College of Agriculture, Engineering and Science

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Carboxamide ruthenium(II) and manganese(II) complexes: structural, kinetic, and mechanistic studies in the transfer hydrogenation of ketones.
(2022) Kumah, Robert Tettey.; Ojwach, Stephen Otieno.
The carboxamide ligands N-(benzo[d]thiazol-2-yl)pyrazine-2-carboxamide (HL1), N-(1H-benzo[d]imidazol-2-yl)pyrazine-2-carboxamide (HL2), were prepared by condensation of pyrazine-carboxylic acid and appropriate heteroaromatic amines. Reactions of HL1 and HL2 with ruthenium(II) precursors, [RuH(CO)Cl(PPh3)3] and [RuH2(CO)(PPh3)3] afforded the mononuclear complexes [RuL1(PPh3)2(CO)Cl] (Ru1), [RuL1(PPh3)2(CO)H] (Ru2), [RuL2(PPh3)2(CO)Cl] (Ru3), [RuL2(PPh3)2(CO)H] (Ru4). The solid-state structures of complexes Ru1, Ru2, and Ru4 reveal bidentate modes of coordination of the ligands and distorted octahedral geometries around the Ru(II) centre. The complexes formed active catalysts in the transfer hydrogenation of ketones and achieved turnover number (TON) up to 530 in 6 h. The ruthenium(II)–hydride complexes, Ru2 and Ru4, were capable of catalysing transfer hydrogenation of ketones reactions under base free reaction conditions and demonstrated higher catalytic activities compared to the corresponding non-hydride analogues (Ru1 and Ru3). An inner sphere monohydride mechanism involving dissociation of one PPh3 group was proposed from in situ 31P{1H} NMR spectroscopy studies. Dipicolinamide ligand system, N,N'-(1,4 phenylene)dipicolinamide (H2L3), N,N'-(1,2-phenylene)dipicolinamide (H2L4), N,N'-(4,5-dimethyl-1,2-phenylene)dipicolinamide (H2L5), N,N'-(4-methoxy-1,2-phenylene)dipicolinamide (H2L6) were synthesised following a similar protocol described for HL1 and HL2. Treatment of the ligands H2L3 and H2L4 with RuH(CO)Cl(PPh3)3 afforded bimetallic complexes [Ru2(H2L3)(PPh3)4(CO)2][2Cl] (Ru5), [Ru2(H2L3)(H)2(PPh3)4(CO)2] (Ru5b), [Ru2(HL4)(PPh3)3(CO)2Cl3] (Ru6) and a mononuclear complex [RuCl2L4(PPh3)2(CO)] (Ru7). The solid-sate structure of the dinuclear ruthenium(II) complexes confirmed a bidentate coordinate mode, with PPh3, CO, and chlorido auxiliary ligands occupying the remaining coordinating sites to afford distorted trigonal bipyramidal geometries (Ru5 and Ru6) while the mononuclear complex Ru7 adopted a distorted octahedral geometry around its ruthenium(II) atom. The reaction of the ligands H2L4-H2L6 with the [RuCl2-η6-p-cymene]2 precursor produces half-sandwich diruthenium complexes [{Ru(η6-p-cymene)}2-μ-Cl(L4)][Ru(η6-p-cymene)Cl3] (Ru8), [{Ru(η6-p-cymene)}2-μ-Cl(L4)][PF6] (Ru9), [{Ru(η6-p-cymene)}2-μ-Cl(L5)][PF6] (Ru10), and [{Ru(η6-p-cymene)}2-μ-Cl (L6)][PF6] (Ru11). The molecular structure of cationic complexes, Ru8-Ru11, was confirmed by single-crystal X-ray crystallography analysis. The complexes Ru8-Ru11 display a bidentate Npyridine ^ Namidate mode of coordination to give pseudo-octahedral geometry (piano-stool-like geometry). The ruthenium(II) complexes demonstrated remarkable enhanced catalytic activity (TON values up to 1.71 x 104) in the transfer hydrogenation of ketones at a very low catalyst loading of 2.75 x10-2 mol% (275 ppm). The dinuclear ruthenium(II) complexes showed higher catalytic activity compared to the corresponding mononuclear complex Ru5. The half-sandwich diruthenium complexes Ru8-Ru11 displayed relatively higher catalytic activity than the ruthenium complexes Ru5 and Ru6 bearing