JPH0632614B2 - Immobilized microorganism and its use - Google Patents

Description

TECHNICAL FIELD The present invention relates to an immobilized microorganism and its use. More specifically, the present invention relates to an immobilized microorganism in which yeast belonging to the genus Lipomyces, which exhibits β-glucosidase activity even in a growth quiescent state, is immobilized, and a method for producing a decomposition product from a β-glucosyl compound using the same.

[Prior Art] Decomposing a β-glucoside bond of a β-glucosyl compound,
The production of decomposition products is often carried out industrially. For example, the case of producing glucose from cellulose can be mentioned.

The general method for decomposing cellulose to produce glucose is a method of chemically hydrolyzing under strong acid and high pressure, but this method is liable to cause damage to the device and also causes decomposition of glucose, There are drawbacks such as browning of the reaction solution.

Therefore, many methods of hydrolyzing cellulose under milder conditions using an enzyme have been reported. The enzyme involved in the degradation of cellulose is not single, but endo-β-1.4
It is well known that it is a cellulase complex enzyme system consisting of various enzyme systems such as glucanase, exo-β-1.4 glucanase and β-glucosidase. Its action is to decompose cellulose and Is to saccharify to glucose. Among them, β-glucosidase is considered to have an action of degrading cellooligosaccharides produced by the action of glucanase into glucose. Therefore, β-glucosidase is an essential component in the cellulase complex for highly converting cellulose to glucose. In addition, when degrading cellulose with a cellulase complex, if β-glucosidase activity is weaker than glucanase activity, cellooligosaccharides accumulate in the reaction solution, which inhibits endo-β-1.4 glucanase activity. Therefore, the cellulose degrading power of the entire enzyme system is weakened, and also from this aspect, it is essential that the β-glucosidase activity is high (Biotechnolo
gy and Bioengineering, Vol.XXIII 473, 1981).

However, in the conventional method in which cellulase is added to a suspension containing cellulose and mainly performed by the batch method, various measures have been made in order to enhance its activity by, for example, adding β-glucosidase in a timely manner ( JP-A-57-15038
However, the use of a large amount of enzyme was unavoidable, and it was difficult to put it to practical use.

For this reason, studies have been conducted to accelerate the hydrolysis rate and continuously separate products, and for example, a method for degrading cellulose while separating glucose cleaved from cellulose using ultrafiltration has been reported. (TK Chose and J.
A. Kostick, Biotechnol, Bioeng., 12,921 (1970)]. This method is excellent in that the produced glucose is removed from the reaction system, and the accumulated glucose does not inhibit the β-glucosidase activity. However, this method has a drawback in that impurities contained in the reaction system and decomposition products adhere to the outer surface of the ultrafiltration material to reduce the over-efficiency, and thus it is not practical.

Further, a method of immobilizing β-glucosidase and packing it in a fluidized bed column, and passing a cellulose saccharified solution by a cellulase complex into this to produce glucose has also been reported, but a method of immobilizing β-glucosidase. Is complicated, and it is not always sufficient in terms of glucose production efficiency and stability of the immobilized enzyme.

[Problems to be Solved by the Invention] Therefore, a microorganism having β-glucosidase activity and more easily immobilized than an enzyme is immobilized and packed in a fluidized bed column, and a saccharified cellulose solution is passed through this column. If the glucosidase activity can be constantly expressed, it is expected that the immobilized enzyme does not need to be repeatedly prepared and added, and glucose can be efficiently produced. Further, this column method is considered to be advantageous in that the enzyme activity is not inhibited by the accumulation of glucose.

As a microorganism having β-glucosidase activity, yeast is widely present in nature, and they grow under aerobic conditions using cellobiose as a single carbon source. However, it is known that yeast having β-glucosidase-producing ability cannot generally assimilate cellobiose under anaerobic conditions, and that β-glucosidase activity is extremely weak even in a growth quiescent state. Therefore, when these yeasts are used as a bioreactor such as immobilized cells to obtain glucose from cellobiose, in the presence of oxygen by adding a nutrient source,
It is necessary to constantly grow yeast to produce enzymes. As a result, (1) the process is complicated, (2) contamination of bacteria is likely to occur, (3) bacterial cells and unpleasant odor of the grown yeast are mixed in the product, and (4) produced glucose is grown in the grown yeast. There are problems such as being consumed. For this reason, no industrial success example of glucose by immobilized microorganisms has been reported yet.

Similar problems were known in the production of genipin, a source of natural colorants. That is, if geniposide, which is an iridoid glycoside obtained from gardenia nuts, or its content is decomposed with β-glucosidase, genipin can be obtained, but when the enzyme treatment is performed by the batch method, it is necessary to use a very large amount of enzyme. In addition, the generated genipin reacts with the primary amine of the enzyme itself, resulting in a decrease in the yield of genipin.

