KR102541634B1 - Methods of producing lactobionic acid by whole-cell bioconversion of microorganism - Google Patents

Abstract

The present invention relates to a method for producing lactobionic acid using whole cells of microorganisms, and more particularly, to Pseudomonas tetrorens capable of overexpressing enzymes involved in lactobionic acid production ( Pseudomonas taetrolens ) Recombinant whole cells are used as a catalyst to shorten the production time compared to the conventional fermentation-based lactobionic acid production method, stably repeat production, and simplify the separation and purification process. .

Description

Methods of producing lactobionic acid by whole-cell bioconversion of microorganisms

The present invention relates to a method for producing lactobionic acid using whole cells of microorganisms.

Lactobionic acid, a type of polyhydroxy bionic acid as a next-generation skin whitening material, has biodegradability and biocompatibility in addition to the dead skin removal, wrinkle improvement, and skin whitening effects of α-hydroxy acid. , anti-aging, anti-oxidation, moisturizing, chelating, etc., and has low skin irritation, attracting attention as a next-generation cosmetic material that can replace glycolic acid and lactic acid. Cosmetics containing lactobionic acid It has been commercialized and is currently being sold.

Lactobionic acid is a substance formed by a β-1,4 bond between galactose and gluconic acid, and has a molecular weight of 358.3 and is a water-soluble white crystalline compound.

Republic of Korea Patent Registration No. 10-2030776, a prior application of the present inventors, provides a method for increasing lactobionic acid productivity by accelerating the lactobionic acid oxidation reaction from lactose through a study on optimization of the cultivation method of a non-pathogenic Pseudomonas taetrolens strain. have been initiated

However, when using a complex medium for lactobionic acid fermentation culture, it can be advantageous for the growth of microorganisms or secretion of enzymes, but it contains a large number of impurities that interfere with the separation and purification of the target substance after the completion of the culture, which increases the cost of separation and purification. it lowers the economy.

Since the problem in the production of lactobionic acid lies in the limitation of fermentation process development, production time can be shortened and stable production is possible during actual mass production compared to the lactobionic acid production method using the existing fermentation of Pseudomonas taetrolens. It is required to develop a method for producing lactobionic acid that can simplify the separation and purification process.

Therefore, the whole-cell bioconversion process through the industrial use of microorganisms is more efficient and environmentally friendly than the traditional chemical synthesis process in mass production and production of refined chemicals (Lorenz et al. 2013; Wachtmeister and Rother. 2016). Research on lactobionic acid production technology is very important.

A number of documents are referenced throughout this specification and citations are indicated. The disclosure contents of the cited documents are incorporated herein by reference in their entirety to more clearly describe the content of the present invention and the level of the technical field to which the present invention belongs.

Republic of Korea Patent Registration No. 10-2030776

Therefore, the present invention has been made to solve the above problems, an object of the present invention is the wild-type strain Pseudomonas tetrorens ( Pseudomonas taetrolens ) Lactobionic acid from lactose using whole cells of a recombinant Pseudomonas tetrorens strain capable of overexpressing enzymes involved in lactobionic acid production in order to solve the economic limitations of mass production of lactobionic acid through culture The present invention was completed by confirming the possibility of production and proving that it is possible to improve the production efficiency and simplify the purification step by optimizing the reaction process.

Objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

One aspect of the present invention is to provide a method for producing lactobionic acid from lactose using whole cells of a recombinant strain capable of overexpressing an enzyme involved in the production of lactobionic acid.

The method for producing lactobionic acid from lactose using whole cells of the present invention includes the following steps:

(i) A recombinant microorganism transformed with a recombinant vector containing a nucleic acid sequence encoding gdh (Quinoprotein glucose dehydrogenase), an enzyme involved in the production of lactobionic acid, is placed in a stationary phase in a nutrient medium (NB) containing lactose. Inducing to a dormant cell state by culturing until; and

(ii) recovering whole-cells from step (i) and culturing the whole-cells in a reaction solution containing lactose to produce lactobionic acid.

In a previous study, the present inventors developed a recombinant strain capable of overexpressing an enzyme involved in lactobionic acid production in Korean Patent Application No. 10-2020-0067897 to accelerate the rate of lactobionic acid conversion reaction to obtain lactobionic acid It has provided a way to increase productivity.

