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Methionine (Met) is the primary limiting essential amino acid in grain legumes. The imbalance in amino acid composition restricts their biological value (BV) to 55 to 75% of that of animal protein. So far improvement of the BV could not be achieved by conventional breeding. Therefore, genetic engineering was employed by several laboratories to resolve the problem. Three strategies have been followed. A) Engineering for increased free Met levels; B) engineering of endogenous storage proteins with increased numbers of Met residues; C) transfer of foreign genes encoding Met-rich proteins, e.g. the Brazil nut 2S albumin (BNA) and its homologue from sunflower, into grain legumes. The latter strategy turned out to be most promising. In all cases the gene was put under the control of a developmentally regulated seed specific promoter and transferred into grain legumes using the bacterial Agrobacterium tumefaciens-system. Integration into and copy numbers in the plant genome as well as Mendelian inheritance and gene dosage effects were verified. After correct precursor processing the mature 2S albumin was intracellularly deposited in protein bodies which are part of the vacuolar compartment. The foreign protein amounted to 5 to 10% of the total seed protein in the best transgenic lines of narbon bean (Vicia narbonensis L., used in the authors' laboratories), lupins (Lupinus angustifolius L., used in CSIRO, Australia), and soybean (Glycine max (L.) Merr., used by Pioneer Hi-Bred, Inc., USA). In the narbon bean the increase of Met was directly related to the amount of 2S albumin in the transgenic seeds, but in soybean it remained below the theoretically expected value. Nevertheless, trangenic soybean reached 100%, whereas narbon bean and lupins reached approximately 80% of the FAO-standard for nutritionally balanced food proteins. These results document that the Met problem of grain legumes can be resolved by genetic engineering.

Methionine is a limiting essential amino acid in human nutrition, to overcome the possible overdosage and improve bioavailability methionine supply should be in protein bound form. So insertion a poly-met encoding DNA sequence results a more efficient solution in increasing methionine content of yeast. Poly-met DNA yeast hybrids were constructed of Saccharomyces cerevisiae CB89, an auxotrophic mutant strain. Synthetic DNA sequence encoding methionine polypeptide was inserted into the polylinker region of pVT-U 100 vector with 2 mu plasmid replicon. After transformation of E. coli HB101 cells the efficiency of the ligation and transformation was checked by digesting the minipreps. S. cerevisiae CB89 was transformed with vector-poly-met insert and with the plasmid vector only as well. Hybrid yeasts were selected on uracilless medium. PVT-U 100 can be used as vector for the expression of DNA sequence in S. cerevisiae. The vector harbours the promoter of ADC1 gene immediately downstream from the promoter lies a polylinker sequence comprising unique restriction enzyme sites for BamHI, HindIII, PvuII, SacI, Xhol. The polylinker sequence is followed by the transcriptional stop site and polyadenylation signal of ADC1 gene. Plasmid pVT-U 100 has selection markers for S. cerevisiae (URA3) and for E. coli (amp, per F). Results show that the poly-met DNA hybrids methionine content is influenced by the length of the insert. Fusion hybrids containing 600 bp oligo insert showed the best values. Distribution of methionine content in the protein subfractions of polymet DNA hybrid and parent strain CB89 was determined in dependence of glucose concentration and aeration intensity. The increase in synthesized methionine appeared in fractions 1 + 2 and residue. Technofunctional properties of parent strains and hybrids were compared for whole cells and cell wall (residue). Results demonstrate that enrichment in methionine in the cell wall fraction resulted improvement of emulsifying ability. Bioavailability of methionine content was better in DNA hybrid yeast than in parent strain and was the best when propagated in whey medium.

Spectroscopic and thermodynamic studies of holo-alpha-lactalbumin folding show that in hydro-ethanolic media this protein structure is subjected to at least three distinct transitions induced by ethanol. As observed by spectrofluorescence and circular dichroism (CD) the first transition is only local, being associated with changes in the state of aromatic chromophores. During this transition overall tertiary structure of alpha-lactalbumin is retained. As shown by high-sensitivity differential scanning calorimetry (HS-DSC) and CD, the second transition involves breakdown of the protein tertiary structure. The final organisation of the protein into highly helical folding may be considered as the third structural transition since the unfolding and the new helix formation are time-resolved processes.

Hen egg white lysozyme was genetically modified to have extreme heat stability and strong antimicrobial activity against Gram negative bacteria and the modified lysozymes were secreted in yeast and tobacco. Complementary DNA encoding lysozyme was subjected to site-directed mutagenesis to have the Asn-X-Thr(Ser) sequence that is the signal for asparagine-linked glycosylation at the positions 49. The glycosyl lysozyme enhanced heat stability was expressed in the yeast carrying the modified lysozyme cDNA. The expression amount of glycosyl lysozyme was about 10 mg/l of yeast culture medium. Using the same yeast expression system, the lysozyme enhanced antimicrobial action by inserting hydrophobic penta-peptide at the C-terminus were secreted in a small amount (less than 100 micrograms/l in the yeast culture medium). These cDNA constructs of modified lysozymes were engineered into tabacco through Agrobacterium-mediated transformation in order to construct antimicrobial plant. The expression of lysozymes was confirmed by the reverse transcriptional PCR, SDS-PAGE analysis and lytic activity of transformants of tobacco. The transformant having the highest lytic activity expressed about 40 micrograms of lysozyme per g of leaf tissue.

