KR20090013617A - Xylitol dehydrogenase-inactivated and arabinose reductase-inhibited mutant of candida tropicalis, method of producing high-yield of xylitol using the same and hydrolyzate, and xyliltol produced thereby - Google Patents

Abstract

A method for producing xylitol by using the mutated Candida tropicalis strain is provided to simplifying the production process by eliminating the additional process for maintaining the dissolved oxygen concentration in culture medium to be very low, and inhibit production of by-products such as ethanol and glycerol generated by cultivating the strain under low dissolved oxygen condition. The method for producing xylitol by using the mutated Candida tropicalis strain BS-xdh1(KCTC 11137BP) in which xylitol dehydrogenase is inactivated and arabinose reductase activity is suppressed comprises the steps of: preparing the xylitol production medium containing the biomass hydrolysate as a substrate; inoculating the Candida tropicalis strain BS-xdh1(KCTC 11137BP) into the production medium; and cultivating the strain with sufficient agitation without the separate process of controlling the dissolved oxygen concentration to the low concentration of 0.5 % or 2 %.

Description

Xylitol DEHYDROGENASE-INACTIVATED AND ARABINOSE REDUCTASE-INHIBITED MUTANT OF CANDIDA TROPICALIS, METHOD OF PRODUCING HIGH-YIELD OF XYLITOL USING THE SAME AND HYDROLYZATE, AND XYLILTOL PRODUCED THEREBY}

The present invention relates to a high yield method of xylitol using candida tropicalis mutant strain and biomass hydrolyzate in which xylitol dehydrogenase is inactivated and arabinose reductase activity is inhibited, and xylitol produced by the method is more specifically. Uses the Candida Tropicalis variant strain BS-xdh1 (KCTC 11137bp), which inactivates xylitol dehydrogenase, instead of expensive pure xylose under conditions that do not require the conventional essential process to maintain the dissolved oxygen concentration of the xylitol production medium extremely low. The production cost can be lowered by using biomass hydrolyzate as a substrate, and when the biomass hydrolyzate is used as a substrate by using the Candida tropicalis mutant ara-89 (KCTC 11136bp) which suppresses the activity of arabinose reductase. Production of arabitol, a byproduct produced The present invention relates to a xylitol production method and a xylitol produced by the method that can significantly reduce the productivity of xylitol.

 Xylitol is an pentose sugar alcohol that has high sweetness and is widely used as a functional sweetener to replace sugar. Xylitol exists naturally in various fruits and vegetables, but in this case only a very small amount, extraction from fruits or vegetables is not economically feasible. Therefore, xylitol is produced by a chemical method of reducing xylose-rich hemicellulose hydrolyzate. However, the chemical method is difficult and expensive to separate and purify xylose or other hydrolysates from the xylitol and semi-fibrous moieties, and the recovery rate of xylose is as low as 50-60%. In addition, there is a disadvantage that the risk and waste problems exist because the process of high temperature and high pressure using alkali.

In recent years, xylitol production methods by biological methods to overcome these disadvantages have been actively studied. Biological methods can use relatively low purity xylose as a raw material compared to chemical methods, and the process itself is a safe and environmentally friendly process because it is made at room temperature and pressure. In order to produce high productivity and high yield of xylitol, research on xylitol production through various bacteria, yeast and fungi, and recombinant yeast was carried out (Winkelhausen, E. et al., J.). Ferment. Bioeng. 86: 1-14, 1998; Granstr.m, TB et al., Appl. Microbiol. Biotechnol. 74: 277-281, 2007). However, bacteria and recombinant yeast strains were not suitable for the production of industrial xylitol due to their weak metabolic pathways or poor efficiency. However, Candida sp strains among yeast strains are known to be suitable for the production of biological xylitol because of higher productivity and yield of xylitol than other microorganisms.

According to the studies so far, Candida genus strains such as C. guillermondi , C. parapsilosis , C. tropicalis , and the like can be found in the extracellular cells as shown in FIG. The absorbed xylose is converted to xylitol by xylose reductase, which is converted to xylulose by xylitol dehydrogenase, and xylulose is again converted to xylulose kinase (xylulokinase) is converted to xylulose-5-phosphate, which is known to continue to be used for cell growth and maintenance through the pentose phosphate pathway (Laplace, JM). et al., Appl. Microbiol.Biotechnol., 36: 158-162, 1991; Hahn-Hagerdal, B. et al., Enzyme Microb.Technol., 16: 933-943, 1994). That is, xylitol producing strains are cell growth using xylose as a carbon source. Xylitol converted from xylose by xylose reductase is converted into xylulose by xylitol dehydrogenase, which is limited to the supply of oxygen in the medium so that the dissolved oxygen concentration remains low at 0.5% to 2.0%. Redox imbalance causes redox imbalance and the supply of NAD (Nicotinamide adenine dinucleotide), which is a cofactor required by xylitol dehydrogenase, is insufficient, and the process of converting xylitol to xylulose is suppressed. As a result, xylitol accumulates in cells and medium, and xylitol is produced from xylose in a yield of 60% to 80%. That is, in the conventional technique of producing xylitol using a xylitol producing strain, it is essential to control the oxygen concentration to a low dissolved oxygen concentration by a limited aeration. Thus, studies have been actively conducted to increase the productivity and yield of xylitol by intentionally inducing redox imbalance in cells by limiting the oxygen supply in the medium to keep the dissolved oxygen concentration low (see Kim, SY et. Al., J.). Ferment.Bioeng., 83 (3): 267-270, 1997; Korean Patent No. 1996-030577).

