Massey Documents by Type

Permanent URI for this communityhttps://mro.massey.ac.nz/handle/10179/294

Browse

Has files: YesSubject: search.filters.subject.Enzymes

Search Results

Now showing 1 - 10 of 30
  • Thumbnail Image
    Item
    Sequential treatment of psychrotrophic pseudomonad biofilms with sodium hydroxide and commercial enzyme cleaners
    (Elsevier Ltd, United Kingdom, 2026-04) Muthuraman S; Palmer J; Flint S
    Pseudomonads are psychrotrophic spoilage bacteria that can form biofilms at the air-liquid interface. Food processing utensils and equipment often facilitate the air-liquid interface biofilm formation. Pseudomonads produce thermostable enzymes and pigments that affect the organoleptic quality of perishable food products. In this study, Pseudomonas lundensis, Pseudomonas cedrina were allowed to form biofilms at 4 °C under continuous flow of nutrients in a CDC reactor (CBR 90; Biosurface Technologies, USA). The mature biofilms were treated with commercial enzyme cleaners, EnduroZyme (protease), DualZyme (protease and lipase), and TriZyme (protease, amylase, and cellulase). The dispersion with EnduroZyme was significantly (p < 0.05) higher than the other enzyme cleaners. Then the biofilms were treated with hot water and sodium hydroxide, and enzyme cleaners (sequential treatment). The cell counts after sodium hydroxide + Enzyme cleaners were below the detection limit. The microscopic observations with epifluorescence microscopy showed that the coupons had less fluorescence after the sequential treatment. FTIR observations showed that the extracellular polymeric substances (EPS) isolated after sodium hydroxide + enzyme cleaners differed from the untreated and sodium hydroxide-only-treated EPS. Biofilm regrowth was significantly (p < 0.05) lower in the biofilms treated with sodium hydroxide + EnduroZyme compared to acid-treated control coupons. The sequential treatment with sodium hydroxide and enzyme cleaners reduced the biofilm footprints, representing a better clean than enzyme treatment alone or sodium hydroxide-only cleaning.
  • Thumbnail Image
    Item
    Characterization of the extracellular polymeric substances matrix of Pseudomonas biofilms formed at the air-liquid interface
    (Elsevier Ltd, 2025-01-27) Muthuraman S; Flint S; Palmer J
    Pseudomonas are common psychotropic food spoilage organisms that affect the quality of aerobically chilled food products. Biofilm formation of these bacteria on food contact surfaces can provide a continuous contamination source, leading to food spoilage. Pseudomonas produce proteolytic and lipolytic enzymes which lead to organoleptic degradation of stored food products. The biofilm extracellular polymeric substances matrix (EPS) protects the bacterial cells from CIP (Cleaning-In-Place) chemicals and adverse conditions. Studies on the composition of the EPS matrix and the molecules present in the EPS matrix are limited. In this study, the EPS composition of mono-species biofilms of Pseudomonas lundensis and Pseudomonas cedrina on polystyrene and stainless-steel surfaces was characterized by chemical analysis and microscopical observations. The biofilms were allowed to grow on polystyrene and stainless-steel surfaces with half-strength TSB for 2 weeks at 30 °C and cold chain temperatures of 7 °C and 4 °C. The EPS was extracted by sonication and centrifugation and chemically analysed for cellulose, total polysaccharides, total proteins, and eDNA. Pseudomonas isolates in this study formed biofilms at the air-liquid interface. The formation of ring-like structures of cells was observed on the polystyrene surface. eDNA formed as a thread-like structure on a polystyrene surface while it formed channels on a stainless-steel surface. The amount of EPS varied at different temperatures. More EPS was formed at 4 °C than 30 °C. Flagellin, Clp protease, Arginine deiminase, and ATP-Binding Cassette (ABC) transporter substrate-binding proteins were the key proteins identified in the biofilm matrix of P. lundensis.