the PPh3 co-ligands. Monohydride inner-sphere catalytic cycle was proposed for the transfer hydrogenation of ketones catalysed by both Ru1 and Ru9, and the formation of the reactive intermediates was supported with low-resolution mass spectrometry data. The dinuclear ruthenium complexes of pyridine and pyrazine-carboxamide bearing quinolinyl motif were synthesised by reacting, N-(quinolin-8-yl)pyrazine-2-carboxamide, (HL7), 5-methyl-N-(quinolin-8-yl)pyridine-2-carboxamide, (HL8), 5-chloro-N-(quinolin-8-yl)pyridine-2-carboxamide, (HL9), and 2-pyrazine-carboxylic acid (HL10) with equimolar [RuCl2(η6-p-cymene)]2 to afford the dinuclear complexes [{Ru(η6-p-cymene)}2Cl3(L10)] (Ru12), [{Ru(η6-p-cymene)Cl}2(L7)] [PF6] (Ru13), [{Ru(η6-p-cymene)Cl}2(L8)][Ru(L8)Cl3] (Ru14), and [{Ru(η6-p-cymene)Cl}2(L9)][PF6] (Ru15), respectively. The solid-state structures of the dinuclear complexes Ru12 and Ru13 reveal a typical piano-stool geometry around the Ru(II) ions. The dinuclear ruthenium complexes Ru12-Ru15 were used as catalysts in the transfer hydrogenation of a broad spectrum of aldehydes and ketones and demonstrated excellent catalytic activity, TON values up to 4.8 x 104, using catalyst loading of 2.0 x10-3 mol% (20 ppm). The catalytic performance of the complexes was affected by the ligand architecture and the substituents on the pyridyl ring. Complexes Ru13-15 exhibited higher catalytic activities compared to the complex Ru12 which could be ascribed to the role of quinoline in stabilising the complexes. The pyridine and pyrazine motifs have a significant impact on the reactivity and the catalytic activity of the complexes. In-situ low-resolution ESI-MS analyses of the reactive intermediates aided in proposing a monohydride inner-sphere mechanism for the transfer hydrogenation of ketones catalysed by Ru15. To develop a more sustainable, environmentally compatible and cost-efficient protocol for transfer hydrogenation of ketones, a new catalytic system based on manganese(II) metal was synthesised. New manganese(II) complexes Mn1-Mn4, ligated on dipicolinamide ligands were synthesized by treating the N,N'-(1,4-phenylene)dipicolinamide (H2L3), N,N'-(1,2-phenylene)dipicolinamide (H2L4), N,N'-(4-methoxy-1,2-phenylene)dipicolinamide (H2L5) and N,N'-(4,5-dimethyl-1,2-phenylene)dipicolinamide (H2L6) with MnCl2.4H2O salt to afford dinuclear manganese(II) complexes [Mn2(H2L3)2Cl4] (Mn1), [Mn2(H2L4)2Cl4] (Mn2), [Mn2(H2L5)2(Cl)4] (Mn3) and [Mn2(H2L6)2Cl4] (Mn4). The solid-state structure of complex Mn2 showed a six-coordinate dinuclear complex with the two Mn(II) ions adopting a distorted octahedral environment surrounded by two tetradentate ligands and chlorido co-ligands, respectively. The Mn(II) complexes formed active catalysts in transfer hydrogenation of ketones to achieve TON values up to 5.12 x 104. The presence of electron-donating substituents -OCH3 and -CH3 in complexes Mn3 and Mn4 displayed minor effects in the transfer hydrogenation of ketones. The new carboxamide-manganese(II) complexes are among the most active manganese-based catalysts capable of hydrogenating a large scope of ketones ranging from aliphatic to aromatic ketones. A dihydride catalytic cycle has been proposed and supported with in-situ low-resolution mass spectrometry data.
Post-quantum cloud security and data exchange using artificial intelligence.
(2023) Mosola, Napo Nathnael.; Blackledge, Jonathan Michael.; Dombeu, Jean Vincent Fonou.
