ResearchSpace :: Browsing by Author "Steenkamp, Lucia H"

Browsing by Author "Steenkamp, Lucia H"

Now showing 1 - 16 of 16

Aldolase: A desirable biocatalytic candidate for biotechnological applications
(2024-01) Mathipa-Mdakane, Moloko G; Steenkamp, Lucia H
The utilization of chemical reactions is crucial in various industrial processes, including pharmaceutical synthesis and the production of fine chemicals. However, traditional chemical catalysts often lack selectivity, require harsh reaction conditions, and lead to the generation of hazardous waste. In response, biocatalysis has emerged as a promising approach within green chemistry, employing enzymes as catalysts. Among these enzymes, aldolases have gained attention for their efficiency and selectivity in catalyzing C-C bond formation, making them versatile biocatalysts for diverse biotechnological applications. Despite their potential, challenges exist in aldolase-based biocatalysis, such as limited availability of natural aldolases with desired catalytic properties. This review explores strategies to address these challenges, including immobilization techniques, recombinant expression, and protein engineering approaches. By providing valuable insights into the suitability of aldolases as biocatalysts, this review lays the groundwork for future research and the exploration of innovative strategies to fully harness the potential of aldolases in biotechnology. This comprehensive review aims to attract readers by providing a comprehensive overview of aldolase-based biocatalysis, addressing challenges, and proposing avenues for future research and development.
Alternative pathway implicated as an influencing factor in the synthesis of theaflavin
(Taylor & Francis, 2016-03) Van der Westhuizen, M; Steenkamp, Lucia H; Steenkamp, P; Apostolides, Z
The principal pigments present in black tea, theaflavins (TF), have been indicated to be of potential clinical significance in various fields of research which has been hampered by the very low levels of TFs from black tea extractions, being the original method employed to acquire TFs. Forelle pear (44µM TF/g dry weight/h) and Yacon leaf (65µM TF/g dry weight/h) homogenates were tested for their TF synthesis capacity and found to have a larger TF synthesis capacity than a green tea leaf homogenate (26µM TF/g dry weight/h) based upon the flavognost method. In an incubation system of green tea leaf extract utilizing endogenous enzymes present in Forelle pear and Yacon homogenates to synthesize TF, the formation of an unknown peak [m/z 563.1349; (23.95)(sup5); C(sub26)H(sub28)O(sub14)] was detected by mass spectrometry with a molecular mass similar to TF. This is in contrast to TF being solely synthesized in an in vitro model incubation system using isolated catechins and purified Forelle pear polyphenol oxidase. The preferential formation of the unknown compound could explain the low levels of TFs in black tea.
Ambrafuran (AmbroxTM) synthesis from natural plant product precursors
(2020-08) Ncube, EN; Steenkamp, Lucia H; Dubery, IA
Ambergris, an excretion product of sperm whales, has been a valued agent in the formulation of perfumes. The composition of ambergris consists of two major components: 40–46% cholestanol type steroids and approximately 25–45% of a triterpenoid known as ambrein. Ambergris undergoes oxidative decomposition in the environment to result in odorous compounds, such as ambraoxide, methylambraoxide, and ambracetal. Its oxidized form, ambrafuran (IUPAC name: 3a,6,6,9a-tetramethyl-2,4,5,5a,7,8,9,9b-octahydro-1H-benzo[e][1]benzofuran), is a terpene furan with a pleasant odor and unique olfactive and fixative properties. The current state of the fragrance industry uses ambrafuran materials entirely from synthetic or semisynthetic sources. However, natural compounds with the potential to be converted to ambergris-like odorants have been extracted from several different types of plants. Here we review plant terpenoids suitable as starting materials for the semisyntheses of ambrafuran or intermediates, such as ambradiol, that can be used in biocatalytic transformations to yield ambrafuran.