Korean Patent Publication No. 0169061 discloses a method for producing xylitol using concentrated Candida parapsilosis strains under dissolved oxygen concentration conditions of 0.8% to 1.2%. However, when producing xylitol using an industrial scale fermenter, it is practically impossible to control the dissolved oxygen concentration as above, and even if it can be adjusted, the production yield is only about 70-80%. In addition, Korean Patent Registration Publication No. 10-0259470 proposes a stirring speed for adjusting the oxygen transfer rate for optimizing xylitol production to a micro aerobic state of DOT (numerical value of oxygen concentration in medium) of less than 1%. As you can see, there is a cumbersome process. In addition, Korean Patent Application No. 95-37516 discloses an optimization of xylitol by controlling oxygen partial pressure during fermentation in a xylitol production method using a variety obtained from a natural species of Candida parapsilosis . No. 95-13638 discloses the optimum medium and culture conditions for the production of the mutant xylitol, but even under these optimum conditions the xylitol yield was not more than 80%.

Therefore, the inventors have focused on the fact that the xylitol produced in the strain of Candida genus is converted back to xylulose by xylitol dehydrogenase and reduced, thereby developing a Candida tropical strain that completely inactivates the activity of xylitol dehydrogenase ( Ko, BS et. Al., Appl. Environ.Microbiol. 72: 4207-4213, 2006). Inhibition of the action of xylitol dehydrogenase, which converts xylitol to xylulose, requires oxygen supply restriction in the medium to cause oxidative redox imbalance in the cells. Mutants that are inactivated by xylitol dehydrogenase are produced from xylose. Xylitol can no longer be used for cell growth, eliminating the need for redox imbalance. Theoretically, the company developed the Candida Tropicalis variant that can bioconvert xylose to xylitol in 100% yield. Thus, the present inventors filed a patent application with Korea on August 30, 2005 about the method for producing xylitol high yield using the xylitol producing strain in which xylitol dehydrogenase was inactivated (Korean Patent Application No. 10-2005-0079751). There is a bar.

As a result of this study, the intracellular metabolic rate of glycerol is affected by the oxygen supply rate, and the higher the oxygen supply rate until the dissolved oxygen is sufficient, the faster glycerol is metabolized (assimilated) and the NADPH supply rate is increased, thus increasing the xylitol production rate It was found to be improved. In addition, the yield of xylitol from xylose was constant from 97% to 98% regardless of the oxygen supply rate, which is almost 100% of the theoretical yield because xylitol dehydrogenase activity was inactivated regardless of the redox imbalance in cells. Up to yield.

Therefore, when xylitol is produced using the Candida tropical strain, it is not necessary to keep the dissolved oxygen concentration as low as 0.5 to 2.0%, and thus it is possible to produce xylitol with high productivity and near theoretical yield even in a large-scale fermenter on an industrial scale. . In addition, when culturing the production strain under low dissolved oxygen conditions, by-products such as ethanol and glycerol, which lowers the yield of xylitol and inhibits the purification and crystallization of xylitol, generate mutant strains from which the xylitol dehydrogenase gene is removed. As a result, by-products such as ethanol and glycerol were not produced, which enabled a more efficient process.

Among the various mixtures of sugars, including xylose, microorganisms that selectively make xylose into xylitol have been attempted to find wild or mutant types of yeast. The Candida tropicalis strain reported by Biotechnol. Lett. 3,130 (1981) was reported to obtain 96% yield of xylitol from xylose when grown in a yeast-malt extract-peptone (YME) medium containing xylose. In the expensive medium containing 3g / L yeast extract, 3g / L malt extract, 5g / L peptone, etc., industrial application is impossible and 250mL flask scale results, it is difficult to apply to industrial production.

Under conditions of sufficient dissolved oxygen concentration, the xylitol yield should be close to the theoretical yield of 100%. However, since xylose reductase of xylitol-producing strains has the same activity as arabinose reductase, arabinose contained in biomass hydrolyzate is inevitably used when biomass hydrolyzate containing a large amount of xylose is directly used as a substrate. Can be converted to arabitol. Arabbitol is a sugar alcohol of pentose sugars such as xylitol, and its molecular structure and physical properties are similar to xylitol, and thus are by-products that hinder the purification and crystallization process during xylitol production, thereby lowering the production yield. The use of biomass hydrolyzate, a low-cost xylose source, for the production of xylitol results in the production of some arabitol, and when the amount is above a certain concentration, it lowers the yield of xylitol crystals, thereby improving productivity and minimizing the production of byproducts. There is a need to develop the system.

The present invention solves the problems of the essential process of low dissolved oxygen concentration according to the prior art and expensive production cost by using expensive media, and the problem of by-products generated by using biomass hydrolyzate as an alternative substrate. In order to solve the problem, a first object of the present invention is to provide a xylitol high yield production method that can simplify the xylitol production process and reduce the production cost.

In addition, a second object of the present invention is to provide a high yield xylitol production method capable of minimizing the production of arabitol, a by-product produced in the xylitol production process using a biomass hydrolyzate.

In addition, a third object of the present invention is to provide a high yield xylitol production method that can solve the disadvantages of low productivity and low final xylitol concentration when using a biomass hydrolyzate directly as a substrate.

Xylitol high yield production method using the Xylitol dehydrogenase-inactivated Candida tropical strain mutant BS-xdh1 (KCTC 11137bp) according to the present invention for achieving the above object as a production strain, (a) biomass hydrolyzate as a substrate Making an added xylitol production medium; And (b) inoculating the mutant strain BS-xdh1 (KCTC 11137 bp) on the production medium, followed by culturing with sufficient agitation without a separate process of adjusting the dissolved oxygen concentration to a low concentration of 0.5% to 2%. Characterized in that it comprises a.

Dissolved oxygen concentration in the production medium is characterized in that more than 5%.

The xylitol high yield production method is characterized in that the fed-batch culture using a mixture of the biomass hydrolyzate and carbon source as a feeding solution (feeding solution).

The hydrolyzate of the biomass is characterized in that any one of corncob hydrolyzate, sugar cane hydrolyzate, coconut by-products, hydrolyzate of birch.

In addition, the carbon source is characterized in that glycerol.