The problem in the production of lactobionic acid is not only the difficulty of process development, but also the difficulty of securing excellent strains. Depending on the type of microorganisms that produce lactobionic acid, the action of the produced enzyme and the ability to oxidize the substrate are very different, so a new enzyme with excellent double lactobionic acid production ability is identified, and a recombinant strain is developed accordingly. Research on the production technology used is very important.

Conventionally, acetic acid bacteria are widely known as microorganisms that produce lactobionic acid, but Komagataeibacter m-GDH enzyme (membrane - bound quinoprotein glucose dehydrogenase) isolated from . When glucose is present in the medium, there is a problem in that the ability to produce lactobionic acid through lactose oxidation is significantly inhibited. Although Gluconobacter suboxydans is an acetic acid bacterium and has various aldose oxidizing abilities, it has been confirmed that the Quinoprotein glucose dehydrogenase enzyme derived therefrom cannot oxidize lactose. In the case of Acinetobacter calcoaceticus , another lactobionic acid-producing strain, it was confirmed that the GDH enzyme derived therefrom had only about 65% of the lactose-oxidizing activity compared to the glucose-oxidizing activity.

Under these circumstances, the inventors of the present invention, among several known lactobionic acid-producing bacteria, especially in the case of the gdh (Quinoprotein glucose dehydrogenase) enzyme isolated from bacteria of the genus Pseudomonas, unlike the Quinoprotein glucose dehydrogenase enzyme isolated from other bacteria, selectively among various substrates was found to be an enzyme capable of oxidizing lactose.

In one embodiment, in the case of the gdh enzyme derived from Pseudomonas tetrorens of the present invention, when the glucose oxidation activity is 100%, the lactose oxidation ability is more than 100%, preferably more than 110%, 120% greater than 130%, greater than 140%, greater than 150%, greater than 160%, greater than 170%, greater than 180%, or greater than 190%, more preferably greater than 200%.

As described above, since the gdh enzyme isolated from the microorganism of the genus Pseudomonas of the present invention has the ability to selectively oxidize lactose among various substrates, the lactose oxidizing ability is not suppressed even in the presence of glucose, and the productivity of lactobionic acid is remarkable through lactose oxidation. There are advantages to showing.

The microorganisms of the genus Pseudomonas are Pseudomonas fluorescens ( Pseudomonas fluorescens ), Pseudomonas morabiensis ( Pseudomonas moraviensis ), Pseudomonas Granadensis ( Pseudomonas granadensis ) , Pseudomonas Coreensis ( Pseudomonas Koreensis ), etc. taetrolens ).

In addition, in an embodiment of the present invention, one or more can be selected from the group consisting of cellobionic acid and maltobionic acid as the type of aldonic acid, and lactobionic acid Not limited.

The nucleic acid sequence encoding the gdh (Quinoprotein glucose dehydrogenase) enzyme derived from a microorganism of the genus Pseudomonas of the present invention may be implemented by synthesizing the gdh gene derived from Tetrolence.

The expression vector containing the nucleic acid sequence encoding the gdh enzyme may be a plasmid vector, preferably pDSK519.

The recombinant microorganism overexpressing the gdh gene into which the recombinant vector is introduced may be a gram-negative bacterium, such as terrabacteria, proteobacteria, helical bacteria, sphingobacteria, and floating bacteria, but is not limited thereto. Preferably, the microorganism is one in which the recombinant vector is introduced into a wild-type Pseudomonas genus microorganism, more preferably a wild-type Pseudomonas tetrorens . The recombinant microorganism may have increased gdh gene expression and lactose oxidation activity compared to wild-type Pseudomonas tetrorens.

In a preferred embodiment of the present invention, the culture solution can be synthesized by synthesizing minimum salts to help the growth of the strain, but considering the ease of purification, a lactose reaction solution containing only CaCO ₃ and inorganic salt NaCl for pH correction is used. characterized by

In the present invention, the use of whole cells has the advantage of eliminating post-treatment processes such as addition of a buffer solution and concentration by using whole cells except for the culture medium after cell culture as an enzyme. In some production methods using whole cells, cells are treated with a permeable agent such as toluene or alcohol to increase the intracellular permeability of the reactive substance, but most of them are organic solvents and there is a risk of remaining toxic substances . Accordingly, the present invention is characterized in that whole cells are used without the use of additional organic solvents.