Application of biocatalysis in synthetic chemistry
(2006-11) Brady, D; Molefe, M; Steenkamp, Lucia H; Parkinson, C; Jordaan, J; Visser, Daniel F; Simpson, C; Chhiba, Varsha P; Mathiba, K
It was found that the nitrile hydratases in microorganisms isolated from around the world had infra-species amino acid sequence differences, and this provided an explanation for the variability of nitrile substrate usage within the species. CSIR Biosciences Enzyme Technology supports the result - South African microbial diversity will include unique biocatalysts
Bacteria and yeast isolation and characterisation from a South African fermented beverage
(Academy of Science of South Africa, 2019-11) Moodley, Sanchia S; Dlamini, Nomusa R; Steenkamp, Lucia H; Buys, Elna M
Spontaneous fermentation of motoho, a southern African non-alcoholic sorghum beverage, results in products with inconsistent microbiological and sensory quality. We aimed to identify the microorganisms involved in the fermentation of motoho by using culture-dependent techniques as well as culture-independent polymerase chain reaction (PCR) screening and matrix-assisted laser desorption/ionisation time-of-flight analysis (MALDI-TOF). Lactobacillus, Candida, Rhodotorula and Geotrichum species were identified. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to evaluate the protein profiles of the isolated Lactobacillus species which produced protein bands of 14 kDa to 160 kDa, similar to those of other lactic acid bacteria isolated from various foods. A sensory panel evaluated and found significant differences (p<0.05) between the mouth feel, aroma and flavour of the traditional and modified motoho, with the latter being preferred. The microorganisms identified in this study could be used as starter cultures to optimise upscaled production of motoho.
Biocatalytic enantiomeric resolution of l-menthol from an eight isomeric menthol mixture
(Elsevier, 2012-03) Brady, D; Reddy, S; Mboniswa, B; Steenkamp, Lucia H; Rousseau, A; Parkinson, CJ; Chaplin, J; Mitra, RK; Moutlana, T; Marais, SF; Gardiner, NS
The four diastereomers of menthol and their enantiomers, namely dl-menthol, dl-neomenthol, dl-neoisomenthol and dl isomenthol, were synthesised by the hydrogenation of thymol to yield an eight isomer liquid menthol. A suitably selective lipase was sought to preferentially esterify l-menthol in hexane, hence simplifying separation from this diasteromeric mix through distillation. From an initial screen of over 70 enzyme preparations, a commercial Pseudomonas fluorescens lipase (Amano AK) was selected, and vinyl acetate was chosen as a suitable irreversible acyl donor for transesterification. The enzyme was recycled a total of 150 times in 5 ml batch reactions using liquid menthol and achieving an overall yield of 184.31 g dl-menthol/g enzyme. An enantiomeric excess of l-menthol of greater than 95% was reproducibly achievable at a conversion of 30% dl-menthol (0.68 M) at =50°C. On the basis of the composition of liquid menthol the reaction had a diastereomeric ratio of 81%. The resolution reaction was scaled up 400 fold to 2 L and the ezyme recycled 38 times with an average conversion of the available l-menthol of 59%.
Decision making in the development of a biocatalytic route for resolution of S-naproxen: from screening to scale-up
(Formatex Publishers, 2010-06) Steenkamp, Lucia H; Brady, D
The non-steroidal anti-inflammatory drug naproxen is most effective as the single S-naproxen enantiomer. However, typical synthetic routes to naproxen yield the racemate of both the R and the S stereoisomers. Biocatalysts can be used to resolve racemic mixtures of naproxen esters using esterases or lipases. During research and development of this process we reached several decision points based on biocatalyst selection, reaction engineering, and process definition. These included reaction type (hydrolytic versus esterification options), substrate selection, biocatalyst selection, and reaction conditions. The study began with identification of a suitable lipase or esterase for biocatalytic enantiomeric resolution of R,S-naproxen to yield the single enantiomer S-naproxen with an enantiomeric ratio (E) in excess of 200. Approximately 650 unidentified fungi, yeasts and bacteria from culture collections were screened and more than 80 commercially available esterases and lipases. From this 9 enzymes were chosen to optimise using statistically designed experiments to find the most important factors which influenced the conversion and enantioselectivity. During the process development, decisions were made regarding hydrolytic versus esterification options, enzyme type, substrate size, co-solvent, and physical parameters. Final considerations were the optimised conversion and enantiomeric excess, reaction productivity, and enzyme cost to give a process which would be feasible on large scale. The result was a commercially viable reaction yielding an E of approximately 500 and enantiomeric excess of 99%. The decisions behind the selection of the route are broadly applicable to other biocatalytic processes.
