Optimal pH for the Alcohol Dehydrogenase Enzyme | 5 Important points

Optimal pH for the Alcohol Dehydrogenase Enzyme

The alcohol dehydrogenase enzyme works by transferring hydrate and proton. Every enzyme works at certain temperatures and pH levels, and the optimal pH for each type of enzyme is the point at which the enzyme operates at its best. For alcohol dehydrogenase enzyme, this pH range is in the range of 7 and 8.

Yarrowia lipolytica

The ideal pH for alcohol dehydrogenase production requires a certain balance of the sugar-acid ratio. It is often impossible to achieve this balance by simply adjusting the pH of the enzyme. Fortunately, a new process developed by Liu et al. has lower material costs and a shorter fermentation period. This method represents a significant breakthrough in biocatalysis, and it may be the key to commercializing alcohol dehydrogenase.

It is not yet known how the Yarrowia lipolytica enzyme regulates citrate production. One theory is that the enzyme regulates ROS accumulation by the NDH2e pathway. Studies have shown that the Y. lipolytic enzyme may protect against ROS accumulation in the stationary phase. In addition, it has been suggested that Yarrowia lipolytica can oxidize long-chain alkanes and release hydrogen peroxide.

The Yarrowia lipolytic enzyme is a non-pathogenic obligate aerobe. It has many industrial applications and has been used to produce single-cell proteins, citric acid, erythritol, omega-3 eicosapentaenoic acid, and lysosomal enzymes.

The Yarrowia lipolytica bacteria cultured in a mineral medium was grown in a nitrogen-limited environment, promoting citrate production. Glucose was used as the carbon source. Experiments have shown two distinct growth phases, one with an exponential growth rate and the other with a stationary growth phase. In the stationary phase, biomass concentration can reach up to 6 gDCW/L.

Yarrowia lipolytica CLIB 122 was obtained from CIRM, France. The Yarrowia lipolytica ADH2 enzyme was cloned, overexpressed in Escherichia coli, and characterized in a laboratory model. Unlike the Yarrowia lipolytica ADH2, the Zn-dependent alcohol dehydrogenase is highly active and can tolerate a broad range of acidic and lipophilic substrates.

Zn-dependent alcohol dehydrogenase

The other subunits coordinate the catalytic zinc of the Zn-dependent alcohol dehydrogenases to a range of residues. This variation reveals a pre-organized active site with a minimal distance. The hydride-delivering NAD(P)H is optimally positioned in this pocket. This variation enables the formation of (R)-alcohol. In the human ADH3 enzyme, the ligands of zinc coordinate to a range of residues, allowing the enzyme to achieve this transition.

To identify a zinc-dependent alcohol dehydrogenase, we identified the protein sequences using the sequence domains PF08240 or PF00107. Next, we used a thermophile annotation and the UniProtKB database for identification. Finally, we combined the corresponding UniProt and PDB sequences. This yielded 808 protein sequences. The sequence alignment was carried out using the MAFFT algorithm and Linsi parameters.

The zinc-dependent alcohol dehydrogenase (ADH) enzyme is part of a large family of biocatalysts. It catalyzes the reversible oxidation of a variety of alcohols. In making beer, the Zn-dependent alcohol dehydrogenase (ADH) can transform various alcohols into aldehydes, ketones, and carbon monoxide.

The pH optimal for alcohol dehydrogenase enzymes is 7.0, but this is not universal. Some variants may occur during the development process and alter the optimal pH value. Thus, further research is necessary to determine the pH optimum for alcohol dehydrogenase enzymes. So, it is important to study these enzymes using isoenzymes and purified forms of these enzymes.

The enzyme also plays a significant role in fermentation. Ethanol is produced when pyruvate (a byproduct of glycolysis) is reduced by alcohol dehydrogenase. The coenzyme nicotinamide adenine dinucleotide (NAD+) also participates in the reaction. The enzyme also has a signaling role and inhibiting it causes organisms to switch metabolic pathways and accumulate metabolic products. Understanding the mechanism behind alcohol dehydrogenase activity in bacteria and yeast will help scientists better understand the role of ethanol in fermentation and how a human alcohol dehydrogenase enzyme works.


The pH of alcohol dehydrogenase is highly sensitive to its substrate, and the number of sulfhydryl reagents it requires is essential to activate it. Several sulfhydryl reagents are used to study alcohol dehydrogenases, including dithiothreitol, mercaptoethanol, and tetrahydrofuran.