Xylitol high-yield production method using a tropical mutant strain and a biomass hydrolyzate in which xylitol dehydrogenase according to the present invention for inactivating the above object to achieve the above object is suppressed, the activity of arabinose reductase is suppressed, inactivated xylitol dehydrogenase Candida is inhibited by arabinose reductase activity, which converts arabinose, which was selected by random mutagenesis, to arabitol by treatment with EMS (Methanesulfonic acid ethylester) from Candida tropical strain BS-xdh1 (KCTC 11137bp). It is a high yield method of xylitol using the Robpicalis mutant ara-89 (KCTC 11136bp) as a production strain.

The xylitol high yield production method includes the steps of: (a) making a xylitol production medium to which the biomass hydrolyzate is added as a substrate; And (b) inoculating the mutant strain BS-xdh1 (KCTC 11137 bp) on the production medium, followed by culturing with sufficient agitation without a separate process of adjusting the dissolved oxygen concentration to a low concentration of 0.5% to 2%. Characterized in that it comprises a.

Dissolved oxygen concentration in the production medium is characterized in that more than 5%.

The xylitol high yield production method is characterized in that the fed-batch culture using a mixture of the biomass hydrolyzate and carbon source as a feeding solution (feeding solution).

In addition, the biomass hydrolyzate is characterized in that any one of corncob hydrolyzate, sugar cane hydrolyzate, coconut by-product, hydrolyzate of birch.

The carbon source is characterized in that glycerol.

In addition, xylitol according to the present invention for achieving the above object is characterized by being produced by the method according to any one of claims 1 to 5.

According to the xylitol production method using the xylitol dehydrogenase activity-suppressed Candida tropical strain BS-xdh1 (KCTC 11137bp) and biomass as a substrate, the dissolved oxygen concentration in the production medium must be kept very low. Since the separate process is unnecessary, it is possible to simplify the production process and to suppress the production of by-products such as ethanol and glycerol generated when culturing the strain under low dissolved oxygen conditions.

In addition, the use of inexpensive biomass hydrolyzate as a substrate instead of expensive pure xylose has the effect of reducing the production cost.

In addition, by using the Candida tropical strain mutant ara-89 (KCTC 11136bp) inhibited the activity of arabinose reductase according to the present invention to suppress the production of arabitol produced from arabinose contained in the biomass hydrolysate to the production of xylitol There is an effect that can increase the productivity. In addition, through fed-batch culturing that separates the xylose substrate of the biomass hydrolyzate and the carbon source for cell growth, there is an effect of improving the productivity of xylitol and the final xylitol concentration.

Hereinafter, with reference to the accompanying drawings, a xylitol high yield production method using a xylitol dehydrogenase-inactivated tropical strain and biomass hydrolyzate of the present invention will be described in detail.

1 is a flow chart showing the process of xylose and arabinose metabolism by xylitol producing strains.

FIG. 2 is a genetic map of a transformed pXYL2-Ura3 vector for constructing a Candida tropical strain VBS-xdh1 (KCTC 11137bp) inactivated by xylitol dehydrogenase according to a preferred embodiment of the present invention.

The procedure for constructing the Candida Tropicalis variant strain BS-xdh1 (KCTC 11137bp) in which the expression of xylitol dehydrogenase is suppressed and producing xylitol using the same is as follows.

Introducing the transgenic pXYL2-Ura3 vector to Candida Tropicalis selectively removes the xylitol dehydrogenase gene and inactivates the activity of xylitol dehydrogenase through an intracellular mechanism called homologous recombination. Transgenic strains that cannot grow in a medium containing only can be constructed.

When the xylitol dehydrogenase-inactivated Candida Tropicalis mutant strain is a production strain, the production strain is used as a carbon source because the xylose in the production medium is no longer used by the cells once converted to xylitol. Lacking and thus no cell growth occurs. Instead, as a carbon source other than xylose, carbon sources such as glycerol and glucose must be added to the medium so that cells can grow using them. Therefore, xylose is used only as a substrate for xylitol production, and a carbon source is used to supply NADPH, a cofactor necessary for cell growth and xylose reductase.

In particular, unlike the conventional strain, the mutant strain inactivated by xylitol dehydrogenase does not need to cause redox imbalance for improving xylitol yield and productivity, so it is expected that the dissolved oxygen concentration should not be adjusted low during the culturing process. In addition, since yeast strains can consume glycerol as a carbon source in aerobic conditions, it is expected that cell growth rate and NADPH supply rate can be affected by oxygen supply rate when glycerol is used as an auxiliary substrate.

Thus, Xylitol dehydrogenase-inactivated Candida Tropicalis mutant BS-xdh1 (KCTC 11137 bp) was substituted with xylose, glycerol, yeast extract, potassium dihydrogen phosphate (KH ₂ PO ₄ ), magnesium sulfate (MgSO ₄ The experiment was performed to inoculate xylitol producing medium consisting of 7H ₂ O) and to produce xylitol under various oxygen supply conditions. The oxygen feed rate was controlled by the stirring speed of the fermentor. As a result of the experiment, the xylitol production rate was the highest when the stirring speed was over a certain level, that is, when the oxygen supply rate was high. As a result, the intracellular metabolic rate of glycerol was affected by the oxygen supply rate, and the higher the oxygen supply rate, the faster the metabolism of glycerol and the NADPH feed rate also increased, indicating that the xylitol productivity was improved. In addition, the yield of xylitol from xylose was constant from 97% to 98%, regardless of the rate of oxygen supply, which produced the theoretical yield of xylitol up to 100% because xylitol dehydrogenase activity was removed regardless of the redox imbalance in cells. Shows that

Biomasses such as corncobs, sugarcane stems, coconut by-products, and birch trees are rich in hemicellulose, consisting of xylose, arabinose, and glucose, and especially because of the high content of xylose. Can be used directly for production. Thus, xylitol was produced using a corncob hydrolyzate having a sugar content of 83.5% of xylose, 5.3% of glucose and 11.2% of arabinose as a substrate and glycerol as an auxiliary substrate. Xylitol-producing strains consumed all of the glucose contained in the hydrolyzate and grew the cells. At this time, the yield of xylitol was 97.2% and the productivity was 3.24g / L / h.

