John R. Whitaker

The biochemistry and control of enzymatic browning

Abstract Half of the worlds fruit and vegetable crops is lost due to postharvest deteriorative reactions. Polyphenol oxidase (PPO), found in most fruit and vegetables, is responsible for enzymatic browning of fresh horticultural products, following bruising, cutting or other damage to the cell. Chemical methods for controlling enzymatic browning include the use of sodium bisulfite, ascorbic acid and/or packaging under controlled atmospheres. Current approaches to understanding and controlling enzymatic browning are presented in this review article, with special focus on the use of antisense RNA as a control method.

Pectic substances, pectic enzymes and haze formation in fruit juices

Abstract The pectic substances, located primarily in the middle lamella between cells in higher plant tissues, are complex polysaccharides. They include the negatively charged rhamnogalacturonans, and the neutral arabinogalactans I and II and l -arabinans. These polysaccharides add viscosity to juices but may also form hazes and precipitates and retard maximum recovery of juices from the fruit. The rhamnogalacturonans are degraded by the enzymes pectin methylesterase and polygalacturonase normally present in plant tissues and by these enzymes and pectate lyase in microbially derived commercial pectic enzymes added during processing. The presence of aŕabinofuranosidase, which degrades l -arabinans, in commercial pectic enzyme preparations, can cause haze formation in juices such as apple and pear.

Chemical modification of papain: I. Reaction with the chloromethyl ketones of phenylalanine and lysine and with phenylmethylsulfonyl fluoride☆

Abstract Reaction of papain with the chloromethyl ketones of tosyl- l -phenylalanine and tosyl- l -lysine is rapid and results in a complete loss of activity. The chloromethyl ketones react specifically with the sole sulfhydryl group (on cysteine residue No. 25) of papain as shown by sulfhydryl group analysis, amino acid analysis, spectrophotometric data, and failure to react with mercuripapain. The limiting second-order reaction rate constant for reaction of α-N-tosyl- l -phenylalanine chloromethyl ketone with papain was found to be 200 m −1 sec−1 at 0 ° and to be dependent upon a single prototropic group of pK 8.90. The rate of the reaction increases with increasing pH, an indication that the reactive species is the thiol anion. Phenylmethylsulfonyl fluoride inhibits papain rapidly at pH 7 and 0 °. The inactive enzyme can be fully reactivated by treatment with dithiothreitol. Attempts to prepare hydroxypapain after reaction with phenylmethylsulfonyl fluoride have not been successful.

Biochemistry of lipoxygenase in relation to food quality

A renewed interest in lipoxygenase has led to detailed studies of its isoenzymes, substrate specificity, and the nature of its reaction products. Lipoxygenase is highly specific for cis,cis-1,4-pentadiene systems such as linoleic, linolenic, and arachidonic acid (or ester) and catalyzes the formation of the corresponding hydroperoxides with a cis,-trans-conjugated diene system. The hydroperoxides can then undergo enzymic or spontaneous degradation, producing a range of carbonyl compounds. This review will discuss the biochemical properties of this enzyme and its contribution to the quality of raw and processed food products. An attempt has been made to discuss both the desirable and undesirable effects associated with the action of lipoxygenase, citing specific food examples where appropriate.

Development of Flavor, Odor, and Pungency in Onion and Garlic

Publisher Summary This chapter introduces us to the development of flavor, odor, and pungency in onion and garlic, which is used as vegetables and spices and home remedies in treatment of illnesses. The chapter briefly describes the sulfur compounds of intact alliurn and biosynthesis of sulfur compounds of AIliurns—such as— alkylcysteines and akylcysteine sulfoxides, which are garlic flavor and odor precursors, y–glutamyl peptides are onion flavor and odor precursors and cysteine, alkylcysteines and alkylcysteine sulfoxides y–glutamyl peptides. The chapter describes the methods of measuring Alliurn pungency, flavor, and aroma. A quantitative method is used to measure pungency and the Chemical Oxygen Demand (COD) method is used to estimate the aroma and flavor. Enzymes in flavor, aroma and pungency development in allium are alliinase and y–glutamyl peptidase and y–glutamyl transpeptidase. The chapter concludes by indicating the need of proper research and various experimental methods to be introduced to maximize the flavor and odor of onion and garlic.

Physical, Chemical, and Nutritional Properties of Common Bean (Phaseolus) Proteins

Publisher Summary This chapter discusses that the family Leguminosae includes approximately 600 genera, with about 13,000 species. In legume seeds, the cotyledons form the bulk of the seed and synthesize most of the proteins. Research on identification and quantification of the storage proteins in beans (Phaseolus) goes back several decades. These investigators referred to three proteins in Phaseolus (phaseolin, phaselin, and conphaseolin). The chapter discusses that bean proteins were first studied by Osborne. He found that the extractable proteins were mostly globulins, a fact which was later confirmed and emphasized by other investigators, who found three globulins in Phaseolus seeds. The biological value of bean proteins in general is low as compared to most other food proteins. According to Boyd, hemagglutinating activity was first described by Stillmark in 1888 in extracts of castor bean (Ricinus communis) and the first plant lectin found to be blood group specific was from lima bean (Phaseolus lunatus). Proteins that inhibit a-amylases from animals and insects have been reported in a variety of plants. Common beans (Phaseolus vulgaris) are normally harvested with about 20% moisture in the seeds and dried to about 10% water content before storage or consumption. The chapter discusses that common beans contain from 18% to 35% protein, however, their biological value is much lower than expected on a protein and amino acid composition basis.

