Mode of pectin deesterification byTrichoderma reesei pectinesterase

Abstract

Summary. Trichoderma reesei pectinesterase (isoelectric point 8.3-9.5; pH-optimum 7.6) catalyzes deesterification of pectin by giving rise to blocks of free carboxyl groups in the macromolecule; a mode similar to that observed with higher plant pectinesteruses which also have |p and pH-optima in the alkaline region.Pectinesterases of higher plants (tomato 1-3, orange 4-7 and alfalfa 3) catalyze deesterification of pectin linearly along the chain of the molecule, giving rise to blocks of free carboxyl groups. On the other hand, deesterification of pectin by alkali and acids results in a statistical distribution of free and esterified carboxyl groups 4, 5. The present study reports the mode of deesterification of pectin catalyzed by Triehoderma reesei pectinesterase, which has its pH-optimum and isoelectric point in the alkaline region. Material and methods. Pectinesterase was prepared from culture filtrate of the fungus Trichoderma reesei QM 9414 cultivated on pectin as a carbon source, after ammonium sulfate precipitation and chromatography on Sephadex G-25 and G-100 columns ~~ This preparation included 5 multiple forms of pectinesterase with isoelectric points in the range 8.3-9.5 and pH-optimum 7.6. Preparation and characterization of pectin samples deesterified by pectinesterase. Purified citrus pectin (Genu Pectin, Medium Rapid Set, Kobenhavns Pektinfabrik, Denmark) was esterified to 94.7% with methanolic 1 M HzSO 4 at 3 ~ This pectin contained 88.2% of highly esterified D-galacturonan and exhibited a limit viscosity number tidus ; O, AspergiUus niger ; 9 tomato ; ~, orange ; I% alfalfa 3.was deesterified by Trichoderma reesei pectinesterase at pH 7.3 under continuous titration with 0.1 M NaOH at 25~ a Radiometer pH-stat and autotitrator set (Denmark). The 1% pectin solution (250 ml) was treated with I ml enzyme (total activity 1.2 x 10 -3 tool sec -~) until the required degree of esterification (d.c.) was obtained. The enzyme was inactivated by 10 min heating on a boiling water bath and after cooling the pectin was precipitated with acidified ethanol (final concentration 0.33 M HC1 in 60% ethanol) and washed several times with 60% ethanol to remove chloride ions. The content of free carboxyl groups of pectin was determined by potentiometric titration with 0.05 M KOH, and the overall content of carboxyl groups by precipitation of insoluble copper pectates ~ ~. lz. The amount of copper bound to pectin carboxyl groups was determined chelatometrically. The limit viscosity number [11] of pectin solutions adjusted to pH ~ 7 with KOH and containing 0.15 mol NaC1 -0.005 tool sodium oxalate in 1 t was measured using an Ubbelohde viscometer at 25.0 -4-0.1 ~ Determination of calcium ion activities in Ca-pectinate solutions. Solutions of pectinic acids (4-5 mmol COOH 1-1) were centrifuged at 20,000 • g for 20 min and the supernatants neutralized with a saturated calcium hydroxide solution (0.021 M Ca(OH)2 ) to pH ~ 7.2. Single-ion activity coefficients 7c,2+ of calcium ions bound to carboxyl groups of pectin were determ...

Structure and Properties of a Non-processive, Salt-requiring, and Acidophilic Pectin Methylesterase from Aspergillus niger Provide Insights into the Key Determinants of Processivity Control

“…To date, the fungal PMEs from Aspergillus (Emericella) nidulans (63) and Trichoderma reesei (Hypocrea jecorina) are reported to be processive (50,64), whereas others are reported to be non-processive with the pattern of de-methylesterification of HG chains being near-random, indicating that these enzymes release their substrate after each de-methylesterification event (38,42,50,52,(65)(66)(67). This mode of action is also referred to as multiple attack (56).…”

Investigation of the Action Patterns of Pectinmethylesterase Isoforms through Kinetic Analyses and NMR Spectroscopy

“…A limited number of investigations of the action pattern of PME have been reported (12)(13)(14)(15)(16). Partially deesterified polymers have been submitted to hydrolysis by endogalacturonases that are known to stop their attack in the vicinity of a methylated galacturonic unit, and the fragments released were subsequently characterized (13).…”

“…Partially deesterified polymers have been submitted to hydrolysis by endogalacturonases that are known to stop their attack in the vicinity of a methylated galacturonic unit, and the fragments released were subsequently characterized (13). In another approach, the activity coefficients ␣ Ca 2ϩ (␣ Ca 2ϩ is a complex function of the linear charge density of the macromolecule) of the calcium pectinates were determined after enzymatic deesterification (14). From these investigations, it was proposed that alkaline PME from higher plants (tomato, orange, alfalfa) catalyze deesterification of pectin linearly along the chain of the molecule, giving rise to blocks of free carboxyl groups, whereas acidic microbial PMEs cause a random cleavage of esterified carboxyl groups (14).…”

“…In another approach, the activity coefficients ␣ Ca 2ϩ (␣ Ca 2ϩ is a complex function of the linear charge density of the macromolecule) of the calcium pectinates were determined after enzymatic deesterification (14). From these investigations, it was proposed that alkaline PME from higher plants (tomato, orange, alfalfa) catalyze deesterification of pectin linearly along the chain of the molecule, giving rise to blocks of free carboxyl groups, whereas acidic microbial PMEs cause a random cleavage of esterified carboxyl groups (14). In the case of the plant PMEs, studies have also pointed to the importance of a free carboxyl group in the vicinity of the active site (13,14,17).…”

“…From these investigations, it was proposed that alkaline PME from higher plants (tomato, orange, alfalfa) catalyze deesterification of pectin linearly along the chain of the molecule, giving rise to blocks of free carboxyl groups, whereas acidic microbial PMEs cause a random cleavage of esterified carboxyl groups (14). In the case of the plant PMEs, studies have also pointed to the importance of a free carboxyl group in the vicinity of the active site (13,14,17). A major drawback to the afore-going methods is that they provide only indirect information on the distribution of the carboxyl units after PME action and do not lead to a precise description of the reaction mechanism.…”