In order to recover the whole cells, the present invention (a) cultures a recombinant microorganism capable of overexpressing an enzyme involved in lactobionic acid production by metabolizing lactose in a nutrient medium (NB) containing lactose until stationary phase to induce a resting cell state; (b) deriving reaction conditions and optimal cell recovery time optimized for lactobionic acid production using the recovered whole-cells; and (c) reacting the whole-cells in a lactose reaction solution to obtain lactobion generating an acid and repeatedly generating it.

In one embodiment of the present invention, step (a) may be performed by a batch culture method. The dormant cells may be suspended to a concentration of 5 to 25 g/L, and the whole cell recovery time is within 9 hours to 48 hours after the start of culture in step (a), and the recovered cells in step (c) The reaction temperature for producing lactobionic acid using whole cells is 25 to 40 ° C, and the inoculation OD value may be 0.5 to 25. The reaction solution may contain NaCl as an inorganic salt, CaCO ₃ and lactose for pH correction, and the incubation temperature in the step (c) may be maintained at 25 to 35 ° C.

내지 37

에서 12시간 내지 48시간 동안 배양시키는 것일 수 있다.In another embodiment of the present invention, in the step of culturing the Pseudomonas tetrorens recombinant strain, the nutrient broth (NB) is 0.2% yeast extract, 0.5% peptone, and 0.1% beef extract ), including 0.5% NaCl, and the culturing step is NB medium 50 mL to 5.0 L, 20

to 37

It may be incubated for 12 hours to 48 hours.

In another embodiment of the present invention, lactobionic acid is repeatedly applied using Pseudomonas taetrolens whole cells transformed with a recombinant vector containing a nucleic acid sequence encoding gdh (Quinoprotein glucose dehydrogenase) derived from Pseudomonas tetrolens. Provided is a method for producing whole cells, wherein the whole cells are at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times. It may be reused up to 15 times, 16 times, 17 times, 18 times, 19 times or 20 times.

A method for improving productivity through cultivation of the Pseudomonas tetrorens strain can be developed as disclosed in Korean Patent Registration No. 10-2030776, and a method for producing a recombinant Pseudomonas tetrorens strain is provided in Korean Patent Application No. 10-2020. Provided in -0067897. The entire contents of the above two patent documents are incorporated herein by reference.

The lactobionic acid production method of the present invention optimizes the production process of converting lactose into lactobionic acid by the whole-cell reaction of a recombinant strain, resulting in higher productivity, easier purification and separation process, and longer reuse than conventional methods using an existing fermentation system. By identifying the possibilities, industrial production efficiency and economic efficiency can be increased.

1 shows a schematic diagram of a recombinant plasmid for expression of gdh in Pseudomonas tetrorens using the pDSK519 vector.
Figure 2 shows a schematic diagram of a process for producing lactobionic acid by obtaining whole cells of a recombinant Pseudomonas tetrorens strain.
3 is a comparison result of lactose oxidation activity and lactobionic acid production after 12 hours of cultivation of wild-type Pseudomonas tetorrence and the recombinant strain.
Figure 4 shows the results of lactose consumption and lactobionic acid production according to temperature during the whole-cell reaction of the Pseudomonas tetrorens recombinant strain.
Figure 5 shows the results of lactose consumption and lactobionic acid production according to the initial OD during the whole-cell reaction of the Pseudomonas tetrorens recombinant strain.
Figure 6 shows the results of lactose consumption and lactobionic acid production according to the cell recovery time during the whole-cell reaction of the Pseudomonas tetrorens recombinant strain.
7 is a result of producing lactobionic acid by reusing whole cells of a Pseudomonas tetrorens recombinant strain.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

The reactants, the ratio of the reactants used, the range of reaction conditions, etc. are for illustrative purposes, and the present invention is not limited thereto, and the subject range is extended to reactions having similar reactants, reaction ranges, and genetic characteristics. Other embodiments applied in the present invention can be easily implemented by those skilled in the art and belong to the scope of the present invention.