Cloning, sequencing, purification, and crystal structure of Grenache (Vitis vinifera) polyphenol oxidase.

The full-length cDNA sequence (P93622_VITVI) of polyphenol oxidase (PPO) cDNA from grape Vitis vinifera L., cv Grenache, was found to encode a translated protein of 607 amino acids with an expected molecular weight of ca. 67 kDa and a predicted pI of 6.83. The translated amino acid sequence was 99%, identical to that of a white grape berry PPO (1) (5 out of 607 amino acid potential sequence differences). The protein was purified from Grenache grape berries by using traditional methods, and it was crystallized with ammonium acetate by the hanging-drop vapor diffusion method. The crystals were orthorhombic, space group C222(1). The structure was obtained at 2.2 A resolution using synchrotron radiation using the 39 kDa isozyme of sweet potato PPO (PDB code: 1BT1 ) as a phase donor. The basic symmetry of the cell parameters (a, b, and c and alpha, beta, and gamma) as well as in the number of asymmetric units in the unit cell of the crystals of PPO, differed between the two proteins. The structures of the two enzymes are quite similar in overall fold, the location of the helix bundles at the core, and the active site in which three histidines bind each of the two catalytic copper ions, and one of the histidines is engaged in a thioether linkage with a cysteine residue. The possibility that the formation of the Cys-His thioether linkage constitutes the activation step is proposed. No evidence of phosphorylation or glycoslyation was found in the electron density map. The mass of the crystallized protein appears to be only 38.4 kDa, and the processing that occurs in the grape berry that leads to this smaller size is discussed.

The effect of oral stimulation on human parotid salivary flow rate and alpha-amylase secretion ☆

Unilateral parotid saliva was collected from ten subjects following oral stimulation with water as baseline, and aqueous solutions of starch (2.5, 5.0, and 10%), sucrose (0.1, 0.2, and 0.4 M) sodium chloride (0.075, 0.15, and 0.30 M), and citric acid (0.005, 0.01, and 0.02 M). Salivary flow rate increased with increasing levels of each taste stimulus. At concentrations of equal taste intensity, citric acid evoked the highest flow rate, followed by sodium chloride and sucrose, while starch, in solution, had a minimal effect. Secretion rate patterns for total protein and alpha-amylase mirrored those of flow rate. The total protein and alpha-amylase concentrations of the saliva, and specific activity of alpha-amylase, were influenced by the type but not the concentration of stimulus, with citric acid stimulation resulting in the lowest concentrations and highest specific activity. Sodium ion (Na+) concentration generally increased with increasing stimulated flow rate, while K+, Ca++, and Mg++ concentrations remained relatively constant. Subjects with lower flow rates had a more concentrated saliva than those with high flow, except for Na+ concentration. Oral stimulation resulted in similar changes in protein and alpha-amylase secretion rates for the two groups.

Chemical and physical modification of proteins by the hydroxide ion

Proteins are exposed to alkaline conditions during solubilization and/or purification, during food storage and processing, in removal of toxic constituents, and for characterization. During alkali treatment, there are changes in solubility and aggregation, hydrolysis, elimination reactions involving the side chains of certain amino acids, racemization of amino acid residues, addition of compounds to proteins, fragmentation of the peptide chain, as well as modification or elimination of nonprotein constituents. The rates of these reactions are affected by pH, temperature, cations (in some cases), ionic strength (in some cases), protein concentration, and to some extent by the specific nature of the protein. The general mechanisms and stoichiometry of these reactions are described. Other constituents of high protein foods also undergo reactions in alkaline solutions and the products of these reactions may in turn react with proteins. We have described the effect of alkali on enediol formation and fragmentation of carbohydrates, the hydrolysis of lipids in alkaline solution and effect on rate of peroxidation of the polyunsaturated fatty acids, the oxidation of amino acid residues, especially methionine, the oxidation of phenols to benzoquinones, and the catalytic effect of metal ions in alkaline solutions. Alkali treatment is also used in the specific modification of proteins to distinguish between O-glycosyl and amide-linked glycosyl groups, to effect specific cleavage of peptide bonds via beta elimination, in the formation of anhydrotrypsin, anhydrochymotrypsin, anhydrosubtilisin and thiol-subtilisin, and in formation of intrachain crosslinking in proteins.

Expression and secretion of rice α-amylase by Saccharomyces cerevisiae

Abstract We report the high level expression and secretion of rice α-amylase isozyme by Saccharomyces cerevisiae . Transcription of this gene was under control of the yeast enolase promoter. The synthesized protein had an approximate molecular size of 45 kDa and a pI of approx 4.7 to 5.0. The rice α-amylase signal peptide was recognized and efficiently processed by yeast and the active, glycosylated enzyme was secreted into the culture media. This enzyme was purified to homogeneity by affinity chromotography and its enzymatic properties were characterized. The K m and V max were found to be similar to those of α-amylase from other organisms. The high level of secretion observed in these studies may be due to the unique features of the rice signal peptide and/or to the glycosylation of the recombinant enzyme.