This thesis investigates the application of plausible modern-day cryptographic solutions for securing the cloud and exchanging confidential data. The context followed is such that the strength of an encryption algorithm is based on the difficulty to cryptanalyse it. This means the more difficult the crypto-system is to cryptanalyse, the stronger and more trusted it is. The success of cryptanalysis on a cryptographic algorithm has been a function of the computational power available at the time of performing the cryptanalysis, without consideration of future innovations, specifically, without careful consideration of Moore’s law. A significant number of public-key crypto-systems can and will be compromised by a quantum computer coupled with the implementation of Shor’s algorithm. This has brought a lot of focus regarding research on cryptographic solutions post quantum computing (PQC) due to the following: ˆ cryptographic algorithms are based on the intractability of prime number factorisation using a conventional computing power; ˆ a quantum computer can factorize prime numbers with relative ease. In the past, the quantum computing paradigm was a hypothetical concept. Thus, cryptanalysis using quantum resources was a theoretical idea. This is no longer the case with the loom of quantum computers eminent. Consequently, prime number based encryption is becoming increasingly irrelevant. Low Qubit quantum computers now exist. Research and development in this area is growing. Hence the existence of the post-quantum cryptography paradigm. This paradigm is based on encryption algorithms developed and considered secure enough to withstand quantum attacks. Thus, the National Institute of Standards and Technology made a call for projects clustered under the Open Quantum Safe project (OQSP), which began in 2016. The ultimate goal of this project is development of future quantum resistant cryptographic algorithms for secure communication and data exchange. The OQSP aims to gather open source libraries which can be standalone or integrated into the public key encryption schemes to improve their security against ii quantum attacks in the quest to achieve quantum resistance. The major focus is placed on quantum key exchange (QKE). It is against this background that the material presented in this thesis reports on a spectrum of algorithms that are thought to be quantum resistant based on a coherence of ideas, methods, models and software implementation, trying to meet the NIST requirements and contributing to new knowledge in the field of cryptography. The aim is to provide confidentiality guarantees on cloud-hosted data as well as secure data exchange between communicating entities, while also tackling the cumbersome key exchange and management problem. The results show that the algorithms presented in this thesis introduce new ideas in cryptography and can be tested to withstand cryptanalytic quantum attacks, while a plausible encryption key distribution and management solution is proposed. In this context, the material presented in this thesis report on a spectrum of algorithms that are proposed to be quantum resistant based on a coherence of ideas, methods and software implementation, aimed at providing security of cloud-hosted data as well as data exchange between communicating entities. The cloud has a flexible, scalable and low cost properties. This is due to two concepts which are fundamental to cloud computing: ˆ virtualization; ˆ multi-occupancy. These above concepts have brought infinitely many benefits which make the cloud an attractive paradigm. Among the benefits are reduced capital and maintenance costs, high processing power, enormous storage facilities etc. However, security concerns affecting confidentiality of cloud-hosted data still plague bring concerns when it comes to cloud adoption. Data confidentiality can be achieved through encryption, which is in turn implemented by cryptographic algorithms. Hence, this thesis proposes and puts into practice cryptographic algorithms to solve issues of confidentiality, specifically in the cloud.
Production and characterization of DNA ligases isolated from Kogelberg Biosphere metagenomics library.
(2021) Zuma, Lindiwe Khumbuzile.; Pooe, Ofentse Jacob.
Microbial enzymes have been described as an underutilized source of novel enzymes with potential economic advantages. Recently discovered enzymes such as DNA ligase from metagenomic studies, have been shown to achieve great potential in transforming the reagent market specifically in the African continent. Reagent proteins are frequently utilized in the research field widely and are prone to protein degradation and shelf-life reduction. Hence, this study sought to improve biological activity, shelf life and stability of the two DNA ligases identified from Kogelberg Biosphere metagenomics library. Two recombinant DNA ligases expression studies were done using E.coli BL21 and purification studies were done subsequently using affinity chromatography. Both recombinant DNA ligases (Ligsv081 & LigpET30) were successfully expressed and purified as homogenous proteins. In this study two approaches were used to enhance the biological DNA ligases, the first approach used was PEGylation. The purified proteins were conjugated to PEG using site-specific PEGylation and non-specific PEGylation. FTIR and UV-VIS spectroscopy were used to analyze the secondary structure of the PEG conjugated DNA ligases. Thermal stability assays were then employed to assess protein stability in the conjugation with PEG. Site-specific PEGylation enhanced ligase activity and reduced the formation of protein aggregates. The second approach involved DNA ligase co-expression in the presence of PfHsp70 or chimeric transcription factor, respectively. Protein co-expression and co-purification assays were conducted. The co-expression and co-purification assays of both proteins with chimeric transcription factor (cTF) were successful, followed by co-expression and co-purification of LigpET30-PfHsp70. Ligation assays were conducted to assess bioactivity of proteins. All DNA ligase complexes were functional and their melting point was increased. Taken together, site-specific PEGylation and protein co-expression with PfHsp70 potentially extended the shelf-life and stability of the proteins. PEGylation strategies and co-expression strategies can potentially be used to enhance reagents in diagnostic and therapeutic tools in molecular biology field.
