pH-optima in lipase-catalysed esterification

Though lipases are frequently applied in ester synthesis, fundamental information on optimal pH or substrate concentration, can almost only be found for the reverse reaction - hydrolysis. This study demonstrates that the pH-optima of lipase-catalysed esterifications differ significantly from the optima of the hydrolysis reaction. In the esterification of n-butanol and propionic acid with lipases of Candida rugosa (CRL) and Thermomyces lanuginosa (TLL) pH-optima of 3.5 and 4.25, respectively, were found. This is about 3-4 units (CRL) and 7 units (TLL) in pH lower than optimum for hydrolysis. Enzyme activity increased with increasing concentrations of protonated acid indicating that the protonated acid rather than the deprotonated form is substrate for esterification. The rate of esterification can be drastically increased by ensuring acid concentrations up to 1000 mmol L^-1 for CRL and 600 mmol L^-1 for TLL in the reaction system.     

pH-optima in lipase-catalysed esterification

Though lipases are frequently applied in ester synthesis, fundamental information on optimal pH or substrate concentration, can almost only be found for the reverse reaction – hydrolysis. This study demonstrates that the pH-optima of lipase-catalysed esterifications differ significantly from the optima of the hydrolysis reaction. In the esterification of n-butanol and propionic acid with lipases of Candida rugosa (CRL) and Thermomyces lanuginosa (TLL) pH-optima of 3.5 and 4.25, respectively, were found. This is about 3–4 units (CRL) and 7 units (TLL) in pH lower than optimum for hydrolysis. Enzyme activity increased with increasing concentrations of protonated acid indicating that the protonated acid rather than the deprotonated form is substrate for esterification. The rate of esterification can be drastically increased by ensuring acid concentrations up to 1000 mmol L^?1 for CRL and 600 mmol L^?1 for TLL in the reaction system.     

Effects of pH, NADH, succinate and malate on the oxidation of glycine in spinach leaf mitochondria

The effect of external pH on several reactions catalyzed by glycine decarboxylase in spinach leaf mitochondria was investigated. Glycine-dependent oxygen consumption showed a pH optimum at 7.6, whereas the release of CO₂ and NH3 from glycine in the presence of oxaloacetate both showed pH maxima at 8.1. Glycine-dependent reduction of 2,6-dichlorophenolindophenol. on the other hand showed a pH optimum at 8.4. It is concluded that these three reactions have different rate-limiting steps. The rate of the glycine-bicarbonate exchange reaction catalyzed by glycine decarboxylase showed no optimum in the pH range investigated, pH 7–9, but increased with decreasing pH. This suggests that CO₂ may be the true substrate in this reaction.
The oxidation of glycine inhibited the oxidation of both malate, succinate and external NADH since the addition of malate, succinate or NADH to mitochondria oxidizing glycine in state 3 resulted in a rate of oxygen consumption which was lower than the sum of the rates when the substrates were oxidized individually. The addition of malate, succinate or NADH did not, however, decrease the rate of CO₂ or NH, release from glycine. It is suggested that the preferred oxidation of glycine by-spinach leaf mitochondria may constitute an important regulatory mechanism for the function of leaf mitochondria during photosynthesis.     

Engineering the pH-optimum of activity of the GH12 family endoglucanase by site-directed mutagenesis

Endo-1,4-β-glucanase from Penicillium verruculosum (PvEGIII) belongs to family 12 of glycoside hydrolases (GH12). Analysis of the enzyme 3D model structure showed that the amino acid residue Asp98 may directly affect the pH-profile of enzyme activity since it is located at the distance of hydrogen bond formation from Glu203 that plays the role of a general acid in catalysis. The gene encoding the PvEGIII was cloned into Escherichia coli. After the deletion of two introns, a plasmid construction was obtained allowing the PvEGIII expression in E. coli. Using site-directed mutagenesis, the Asp98Asn mutant of the PvEGIII was obtained. Both the wild type and mutant PvEGIIIs were expressed in E. coli with a yield of up to 1 g/L and then isolated in a highly purified form. The enzyme specific activity against soluble carboxymethylcellulose was not changed after a single amino acid substitution. However, the pH-optimum of activity of the mutant PvEGIII was shifted from pH 4.0 to 5.1, compared to the wild type enzyme. The shift in the enzyme pH-optimum to more neutral pH was also observed on insoluble cellulose, in the process of enzymatic depigmentation of denim fabric. Similar situation featuring the effect of the Asp/Asn residue, located near the Glu catalytic residue, on the enzyme activity pH-profile has previously been described for xylanases of the GH11 family. Thus, the glycoside hydrolases belonging to the GH11 and GH12 families function by a rather similar mechanism of catalysis.     

芒果过氧化物酶活性的研究

从新鲜芒果中提取酶液,进行过氧化物酶活性的测定,结果表明：芒果酶提取液中过氧化物酶最适ＰＨ值为４．７５,米氏常数Ｋｍ值为３．２３×１０＾－３ｍｍｏｌＨ２Ｏ２／ｍＬ最大反应速度Ｖｍａｘ＝０．２４４Ｏ．Ｄ／ｍｉｎ。在７５℃下加热处理２０ｍｉｎ或８０℃下处理５ｍｉｎ,可使过氧化物酶完全失活。     

Biocatalytic properties of recombinant tobacco peroxidase in chemiluminescent reaction

The wild-type anionic tobacco peroxidase and its Glu141Phe mutant have been expressed in Escherichia coli, and reactivated to yield active enzymes. A Glu141Phe substitution was made with the tobacco anionic peroxidase (TOP) to mimic neutral plant peroxidases, such as horseradish peroxidase (HRP). Both recombinant forms of tobacco peroxidase show extremely high activity in luminol oxidation with hydrogen peroxide, and thus, preserve the unique property of the native tobacco peroxidase, a superior chemiluminescent reagent. The chemiluminescent signal intensity for both recombinant forms of TOP is orders of magnitude higher than that for wild-type recombinant HRP. The substitution slightly increases TOP activity and stability in the reaction course, but has almost no effect on the optimal parameters of the reaction (pH, luminol and hydrogen peroxide concentrations) and calibration plot. Comparison of substrate specificity profiles for recombinant TOP and HRP demonstrates that Glu141 has no principal effect on the enzyme activity. It is not the presence of the negative charge at the haem edge, but the high redox potential of TOP Compounds I and II that provides high activity towards aromatic amines and aminophenols, and luminol in particular.     

Biocatalytic properties of recombinant tobacco peroxidase in chemiluminescent reaction

The wild-type anionic tobacco peroxidase and its Glu141Phe mutant have been expressed in Escherichia coli, and reactivated to yield active enzymes. A Glu141Phe substitution was made with the tobacco anionic peroxidase (TOP) to mimic neutral plant peroxidases, such as horseradish peroxidase (HRP). Both recombinant forms of tobacco peroxidase show extremely high activity in luminol oxidation with hydrogen peroxide, and thus, preserve the unique property of the native tobacco peroxidase, a superior chemiluminescent reagent. The chemiluminescent signal intensity for both recombinant forms of TOP is orders of magnitude higher than that for wild-type recombinant HRP. The substitution slightly increases TOP activity and stability in the reaction course, but has almost no effect on the optimal parameters of the reaction (pH, luminol and hydrogen peroxide concentrations) and calibration plot. Comparison of substrate specificity profiles for recombinant TOP and HRP demonstrates that Glu141 has no principal effect on the enzyme activity. It is not the presence of the negative charge at the haem edge, but the high redox potential of TOP Compounds I and II that provides high activity towards aromatic amines and aminophenols, and luminol in particular.