  • Thumbnail Image
    Item
    The analysis of inquiry in students' conversations in the biochemistry laboratory : the elucidation of proton-coupled electron-transfer reaction mechanism in manganese superoxide dismutase through structural analysis of mutants : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2023) Hermawan, Jatnika
    Superoxide dismutases (SODs) have very significant biological importance, protecting organisms against reactive oxygen species such as superoxide. They are also known as the fastest enzyme with the largest kcat/Km of any known enzyme. To perform super-fast enzymatic function, SOD must shuttle proton-coupled electrons in an efficient systematic way. However, since its discovery in 1968, the mechanistic nature of SOD catalytic function remains vague. Wide-ranging approaches have attempted to uncover the catalytic mechanism of the manganese-containing SOD, MnSOD, but there were experimental limitations that obstructed the investigations. Here, the structural analyses of two dimer interface mutants of MnSOD, S126D and S126W, explored possible changes in water structure near the active site providing new information to examine the hypothesis of the Glu170 bridge as a key player in the proton shuttle in the outer-sphere mechanism. To gain insight into the mechanism of the proton-coupled electron-transfer (PCET) reaction mechanism, the technique of single-crystal X-ray crystallography was used to observe the three-dimensional structure of Escherichia coli MnSOD mutants, analytical ultracentrifugation was used to observe quaternary association in solution, and protein stability was assessed by differential scanning calorimetry. The key residue Ser126 at the conserved but asymmetric dimer interface of the MnSOD was mutated with the initial intent to generate a monomeric species. Ser126 is not essential for activity and is not part of the active site, whereas Glu170 forms part of the dimer interface where Glu170 from one subunit forms part of the active site of the second subunit of the dimer. The loss of activity occurring in a monomeric MnSOD may indicate an alternative catalytic mechanism of the MnSOD enzyme. The substitution of Ser126 to Asp, intended to produce a monomeric species by charge repulsion, surprisingly produced a dimer at pH>7.5 with little change in structure at the Mn active site, but there was a 94 % reduction in catalytic activity. Partial loss of activity in Ec-MnSOD-S126D may be due to electrostatic effects of the negative charge ~7 Å from metal centre perturbing the Mnᴵᴵᴵ/Mnᴵᴵ redox couple. The substitution of Ser126 to Trp, intended to produce a monomeric species by steric bulk, enforces mostly monomeric Ec-MnSOD S126W in solution form, coupled with a 99.9 % reduction in catalytic activity. Here one mutation to a conserved dimer interface led to altered tertiary structure and a completely different dodecameric domain-swapped quaternary association in the crystalline state and complete loss of activity in Ec-MnSOD-S126W in the solution state. In the course of evolution, higher and less often lower degrees of oligomerisation have arisen. Evolving complexity does not require multiple mutations. As part of the scholarship requirements, this dissertation contains a pedagogical component. Student conversations in a guided inquiry third-year biochemistry laboratory were recorded and analysed to discover the extent of higher-order critical thinking that might occur. Although students initially struggled to move beyond core first-year laboratory skills, they were at all times strongly engaged in the project-style experiment, which ran over three five- to eight-hour sessions. Some progress in the level of inquiry was captured from their conversations from the first to the third laboratory session. A simple diagram and table were developed to help guide teachers in a guided inquiry-based learning in higher education.
  • Thumbnail Image
    Item
    Characterisation of pseudogene-like EP400NL in chromatin remodelling and transcriptional regulation : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Ph.D.) in Biochemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2023) Li, Zidong
    EP400 is an ATP-dependent chromatin remodelling enzyme that has been implicated in DNA double-strand break repair and transcription regulation including Myc-dependent gene expression. It was previously shown that the ectopic expression of the N-terminal domain of EP400 increases the efficacy of chemotherapeutic drugs against cancer cells. This prompted the question of whether the EP400 N-terminal-Like (EP400NL) gene, which resides next to the EP400 gene locus, also plays a similar role in epigenetic transcriptional regulation to the full-length EP400 protein. To characterize the function of the EP400NL nuclear complex, a stable cell line expressing TAP-tagged EP400NL was established, and the EP400NL complex was affinity purified and analyzed by mass spectrometry. EP400NL was found to form a human NuA4-like chromatin remodelling complex that lacks both the TIP60 histone acetyltransferase and EP400 ATPase. However, despite no histone acetyltransferase activity being detected, the EP400NL complex displayed H2A.Z deposition activity on a chromatin template comparable to the human NuA4 complex, suggesting another associated ATPase such as BRG1 or RuvBL1/RuvBL2 catalyses the reaction. In addition to a role in H2A.Z deposition, it was also determined that the transcriptional coactivator function of EP400NL is required for serum and IFNγ- mediated transcriptional activation of the immune checkpoint gene PD-L1. EP400NL, cMyc and multiple identified ATPases such as BRG1, RuvBL1/RuvBL2 were shown to be recruited to the promoter region of PD-L1. To further demonstrate the importance of EP400NL in regulating Myc and IFNγ-mediated PD-L1 expression, CRISPR/Cas9 mediated EP400NL indels were introduced in H1299, a human non-small cell lung carcinoma cell line. These EP400NL indel cell lines show compromised gene induction profiles with significantly decreased PD-L1 expression from both Myc and IFNγ stimulation experiments. In contrast to full-length EP400NL, two deletion mutants (Δ246- 260 and Δ360-419) lacked the ability to enhance the expression level of PD-L1 mRNA or protein, indicating that these regions are important for coactivator activity. Collectively, these data show that EP400NL plays a role as a transcription coactivator for cMyc-mediated gene expression and provides a potential target to modulate PD-L1 expression in cancer immunotherapy.
