Glucose isomerase (GI), also known as xylose isomerase, refers to the ability to convert D-xylose,D-glucose, D ribose and other aldoses are isomerized into corresponding ketose isomerase.GI comes from a wide range of microorganisms, such as bacteria, fungi and actinomycetes, as well as plants andAnimal cellGI exists in all of them. It is produced in industryHigh fructose syrupIt plays a key role in the industrial production of high fructose syrup and fuel ethanol.
Glucose isomerase (GI), also known as D-xylose isomerase (D-xy lose isomerase).In 1957, he was first addicted to waterPseudomonasIts activity was found inActinomycesIdentified as a strain producing GI[1]It comes from a wide range of microorganisms, such as bacteria, fungi and actinomycetes, as well as plants andAnimal cellBoth exist in.
GI is an intracellular enzyme, which is involved in the utilization of xylose entering the body. Its optimal natural substrate is D-xylose[2]。Later, it was found that it couldD-glucoseConvert toD-fructose。With this enzyme, more than 90% of the sugar in glucose syrup can be converted into fruit essence, which greatly improves the sweetness. Since then, GI has been widely used inHigh fructose syrup(HFS) industrial production.[3]
Structural characteristics
Announce
edit
Although the primary structure of GI from different species and genera has some differences, they are similar in spatial structure.GIases are all non glycoproteins and generally exist in the form of tetramers or dimers.The molecular weight of the subunit monomer is 19~52 kD.The tetramer subunits are bound by non covalent bonds without disulfide bonds. The binding force between the dimers is stronger than that between the subunits in the dimer. The subunit monomer conforms to 222 point group symmetric distribution, and each subunit monomer is divided into two domains[4]。The main domain at the N-end: 8 strandsα /β The spiral folded structure forms a catalytic "catalytic pocket", and the inner layer is composed of 8 parallelβ The outer layer consists of 8 strands andβ Alternating adjacent folding sheetsα Spiral composition,α The trend of the spiral peptide chain andβ The folded sheets are antiparallel, and the active center is located atβ Folded near port C.Small domain at C end: composed of several segmentsα The helix curls irregularly into an irregular ring structure far from the N-terminal, and this domain is involved in the interaction between subunits and the construction of active centers.
Tetramer grape isomerase has four active centers, which are pocket shaped, and the active centers are located in the subunit catalytic domainβ Near port C of the barrel.Each active center consists of two adjacent subunits, including two divalent metal ion binding sites, and conservative residues related to substrate binding and catalytic processes.
Biological properties
Announce
edit
GI is a key enzyme for large-scale industrial production of high fructose syrup from starch, and this enzyme can isomerize xylan into xylulose, and thenMicrobial fermentationProduce ethanol.
thermal stability
LactobacillusAnd Escherichia coli GI have poor thermal stability,StreptomycesandBacillus subtilisGI is quite stable at high temperatures.Thermophilic bacteria (Thermophilus lus) GI has the highest thermal stability, which may be due to its preference for Val, Pro and other amino acids, so that it has a closer spatial structure[1]。
substrate specificity
In addition to D-glucose and D-xylose, GI canD-ribose、L-arabinose、L-rhamnose、D-allose andDeoxyglucoseAnd glucose C-3, C-5 and C-6 modified derivatives as catalytic substrates.But GI can only catalyze D-glucose or D-xyloseα-Conversion of optically active isomersβ-The optically active isomer is the substrate.
Optimum pH and temperature
The optimum pH of GI is generally slightly alkaline, between 7.0 and 9.0.Under acidic conditions, the GI activity of most species is very low, and the optimum reaction temperature of GI is generally 70~80 ℃.
Influence of metal ions
The activity and stability of GI are significantly related to divalent metal ions, Mg2+、Co2+、Mn2+And so on can activate the enzyme, Ca2 +、Hg2 +、Cu2+And so on.Metal ions also affect the activity of GI on different substrates. For example, Bacillus coagulans GI has the highest activity on xylose when combined with Mn2+, and Co2+The activity of glucose is the highest when combined.
Action mechanism
Announce
edit
The catalytic process of GI is mainly divided into four steps: substrate binding, substrate ring opening, hydrogen migration reaction (isomerization) and closed loop of product molecules. Hydrogen migration reaction is considered as the rate limiting step of the whole reaction process.
Catalytic Mechanism of Alkene Glycol Intermediates
This catalytic mechanism first proposed that the substrate was linked to the enzyme by ring opening.H54 interacts with substrate C1 as an alkaline catalyst.Substrate OoneAnd OtwoThe nearby water molecule may be a catalytic acid, which polarizes the carbonyl group of the substrate to promote the formation of olefin glycol intermediates[1]。
Negative hydrogen ion transfer mechanism
Crystallography andEnzyme kineticsThe evidence shows that GI is a metal ion mediated negative hydrogen ion transfer mechanism.There are two views on the form of negative hydrogen ion transfer intermediates. One is the cationic form. In the isomerization process, the carbonyl group of Mg-2 polarization substrate C1 produces carbopositive ions. Mg-1 and K183 act as Lewis acid to stabilize carbopositive ions, while two metal ions stabilize the negative charge of O2.The other is in the form of anion. It is proposed that the water molecule, which forms hydrogen bond with the carboxyl group of O1, O2 and D257 of the substrate and coordinates with the catalytic ion Mn2+, transfers the proton to CO1 of D257 and forms OH ion itself. This OH ion grabs the proton of the substrate and makes it negatively charged. The proton transfer is made by the water molecule/Hydroxyl ionCompleted[1]。
HFCS preparation
Announce
edit
Industrial production of HFCSα－amylase、β－The price of glycosidase is relatively cheap, while the production cost of GI is high, which is the most critical step in the production of HFCS and directly affects the output and production cost of HFCS. Therefore, the production cost of GI is of great significance to the production of HFCS.At present, the enzyme production of commercial production bacteria is between 1000 and 35000UL-1Therefore, it is an important development trend for HFCS production to screen better GI, improve the catalytic performance and process conditions of GI to improve conversion and yield[5]。Such as high temperature resistant GI, acid resistant GI, GI with increased affinity for substrate, GI with altered dependence on metal ions, and GI insensitive to inhibitors.
Immobilization
Announce
edit
In order to increase the batch number of GI used in catalytic reaction and reduce the production cost of HFCS, the industry generally adopts the method of immobilizing free GI or GI producing microbial cells to produce HFCS.The research on GI immobilization abroad is earlier and the technology is more mature.In the 1970s, six kinds of GI were immobilized through cross-linking, adsorption, embedding and other immobilization technologies, and were commercially sold.For example, the IGI produced by Jeroenke isPolyethyleneimine/Glutaraldehyde crosslinks the cells of GI producing bacteria, and adds inorganic carrier bentonite and diatomite to mix. The immobilized GI has excellent stability, and the half-life in a packed bed reactor at 60 ℃ can reach more than one year[5]。
There are also many domestic studies on GI immobilization, but the stability and enzyme activity are still lower than those of foreign countries.Deng Hui et al[6]Using chitosan flocculation and glutaraldehyde cross-linking method, the recombinant GI expressing and producing brown thermophilic SchizosporumEscherichia coliImmobilize to make the immobilized enzyme activity reach 356U·
g-1，half life61 days, basically meeting the industrial production requirements of HFCS.