Star formation as a function of environment in the MeerKAT Galaxy Clusters Legacy Survey=Ulwazibunkanyezi Njengethuluzi Lokuhlola isiqoqzinkanyezi iMeerKAT Galaxy Clusters Legacy.
(2023) Kesebonye, Kabelo Calvin.; Hilton, Matthew James.; Knowles, Kenda Leigh.
Probing the star formation (SF) activity of cluster galaxies paves an important path towards the understanding of cluster evolution. This thesis presents the study of star formation rates (SFR) in clusters using dustunbiased radio luminosities from the MeerKAT Galaxy Clusters Legacy Survey (MGCLS). Our radio data is complemented by optical data from the Dark Energy Camera Legacy Survey (DECaLS), for photometric redshifts, and also Sunyaev-Zel’dovich (SZ) effect-derived cluster masses from the Atacama Cosmology Telescope (ACT). We present the first statistical study of SFR in clusters using MeerKAT-detected galaxies which takes advantage of MeerKAT’s large field of view to investigate the relation between SF activity and cluster environments out to 2R200. Using radio diffuse emission in the form of haloes and relics as a proxy for cluster merger activity, we divide our cluster sample between disturbed/merger clusters and relaxed clusters. We observe a higher fraction of star-forming galaxies (fSF) in disturbed clusters than in relaxed clusters. Disturbed clusters also have higher masses (M200) and total SFR (ΣSFR) in contrast to relaxed clusters. On analysing the redshift evolution of the massnormalised ΣSFR, we observe a ≈ 4× decline in the SF activity of clusters from the redshift of 0.35 to 0.15, corresponding to ≈ 2 Gyr in look-back time. Our result is roughly consistent with the one from cluster studies that used infrared-derived SFR (≈ 5× decline) at a similar redshift slice as our sample. We use a subsample of double relic-hosting clusters to investigate the relation between cluster SF activity and the time that has passed since the merger started (tmerger) estimated from the relic distances from cluster cores. We observe an anti-correlation between ΣSFR and merger, suggesting that younger mergers have a higher SF activity. However, we see no clear correlation in the mass-normalised ΣSFR with tmerger. We also investigate for differences in the SF activity of galaxies closer to radio relics and those away from the relics and observe no significant differences between the two populations. Iqoqa Ukuhlola kokusebenza kolwazi-bunkanyezi ( star formation -SF) kwesixhobo sezinkanyezi cluster galaxies kucabe indlela emqoka ukuqonda kokukhula kwalamaqoqo (cluster evolution). Lolu cwaningo lwethula ukuhlolwa lwe star (formation rates -SFR) kuma-clusters kusetshenziswa i dust-unbiased radio luminosities kuMeerKAT Galaxy Clusters Legacy Survey (MGCLS). Imininingo ye-radio isebenzisana ne-optical data yeDark Energy Camera Legacy Survey (DECaLS), ukuhlola iphotometric redshifts, ne Sunyaev-Zel'dovich (SZ) nomthelela odalwa yicluster masses esuka kwiAtacama Cosmology Telescope (ACT). Kube sekwethulwa ucwaningo lokuqala lwezibalo lweSFR in clusters kusetshenziswa iMeerKAT-detected galaxies. Kusetshenziswe ithuba lobukhulu bomkhakha lokubonakala kahle kweMeerKAT ukuhlola ubudlelwane phakathi kwe-SFR nemvelo yamaclusters ezinhlobonhlobo zedynamical states. Kusetshenziswe iradio diffuse emission eyizinhlobo zehaloes nerelics njengegunya lokuhlola ukuhlangana kwamacluster , amasampula ama-cluster ahlukaniswe phakathi kwamadisturbed/merger clusters namarelaxed clusters. Okutholakele ukuthi ihigher fraction of star-forming galaxies (fSF) kumaclusters aphazamisekile (disturbed clusters) kunama clusters akhululekile (relaxed clusters). Amaclusters aphazamisekile anama masi aphezulu (M200) kanti isamba sonke siwuSFR (∑SFR) uma eqhathaniswa namaclusters aphazamisekile . Sekucutshungulwa iredshift evolution yemass-normalised ∑SFR, kutholakale ukuthi aphindwe kawu ≅4 times decline ekusebenzeni kwe SF esukela kwiredshift of 0.35 to 0.15, ahlobana ne ≅2 Gyr kwi look-back time. Imiphumela ilinganiselwe ngokuhambisana nezifundo zamacluster kusetshenziswa i-infrared-derived SFR (≅5 times decline) ngokufanayo neredshift slice njengamasampula ocwaningo lolu .Kusetshenziswe elinye isampula esigabeni sesibili ukuze kwenziwe idouble relic-hosting clusters ukuhlola ubudlelwane okusebenza kwama clusters (SF activity) kanye nesikhathi esisuke sidlulile ehlanganiswe (t_merger) kulinganiselwe kusukela ebangeni lerelic distances kucluster ewumsuka (cluster cores). Kutholakale ukungadlelani phakathi kwe ∑SFR ne t_merger, okuchaza ukuthi ukuhlanganiswa kwalawo asemancane kusebenzisa izinga eliphezulu (SF activity). Yize-kunjalo , akubonakali ubudlelwane obucacile kumass-normalised ∑SFR ne t_merger. Kuphinde kwahlolwa ukusebenza okungefani kumagalaxies asondelene neradio relics nalawo aqhelile kumarelics kwase kwangatholakala mehluko otheni kule miphakathi yomibili.