Development of a two-step “green” synthesis for (-)-ambafuran production
(CSIR, 2010-09-01) Steenkamp, Lucia H
The main objective of the development of a two-step “green” synthesis for (-)-ambafuran production is to find an alternative synthesis of (-) Ambrox from sclareol, to use a bioconversion or biocatalysis route, and that it results in a natural or nature-identical product. The final aim is to scale the process to bench scale, to finalise a technology package which the client can use and to commercialise the technology.
Gas chromatographic profiling of the biocatalytic conversion of sclareol to ambradiol by Hyphozyma roseoniger
(2021-10) Ncube, EN; Mathiba, Kgama; Steenkamp, Lucia H; Dubery, IA
Hyphozyma roseoniger is a filamentous yeast used for its biocatalytic ability to convert sclareol, a plant diterpenoid to ambradiol, an intermediate to ambrafuran, which is a sought-after fixative in the perfume industry. Metabolite profiling is a suitable investigative tool to dissect the biochemical steps involved in the biocatalytic reaction by H. roseoniger from sclareol to ambradiol. H. roseoniger suspensions were grown in batch culture to simulate growth in a bioreactor over a 14-day period. Cells were harvested at stipulated days (phases of growth) using an ethyl acetate extraction procedure. The progress of the bioconversion was monitored using gas chromatography with flame ionisation detection (GC–FID) profiling. Here, decreasing concentrations of sclareol with concomitant increasing concentrations of ambradiol as product and with sclareolide as a putative intermediate, were recorded. While the presence of unidentified peaks were noted in the GC chromatograms, no previously unknown intermediates could be identified in the sequence of sclareol to sclareolide to ambradiol.
Naproxen: from skunk work to scale-up
(Catalysis Society of South Africa (CATSA), 2008-11) Steenkamp, Lucia H; Brady, D
The presentation starts with a comparison between Naproxen and Aspirin. The characteristics and properties of Naproxen are explained, as well as the biocatalysis and the screening of the enzyme activity and selectivity. It ends with a discussion of the product quality.
Optimisation of stabilised carboxylesterase NP for enantioselective hydrolysis of naproxen methyl ester
(Elsevier, 2008-12) Steenkamp, Lucia H; Brady, D
Although the enantioselective kinetic resolution of ester racemates of the non-steroidal antiinflammatory drug naproxen ([2-(6-methoxy-2-naphthyl) propionic acid]) is a common demonstration for biocatalysis, the enantiomeric excess of the reactions is usually insufficient to warrant commercialisation. However, optimised reactions using heterologously expressed Carboxylesterase NP provided highly enantioselective hydrolysis of racemic naproxen methyl ester. Up to 46.9% conversion was achieved in 5 h in the presence of 10 units enzyme/g ester with a product ee of 99% and an E of approximately 500. The final optimised reaction conditions were 150 g/l of substrate slurry in 0.01 M sodium phosphate buffer pH 8.75 (maintained with 2.5 M NaOH) at 45 8C and in the presence of 1% surfactant (Tween 80). Stabilisation of the enzyme with >2000 ppm formaldehyde prior to use resulted in an optimal volumetric productivity of 22.2 g/l/h substrate at an enzyme loading of 18 units enzyme/g ester. Reaction kinetics revealed that the product S-naproxen formed causes product inhibition, but not enzyme toxicity, and resulted in the decrease in reaction rate with conversion. The R-NME (naproxen methyl ester) (unwanted enantiomer) did not have a significant influence on the reaction rate. DBU (diazobicycloundecene), used for the racemisation of the unwanted enantiomer, was recycled with the substrate but did not influence the conversion rate
Optimisation of the enantioselective biocatalytic hydrolysis of naproxen ethyl ester using ChiroCLEC-CR
(Elsevier Science Inc., 2004-03-04) Brady, D; Steenkamp, Lucia H; Skein, E; Chaplin, JA; Reddy, S