In addition, since high concentrations of xylose above 100 g / L inhibit cell growth and xylitol production, high concentration of xylitol was produced using fed-batch culture while maintaining the concentration of xylose in the medium to be 100 g / L or less. Xylitol was produced while continuously supplying a mixture of corncob hydrolysate as a xylose raw material and glycerol as a carbon source for cell growth and NADPH. As a result, the yield of xylitol was 97% and the productivity was 2.67 g / L / h. Glucose, one of the components of corncob hydrolysates, was completely consumed during the cell culture process, and 15.2 g / L of 57% of the total added arabinos 26.5 g / L remained arabinose and 43% of 11.3 g. / L was converted to arabitol.

Hereinafter, referring to the accompanying drawings, the xylitol dehydrogenase of the present invention is inactivated and the activity of abinos reductase is inhibited, and a detailed description of the xylitol high yield production method using the Candida tropicalis mutant ara-89 (KCTC 11136bp) is provided. Shall be.

Figure 3 is a process diagram showing a method for producing xylitol using a Candida Tropicalis mutant strain (KCTC 11136bp) in which xylitol dehydrogenase is inactivated and arabinose reductase activity is inhibited by a preferred embodiment of the present invention.

Reduction of arabinose reductase activity from the Candida tropicals variant BS-xdh1 (KCTC 11137bp) inactivated by xylitol dehydrogenase using EMS (methanesulfonic acid ehtylester), a mutagenesis-causing agent, as an alkylating agent Mutant ara-89 (KCTC 11136 bp) was selected. That is, mutant strains with reduced arabitol production capacity were developed by inhibiting cellular metabolic process in which arabinose is converted to arabitol by mutation. As a result of measuring the enzymatic activity of the mutant strain, the activity of xylose reductase converting xylose to xylitol was the same as that of the original strain, while the activity of arabinose reductase converting arabinose to arabitol was reduced by 63%.

In addition, as a result of producing xylitol through fed-batch culture using the mutant strain, the yield of xylitol was 97%, and the productivity was 2.80 g / L / h. Among the components of corncob hydrolyzate, glucose was completely consumed in the cell culture process, and 23.7 g / L of 85% of 27.9 g / L of total arabinos remained as arabinose and 4.2 g / 15 of 15%. L was converted to arabitol.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1

To clone the unknown Candida Tropicalis xylitol dehydrogenase, genes belonging to the already known group of medium-chain alcohol dehydrogenases, Pichia stipitis xylitol dehydrogenase and similar enzymes Gene homology between (sorbitol dehydrogenase, alcohol dehydrogenase, threonine dehydrogenase, etc.) was compared. As a result, PCR (94 ° C. 1 minute, 25 repetitions (94 ° C. 30 seconds, 58 ° C. 30 seconds, 72 ° C. 30 seconds) and 72 using the following primers as a template using a highly homologous region of the gene as a template 3 min), yielding 1,095 bp of xylitol dehydrogenase gene of Candida Tropicalis. After cloning the amplified Candida Tropicalis xylitol dehydrogenase gene into pGEM-T easy vector (BIONEX, Korea), a BamHI locus was introduced in the middle of the xylitol dehydrogenase gene, and the BamHI locus was introduced. By introducing the ura3 gene, as shown in FIG. 2, a 4,7 kb transformation vector pXYL2-Ura3 was obtained.

primer F: 5'-aatggtcttgggtcacgaatcc-3 '(SEQ ID NO: 1)

primer R: 5'-gctctgaccaagtcgtaggcttc-3 '(SEQ ID NO: 2)

The obtained vector pXYL2-Ura3 was introduced to Candida Tropicalis, and this was selected as a solid medium for selection (Yrasil) lacking (yield nitrogen base (amino acid free) 6.7 g / L, xylose 20 g / L, agar powder 15 g). / L) and incubated at 30 ° C. for 2 days.

Next, the colonies formed in the solid medium were xylose-containing solid medium (yeast nitrogen base (non-amino acid) 6.7g / L, xylose 20g / L, agar powder 15g / L) and glucose-containing solid medium (yeast nitrogen) Inoculated at 6.7g / L base, 20g / L glucose, and 15g / L agar, respectively, and incubated at 30 ° C for 2 days.The solid medium containing glucose was not grown in solid medium containing xylose. Candida tropicals from which xylitol dehydrogenase was removed were obtained by selecting strains that grew only in.

Candida Tropicalis variant strain BS-xdh1 (KCTC 11137bp) from which the final screened xylitol dehydrogenase was removed was deposited on June 12, 2007 to KCTC, an international microbial deposit institution (KCTC 11137bp).

Effects of Aeration to Oxygen Transfer Rate in the Medium on Xylitol Yield and Productivity When Xylitol is Produced Using the Xylitol Dehydrogenase-Inactivated Candida Tropicalis Variant BS-xdh1 (KCTC 11137 bp) Was investigated.

Xylitol dehydrogenase-inactivated Candida tropical strain VBS-xdh1 (KCTC 11137bp) was inoculated in 3 ml of YM medium (20 g / L glucose, 3 g / L yeast extract, 3 g / L malt extract, 5 g / L bacto-peptone) After incubation at 30 ° C. at 200 rpm for 12 hours in a shake incubator, 1 ml of the culture was inoculated again in 50 ml of YM medium and incubated at 30 rpm at 200 rpm for 12 hours in a shake culture.

Subsequently, 50 ml of the gastric culture solution was prepared with xylitol production medium (xylose 50 g / L, glycerol 20 g / L, yeast extract 10 g / L, potassium dihydrogen phosphate (KH ₂ PO ₄ ) 5 g / L and magnesium sulfate (MgSO ₄ · 7H). ₂ O) 0.2 g / L, pH 4.5) inoculated into a fermenter containing 1.0 L, incubated for 24 hours with agitation at 300, 400, and 500 rpm at 30 ° C., and then over time Changes in dry cell weight, xylose, glycerol and xylitol concentrations were measured. That is, the dry cell concentration was calculated by measuring the absorbance at 600 nm using a spectrophotometer (Shimadzu, Japan) and converting the sample into a standard curve analyzed beforehand. In addition, the sample was centrifuged and the supernatant was purified by HPLC (Shimazu, Japan) system (Sugar-Pak I column (Waters, USA), refracive index detector (Waters, USA), solvent: water, flow rate: 0.5 ml / min, column temperature: 90 ° C.) to determine the concentration of xylose and glycerol xylitol. The results are shown in Table 1 below.