The hydrolysis of whole casein and isolated casein components were investigated with the purpose of obtaining information concerning the kinetic and specifty of aspartic proteases in rennin, pepsin and 4 microbial rennet substitutes. The velocity of hydrolysis decreased rapidly within the first hour. However, the hydrolysis was not completed after 2 days. A mathematical description of the slope of hydrolysis is possible by use of exponential equations. More than 40 peptides were detected by capillary electrophoresis or PAGE. The characterization of the C- and N-terminal amino acids of peptides shows that the hydrolysis of any peptide bond depends mainly on the structure of the C-terminal side chains of the amino acids. The detection of the basic amino acids lysin and arginin in the C-terminal position of peptides is a new result, furthering the knowledge about the specificity of aspartic proteases. Differences in the reaction velocity or in the extent of hydrolysis are one of the possible explanations for the described differences in the rennet curd yield. It was concluded that the rennet enzymes are active also in the later phases of cheese ripening and are able to support the action of cheese ripening flora.

Efforts have been done to recover proteins from waste liquors rich in protein in a soluble form. Cheese whey and animal bloods are by-products from the manufacture of cheese and meat. It contains a variety of proteins which can be reclaimed. The efficiency of protein precipitation from the sweet-cheese whey by the use of hydroxyethyl cellulose (HEC) was similar to that precipitated by the use of carboxymethyl cellulose (CMC). Both are greater than that precipitated by trichloro acetic acid. The same results of the efficiency of precipitation were attained when the plasma was precipitated. It was found that cheese-whey protein-HEC-complex and plasma protein-HEC-complex contain a large amount of essential amino acids. Electrophoretic separation of whey protein complex showed that beta-Lactoglobulin forms the major fraction while in case of plasma protein complex albumin forms the major fraction. The fractionation patterns of different complexes with HEC, CMC or TCA gave the same components and about the same ratio. It appears from these results that HEC-protein complexes are preferable than CMC-protein complexes or proteins precipitated by TCA. Chemical analysis of whey protein complexes revealed that lactose content of whey protein-HEC-complex was higher than that of CMC-complex or protein precipitated by TCA. Elemental analysis of protein complexes showed that the level of sodium, phosphorus, and potassium was increased while that of copper or zinc decreased. Cellulose derivative protein complexes showed no significant effects on the liver or kidney function of albino rat and these results indicted that no toxic effect was observed from the uses of these protein complexes in feeding.

A method for determination of zinc, cadmium, lead and copper in wines by means of potentiometric stripping analysis (PSA) is described. Cadmium, lead and copper are determined directly, whereas the zinc determination is possible only after the wine samples decomposition. The results for five red and white Yugoslav wines are given. The content of zinc, cadmium, lead and copper in analyzed samples were in the range of 0.16-0.79, 0.010-0.045, 0.13-0.27 and 0.10-0.46 mg/l, respectively. The contents of the all analyzed metals were below the maximum ordered by the Yugoslav law.

Manzala Lake exposed to many pollutants including untreated sewage, agricultural and industrial wastes which increase the concentration of heavy metals, and compromise the health state of the fishermen. This study investigated 100 fishermen and 100 males of other occupations as controls. Both groups work in and live on and around the lake. Clinical examination revealed no significant changes between the fishermen and control group as regards the cardiovascular, gastrointestinal and dermatological systems. However, the urinary, musculoskeletal and respiratory symptoms were significantly higher in fishermen than in control males. There was a significant decrease in neutrophils (48.8%) and a significant increase in lymphocytes and eosinophils (35.4% and 9%), respectively. Hepatotoxicity was evidenced by an increase in serum aspartate transaminase and alanine transaminase. There were no significant differences in serum creatinine and urea between fishermen and control. Levels of lead, cadmium and mercury in water and sediment were 0.26, 0.014, 0.002 mg/l, and 33.5, 1.37, 0.28 micrograms/kg, respectively. Levels of the three heavy metals in the fish samples and serum of fishermen and control males in average were 1.06, 0.18, 0.00025 ppm, 523, 33.5, 13.7 micrograms/l and 374, 12.8 11.2 micrograms/l, respectively. This study aimed to establish the relation between the environmental pollution and the health status of the population inhabiting the contaminated areas.

Edible fats and oils are an important component of human nutrition. There are many kinds of changes and destruction of lipids. One of the most important processes is the autoxidation of essential fatty acids. Primary products of oxidation are mono-hydroperoxides of fatty acids formed by three different pathways. Thermal activation of fatty acids will be followed by thermodynamically controlled distribution of the hydroperoxide isomers. With enzymatic or photochemical oxidation specific distribution of isomeric hydroperoxides is observed. These products can be also used for the determination of the type of oxidation which took place. The hydroperoxides formed in the process of oxidation cause a radical chain reaction with a powerful progress of oxidation. The products formed by chain termination are characterized by different stability. These products include the possibility of decomposition and consequent reactions.

A semimicro method for the quantitative determination of chloro-organic insecticides (COI) and polychlorinated biphenyls (PCB) in cereals, feed-pellets and water is presented. The extraction of the active compounds is carried out with n-hexane or dichloromethane. The extracts of cereals and pellets are purified by column chromatography with aluminum oxide. A silicagel column is used for the separation of the compounds into COI as well as PCB and hexachlorobenzene. The determination of the active compounds is carried out by gas chromatography using an electron capture detector. Recoveries range between 70 and 108%, except for beta-endosulphan.