Syntheses of mixed donor homogeneous and immobilized palladium(II) complexes catalysts for methoxycarbonylation and hydrogenation reactions.
(2021) Akiri, Saphan Owino.; Ojwach, Stephen Otieno.
Reactions of ligands (E)-N'-(2,6-diisopropylphenyl)-N-(4-methylpyridin-2-yl)benzimidamide (L1), (E)-N'-(2,6-diisopropylphenyl)-N-(6-methylpyridin-2-yl)benzimidamide (L2), (E)-N'-(2,6-dimethylphenyl)-N-(6-methylpyridin-2-yl)benzimidamide (L3), (E)-N'-(2,6-dimethylphenyl)-N-(4-methylpyridin-2-yl)benzimidamide (L4) and (E)-N-(6-methylpyridin-2-yl)-N'-phenylbenzimidamide (L5) with [Pd(NCMe)2Cl2] furnished the corresponding palladium(II) pre-catalysts (Pd1-Pd5), in good yields. Molecular structures of Pd2 and Pd3 revealed an N^N bidentate coordination mode to afford square planar compounds. Activation of the palladium(II) complexes with para tolyl sulfonic acid (PTSA) afforded active catalysts in the alkenes methoxycarbonylation. The resultant catalytic activities were controlled by both the complex structure and alkene substrate. While aliphatic substrates favoured the formation of linear esters (>70%), styrene substrate resulted in predominantly branched esters of up to 91%. The water-soluble ligands; sodium 4-hydroxy-3-((phenylimino)methyl)benzenesulfonate (L6), sodium 3-(((2,6-dimethylphenyl)imino)methyl)-4-hydroxybenzenesulfonate (L7) and sodium 3-(2,6-diisopropylphenyl)imino)methyl)-4-hydroxybenzenesulfonate (L8) reacted with with Pd(OAc)2 afford their respective palladium(II) complexes [Pd(6)2] (Pd6), [Pd(L7)2] (Pd7) and [Pd(L8)2] (PdL8). In addition, treatment of the non-water-soluble ligands 2-((phenylimino)methyl)phenol (L9), 2-(((2,6-dimethylphenyl)imino)methyl)phenol (L10) and 2-((2,6 diisopropylphenyl)imino)methyl)phenol (L11) with Pd(OAc)2 yielded complexes [Pd(L9)2] (Pd9), [Pd(10)2] (Pd10) and [Pd(L11)2] (Pd11), respectively in good yields. Solid-state structures of compounds Pd6 and Pd9 revealed bis(chelated) square planar neutral compounds. All the complexes formed active catalysts in the methoxycarbonylation of 1- hexene, affording yields of up to 92% within 20 h and regioselectivity of 73% in favour of linear esters. The activities and selectivities of the compounds depended on the steric encumbrance around the coordination centre. The water-soluble complexes displayed comparable catalytic behaviour to the non-water-soluble systems. The complexes could be recycled five times with minimal changes in both the catalytic activities and regio-selectivity. Reactions of (amino)phenyl ligands, (E)-N-((Z)-4-(phenylamino)pent-3-en-2-ylidene)aniline (L12) and N,N'E,N,N'E)-N,N'-(3-(3 (triethoxysilyl)propyl)pentane-2,4-diylidene)dianiline (L13) with [Pd(NCMe)2Cl2] led to the formation of homogeneous complexes Pd13 and Pd14. Besides, supporting of complex Pd14 with either MCM-41, SBA-15, or Fe3O4 magnetic nanoparticles gave immobilized complexes P15-Pd17, respectively. Using varying metal loading in the MCM-41 immobilization of complex Pd14 produced complexes Pd18 and Pd 19. In addition, calcination of complex Pd16 at 150oC and 200oC led to the formation of complexes Pd20 and Pd21, respectively. All the complexes were received in good yields. The catalytic activities and selectivities of the homogeneous complexes were influenced by the coordination sphere, with the complexes predominantly forming linear esters. On the other hand, the catalytic behaviours of the immobilized catalysts depended on the nature of support and calcination temperatures. In addition, the catalytic activities were observed to depend on the reaction temperature, catalyst loading, amounts of PPh3 and acid promoters. The immobilized complexes Pd15, Pd16 and Pd17, were recycled up to five times. The homogeneous and silica immobilized palladium(II) complexes