Example

Example 1: Preparation of recombinant Pseudomonas tetrorens

Experimental Example 1: Preparation of recombinant plasmid

gdh3 (Quinoprotein glucose dehydrogenase: 1.1.5.2, GenBank accession number WP048384179), which is expected to be essential for lactobionic acid production, was extracted from Pseudomonas tetrorens, which has excellent lactobionic acid production ability through lactose oxidation reaction. Polymerase chain reaction (5'-CTGCAGAGGAATATGATATGAGTACGCAAGCGAAAGGCTCAG-3', SEQ ID NO: 1) and reverse (5'- GAATTCTCATTTCTCTTTAGGATCGGGCAGCGCG-3', SEQ ID NO: 2) primers from the genomic gene template were used for polymerase chain reaction (once at 94°C for 5 minutes; 30 reactions at 94°C for 30 seconds, 55°C for 2 minutes and 20 seconds, and 72°C for 30 seconds; once for 72°C for 7 minutes), the EcoR ° recognition site was present in the forward primer and the Pst ° recognition site was present in the reverse primer, resulting in gdh3 gene amplified. The amplified gene was separated through agar gel electrophoresis, the amplified separated DNA fragment was digested at EcoR ° and Pst °, and then introduced into the Pseudomonas plasmid vector pDSK519 cut at EcoR ° and Pst ° to obtain a recombinant plasmid shown in FIG. was produced.

Experimental Example 2: Preparation of recombinant Pseudomonas tetrorens

A recombinant Pseudomonas tetrorens strain transformed with the plasmid pDSK519-gdh prepared in Experimental Example 1 was prepared.

Specifically, in the transformation method of Pseudomonas tetrorens, a transformation method by electroporation was used to increase stable production and efficiency. Cells for electrical transfection were prepared through the following process.

Pseudomonas tetrorens (received P. taetrolens KCTC 12501 strain from the Center for Biological Resources, Korea Research Institute of Bioscience and Biotechnology) was prepared on an LB (Luria-Bertani) plate (1% tryptone, 0.5% yeast extract, 1% NaCl, 1,5% agar). Smeared and incubated for 24 hours in a 25 ℃ stationary incubator. The colony of the strain grown on the LB plate was inoculated into 200 mL of LB broth, and cultured at 150 rpm in a 25° C. stirred incubator until the OD (600 nm) reached 0.6 (measured by a spectrophotometer). The culture medium and strain cells were separated by centrifugation, and the culture medium was discarded, and then the strain cells were washed with 30 mL of 10% glycerol. 10% glycerol and strain cells were separated through centrifugation, and the separated 10% glycerol was discarded. The above process was repeated three times, and the washed strain cells obtained through centrifugation were released in 3 mL of 10% glycerol. When the pDSK519-gdh plasmid was introduced into microbial cells by electric shock, 80 ul of the cells for electric transformation and 5 ul of the plasmid were put in a cuvette for electric shock transformation, a voltage of 1.8 kV was applied for 5 ms, and SOC medium (2% tryptone , 0.5% yeast extract, 0.05% NaCl, 0.0186% KCl, 0.095% MgCl2, 0.6% glucose) was added and cultured for 1 hour at 100 rpm in a 25° C. stirring incubator to prepare a recombinant strain. The prepared recombinant strain was plated on an LB plate and incubated at 25°C. Through this, a recombinant strain ( Pseudomonas taetrolens /pDSK519-gdh) was developed by transforming Pseudomonas tetrolens with the recombinant plasmid pDSK519-gdh.

Experimental Example 3: Evaluation of substrate-specific oxidation ability

In the case of Komagataeibacter medellinensis , known as an acetic acid bacterium that produces lactobionic acid, the m-GDH enzyme (membrane-bound quinoprotein glucose dehydrogenase) isolated from it is lactose (lactose) in preparation for this when the glucose oxidation activity is 100%. ) It is known that the oxidation ability is only around 5%, so the lactose oxidation ability is insignificant, and when glucose is present in the medium, the lactobionic acid production ability through lactose oxidation is significantly inhibited (Kiryu et al., Biosci Biotechnol Biochem.2019 Jun;83(6):1171-1179).

