pH 10 and ee % reached 16.7 % at pH 11. In enzyme enhanced ultrafiltration (EEUF) experiments, specific to benzoin, apo form of Benzaldehyde Lyase (BAL, E.C. 4.1.2.38) was used as ligand. These experiments were performed with constant ~ 10 ppm benzoin concentration in only 15% (v/v) PEG 400 &ndash / water solvent. Effect of BAL concentration on total benzoin retention and ee% was investigated. It was found that / for all the studied BAL concentrations in the range of 650- 1936 ppm total benzoin retention and ee % were kept almost constant at ~75% and ~60%, respectively.

Benzaldehyde lyase (BAL, EC 4.1.2.38) from Pseudomonas fluorescens Biovar I, a thiamine pyrophosphate (ThDP) dependent enzyme, catalyzes the enzymatic kinetic resolution of racemates by C-C bond cleavage and concomitant C-C bond formation. In this study, benzaldehyde lyase gene from Pseudomonas fluorescens Biovar I was cloned into Pichia pastoris, with the aim of the extracellular production of the enzyme. For this purpose, firstly, PCR amplified bal gene was cloned into an integration vector pPICZalphaA. Thereafter the recombinant plasmid pPICZalphaA::bal was transformed into P.pastoris. Extracellular benzaldehyde lyase enzyme was expressed under the control of the strong AOX promoter and the secretion of the enzyme in the fermentation medium was achieved by means of S. cerevisiae alpha factor signal sequence. The recombinant cells were grown for 48-72 hours in solid medium then the cells inoculated in glycerol containing medium. After being separated by centrifugation cells were transferred into methanol containing production medium. In methanol containing medium cells were grown for 72 h. Starting from t=24 h methanol was added to medium as an inducer of AOX promoter and the carbon source in order to produce BAL in every 24 hour. SDS-PAGE analyses illustrated that extracellular benzaldehyde lyase enzyme produced by the recombinant P.pastoris strain had the size of 59 kDa, which is the size of benzaldehyde lyase monomer. FPLC analysis showed that concentration of the tetrameric form of benzaldehyde lyase enzyme, active form, was much less than the monomeric form of the enzyme indicating that the enzyme produced by recombinant P.pastoris mostly could not fold into multimeric form in the fermentation medium.

In this study, the effects of oxygen transfer conditions on the synthesis of the enzyme benzaldehyde lyase as intracellular in recombinant E. coli BL21 (DE3) pLysS was investigated sistematically and a comprehensive model was developed to determine benzaldehyde lyase activity. For this purpose, the research program was carried out in mainly two parts. In the first part of study, the effects of oxygen transfer together with the mass transfer coefficient (KLa), enhancement factor E (=KLa/KLao), volumetric oxygen transfer rate, volumetric and specific oxygen uptake rates, mass transfer and biochemical reaction resistances / moreover, the variation in product and by-product distribution, specific substrate uptake rates, yield and maintenance coefficient were investigated in the pilot scale batch bioreactor at QO/VR = 0.5 vvm and agitation rates of N= 250, 500, 625, and 750 min-1, and dissolved oxygen levels DO= 20%, 40% conditions, while medium components were CGlucose= 8.0 kg m-3, C(NH4)2HPO4= 5.0 kg m-3 and salt solution at controlled pHc=7.2. The highest cell concentration and benzaldehyde lyase activity were obtained at DO=40% condition as 3.0 kg m-3 and A=1095 Ucm-3, respectively. v Then a mathematical model was proposed to estimate benzaldehyde lyase activity as function of time, agitation rate, cell concentration, dissolved oxygen concentration, and by-product concentration with reasonable accuracy.

This study focuses on the molasses based complex medium design for benzaldehyde lyase production by recombinant E. coli BL21 and development of a feeding strategy based on the designed complex medium. For this purpose, firstly, the effects of molasses were investigated in laboratory scale bioreactors. As E. coli BL21 was not able to utilize sucrose, molasses was pretreated and hydrolyzed to fructose and glucose. Thereafter, effect of pretreated molasses concentration was investigated in the range of 16 to 56 kg m-3 by batch-bioreactor experiments / and the highest cell concentration and benzaldehyde lyase activity were obtained as CX=5.3 kg m-3 and A=1617 U cm-3, respectively, in the medium containing 7.5 kg m-3 glucose and 7.5 kg m-3 fructose. Then, different feeding strategies were developed to produce efficient cells with high concentration and BAL activity. In the first strategy, after 10 hours of batch-cultivation with molasses based medium having 7.5 kg m-3 glucose and 7.5 kg m-3 fructose concentration, based on the airflow rate, pretreated molasses was fed to the system. When air flow rate decreased considerably, fed was given to the system that results in increase in glucose and fructose concentration in the medium to 2.5 kg m-3. At the end of the process, the highest cell concentration obtained was CX=7.4 kg m-3. The maximum activity was reached at 20th hour as A=2360 U cm-3. On the other hand, as air flow variation only demonstrated the absence of glucose not fructose, a second strategy, based on the detection of the fructose and glucose concentrations during the process, was applied. In this strategy when glucose and fructose were depleted, fed was given to the system that results in increase in glucose and fructose concentration in the medium to 2.5 kg m-3 / and the highest BAL activity was obtained as 2370 U cm-3 at t= 26 h where the cell concentration was 7.5 kg m-3. At the last strategy, when glucose and fructose were depleted, fed was given to the system that results in increase in CGlucose=1.5 kg m-3 and CFructose=1.5 kg m-3 in the production medium to decrease the accumulation of acetic acid. By this strategy highest cell concentration was obtained as 8.04 kg m-3 at t=24 h and the highest BAL activity was 2315 U cm-3. These strategies could be accepted having the same BAL activity with little distinctions. However, cell concentration of the last one was higher than others and also the lowest amount of carbon source was used. Thus, last one could be chosen as the most favorable strategy.

The objective of the study presented here was to develop either enzymatic or whole cell mediated green procedures for the syntheses of a-hydroxy ketones. Production of optically active synthons is crucial for the preparation of fine chemicals. Enzymes and whole-cell biocatalysts have proven to be excellent vehicles with their chiral nature for the biotransformations. Under the light of this discussion, firstly benzaldehyde lyase [BAL, (EC 4.1.2.38)] was used in novel C-C bond formation reactions to obtain interesting and biologically important precursors / 2-Hydroxy-1-arylethan-1-ones and functionalized aliphatic acyloin derivatives. All the compounds were obtained with high yields and in the case of aliphatic acyloin derivatives with high enantiomeric excesses (ee&rsquo / s). Another strategy was to use whole cell biocatalysis. A.flavus 200120 was found to be a promising biocatalyst with the ability to catalyze a broad range of reactions / reduction, hydrolysis and deracemization, while another fungus / A. oryzae 5048 was utilized in bioreduction reactions of benzil and its derivatives. Each reaction was investigated, optimized and thus enhanced via medium design. Products were obtained with high yields and ee&rsquo / s. To sum up, in this study novel efficient green procedures were developed to synthesize various ahydroxy ketones with high yield and stereoselectivity. These newly established methods present promising alternatives to classical chemical methodologies.