of ligands (2-phenyl-2-((3(triethoxysilyl)propyl)imino)ethanol) (L14), (4-methyl-2-((3(triethoxysilyl)propyl)imino)methyl)phenol ) (L15 ), [L14-MCM-41 (L16), and [L15- MCM-41 (L17)]. The homogeneous complexes [Pd(L14)2] (Pd22), [Pd(L14)2] (Pd23), [Pd(L14)(Cl2)] (Pd24), [Pd(L15)(Cl2)] (Pd25) were obtained from homogenous ligands L14, L15, L16 and L17 respectively. In addition, the silica immobilized compounds [Pd(L14)2]-MCM-41] (Pd26) and [Pd(L15)2)-MCM-4] (Pd27) were obtained through convergence immobilization of complexes Pd22 and Pd23, respectively. Comparatively, immobilized complexes [Pd (L14)(Cl2)-MCM-41] (Pd28) and [Pd(L15)(Cl2)]-MCM-41] (Pd29) were obtained from the complexation of immobilized ligands L16 and L17. Both sets of complexes gave active catalysts in molecular hydrogenation of alkenes, alkynes and functionalized benzenes. The catalytic activities and product distribution in these reactions were largely dictated by the nature of the substrate. The kinetic studies revealed reaction orders dependence on styrene for both the homogeneous and supported catalysts. Significantly, the selectivity of both sets of catalysts was comparable in the hydrogenation of alkynes and multi-functionalized benzenes. The supported catalysts could be recycled up to four times with minimum reduction in catalytic activities and showed the absence of any leaching from hot filtration experiments. Kinetics and poisoning studies established the presence of active homogeneous species for complexes Pd22-Pd5 and Pd(0) nanoparticles for the immobilized complexes Pd26-Pd29, respectively.
The isolation and characterisation of proteases from Euphorbia tirucalli, E. triangularis, and Carica papaya latex.
(2023) Boodhoo, Akira.; Coetzer, Theresa Helen Taillefer.
Plant proteases play an important role in the food and industrial sectors from meat tenderisers to milk clotting agents and even anti-parasitic agents. Proteases have been identified in plant latex, but many proteases have not been isolated and characterised. This research aimed to isolate and characterise proteases from the latex of Euphorbia tirucalli, E. triangularis, and Carica papaya. Three-phase partitioning (TPP) of E. tirucalli plant latex revealed the presence of two active proteases on gelatin-containing zymograms, that were subsequently separated by size exclusion chromatography. These proteases were classified as a 75 kDa serine protease (E. tiru SP), inhibited by PSMF, and SBTI and a 37 kDa cysteine protease (E. tiruCP), inhibited by E-64. Analysis of E. triangularis latex by TPP and p-aminobenzamidine affinity chromatography showed the presence of three serine proteases inhibited by PMSF and SBTI, E. laris SP1 (>97.4 kDa), E. laris SP2 (68 kDa) and E. laris SP3 (38 kDa). These proteases showed stability in constant ionic strength buffers from pH 4 to 9, both with and without the presence of the reducing agent cysteine. Papain was isolated from the latex of Carica papaya for use as a control protease in zymograms on account of the anomalous behaviour of commercial papain preparations on these gels. Papain was isolated by two methods: ammonium sulfate precipitation followed by TPP and CM-cellulose cation exchange chromatography. The isolated papain was detected by rabbit anti-papain antibodies in a dot blot and western blot and showed inhibition by E-64. The latex from all three plant species showed milk clotting activity with higher activity in the presence of calcium chloride. These findings suggest that isolated plant latex proteases from the Euphorbia species can be used in the food industry as milk clotting agents. Further characterisation of these isolated proteases should identify further uses in biotechnology.

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