In a biocatalytic reaction the immobilized lipase ChiroCLEC-CR enantioselectively hydrolysed a naproxen ethyl ester racemate, yielding (S)-naproxen with an enantiomeric excess of more than 98%, an enantiomeric ratio (E) of more than 100, and substrate conversion in excess of 40%. Statistically designed experiments were performed to optimise temperature, enzyme to substrate ratio, substrate concentration, agitation, reaction time, pH, buffer concentration and co-solvent addition. Optimisation efforts resulted in more than 20-fold improvement of activity, while the excellent enantioselectivity of the enzymes was maintained. In particular, the addition of PEG 1000 as a co-solvent improved conversion rates 10-fold. The kinetic parameters V-max and K-M were determined to be 0.359_mol/min/mg and 17.6 mM, respectively. The optimised reaction conditions were 10% (m/v) substrate, and enzyme to substrate ratio of 1:50, at 50 degrees C and pH 5 with addition of 41% PEG 1000. In spite of these kinetic improvements, the stability of the biocatalytic activity under these conditions was poor, limiting the number of possible recycles.
Profiling of botanical extracts for authentication, detection of adulteration and quality control using UPLC-QTOF-MS
(Springer, 2018-01) Steenkamp, Paul A; Steenkamp, Lucia H; Mancama, Dalubuhle T
Herbs and plant extracts are very popular and are being used by all cultures for healthcare and as food supplements. The correct identification of the required plant material is vital for formulation of final formulas for use by mankind. The use of instruments such as UPLC-QTOF-MS can be advantageous in the identification and authentication of plant extracts and formulated products to ensure the safe use of these products. Furthermore, it is also imperative that the extracts are not contaminated with other chemicals or pesticides as this can be detrimental to humans during the consumption of these products. Organic extracts of six example plants were made as part of the PlantLIBRA collaboration. The development of UPLC-QTOF-MS profiling methods showed that separation of the major compounds found in the extracts was possible and allowed for high resolution mass spectral evaluation of the compounds detected. By using reference standards and published literature, the presence of active or marker compounds could be confirmed in the different plant extracts. The high mass accuracy of the TOF data also allowed for the tentative identification of extra compounds as well as the ability to differentiate between different formulations. Establishing the correct chemical profile of a plant before use as a food supplement or herbal formulation, would ensure the use of the correct plant species, but also detect any new compounds or contaminants such as pesticides or toxins which may be identified using UPLC- QTOF-MS technologies.
Real time PCR of Nor~1 (aflD) gene of aflatoxin producing fungi and its correlative quantization to aflatoxin levels in South African compound feeds
(Elsevier, 2014-02) Iheanacho, HE; Dutton, MF; Steenkamp, PA; Steenkamp, Lucia H; Makund, HA; Swart, A; Mthombeni, JQ
Aflatoxins (AFs) are naturally occurring secondary metabolites. This toxin is principally produced by Aspergillus flavus and Aspergillus parasiticus in compound feeds worldwide. Compound feeds are feeds blended from various raw materials and additives. Contaminations of these feeds by AFs and its possible transmission into edible materials like milk, egg and organs of the body, are a serious problem. Expression of the Nor~1 (aflD) gene is the main factor responsible for AFs production. For this reason, a study was carried out to establish a correlation between levels of AFs and determinant gene (Nor~1) in South African compound feeds. To achieve this, compound feeds (n=30) were analyzed for Nor~1 gene using real time polymerase chain reaction (RT-PCR), while AFs levels in similar samples were estimated using high-performance liquid chromatography (HPLC) after an immune-affinity clean-up extraction procedure. Results indicated that AFs levels in positive samples ranged from 0.7 to 33.0ppb. These levels generally did not correlate (R(sup2)=0.093) with those of Nor~1 gene in similar samples. Consequently, Nor~1 gene levels established via RT-PCR cannot be used as a predicting model for AFs in compound feeds. Only four of the feeds analyzed, specifically poultry feeds, contained levels of AFs above the regulatory limits of 10ppb established in South Africa (S.A.). This should be considered unsafe when consumed on a continuous basis and may pose some health related problems especially when AFs are found together with other significant mycotoxins such as ochratoxins (OTs) and/or fumonisins (FBs).
