Enzyme citrus

The Citrus enzyme catalyzes 2"-0-rhamnosylation of the 7-O-glucoside of flavanones and flavones but not flavonols. 

ENZYMATIC ANALYSIS WITH CARBOXYPEPTIDASES. Carboxypeptidases are enzymes that cleave amino acid residues from the C-termini of polypeptides in a successive fashion. Four carboxypeptidases are in general use A, B, C, and Y. Carboxypeptidase A (from bovine pancreas) works well in hydrolyzing the C-terminal peptide bond of all residues except proline, arginine, and lysine. The analogous enzyme from hog pancreas, carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues. Thus, a mixture of carboxypeptidases A and B liberates any C-terminal amino acid except proline. Carboxypeptidase C from citrus leaves and carboxypeptidase Y from yeast act on any C-terminal residue. Because the nature of the amino acid residue at the end often determines the rate at which it is cleaved and because these enzymes remove residues successively, care must be taken in interpreting results. Carboxypeptidase Y cleavage has been adapted to an automated protocol analogous to that used in Edman sequenators. 

Pectin is a long chain of pectic acid and pectinic acid molecules. Because these acids are sugars, pectin is categorized as a polysaccharide. It is prepared from citrus peels and the remains of apples after they are squeezed for juice. In the plant, pectin is the material that joins the plant cells together. When fungus enzymes break down the pectin in fruit, the fruit gets soft and mushy. 

When cell-wall fragments are incubated in molar NaCl, ionically bound proteins are released into the incubation medium. All investigated crude cell extracts deesterified Citrus pectin (Table 2) but the deesterification rates were clearly higher when the enzymes were still bound to the cell walls, indicating a major loss of activity during the solubilization process. 

Indeed, apple pomace, beet pulp or citrus peels contain pectins (3). Chemicals, enzymes, microorganisms, or physical treatments can be used for thextraction (4). 

Activation studies were conducted at pH 7.5 at 30°C in 20 mL of 0.5% high methoxyl citrus pectin (Citrus Colloids, Hereford, U.K.). Final cation concentration in PE extracts used for activation studies was less than 2 mM as measured by potentiometry. Controls were conducted to correct for non-enzymic alkali consumption, with no polyamine/no PE and polyamine added/no PE. PE activity was normalized as a percentage of activity with no cation addition. 

The basal medium of Mandels (Mandels et al., 1976) was used with the following modifications it was buffered with 3 g/1 of sodium nitrate to pH 5.5 and supplemented with 1% w/v citrus pectin " Sigma" or other carbon sources. For enzyme production, 50 ml medium in 250 ml erlemneyer flasks were inoculatedwith spores (10 spores /ml ) exept for the non sporulating Pol 6 strain, where mycelium was used. The culture were incubated at 30° C on a rotary shaker (150 rev mn -1) for 5 days. The culture broth was filtered (Millipore 0.45 pm ) and the supernatant was analysed for pectinolytic activities, reducing sugars and proteins. 

Enzymes produced by parental (CLIOO) and mutant (CTl) strains cultured on citrus pectin... 

One of the important criteria taken into account for the choice of an industrial producer strain is its ability to secrete enzymes on cheap and local substrate. Thus, we cultured our mutant as well as the Pol6 mutant on a local substrate milled "orange peel", at the same concentration as citrus pectin in the liquid medium. The results summarized on table 3 show a net difference between both strains the CTl mutant is able to produce high amounts of endo and exopectinases on both substrates whereas Pol6 is unable to hyper-produce both pectinases on "orange peel". 

Comparison of Enzymes production by CTl and Pol6 strains cultured on citrus pectin and orange peel... 

Enzyme.—Polygalacturonase (PG) was obtained from a culture of Rhizopus nigricans using Citrus pectin as carbon source [11,12], The enzyme used for oligouronides obtention was the residual activity after a thermal treatment (100°C, 60 sec) of the native Rh. nigricans endo and exoPG, since the endo-enzyme was thermoresistant while the exo-enzyme was thermolabile [13]. 

Lineweaver and Ballou49 have proposed a pectinesterase unit ( PE. u. ) for expressing PM activity. One such unit is equivalent to 1/930 PMU under the same experimental conditions or the quantity of enzyme that, at 30° and optimum pH, will catalyze the hydrolysis of pectin at an initial rate of one milliequivalent ester bonds per minute in a standard substrate (0.5% citrus pectin containing 8-11% methoxyl) and 0.15 M sodium chloride. The use of the latter unit is unfortunate since the values obtained for the activity in ordinary plant materials are obtained in the third decimal place and because the experimental conditions are so... 

Similarly, there are present in citrus juices active enzyme systems that destroy the important pectic constituents. These enzymes have to be rapidly inactivated to protect the desirable cloud (turbidity) which is apparently stabilized by pectic substances. ... 

With the exception of tomato and perhaps citrus fruits, there is no instance in which the presence of a PG in higher plants or macerates has been conclusively demonstrated. Often the types of changes reported suggest 1 that infection by microorganisms rather than naturally occurring enzymes was the cause of the observed chemical transformations. This is especially true with fruit juices, where the rapidly increasing bacterial and mold flora may eventually cause reactions which do not occur when a sterile macerate is incubated. 

Table 6.1 Citrus phenylpropanoid and core flavonoid biosynthetic enzymes...

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