Therefore, also in the production of genipin, there is a strong demand for the development of a method in which a β-glucosidase-producing microorganism is directly immobilized and used. However, if a conventionally known β-glucosidase-producing microorganism is used,
As described above, genipin must be produced while constantly growing the cells. Therefore, in addition to the same problem as in the case of glucose production, the product genipin reacts with an enzyme such as β-glucosidase of yeast which is released from the immobilization carrier and proliferated, and there is a problem of decreasing production efficiency. there were.

The present invention is intended to solve the above problems by finding a microorganism that exhibits β-glucosidase activity against β-glucosyl compounds even in a growth quiescent state.

[Specific Means for Solving Problems] The present inventors have found that certain yeasts, particularly certain yeasts of the genus Lipomyces, are not only in a growth state (growth system) but also in a growth quiescent state (non-growth state). System) has also found a new fact that it exhibits β-glucosidase activity. further,
When these yeasts were immobilized on a suitable carrier and allowed to act on a β-glucosyl compound, it was found that the β-glucosidase activity in the non-growing system was stably maintained for a long period of time even after immobilization.

Therefore, the present invention provides an immobilized microorganism in which yeast belonging to the genus Lipomyces which exhibits β-glucosidase activity is immobilized on a carrier even in a growth quiescent state. Furthermore, the present invention provides a method for efficiently producing a target decomposition product by causing this immobilized microorganism to act repeatedly on a β-glucosyl compound by a continuous method or a batch method.

Immobilized Microorganism The yeast used in the present invention is selected from any yeast belonging to the genus Lipomyces and exhibiting β-glucosidase activity in a non-growing system. As an example, Lipomyces Starkey HUT
7263, Lipomyces starkey IFO 0678, Lipomyces lipophore IFO 1288, and the like, but are not limited thereto, and can be selected from cellobiose-assimilating Lipomyces yeasts according to the method of Example 1 described below. . Preferably, a yeast having a β-glucosidase activity of about 5 units / g dry cells or more, more preferably about 20 units / g dry cells or more in a non-growing system is selected.

Furthermore, the yeast used in the present invention must exhibit β-glucosidase activity in the immobilized state in a non-growing system. The confirmation can be performed according to Example 2 described later.

In the present invention, the carrier used for the immobilization of yeast is any carrier that holds the yeast inside or on the surface thereof and that does not prevent the held yeast from contacting the β-glucosyl compound that is the substrate thereof. You can Examples thereof include gel-like substances such as calcium alginate, polyacrylamide gel, agar gel, and sepharose gel, which consist of a matrix that encapsulates yeast but allows a β-glucosyl compound as a substrate to pass through. The amount of yeast immobilized on the carrier is not particularly limited, but it is preferable to use a large amount as long as the immobilization is not hindered. The immobilized microorganisms may have any shape such as beads, granules, plates and blocks depending on the purpose, but beads are convenient for use in a column.

Production of Target Product from β-Glucosyl Compound by Immobilized Microorganism When the immobilized microorganism of the present invention is used, contact between yeast and β-glucosyl compound is carried out under conditions of growth suspension of yeast, and the target decomposition product is produced. Can produce things. As described above, since it is not necessary to add a nutrient source to the reaction system, contamination of various bacteria can be prevented, waste of raw materials can be omitted, and labor for purifying the target product later can be reduced.
Furthermore, if the growth is carried out under resting conditions, free yeast cells and unpleasant odor do not contaminate the target product, and the target product by the growing yeast released from the immobilization carrier, for example, consumption of glucose does not occur. There is an advantage.

The method of the present invention for obtaining a decomposition product from a β-glucosyl compound using an immobilized microorganism may be performed by a batch method or continuously by a column. To perform the batch method, the immobilized microorganisms are added to an aqueous solution of a β-glucosyl compound at an appropriate concentration and gently stirred until the desired decomposition product is obtained. The amount of the immobilized microorganism used, the concentration of the β-glucosyl compound, the reaction time, and the like can be selected as appropriate by experiments. The reaction temperature is 20 to 45 ° C, preferably about
The temperature is 30 to 40 ° C., and the preferable reaction pH is weakly acidic, more preferably pH 3 to 5. When glucose is produced from cellobiose, for example, about 50 mM bead-shaped immobilized microorganisms with a diameter of about 4 mm are added to a cellobiose solution of about 100 mM, and a pH of about 5 is added.
Good results are obtained by reacting at 35 ℃ for 30 minutes.