To determine the pH of alcohol dehydrogenase, enzyme activity was measured in various human tissues and biological fluids. Crude enzyme preparations were prepared from human livers from both fetal and adult animals. Fetal livers were obtained by legal abortion, and liver tissue from children and adults was acquired by abdominal surgery free of macroscopic abnormalities. Enzyme activity was stable at -20 degrees Celsius for up to 6 days.

The pH and temperature results from the present study agree with the literature data for both substrates and enzymes in solution. These results help maintain enzyme activity in the future. Furthermore, they are consistent with enzymes grown in culture under physiological conditions. These studies will help understand how the pH of alcohol dehydrogenase enzyme is affected by ambient temperature. However, the layered structure of alcohol dehydrogenase enzymes facilitates diffusional processes and contributes to the system’s overall performance.

The enzymes were immobilized on a carbon platform, where they could be studied in solution. These immobilized proteins were used for specific studies. The pH of alcohol dehydrogenase enzymes was measured at various temperature ranges between 35 and 40 degrees Celsius. Its optimum activity was also found to be 7.0 to 8.0. Its temperature optimum was found to be in the range of 35 to 40 degrees Celsius.


The temperature of alcohol dehydrogenase enzymes affects their activity. The expression of this gene in a strain of bacteria allowed it to produce 35% theoretical ethanol. Similarly, the expression of the same gene in hyperthermophilic bacteria resulted in methanol and ethanol production. However, the temperatures at which these enzymes function are different from those of hyperthermophilic bacteria.

Most enzymes in this class are hyperthermophilic, which means they thrive in hyperthermic conditions. A thermophilic enzyme is the most common type of alcohol dehydrogenase and has a variety of physiological functions. The temperature at which alcohol dehydrogenase enzymes function largely determines their efficiency in dehydrating organic acids. The temperature at which these enzymes are most active is essential to the process, as a higher temperature causes the reaction rate to be faster.

The temperature at which alcohol dehydrogenase enzymes function in hyperthermophilic bacteria is crucial to the activity of the enzymes. ADH gene manipulation in hyperthermophilic bacteria has increased their ALDH activity by three to fourfold and significantly increased ethanol production. The bacterial strain Caldicellulosiruptor bescii, for instance, was engineered to express the adhE enzyme from C. thremocellum. Its ethanol production amounted to 33% of the theoretical yield. Similarly, P. furiosus, a hyperthermophilic bacterium, was engineered to express Adha from Thermoanaebacter sp. X514 showed a threefold increase in inactivity.

In addition to its role in ethanol biosynthesis, alcohol dehydrogenase enzymes play an essential role in fermentation. Alcohol dehydrogenase breaks down pyruvate, an intermediate derived from glycolysis, into ethanol. The enzyme regenerates NAD+, which allows the enzyme to perform its other functions. Humans have exploited the ADH enzyme to produce alcoholic beverages.

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The stability of enzymes is one of the most significant obstacles to further progress in biotechnology. For this reason, any method that enhances stability is an attractive prospect. The alcohol dehydrogenase enzyme has been engineered with the help of the FRESCO computational approach. Twenty-five mutations increased the clear melting point of the enzyme. One mutation was so significant that it reached the boiling point of water. The mutation that impairs the enzyme’s activity was identified in its structure.

The optimal temperature for immobilization is 4 deg C, and the enzyme concentration is 0.02 mg/mL. Rotation speed was varied for the best immobilization yield. Moreover, the higher the rotation speed, the higher the contact between the enzyme and the support. ADH immobilized on CMD-MNPs was activated using 4% (v/v) EClH in a pH 7.5 sodium phosphate buffer. Rotation speed was manipulated between 100 and 400 pm. The immobilization yield was reduced to approximately half.

The activity of the enzyme was most significant at pH 9.6. The activity remained high when ethanol concentration was 500 mmol/L. The apparent Km value for ethanol was 662 mmol/L, while the Ki value for pyrazole was 1.7 mmol/L. The optimum pH for alcohol dehydrogenase enzyme was determined using blue native PAGE and gel filtration chromatography.

The electrophilic nature of the enzyme’s catalysis makes it challenging to exclude protonic acid and zinc electrocatalysis. However, if the pKa of the acid catalysts is smaller than 6.0 or 10, complex ternary forms, and the rate of aromatic alcohol dissociation changes dramatically. Thus, both enzymes may be responsible for benzyl alcohol oxidation.

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