Screening of commercial enzymes for the enantioselective hydrolysis of R,S-naproxen ester
(Elsevier Science Inc, 2003-03-03) Steenkamp, Lucia H; Brady, D
This study focused on the identification of suitable lipase or esterase activity for enantiomeric resolution of (R, S)-naproxen. For an economically viable reaction the enantiomeric ratio (E) should preferably be >100, while maximising the conversion will reduce the mass of material that requires racemisation and recycling. Hence the aim was to find an enzyme that yields (S)-naproxen with an enantiomeric excess of more than 98%, a substrate conversion in excess of 40% of the race mate, and an E of >100. (R, S)-Naproxen ethyl ester (NEE) (50 mg) was used as substrate for enzyme hydrolysis reactions at 37 degrees for 4 h. Biocatalyst screening was performed in buffered aqueous solvent on a I ml scale. The reactions were stopped with 2 ml MeCN, filtered through cotton wool and analysed by HPLC to determine the percentage m/m and R/S ratio. Eight commercially available enzymes were selected for optimisation of enantioselectivity through statistically designed experiments where the reaction conditions were varied. ChiroCLEC-CR from Altus and ESL001-01 from Diversa provided acceptable enantiomeric excess, but only ChiroCLEC-CR met the specification set for the enantiomeric ratio (E).
UPLC-MS profiling, identification of major peaks and comparison of Harpagophytum procumbens extracts from different locations
(Elsevier Science BV, 2019-08) Steenkamp, Paul A; Steenkamp, Lucia H
Organic extracts (methanol:acetonitrile (1:1 v/v)) of the dried and grounded secondary tubers of Devil's Claw, Harpagophytum procumbens (Burch.) DC. ex Meisn., produced extracts rich in iridoid glucosides. An UPLC-MS profiling method was developed that could separate the major compounds found in the extracts and allowed for high resolution mass spectral evaluation of the compounds detected. By using reference standards, the presence of six compounds could be confirmed namely harpagide, verbascoside, isoverbascoside, harpagoside and 6-acetylacteoside. The high mass accuracy of the TOF data also allowed for the tentative identification of three compounds, namely dihydrichinatrienone, totaratrienediol and a possible isomer of 6-acetylacteoside based on the observed accurate mass and fragmentation pattern. By using the extracted mass chromatogram of the known compounds of H. procumbens, a rapid assessment of the identity of the plant extract can be made. A statistical evaluation of H. procumbens plant material obtained from two different sources using Scores Plot indicated similarity as well as differences between the CSIR and PlantLIBRA samples. A further evaluation of the data via a Hotellings T2 Range comparison indicated that the two samples were > 95% similar, while the Scores Plot data revealed that the indicated differences were caused by concentration variations between the CSIR and PlantLIBRA sample. The developed analytical method was applied to the comparison of 12 PlantLIBRA samples and a CSIR sample. The method was found suitable for profiling H. procumbens extracts and can also be used to manually or statistically evaluate samples for similarities/differences. It would thus be suitable to screen for batch to batch reproducibility of crude plant material as well as processed samples.

Browsing by Author "Steenkamp, Lucia H"

Results Per Page

Sort Options