Effect of Aeration on the Production of Xylitol Using Xylitol Dehydrogenase-Inactivated Candida Tropicalis Mutant BS-xdh1 (KCTC 11137bp) Stirring Speed 300 (rpm) Stirring Speed 400 (rpm) Stirring Speed 500 (rpm) Hours (h) Xylitol Concentration (g / L) Xylitol Concentration (g / L) Xylitol Concentration (g / L) 0 0.0 0.0 0.0 6 1.6 1.9 1.1 12 8.4 13.9 38.3 15 13.6 35.4 48.4 18 18.1 49.0 48.4 24 30.4 47.5 48.2

As shown in Table 1, 48.4 g / L xylitol was produced in 15 hours of incubation time at the agitation speed of 500 rpm with the highest oxygen feed rate, whereas 400 rpm and 300 rpm were 35.4 g / L and 13.6, respectively. At g / L, xylitol productivity was proportional to the stirring speed. Dissolved oxygen concentration during the culture is preferably maintained at 5% or more. Although not shown in Table 1, the output of xylitol no longer increased at a stirring speed of 500 rpm or more.

As a result, the intracellular metabolic rate of glycerol is affected by the oxygen supply rate, and until the dissolved oxygen is sufficient, the higher the oxygen supply rate, the faster glycerol is metabolized (assimilated) and the NADPH supply rate is increased, thereby improving the xylitol production rate. Could know. In addition, the yield of xylitol from xylose was constant from 97% to 98% regardless of the oxygen supply rate, which is almost 100% of the theoretical yield because xylitol dehydrogenase activity was inactivated regardless of the redox imbalance in cells. Up to yield.

Therefore, when xylitol is produced using the xylitol producing strain, it is not necessary to keep the dissolved oxygen concentration as low as 0.5 to 2.0%, and thus it is possible to produce xylitol with high productivity and near-theoretical yield even in an industrial scale large-scale fermenter. In addition, when yeast strains are cultured under low dissolved oxygen conditions, by-products such as ethanol and glycerol, which reduce xylitol production yield and inhibit xylitol purification and crystallization, are produced. Mutant strains from which xylitol dehydrogenase gene is removed have high dissolved oxygen concentration. Because it can maintain the by-products such as ethanol, glycerol was not produced it was able to build a more efficient process.

<Example 2>

Xylitol was produced using corncob hydrolyzate as substrate.

Under the same conditions as in Example 1, xylitol dehydrogenase-inactivated Candida tropical strain mutant BS-xdh1 (KCTC 11137 bp) was cultured, and 50 ml of the culture solution was subjected to corncob hydrolyzate (xylose 83.5%, glucose 5.3%). , Xylitol production medium using arabinose 11.2% (xylose 100g / L, glucose 6.6g / L, arabinose 13.4g / L, glycerol 30g / L, yeast extract 10g / L, potassium dihydrogen phosphate (KH ₂ PO) ₄ ) Inoculate a fermenter containing 5 g / L and 1.0 g of magnesium sulfate (MgSO ₄ · 7H ₂ O), 1.0 L, and incubate for 30 hours with stirring at 500 ° C. at 30 ° C. Changes in dry cell weight, xylose, glucose, arabinose, glycerol, and xylitol concentrations over time were measured. The measurement of dry cell weight, xylose, glucose, arabinose, glycerol, and xylitol concentrations was determined in the same manner as described in Example 1. The results are shown in Table 2 below.

Xylitol was produced in high yield and high productivity through batch culture using a hydrolyzate of corncob Hours (h) Dry cell concentration (g / L) Xylose Concentration (g / L) Glycerol Concentration (g / L) Arabinose concentration (g / L) Arabitol concentration (g / L) Xylitol Concentration (g / L) 0 0.2 100.0 30.0 13.4 0.0 0.0 6 1.5 100.0 29.8 13.4 0.0 0.0 12 12.2 96.7 28.3 12.2 1.0 3.2 18 16.3 62.2 18.7 10.8 2.1 35.7 24 15.0 20.9 9.0 9.6 3.1 78.5 30 15.2 0.0 2.3 9.2 3.8 97.2

In the results of Table 2, the xylitol producing strain consumed all the glucose for cell growth and used glycerol as an auxiliary substrate. Subsequently, 97.2 g / L xylitol was produced for 30 hours using xylose contained in a large amount of corncob hydrolyzate as a substrate, and a part of arabinose was converted into abaritol. The yield of xylitol was 97.2%, and the productivity was 3.24 g / L / h.

Yield of using xylitol as a substrate when producing pure xylose as a substrate, i.e., yield of 97.2% at a stirring speed of 500 rpm and productivity of 3.24 / L / h in Example 1 as a substrate. And because the productivity is similar, using inexpensive viromass hydrolyzate as a substrate for xylitol production without using purely purified xylose will not only increase the economics of the production technology, but also reduce waste biomass. It is environmentally friendly as it is recycled.

<Example 3>

Since high concentration of xylose above 100 g / L inhibits cell growth and xylitol production, high concentration of xylitol was produced using fed-batch culture while maintaining the concentration of xylose in the medium to be 100 g / L or less.

