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Subject: search.filters.subject.Hydrolases

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    Beef hydrolysis by Zyactinase™ enzymes : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Auckland, New Zealand.
    (Massey University, 2016) Ahmad, Noriza Binti
    Protein hydrolysis is the term that applies to all possible ways of splitting proteins to produce products with lower molecular weight. There is a continuous search for novel products derived from waste materials. In the developed nations considerable amount of meat off-cuts are discarded each year. Utilizing these leftovers by developing new technology for protein recovery and modification and production of a broad spectrum of food ingredients greatly enhances its final value. The aim of this research was to partially hydrolyse beef meat protein with a commercial kiwifruit product called ZyactinaseTM, which is essentially freeze-dried kiwifruit to determine the effect of various processing conditions that influence the extent of beef meat hydrolysis. Secondly to determine the peptide and amino acid profile of the beef meat sample after hydrolysis. Thirdly to determine the relative reaction of ZyactinaseTM on various beef meat protein fractions. This study also aimed to evaluate the rate and the extent of partial enzymic hydrolysis of lean beef using ZyactinaseTM enzymes in order to obtain a better understanding of protein hydrolysis reaction. Lean beef minced was partially hydrolysed using the Zyactinase enzymes for different processing times (up to 360 minutes), temperatures (27°C to 70°C) and varying enzyme concentrations. No pH adjustment on the raw material was carried out except for pH studies. The hydrolysates were collected and analysed for total nitrogen content and degree of hydrolysis. The method used to characterize the extent of protein hydrolysis was SN-TCA index (fraction of nitrogen soluble in trichloroacetic acid) also called non-protein nitrogen NPN. Peptide and amino acid in protein hydrolysates were analysed by HPLC and different protein fractions in the hydrolysates were characterised by SDS-PAGE. The relationship between the reaction temperature, enzyme concentration and processing time to the total nitrogen and NPN were determined. The total nitrogen content remained relatively constant throughout the hydrolysis process. In addition, the NPN content increased as the temperature, processing time and enzyme concentration increased. The optimum pH range for the enzyme’s activity was 4 – 5.6 and optimum temperature was 60°C. Furthermore, most of the higher molecular weight protein bands on SDS- PAGE disappeared after hydrolysis and lower molecular weight protein bands increased in intensity. Zyactinase was also found to digest protein in the myobrilla and sarcoplasmic meat fractions at similar rates as whole beef meat. The results provide basic understanding of the kiwifruit enzymes action toward protein that may lead to improved methods for recovering meat protein or developing new food materials.
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    Biochemical characterization of metal-dependent 3-deoxy-D-manno-octulosonate 8-phosphate synthases from Chlorobium tepidum & Acidithiobacillus ferrooxidans : a thesis presented in partial fulfillment of the requirements for the degree of Masterate of Science in Biochemistry at Massey University, Turitea, Palmerston North, New Zealand
    (Massey University, 2007) Yeoman, Jeffrey Aaron
    3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase is the enzyme responsible for catalyzing the first reaction in the biosynthesis of KDO. KDO is an essential component in the cell wall of Gram-negative bacteria and plants. This compound is not present in mammals; therefore the enzymes responsible for its biosynthesis are potential targets for the development of new antibiotic agents. KDO8P synthase catalyzes the condensation reaction between phosphoenol pyruvate (PEP) and D-arabinose 5-phosphate (A5P) to form KDO8P. Two types of KDO8P synthase have been identified; a metal-dependent type and a non metal-dependent type. KDO8P synthase from the organism Chlorobium tepidum (Cte) has been partially purified and partially characterized. In line with predictions based on sequence alone, the activity of this enzyme is dependent on the presence of a divalent metal ion and is sensitive to the presence of the metal chelating agent EDTA. Cte KDO8P synthase was found to have the highest activity in the presence of Mn2+ or Cd2+. KDO8P synthase from the organism Acidithiobacillus ferrooxidans (Afe) has also been cloned, purified and biochemically characterized. Afe KDO8P synthase was also found to be a metallo enzyme and the catalytic activity is highest in the presence of Mn2+ or Co2+. Afe KDO8P synthase was found to exist as a tetramer in solution and is most active within the pH range of 6.8 to 7.5 and within a temperature range of 35 ºC to 40 ºC. Sequence analysis suggests that this enzyme has characteristics conserved throughout the metallo and the non-metallo KDO8P synthases and is closely related to the metal-dependent 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) synthases. The role of several active-site residues of Afe KDO8P synthase has been investigated. A C21N mutant of Afe KDO8P synthase was found to retain 0.5% of wildtype activity and did not require a divalent metal ion for catalytic activity. This suggests that the metallo and non-metallo KDO8P synthases have similar catalytic mechanisms.