Glucose Dehydrogenase

Glucose dehydrogenase catalyzes the oxidation of β-D-glucose to β-D-glucose-1,5-lactone while reducing the cofactor NADP + to NADPH, or to a lesser extent NAD + to NADH The enzyme is present in a variety of organisms, such as Bacillus megaterium, Bacillus subtilis, Gluconobacter suboxydans, Halobacterium mediterranean, Acidophilus and Thiobacillus. Glucose dehydrogenase accepts NADP⁺ and NAD⁺ as cofactors. Therefore, this enzyme is a good candidate for NADPH or NADH regeneration. This is of interest for the asymmetric reduction of prochiral ketone compounds to produce chiral hydroxyl groups or amino acids or alcohols, which are important raw materials and starting materials for the synthesis of important substances. Most oxidoreductases currently in use depend on nicotinamide coenzymes. For NADH, regeneration based on formate/formate dehydrogenase (FDH) has entered the highest stage of development.

Figure 1. Protein structure of glucose dehydrogenase.

Relationship between structure and function of glucose dehydrogenase

Although glucose dehydrogenase from different sources has similar functions, there are certain differences in its primary structure and spatial structure. Comparing the homology of glucose dehydrogenase derived from Bacillus megaterium and Bacillus subtilis, it was found that 23 structural genes had transitions, 30 transversions, 1 inversion, and 3 insertions or deletions, but no shifts were found. code. The homology of the two enzyme subunits is 85%, reflecting the similar evolutionary time of the two.

Source of glucose dehydrogenase

At present, researchers at home and abroad mainly obtain this enzyme from the fermentation of cattle heart and various microorganisms, such as Bacillus megaterium, Bacillus sub2tilis, Bacillus pum ilus, calcium acetate immobilized Bacillus (Acinetobacter calcoaceticus) halophilic archaebacteria (hala. (Thermoacidophilis archaebacterium), etc. Transketolase-deficient (thermoacidophilis archaebacterium), etc. Transketolase-deficient Bacillus subtilis-derived DH is easily purified due to its high enzyme production capacity It is widely accepted and applied by people.

Separation, purification and kinetic properties of glucose dehydrogenase

At present, foreign high-yield QH is generally obtained by cloning the gene, transformed into E. coli expression host bacteria, and obtained by fermentation of microorganisms. As an intracellular enzyme, to study DH, the cell-free extract must be prepared by breaking the cells to determine the enzyme activity. Considering the crushing efficiency and the particularity of the enzyme protein, the domestic freeze-thaw method or ultrasonic crushing method is often used. Some people abroad use advanced high-pressure cell disruptor's glucose dehydrogenase from different sources. Although their catalytic activities are very similar, their structures are different, and of course their properties will be different. Therefore, it is necessary to select the appropriate purification method for the enzyme. β2D2 glucose dehydrogenase in porcine liver is a multifunctional enzyme, which can be separated from the endoplasmic reticulum with TritorX2114, and NAD⁺ is used to purify the enzyme bound to NADP⁺ on the agarose gel column.

Applications

One of the application areas of GDH is the regeneration of NADH. There are some examples in the literature. GDH of the genus Bacillus when synthesizing and converting 2-keto-6-hydroxyhexanoic acid to L-6-hydroxynorleucine by reductive amination using bovine liver glutamate dehydrogenase. Regenerate NADH. The same purpose is used for GDH from Bacillus. The L-carnitine dehydrogenase of Pseudomonas putida IAM12014 produces L-carnitine from three dehydrocarnitines. Another application of GDH is the regeneration of NADPH. In order to asymmetrically reduce 4-chloro-3 oxobutyric acid ethyl ester, the aldehyde reductase of S. salmonella was coupled with GDH of Bacillus megaterium and co-expressed it as a NADPH regenerant. Gluconobacter sclerotiorum KY3613 from GDH is used for co-factor recovery of L-folate production. 6-phosphate glucose dehydrogenase can also regenerate NADPH. Commercially available glucose 6-phosphate dehydrogenase and regeneration of glucose 6-phosphate dehydrogenase from Leptobacterium enteritidis were used. We performed stereoselective production of glucose dehydrogenase of Bacillus subtilis and NADP + -dependent (R) -specific alcohol dehydrogenase (overexpressed in E. coli) overexpressed in E. coli. Since E. coli cells do not have sufficient cofactor regeneration systems, we have developed a co-expression system to express two genes in E. coli and perform whole-cell biotransformation.

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