Enzymes pectate lyase

In the first edition of this book this chapter was entitled "Antiparallel Beta Structures" but we have had to change this because an entirely unexpected structure, the p helix, was discovered in 1993. The p helix, which is not related to the numerous antiparallel p structures discussed so far, was first seen in the bacterial enzyme pectate lyase, the stmcture of which was determined by the group of Frances Jurnak at the University of California, Riverside. Subsequently several other protein structures have been found to contain p helices, including extracellular bacterial proteinases and the bacteriophage P22 tailspike protein. 

Figure 5.30 Schematic diagrams of the structure of the enzyme pectate lyase C, which has a three-sheet parallel P-helix topology.

A single P strand can also be wound into a cylinder with the hydrogen bonds running parallel to the helix axis. A right-handed parallel P helix of this type has been found in the bacterial enzyme pectate lyase.121122 The polypeptide chains of the 353-residue protein contain seven complete turns of about 22... 

Two molecules of the ion-channel-forming peptide gramicidin, which contains alternating L- and D-amino acids, have been shown to form a membrane-spanning P-helix. The alternation of L- and D-amino acids is a feature of a series of cyclic peptides that self-assemble into membrane spanning nanotubes. P-Helices have now also been shown to occur as a structural element in proteins consisting only of L-a-amino acids, viz. in the enzyme pectate lyase. Related to the naturally... 

Improvements in detergent efficacy will continue to capture the undivided attention of detergent manufacturers. There are multiple approaches that encompass a wide variety of current and novel technologies. Examples of these types of approaches range from unique combinations of enzymes (pectate, lyase, and mannanase) to facilitate the removal of food soil residues, to ethoxylated quaternized amines to improve soil suspension and cleaning of outdoor soils/stains, to nanoparticle technologies to deliver crease resistance properties in tumble dry additives and aqueous ironing formulations [180],... 

The PelX protein contains 749 amino-acids including an amino-terminal signal sequence of 26 amino-adds. The celliilar localisation of the enzyme has not been rigorously determined. In E. chrysanthemi CUCPB1237, the exo-pectate lyase activity was cell-bound (14). The presence of this type of enzyme in the bacterial periplasm appeared normal since it acts better on oligomers produced by endo-pectate lyases than on long polymeric substrates (15). 

Digestion of PGA by the PelL enzyme yielded a mixture of unsaturated ohgogalacturonides, giving evidence that PelL is an endo-deaving lyase (17). An exo-enz3mie, such as the EC 16 PelX, would generate a single product (15). The PelL protein differs from the major E. chrysanthemi pectate lyases in its ability to cleave both PGA and methylated pectin (17). The PelL activity has a basic optimum pH and an absolute requirement for Ca + ions. Analysis of culture supernatants demonstrated that PelL is an extracellular enzyme, such as the other secondary pectate lyases (17). 

Among the five different species of Azospirillurn, only A. irakeme shows clearly pectinolytic activity on solid and in liquid medium. Moreover, this species can grow under non-diazotrophic as well as diazotrophic conditions when pectin is the sole carbon source (Khammas and Kaiser, 1991). Khammas and Kaiser (1991) analysed the pectinolytic activity of seven A. irakense isolates, and gave evidence for the presence of two types of pectinolytic enzymes. All strains tested have inducible Ca dependent pectate lyase activity. Six strains, showed also pectin methylesterase activity. So far, none of the corresponding enzymes have been purified. 

In liquid medium, the thiobarbuturic acid test was used to determine polygalacturonase and pectate lyase activity (Sherwood, 1965). 1 ml of the crude enzyme preparation was added to 2 ml of 0.5 N HCl in a test tube. 4 ml of 0.01 M thiobarbuturic acid, dissolved in distilled water, were added. The tubes were heated in a boiling water for Ih and centrifuged. The absorption of the supernatant was determined in the spectrophotometer over the range 480-580 nm. Reaction mixtures without enzyme, which showed no reaction with thiobarbuturic acid, were used as controls. 

In order to characterize the pectinolytic enzymes encoded by these clones, the culture supernatants of all these clones were tested for pectate lyase and polygalacturonase activity, using thiobarbutiric acid as described in materials and methods. Absorption at 550 nm indicates the activity of pectate lyase whereas absorption at 510 nm indicates the activity of polygalacturonase. 

The enzyme had a requirement for calcium. The addition of EDTA to the reaction mixtures, resulted in complete loss of activity, whereas the addition of CaCl2 increased the activity (figure 8). Presumably, sufficient contaminating calcium ions were present in the dialyzed enzyme and substrate mixture to permit the limited activity of the controls, but apparently these were removed by chelation with EDTA. The optimum concentration was in the range of 5 to 15 M, and higher concentration resulted in a decrease in activity. Phoma medicaginis var. pinodella synthesizes a pectin lyase that lacked an absolute requirement for calcium ions but maximum enzyme activity required the presence of 1 mM Ca [25]. The lyase from Fusarium solani f sp. phaseoli, that is active on pectin and pectic acid, is calcium-dependent [30]. Most of the pectate lyases characterized are calcium-dependent the pectate lyase from Rhizoctonia solani [34] and the endopectate lyase fi om Fusarium solani f sp. pisi [31]. 

Figure 2. CM-cellulose chromatography of pectolytic enzymes. The activity peaks of the flow-through of a DEAE-cellulose chromatography was applied to a CM-cellulose column. The column was eluted with a NaCl (0-0.5M) continuous gradient at a flow rate of 34 ml/h. 10 ml fractions were collected and assayed for pectolytic activities Symbols (0) pectate lyase ( ) polygalacturonase (reducing sugar-releasing activity) (x) protein. Other details in Methods.

Table 1 shows some biochemical properties of the pectolytic enzymes present in pool 1. The pectin lyase/pectate lyase activities (pool I) and polygalacturonase activity (pool II) were not significantly affected by NH4+, Na+ and K+ (0,25 - 2,5mM), while Al +, p-mercaptoethanol, Hg2+, EDTA, Ba + and Zn+2 (2,5mM) inhibited 30-100% these activities. On the other hand, Ca2+, Mg + and Mn + at 2,5mM concentration activated 20-100% pectin/pectate lyases but Ca " " and Cu " " (2,5mM) inhibited polygalacturonase activity about 42 - 70%. 

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