  • Thumbnail Image
    Item
    Biophysical and biochemical characterisation of DNA-based inhibitors of the cytosine-mutating APOBEC3 enzymes : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand
    (Massey University, 2020) Barzak, Fareeda Maged Yahya Mohammad
    With the rise of antiviral and anticancer drug resistance, a new approach must be taken to overcome this burden. The APOBEC3 (A3) family of cytosine deaminases hypermutate cytosines to uracils in single-stranded DNA (ssDNA). These enzymes act as double-edged swords: on one side they protect humans against a range of retroviruses and other pathogens, but several A3s are exploited by viruses and cancer cells to increase their rate of evolution using the enzyme’s mutagenic actions. This latter mode permits escape of cancer cells from the adaptive immune response and leads to the development of drug resistance. In particular, APOBEC3B (A3B) is considered to be a main driving source of genomic mutations in cancer cells. Inhibition of A3B, while retaining the beneficial actions of the other A3 in the immune system, may be used to augment existing anticancer therapies. In this study, we showed for the first time that short ssDNAs containing cytosine analogue nucleosides, 2'-deoxyzebularine (dZ) or 5-fluoro-2'-deoxyzebularine (5FdZ) in place of the substrate 2'-deoxycytidine (dC) in the preferred 5'-TC motif, inhibit the catalytic activity of A3B. However, as most A3 enzymes (except A3G) prefer to deaminate ssDNA with a 5'-TC motif, selective A3B inhibition was uncertain. We noted that nucleotides adjacent to the 5'-CCC motif influence the dC deamination preference of A3A, A3B, and A3G’s. Replacement of the A3B’s preferred dC in the 5'-CCC motif with dZ (5'-dZCC) led to the first selective inhibitor of A3B, in preference to A3A and A3G. Furthermore, using small-angle X-ray scattering (SAXS) we obtained the first model of a full-length two-domain A3 in complex with a dZ-ssDNA inhibitor. Our model showed that the ssDNA was largely bound to the C-terminal domain (CTD) with limited contact to the N-terminal domain in solution, due to the high affinity of the dZ for the CTD active-site. Our work provides a new platform for use of ssDNA-based inhibitors in targeting the mutagenic action of the A3B. Further developments using more potent inhibitors will help to achieve inhibition in cellular studies, with the ultimate goal to complement anti-cancer and antiviral treatments.
  • Thumbnail Image
    Item
    Removal of Cronobacter sakazakii and Listeria monocytogenes biofilms using enzymes : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology at Massey University, Manawatū, New Zealand
    (Massey University, 2019) He, Yang
    C. sakazakii and L. monocytogenes are pathogens that may occur in dairy manufacturing plants with the potential cause serious diseases in neonates if dairy products containing these bacteria are consumed. Control of these pathogens is through cleaning with chemicals such as caustics and acids. Enzymes have been considered as alternative cleaning agent as they are more environmentally friendly compared with traditional chemical-based cleaning. However, the use of enzymes to remove biofilms of these pathogens has not been well studied. The objectives of this study were (1) to explore the biofilm formation of C. sakazakii and L. monocytogenes on hydrophilic and hydrophobic surfaces; (2) test the efficacy of α-amylase, proteases and endoglucanase on removal of these biofilms. The first part of this study focused on screening for the robust strains that can form strong biofilm in reconstituted infant milk formula using a microtiter plate assay and stainless steel coupons. The second part evaluated the efficacy of enzymes on the removal of biofilms by impedance detection and epifluorescence microscopy. The results showed that 12 of 14 strains of C. sakazakii strains and half of the L. monocytogenes strains formed strong biofilm. The ability to form biofilm varied with the origin of the isolates with clinical isolates of C. sakazakii and food isolates of L. monocytogenes forming strong biofilm on microtiter plates and stainless steel surfaces. For the evaluation of enzyme efficacy, the biofilms were treated with α-amylase, proteases and endoglucanase at 85°C, pH 3 for 15 min. The results from the microtiter plate assay based on the absorbance at 550 nm showed that the enzymes especially proteases and endoglucanase were effective in removing biofilms. The effectiveness of cleaning by enzymes demonstrated by plate counting and impedance detection was supported by epifluorescence microscopy results. In conclusion, these results demonstrated the efficacy of enzymes on removal of biofilms of C. sakazakii and L. monocytogenes. Compared with traditional method of cleaning, enzymes are more effective in removing extracellular polymeric substance of biofilms.