Xylitol dehydrogenase-inactivated Candida tropical strains BS-xdh1 (KCTC 11137bp) were incubated under the same conditions as in Example 1, and 50 ml of the culture solution was used for xylitol production medium using xylitol hydrolyzate (xylose 50 g / L, glucose 3.3 g / L, arabinose 6.7 g / L, glycerol 20 g / L, yeast extract 10 g / L, potassium dihydrogen phosphate (KH ₂ PO ₄ ) 5 g / L and magnesium sulfate (MgSO ₄ · 7H ₂ O) 0.2 g / L, pH 4.5) was inoculated into a fermenter containing 1.0 L and incubated with stirring at 30 ° C. at 500 rpm. From 12 hours of cultivation, a feeding solution using hydrolyzate (xylose 460g / L, glucose 30.4g / L, arabinose 61.6g / L, glycerol 138g / L) was added and the concentration of xylose in the medium was 100g /. The fed-batch culture was performed for 72 hours while adjusting to below L. Changes in dry cell weight, xylose, glucose, arabinose, glycerol, and xylitol concentrations over time were measured. The measurement of dry cell weight, xylose, glucose, arabinose, glycerol, and xylitol concentrations was determined in the same manner as described in Example 2. The results are shown in Table 3.

Production of xylitol with high yield and high productivity through fed-batch cultivation using corncob hydrolyzate Hours (h) Dry cell concentration (g / L) Xylose Concentration (g / L) Glycerol Concentration (g / L) Arabinose concentration (g / L) Arabitol concentration (g / L) Xylitol Concentration (g / L) 0 0.6 50.0 20.0 6.7 0.0 0.0 12 17.5 24.5 7.5 5.3 1.0 24.5 24 26.1 29.8 13.1 11.8 5.3 92.5 36 30.8 29.0 9.9 14.4 7.5 134.2 48 28.4 13.2 4.0 11.8 9.1 160.3 60 30.4 17.5 1.7 13.6 9.9 175.0 72 30.8 21.9 6.7 15.2 11.3 192.3

According to the results of Table 3, when xylitol was produced through fed-batch culture using corncob hydrolyzate, 192.3 g / L of xylitol was produced for 72 hours, wherein the yield of xylitol was 97% and the productivity was 2.67. g / L / h. Glucose, a component of corncob hydrolyzate, was completely consumed during cell culture, and 15.2 g / L of 57% of 26.5 g / L of arabinos added remained as arabinose and 11.3 g / 43 of 43%. L was converted to arabitol. At this time, the concentration of arabitol was 11.3 g / L, and the ratio of the arabitol concentration to the final xylitol concentration was 100: 5.9.

<Example 4>

According to the results of Example 3, arabinose from the Candida tropicalis mutant BS-xdh1 (KCTC 11137 bp) was inactivated by xylitol dehydrogenase using EMS (Methanesulfonic acid ethylester) as an alkylating agent. A new xylitol producing strain with suppressed cellular metabolism was developed.

Xylitol dehydrogenase-inactivated Candida tropical strain VBS-xdh1 (KCTC 11137bp) was inoculated in 3 ml of YM medium (20 g / L glucose, 3 g / L yeast extract, 3 g / L malt extract, 5 g / L bacto-peptone) After culturing for 24 hours with shaking culture at 30 ° C. at 200 rpm, 30 ml of the culture solution was transferred to a 50 ml conical tube, followed by 30 ml Minimal A buffer (K ₂ HPO ₄ 10.5 g / L, potassium dihydrogen phosphate (KH 2 PO 4). ) Wash cells twice with 4.5 g / L, ammonium sulfate ((NH ₄ ) ₂ SO _{4 )} 1.0 g / L, sodium citrate 0.5 g / L), and again wash cells with 15 ml Minimal A buffer. After resuspending, 450 μl of EMS (Methanesulfonic acid ethylester) was added.

Next, the reaction was carried out for 90 minutes with shaking culture at 30 ° C. at 200 rpm, and the cells were washed twice with 5 ml Minimal A buffer, and then resuspended with 2 ml Minimal A buffer, followed by YM agar plate. Smeared. Then, single colonies (single colonies) after incubation at 30 ° C. for 2 days were inoculated in 5 ml of arabitol production medium (arabinose 20g / L, glycerol 20g / L, yeast extract 10g / L) and incubated for 24 hours. It was.

The HPLC analysis method described in Example 1 was used to quantify the concentration of arabitol produced by the mutant strains to select mutant strains with arabitol production of 50% or less than the original strain. These mutants were inoculated in 5 ml of xylitol production medium (xylose 20 g / L, glycerol 20 g / L, yeast extract 10 g / L) and incubated for 24 hours, resulting in the final mutants having 90% or more of the resulting xylitol concentration. Screened. The results are shown in Table 4 below.

Selection of Mutant strains with Inhibited Production of Arabbitol Mutant Dry cell concentration (g / L) Production of arabitol (g / L) Xylitol Production Concentration (g / L) Won Kyun 5.5 3.9 19.7 ara-06 4.9 1.9 16.6 ara-26 5.1 1.8 17.9 ara-51 3.9 1.3 15.4 ara-77 5.1 1.7 18.1 ara-89 5.3 1.7 19.6

As shown in Table 4, the mutant strain 'ara-89' had almost similar cell growth and xylitol production concentrations compared to the control strain, but the production concentration of arabitol was reduced by 65% compared to the original strain. In order to measure the enzyme activity of the mutant ara-89, 50 ml of arabitol-producing medium (arabinose 20g / L, glycerol 20g / L, yeast extract 10g / L) was inoculated and incubated for 12 hours, and then 50mM potassium phosphate ( After the cells were resuspended in potassium phosphate buffer, sonication was repeated 5 times for 20 seconds, and the cells were crushed and centrifuged, and the supernant was transferred to a 1.5 ml tube. The activity of xylose reductase and arabinose reductase was measured using enzyme stock, coenzyme 0.2 mM NADPH, and xylose and arabinose as substrates. The enzyme activity was analyzed by measuring the rate of change of absorbance at 340 nm using a spectrophotometer (Shimadzu, Japan). The results are shown in Table 5 below.