Accordingly, the recombinant Pseudomonas tetrorens prepared above ( Pseudomonas taetrolens / pDSK519-gdh) was cultured using a shake flask culture with 20 g/L each of glucose and lactose to evaluate the lactose oxidation ability against glucose. Amounts of substrate and products were determined by HPLC analysis. Pseudomonas in shake flask culture controlled at 30°C for 48 hours taetrolens / As a result of evaluating the ability to produce acidic substances from 20 g/L glucose and 20 g/L lactose using pDSK519-gdh, it was confirmed that the enzyme does not oxidize glucose and has the ability to selectively oxidize lactose. .

Example 2: whole cell Recombinant Pseudomonas for reaction Tetrolence Cell growth and recovery

In this experiment, recombinant Pseudomonas tetrorens ( Pseudomonas taetrolens / pDSK519-gdh) strains were mixed with plate medium or 20 to 40% glycerol, stored and used after being frozen in a low-temperature refrigerator at -80 ° C, and colonies of strains grown on agar plate medium containing LB (Luria-Bertani) was inoculated in 10 mL of LB liquid medium, and the antibiotic kanamycin was added and cultured for 18 to 24 hours at 200 rpm in a 25 ° C. stirred incubator. Inoculated as % (v/v).

Finally, a reactor was used to recover resting-phase recombinant Pseudomonas tetrorens whole cells, and proceeded in the same manner as in FIG. 2 . Inoculation of the strain in the reactor was adjusted using a spectrophotometer to measure the initial OD ( _{600 nm} ) to a value of 0.15 to 0.2.

The medium used was a nutrient broth (NB) containing 0.2% yeast extract, 0.5% peptone, and 0.1% beef extract based on 1 L of distilled water, and 0.5% NaCl. It contains, and it was made to contain 0.2% to 20% of lactose.

The culture volume was 2 L to 3 L, and cultured for 12 to 48 hours in a 5 L fermentor (CNS, Korea) incubator. The dissolved oxygen concentration (Dissolved Oxygen, DO) was set to 30%, and fermentation was performed while adjusting the stirring speed to 200 ~ 500 rpm to maintain the dissolved oxygen concentration.

When the lactose in the culture medium was exhausted, the culture medium was placed in a supra 500 mL bottle, and cell separation was recovered from the culture medium in a reaction at 8,000 rpm, 4° C., and 30 minutes using a centrifuge. The recovered cells were immediately stored on ice and washed twice with a 0.8% NaCl solution at 4°C, and the whole cells were stored at 4°C for use.

As a result of comparing lactose oxidation activity and lactobionic acid productivity through 200 g/L lactose fermentation of the wild-type strain and the recombinant strain (FIG. 3), the lactose oxidation activity of the recombinant strain was 215% higher than that of the wild-type strain at 12 hours of culture, Lactobionic acid production also improved by 168%.

At this stage, enzyme activity was measured in 1.350 mL of 100 mM acetate buffer solution (pH 5.5) was measured by reacting 0.15 mL of enzyme solution at 30 ° C for 10 minutes, and protein concentration was measured by the Bradford method (Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle using albumin as a standard). of protein-dye binding. Anal. Biochem. 1976, 72, 248-254).

For the analysis of lactose and lactobionic acid, Agilent's high-performance liquid chromatography (HPLC 1260 model) equipped with a Refractive Index Detector (RID) was used. A Coregel ION 300 column was used as the column, and the column temperature was maintained at 70°C. The mobile phase was 0.5 mM H ₂ SO ₄ and the flow was 0.3 mL/min. Under the above conditions, the retention times of lactose and lactobionic acid were confirmed to be 19.2 minutes and 20.4 minutes, respectively.

Example 3: Optimal conditions for lactobionic acid production through whole-cell reaction of recombinant strains

In order to improve productivity in the lactobionic acid production process through the recombinant Pseudomonas tetrorens whole-cell reaction of the present invention, optimal reaction conditions were derived. In order to determine the optimal reaction temperature, reaction conditions were set at 5 ° C intervals in the range of 25 ° C to 40 ° C, and lactobionic acid productivity was confirmed by setting the initial cell inoculation OD value (OD _{600 nm} ) to be 0.5 to 25.

The whole cells recovered in Example 2 were washed with NaCl, and the cells were suspended in 50 mL of a reaction solution containing lactose (200 g/L), CaCO ₃ (30 g/L), and NaCl (8 g/L), and then placed in a 300 mL baffle ( baffled) flasks, shaken at 200 rpm.