  • Thumbnail Image
    Item
    On the mechanism of Dehydroquinate synthase : a thesis presented to Massey University in partial fulfilment of the requirements for the degree of Master of Science in Chemistry
    (Massey University, 2001) Kathirgamanathan, Kalyani
    The aim of this thesis is to investigate the influence of fluorine substitution on the second reaction of the shikimate pathway catalysed by the enzyme 3-dehydroquinate synthase. The shikimate pathway is an essential pathway that is required for the synthesis of aromatic compounds in bacteria, microbial eukaryotes and plants. The enzyme, 3-dehydroquinate synthase, catalyses the second step of the shikimate pathway, the conversion of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) into 3-dehydroquinate (DHQ). Item_03771-1.jpg It has been reported that when (3S)-3-fluoro DAHP (where fluorine occupies the C3 axial position) is treated with the enzyme 3-dehydroquinate synthase, two products, the expected (6S)-6-fluorodehydroquinate (5) and its C1 epimer, (6S)-6-fluoro-1-epideydroquinate (6) are formed in a ratio of 2 : 1. Item_03771-2.jpg The C1 epimer of 3-dehydroquinate was reported to be formed from the natural substrate DAHP in a solution reaction, but not in the enzyme catalysed reaction. Therefore, it has been suggested that fluorine substitution at the axial position on C3 stabilises the fluoroenolpyranose intermediate allowing the intermediate to dissociate from the enzyme and cyclise to complete the formation of (6S)-6-fluoro-1-epideydroquinate free in solution. The results reported in this thesis are from an investigation carried out to understand further the influence of fluorine orientation on the stereochemical outcome of the products in the dehydroquinate synthase reaction. (3S)-3-Fluoro DAHP was synthesised in large amounts using both chemical and enzymatic synthesis. This was achieved by treating the isomers of 3-fluoro phosphoenolpyruvate and D-erythrose 4-phosphate with DAHP synthase, the first enzyme of the shikimate pathway. The erythrose 4-phosphate was prepared by lead tetraacetate oxidation of D-glucose 6-phosphate. The isomers of 3-fluoro phosphoenolpyruvate were prepared from 3-bromo, 3-fluoropyruvic acid by the Perkow reaction. Then (3S)-3-3-fluoro DAHP was purified by anion exchange chromatography. The chemical synthesis of erythrose 4-phosphate and the isomers of 3-fluoro phosphoenolpyruvate and the enzymatic synthesis of (3S)-3- fluoro DAHP and its purification are discussed in Chapter Two. A recombinant Escherichia coli strain (pJB 14) was used to over-express the enzyme dehydroquinate synthase, and partial purification of the enzyme was achieved by anion exchange chromatography. Chapter Three describes the production and purification of the enzyme 3-dehydroquinate synthase. Purified (3S)-3-fluoru DAHP was treated with the E. coli enzyme 3-dehydroquinate synthase. Formation of both (6S)-6-fluorodehydroquinate and its C1 epimer was observed. The reaction was followed at different pH and temperature values. The ratio of products produced in the enzyme-catalysed reaction was monitored by 19 F NMR spectroscopy. No significant change in the ratios was observed with the different conditions employed. The results from these experiments are discussed in Chapter Four. Our results are consistent with the hypothesis that the fluoroenolpyranose intermediate is released to the solution, where it cyclises without the constraint of an enzymatic template. To test this hypothesis unequivocally, further investigations are required and these are discussed in Future Directions. [NB: Mathematical/chemical formulae or equations have been omitted from the abstract due to website limitations. Please read the full text PDF file for a complete abstract.]