Analysis of Xylose and Arabinos Reductase Activity of Mutant Strains with Inhibition of Arabbitol Production Mutant Xylose Reductase Activity (mU) Arabinose reductase activity (mU) Won Kyun 324.9 354.7 ara-89 317.5 131.2

According to the results of Table 5, mutant ara-89 has the same activity of xylose reductase that converts xylose to xylitol as compared to the original strain, whereas arabinose reduction that converts arabinose to arabitol is the same as that of the original strain. The activity of the enzyme was reduced by 63%.

Candida C.tropicalis mutant ara-89, which inhibited the activity of the finally selected arabinose reductase, was deposited on June 12, 2007 to KCTC, an international microbial deposit institution (KCTC 11136bp).

Example 5

Ara-89 (KCTC 11136 bp) with suppressed arabitol production was inoculated in 3 ml of YM medium (20 g / L glucose, 3 g / L yeast extract, 3 g / L malt extract, 5 g / L bacto-peptone), 30 After incubation for 12 hours in a shaker at 200rpm at ℃, 1ml culture medium was inoculated again in 50ml of YM medium, and incubated for 12 hours in shaker at 30rpm at 200 ℃.

Subsequently, 50 ml of gastric culture solution was prepared using a xylitol production medium using corncob hydrolysate (xylose 50g / L, glucose 3.3g / L, arabinose 6.7g / L, glycerol 20g / L, yeast extract 10g / L, phosphoric acid). 5 g / L potassium dihydrogen (KH ₂ PO ₄ ) and 1.0 L of magnesium sulfate (MgSO ₄ · 7H ₂ O), pH 4.5) were inoculated into a fermenter and incubated with stirring at 30 ° C. at 500 rpm. From 12 hours of cultivation, a feeding solution using hydrolyzate (xylose 460g / L, glucose 30.4g / L, arabinose 61.6g / L, glycerol 138g / L) was added and the concentration of xylose in the medium was 100g /. The fed-batch culture was performed for 72 hours while adjusting to below L. Changes in dry cell weight, xylose, glucose, arabinose, glycerol, and xylitol concentrations over time were measured. The measurement of dry cell weight, xylose, glucose, arabinose, glycerol, and xylitol concentrations was determined in the same manner as described in Example 1. The results are shown in Table 6 below.

Xylitol was produced by fed-batch culture using ara-89 (KCTC 11136bp), a Candida Tropicalis mutant strain that suppressed arabitol production Hours (h) Dry cell concentration (g / L) Xylose Concentration (g / L) Glycerol Concentration (g / L) Arabinose concentration (g / L) Arabitol concentration (g / L) Xylitol Concentration (g / L) 0 0.7 50.0 20.0 6.7 0.0 0.0 12 16.3 26.1 9.2 5.5 0.2 22.7 24 25.1 28.1 12.5 12.8 1.3 90.9 36 28.7 26.0 10.4 16.4 1.9 137.6 48 28.9 12.2 8.5 18.4 2.4 169.5 60 29.1 13.5 7.6 20.2 3.0 175.0 72 29.0 5.8 3.3 23.7 4.2 201.3

  According to the results of Table 6, the production of xylitol through fed-batch culture using the new mutant strain produced 201.3 g / L of xylitol for 72 hours, wherein the yield of xylitol was 97% and the productivity was 2.80 g. / L / h. Glucose, a component of corncob hydrolyzate, was completely consumed in the cell culture process, and 23.7 g / L of 85% of 27.9 g / L of arabinos added remained as arabinose and 4.2 g / 15 of 15%. L was converted to arabitol. The ratio of the final arabitol concentration of 4.2 g / L to the final xylitol concentration of 201.3 g / L was 100: 2.1.

In addition, xylitol productivity was improved by 5% compared to the original strain, which can be explained by the fact that NADPH required for producing arabitol was used to generate xylitol additionally as much as the portion where arabitol was suppressed. By using the newly developed xylitol producing strain with suppressed arabinose reductase activity, it is possible to complete a high-yield, high-productivity and efficient xylitol production process that significantly suppresses the production of arabitol, a byproduct that interferes with xylitol purification and crystallization. there was.

<Example 6>

Ara-89 (KCTC 11136 bp) with suppressed arabitol production was inoculated in 3 ml of YM medium (20 g / L glucose, 3 g / L yeast extract, 3 g / L malt extract, 5 g / L bacto-peptone), 30 After incubation for 12 hours in a shaker at 200rpm at ℃, 1ml culture medium was inoculated again in 50ml of YM medium, and incubated for 12 hours in shaker at 30rpm at 200 ℃.

Subsequently, 50 ml of the above culture broth, xylitol production medium using a hydrolyzate of bagasse of sugarcane (xylose 50g / L, glucose 5.2g / L, arabinose 3.8g / L, glycerol 20g / L, yeast 10 g / L of extract, 5 g / L potassium dihydrogen phosphate (KH ₂ PO ₄ ) and 0.2 g / L magnesium sulfate (MgSO ₄ · 7H ₂ O) 0.2 g / L, pH 4.5) were inoculated into a fermenter containing 1.0 l, and 500 rpm at 30 ° C. Incubated with stirring. Dissolved oxygen concentration during the culture is preferably maintained at 5% or more. From 9 hours of incubation, the feeding solution using hydrolyzate (xylose 460g / L, glucose 47.8g / L, arabinose 35.4g / L, glycerol 121g / L) was added and the concentration of xylose in the medium was 100g /. Oil culture was carried out for 72 hours while adjusting to below L. Changes in dry cell weight, xylose, glucose, arabinose, glycerol, and xylitol concentrations over time were measured. The measurement of dry cell weight, xylose, glucose, arabinose, glycerol, and xylitol concentrations was determined in the same manner as described in Example 1. The results are shown in Table 7 below.