In order to set the optimal reaction temperature conditions in the lactobionic acid production reaction using recombinant Pseudomonas tetrorens whole cells as a catalyst, the initial inoculation OD value was set to 20, and the reaction temperature conditions were 25 ℃ ~ 40 ℃. When reacted in, the fastest lactobionic acid production rate was shown when the temperature was 30 ° C, as shown in FIG. 4, which was completed within 12 hours, confirming the lactobionic acid productivity of about 16.7 g / L / h .

In addition, in order to set the optimal initial OD of the reaction in the lactobionic acid production reaction using recombinant Pseudomonas tetrorens whole cells as a catalyst, when the reaction was performed under the conditions of OD from 0.5 to 25 as shown in FIG. 5, inoculation OD value Under the condition set to 10, 16.7 g / L / h per hour was obtained, and it was confirmed that there was no difference in the production rate of lactobionic acid under the condition of OD value of 10 or more. This represents the reaction rate limit value according to the saturation of the whole cell-derived lactose oxidase.

Since the LBA conversion rate rapidly increases around 12 hours in the production of lactobionic acid through the cultivation of Pseudomonas tetrorens, experiments were performed to optimize LBA production according to the growth period of the cells in order to obtain whole cells with the highest activity. As shown in FIG. 6, when the cells were collected based on the logarithmic growth phase, stationary phase, and death phase, it was confirmed that the cells obtained in the stationary phase were the most active.

Example 4: the present invention of recombinant strains whole cell According to reuse in reaction lactobionic acid production potential

For industrial use of the method for producing lactobionic acid through whole-cell culture, the Pseudomonas tetrorens resting-phase cells were collected to confirm the stability of the whole-cell reaction. As shown in FIG. 7, as a result of the first reaction, 200 g/L of lactose was converted to 200 g/L of lactobionic acid for 12 hours. The cells were collected again, washed once with NaCl, centrifuged to recover the cells, and then reacted again by adding only lactose and CaCO ₃ . As the number of times repeated, the production rate decreased when reused more than 5 times, but the productivity was 12.5 g/L/h even when reused 7 times, confirming that whole-cell reuse of the recombinant Pseudomonas tetrorens strain is possible. The economic feasibility of culture can be confirmed.

Claims (11)

A method for producing lactobionic acid from lactose using whole cells comprising the following steps:
(i) A recombinant microorganism transformed with a recombinant vector containing a nucleic acid sequence encoding gdh (Quinoprotein glucose dehydrogenase), an enzyme involved in the production of lactobionic acid, is placed in a stationary phase in a nutrient medium (NB) containing lactose. Inducing to a dormant cell state by culturing until; and
(ii) recovering whole-cells from step (i) and culturing the whole-cells in a reaction solution containing lactose to produce lactobionic acid;
Here, the enzyme gdh involved in the production of lactobionic acid is derived from Pseudomonas taetrolens . The method according to claim 1, wherein the recombinant microorganism is Pseudomonas taetrolens . delete The method of claim 2, wherein the recombinant microorganism is gdh compared to wild-type Pseudomonas tetrorens A method characterized by increased gene expression and lactose oxidation activity. The method according to claim 1, wherein step (i) is performed by a batch culture method. The method of claim 1, wherein the dormant cells are suspended at a concentration of 5 to 25 g/L. The method of claim 1, wherein the whole cell recovery time is within 9 hours to 48 hours after the start of the culture in step (i), and the reaction temperature at which lactobionic acid is produced using the recovered whole cells in step (ii) Is 25 ~ 40 ℃ and characterized in that the inoculation OD value is 0.5 ~ 25. The method according to claim 1, wherein the reaction solution contains NaCl as an inorganic salt, CaCO ₃ for pH correction and lactose. The method of claim 1, wherein the culture temperature in step (ii) is maintained at 25 to 35 ° C. A method for repeatedly producing lactobionic acid using Pseudomonas taetrolens whole cells transformed with a recombinant vector containing a nucleic acid sequence encoding gdh (Quinoprotein glucose dehydrogenase) derived from Pseudomonas tetrorens . 11. The method of claim 10, wherein the whole cells are reusable at least 7 times.

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