  • Thumbnail Image
    Item
    Lipolytic enzymes from the bovine rumen : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University
    (Massey University, 1970) Faruque, Abu J M Omar
    1.1. Lipid metabolism in the ruminants and significance of hydrolysis and hydrogenation in the rumen. The digestion pattern of ruminants differs from other mammals in that the food of ruminants is subjected to a microbial fermentation in the rumen before passing into the true stomach. Carbohydrates, proteins, organic acids and many other food constituents are attacked by the micro organisms in the rumen and as a result short-chain fatty acids (VFA), CO2 , CH4 , NH3 etc. are produced as the end product of microbial metabolism. This ruminal fermentation has a considerable effect on the metabolic processes of the animal and moreover the functions of the rumen microorganisms are intimately associated with certain metabolic disorders of the ruminant (Bryant, 1959) e.g. Ketosis, bloat etc. It is now generally believed that the organisms of functional significance in the rumen are protozoa and bacteria which are capable of growth under the anaerobic conditions prevailing. The rumen provides an ideal anaerobic environment for a large and diverse microbial population at a temperature of 39°-40°C. The pH of the ingesta is slightly acid and the bacteria are adapted to live between pH 5.5 and 7.0 (Hungate, 1966). It is apparent from earlier studies that the lipids of ruminants differ in several respects from those of non-ruminant herbivorous animals in particular. Occurrence of unusually high proportions of stearic acid and the presence of trans acids and isomeric forms of oleic, linoleic and linolenic acid in depot fats and tissue lipids are peculiar to ruminants. [From Introduction]
  • Thumbnail Image
    Item
    Mutants of escherichia coli with abnormal patterns of repression of arginine biosynthetic enzymes : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University
    (Massey University, 1973) Morrison, Jeffrey
    Experimental work was done to attempt to isolate further mutants of the arginine regulatory gene in Escherichia coli W of the same type as a strain known as W2-250, This mutant carries the argR* regulatory gene which confers upon it the diminished ability to repress or derepress its arginine biosynthetic enzymes. Experimental work involved establishing a suitable method of exaggerating the slow growth rate of this strain on the arginine intermediate N -acetyl-L-ornithine, such that this could be the basis of protecting similar mutants when selecting them from a parent strain (W2-40) in a penicillin select-ion procedure. This was achieved by growing both strains in diphasic a medium containing 15ug/cm3 of L-arginine and an excess (30ug/cm3 ) of acetylornithine, in which it was found that the argR strain entered & premature stationary phase when the arginine was exhausted but the argR strain continued growth at a slower rate than normal in the acetylornithine. It was also found that a culture of W2-250 which had entered this stationary phase could be diluted 1:1 in fresh minimal medium + acetylornithine and still remain in a stationary condition for up to 24 hours. Mutants of strain W2-40 produced using ultraviolet light were cycled twice through a system involving this stationary phase in diphasic medium and dilution 1:1 in fresh medium + 500 units of benzyl-penicillin/cm3 in order to select against the parent-type. Further selection was carried out on minimal agar plates supplemented with acetylornithine and canavanine on which the parent strain will grow, but the argR* type is inhibited until arginine was added to the agar. The colonies which appeared at this stage were screened on variously supplemented solid media to select those most like W2-250. Those selected on these criteria were then screened using the acetylornithinase assay and the diphasic medium, with W2-250 included for comparison. A total of nine possible mutants of the desired type were isolated, but problems with the preparation of enzyme samples precluded a definite decision on their identity. In the course of this work it was also discovered that an arg8 bradytroph (W2-25/8) could behave similarly to W2-250 in diphasic media. It was also found that there was some transferable "factor" in the medium of cells which had reached the premature stationary phase which could prevent further growth of either W2-250 or W2-25/8 in non-repressive minimal medium. This is discussed in detail.
  • Thumbnail Image
    Item
    An investigation into the extracellular enzymes produced by Dothistroma pini and their relation to pathogenicity : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry.
    (Massey University, 1970) Brunt, Peter John
    The aim of this chapter is to provide a background against which the pathogenic action of Dothistroma pini can he discussed. Plant Diseases. There are three principal groups of organisms implicated as causal agents of diseases in planto; fungi, bacteria and viruses. These pathogens cause diseases which in general can be divided into three kinds: necrosis, hypertrophy and hypoplasia. Necrosis Necrotic diseases can be general or local in nature. General necrosis is called rotting or decay and is usually caused by fungi or bacteria. An example is Rhizoctonia disease of sugar beet. Local necrosis is more limited in its extent. Examples are leaf spots, fruit spots, anthracnoses and certain types of cankers e.g. the leaf spot of maize caused by Cochliobolus carbonum and red band blight of pines caused by Dothistroma pini. Hypertrophy. [FROM INTRODUCTION]