Production of xylitol in fed-batch culture using candida tropicalis mutant ara-89 (KCTC 11136bp) and sugar cane vargas with suppressed arabitol production Hours (h) Dry bacteria concentration (g / L) Xylose Concentration (g / L) Glycerol Concentration (g / L) Arabinose concentration (g / L) Arabitol concentration (g / L) Xylitol Concentration (g / L) 0 0.9 50.0 20.0 3.8 0.0 0.0 12 21.0 52.3 18.8 4.1 0.0 15.4 24 28.9 57.3 16.7 6.7 0.7 84.5 36 31.4 38.0 13.1 8.8 1.2 128.9 48 32.1 17.6 10.5 11.1 1.7 168.8 60 32.0 9.5 7.2 12.8 1.9 181.4 72 31.8 3.4 5.5 13.9 2.1 203.2

According to the results of Table 7, the production of xylitol through fed-batch culture using the new mutant strain produced 203.2 g / L xylitol for 72 hours, at which the yield of xylitol was 97% and the productivity was 2.82 g. / L / h. Glucose, a component of sugarcane vargas hydrolysates, was completely consumed in the cell culture process, and 13.9 g / L of 87% of the total added arabinose 16.0 g / L remained arabinose and 11% of 2.1. g / L was converted to arabitol. The ratio of the final arabitol concentration 2.1 g / L to the final xylitol concentration 203.2 g / L was 100: 1.0.

In addition, the sugar cane vargas hydrolyzate has a 43% lower arabinos content than the corncob hydrolyzate, resulting in a 50% reduction in arabitol production. There is also an advantage.

Thus, by using the Candida tropical mutant strain inactivated xylitol dehydrogenase and suppressed arabitol reductase activity in the xylitol production by inhibiting the conversion of xylulose in xylitol to suppress the production of by-product arabitol By using the biomass hydrolyzate as a substrate and by the fed-batch culture method, an efficient xylitol production process with high yield and high productivity can be completed. In addition, it is not necessary to control the low dissolved oxygen concentration through the oxygen supply restriction, which was an essential condition in the conventional xylitol production method using microorganisms, so it can be applied as a more simple, economical and safe environment-friendly xylitol production process.

Korean Patent Publication No. 10-0643793 discloses a production method with optimum conditions for producing xylitol using a novel coherent yeast Candida Tropicalis HY200 as a production strain and using a repeated batch culture method. By fundamentally disrupting or inhibiting the genes of enzymes involved in the production of xylitol, high yields of xylitol can be produced without any additional process or optimization.

The present invention is not limited to the above-described embodiments, but can be variously modified and changed by those skilled in the art, which should be considered as being included in the spirit and scope of the present invention as defined in the appended claims. will be.

1 is a metabolic flow chart of xylose and arabinose by xylitol producing strains

Figure 2 is a genetic map of the transformed pXYL2-Ura3 vector for constructing the Candida tropical mutants BS-xdh1 (KCTC 11137bp) inactivated xylitol dehydrogenase according to a preferred embodiment of the present invention

Figure 3 is a process diagram showing a method for producing xylitol using a Candida tropical strain (KCTC 11136bp) according to a preferred embodiment of the present invention

<110> Korean Advanced Institute of Science and Technology          LPBio Co.Ltd <120> Xylitol dehydrogenase-inactivated and arabinose          reductase-inhibited mutant of Candida tropicalis, method of          producing high-yield of xylitol using the same and hydrolyzate,          and xylitol produced therby. <130> DPP-2007-0141-KR <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 aatggtcttg ggtcacgaat cc 22 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gctctgacca agtcgtaggc ttc 23  

Claims (13)

In the xylitol production method using xylitol dehydrogenase-inactivated Candida Tropicalis mutant BS-xdh1 (KCTC 11137bp) as a production strain, (a) making a xylitol production medium with the biomass hydrolyzate added as a substrate; and (b) inoculating the mutant strain BS-xdh1 (KCTC 11137bp) on the production medium, followed by culturing with sufficient agitation without a separate process of adjusting the dissolved oxygen concentration to a low concentration of 0.5% to 2%; Xylitol high yield production method comprising a The method of claim 1, wherein the oxygen concentration in the production medium is: Xylitol high yield production method, characterized in that more than 5% The method of claim 1 wherein the xylitol high yield production method is: Xylitol high yield production method characterized in that the fed-batch culture using a mixture of the biomass hydrolyzate and carbon source as a feed solution (feeding solution) The hydrolyzate of claim 1, wherein the hydrolyzate of the biomass is: Xylitol high yield production method characterized in that any one of the hydrolysates of corncob, sugar cane hydrolyzate, coconut by-product, hydrolyzate of birch The method of claim 3, wherein the carbon source is: Xylitol high yield production method characterized in that the glycerol Reduction of arabinose that converts arabinose to arabitol by mutagenesis by treatment with EMS (Methanesulfonic acid ethylester) from Candida Tropicalis mutant BS-xdh1 (KCTC 11137bp) inactivated xylitol dehydrogenase Candida tropicicalis mutant ara-89 (KCTC 11136bp) Xylitol high yield production method using the Candida tropicalis mutant ara-89 (KCTC 11136bp) of claim 6 as a production strain The method of claim 7 wherein the xylitol high yield production method is: (a) making a xylitol production medium with the biomass hydrolyzate added as a substrate; and (b) inoculating the production medium with the mutant ara-89 (KCTC 11136 bp), and then cultured by sufficiently agitation without a separate process of adjusting the dissolved oxygen concentration to a low concentration of 0.5% to 2%. Xylitol high yield production method comprising the; The method of claim 8 wherein the dissolved oxygen concentration in the production medium is: Xylitol high yield production method, characterized in that more than 5% The method of claim 8 wherein the xylitol high yield production method is: Xylitol high yield production method characterized in that the fed-batch culture using a mixture of the biomass hydrolyzate and carbon source as a feed solution (feeding solution) The method of claim 8, wherein the biomass hydrolyzate is: Xylitol high yield production method characterized in that any one of the hydrolysates of corncob, sugar cane hydrolyzate, coconut by-product, hydrolyzate of birch The method of claim 10, wherein the carbon source is: Xylitol high yield production method characterized in that the glycerol Xylitol produced by the method according to any one of claims 1 to 5 and 7 to 12.

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