β-Glucosidase

In 1837, Liebig and Wohler first discovered the β-glucosidase in bitter almond juice. β-glucosidases (EC 3.2.1.21) belong to hydrolases, also known as β-D-glucosidase. It catalyses the hydrolytic of non-reducing β-D-glycosidic bond binding to the terminal, releasing both the ligand group and the glucose.

Sources

β-glucosidase is widely found in nature, it can be derived from plants, microorganisms, as well as animals. Plant sources of β-glucosidases include ginseng and soybeans. Microbial sources have more reports, such as Flavobacterium meningosepticum and Flavobacterium johnsonae. Eukaryotes sources include Candida peltata, Phanerochaete chrysosporium, etc. Animal sources of β-glucosidase include bees, pig liver and pig intestine and so on.

Classification

β-glucosidases can be classified into three types according to their substrate specificity. The first type is an enzyme that hydrolyzes a hydrocarbyl-β-glucoside or an aryl-β-glucoside, of which the substrate is cellobiose, p-nitrobenzene-β-D-glucoside, etc. The second type can only hydrolyze hydrocarbyl-β-glucoside, this type of β-glucosidase can hydrolyze cellobiose, etc. The third type is an enzyme that hydrolyzes only the aryl-β-glucosidases, which hydrolyze p-nitrophenyl-β-D-glucoside and the analogues.

Properties

Due to the different origins, β-glucosidases may have different relative molecular weights, and their structures and compositions may vary widely. The relative molecular weight of β-glucosidase is generally between 40 and 250 kDa, and its structure may be composed of a single subunit, double subunit or multi-subunit. In addition, some strains contain intracellular and extracellular β-glucosidases, so sometimes β-glucosidase derived from the same strain is a mixture of two different molecular weight enzymes, or even a mixture of many different molecular weight enzymes. At present, many studies have shown that β-glucosidase is an acidic protease, and its optimum pH value is usually in the range of 3.0-7.0. Among them, the optimum pH value for many intracellular β-glucosidases of yeast and bacteria is close to 6.0. For most β-glucosidases, their isoelectric point (pI) values are in the acidic range and do not change much, generally between 3.5 and 5.5. The optimum reaction temperature of β-glucosidase is 40 to 110°C. Most of β-glucosidase have less specific on the substrate structure of glycosyl. They can cleave the C-O glycosidic bond, C-S bond, C-N bond, C-F bond, etc. β-glucosidase is not also specific to substrate glycosyl moiety C4 and C2 configuration, which can simultaneously hydrolyze β-glucosidic bond and β-galactosidic bond.

Structure

According to the amino acid sequence of glycosidase, most β-glucosidases belong to glycosidase family 1. Glycosidase family 1 has a clear barrel structure. However, some β-glucosidases belong to glycosidase family 4 and often require the involvement of dehydrogenase and cofactors in the catalytic process. It has been reported that 6-phospho-β -D-glucosidase requires the participation of Mn²⁺ and coenzyme NAD+ during the catalysis. Studies have shown that the catalytic role of β-glucosidase residue is two glutamic acid residues. The method of site-directed mutagenesis proved that glutamic acid near the N-terminal is a caustic group and the other is a nucleophilic group.

Catalytic Mechanism

In the catalytic reaction, two important amino acid residues are required as proton donors and nucleophiles. The basic process of hydrolysis reaction can be divided into three steps. The first step is the enzyme and substrate bonding to form Michaelis complex ES. The second step is the nucleophilic group of the enzyme attack the glycosidic bond O atoms of the substrate to form a covalent glycosylase intermediate ES with the acid-base catalyst help (to provide a proton). In this process, the active center of β-glucosidase may undergo a certain degree of structural change according to different types of substrates, so β-glucosidase can be combined with a variety of carbohydrate substrate. This step determines the β-glucosidase substrate specificity. The third step is the enzyme substrate hydrolysis. Acid or base group catalyzes water molecule to attack the intermediate ES, cleaving the glycosidic bond and releasing the β-glycosyl product and allowing the enzyme to restore its original protonation state. The above three steps can basically explain the catalytic mechanism of β-glucosidase.

However, the β-glucosidases from different families are slightly different in reaction mechanism. For example, the catalytic reaction of glycosidase 4 requires the participation of bivalent metal ions and NAD+. The dehydrogenase NAD (H) and the substrate bind through the covalent bonding with Mn²⁺ to form a "V" shape. Under the action of dehydrogenase, the substrate becomes a keto structure, and then the enzyme and the substrate form an intermediate transition state under the action of acid catalysis and base catalysis. Finally, the water molecule attacks the double bond of the glycosyl group in the glycosylase intermediate while the keto structure of the substrate disappears, releasing the β-glycosylation product and NAD+.

Applications

In the feed industry, β-glucosidase breaks down fiber-rich cell walls to release and use nutrients such as proteins and starch that it contains. At the same time, it degrades the fiber into reducing sugar that can be digested and absorbed by livestock and poultry, thereby improving feed utilization. In addition, in the food development, β-glucosidase as a special flavor enzyme has been applied. For example, the β-glucosidases from Aspergillus niger are used in fruit juices, tea juice, wine and other preparations, which can play a better fragrance effect.