In order to carry out the method of the present invention continuously using a column, the immobilized microorganism is packed in a column having an appropriate diameter and height,
It is carried out by flowing an aqueous solution containing a β-glucosyl compound at an appropriate flow rate. The reaction conditions such as temperature, pH, and β-glucosyl compound concentration at that time are the same as above, and other conditions can be determined by experiments.

Since the β-glucosidase activity of the immobilized microorganism of the present invention is stable for a long period of time, even when used in the batch method, it can be recovered and repeatedly used, and a large amount of enzyme is required when the enzyme is used as it is. It is much more advantageous than doing. When used in the column method, it can be used continuously for a long period of time. Furthermore, when the β-glucosidase activity of the immobilized microorganism is decreased, the immobilized microorganism is allowed to stand or shake culture in a suitable growth medium at 20 to 40 ° C for several hours to several days, preferably at 30 to 35 ° C. The activity can be recovered by shaking culture for 24 hours and reused.

The method of the present invention can be applied to any β-glucosyl compound in aqueous solution to produce its degradation product.

For example, according to one aspect of the method of the present invention, cellobiose can be contacted with an immobilized microorganism to produce the decomposition product glucose. At that time, as cellobiose,
If a saccharified solution obtained by partially saccharifying cellulose with a soluble cellulase complex in advance is used, continuous production of glucose from cellulose is also possible. Furthermore, continuous fermentation of ethanol from cellulose is also possible if the effluent from the immobilized microbial column is subsequently contacted with alcohol-fermenting yeast.

Another preferred embodiment of the method of the invention is the production of the aglycone genipin from geniposide. In this production, it is particularly efficient to carry out the method of the invention on a column. This is because the desired genipin can be recovered in crystalline form based on the difference in solubility with geniposide, simply by cooling the solution flowing out from the immobilized microorganism column. The genipin thus obtained can be further reacted with a primary amino group-containing compound to obtain a blue dye.

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

Example 1 Selection of Yeast Having β-Glucosidase Activity in Non-Proliferation System Eleven cellobiose-assimilating yeasts shown in Table 1 (all of which show β-glucosidase activity in a growth system)
The cells were cultured with shaking at 30 ° C. for 36 hours in a medium containing cellobiose as a sole carbon source shown in Table 2. The obtained bacterial cells (the amount of bacterial cells are shown in Table 1) were washed with 100 mM acetate buffer of pH 5 and then kept in the same buffer overnight at 4 ° C.

Next, the cellobiose-degrading activity at 37 ° C. for 30 minutes in an acetate buffer containing 3.4% cellobiose and having a pH of 5 was measured using the cells in the growth quiescent state. Degradation activity determines the amount of cellobiose remaining and the amount of glucose produced by HP.
It was performed by quantifying by LC.

As a result, as shown in Table 1, lipomyces (Lipomyc
es) several yeast strains (especially Lipomyces starkey H
UT 7263, IFO 0678 and Lipomyces Lipopher IFO
1288) was found to have a high cellobiose degrading activity even in the growth quiescent state.

In Table 1, HUT and I assigned to these yeasts
FO is a preservation number of Faculty of Engineering, Hiroshima University and Fermentation Research Institute, respectively, and can be obtained by those skilled in the art. In addition, Lipomyces Starkey HUT 7263 is named SAM 0433,
Deposited with the deposit number FERM P-9037 at the Institute of Mechanical Engineering.

Example 2 (i) Preparation of Immobilized Cells and Degradation of Cellobiose Lipomyces Starkey HUT 7263 (FERM P-9037) One platinum loop was treated with 90 ml of cellobiose medium (cellobiose 1%, yeast extract 0.5%, pH 5.7). After culturing with shaking at 30 ° C. for 48 hours, the cells were centrifugally precipitated, washed, and then suspended in a 2% sodium alginate solution, and the suspension was added dropwise to a 1% CaCl ₂ solution to prepare an immobilized yeast.

Next, 50 beads of immobilized bead-shaped yeast having a diameter of about 4 mm prepared in this way were added to a 100 mM cellobiose solution (acetate buffer, pH 5), and after reacting at 35 ° C for 30 minutes, the amount of glucose produced was measured by HPLC. Then, the β-glucosidase activity of the immobilized yeast (Lipomyces) was calculated. As a result, the activity of the present immobilized yeast was 1.5 unit / ml gel.

Next, in order to investigate the possibility of repeated use of the immobilized yeast, an experiment in which the supernatant after the reaction (30 ° C, 30 minutes) was replaced with a new cellobiose solution, and the reaction was repeated under the same conditions as above. Was repeated 15 times. As a result, as shown in FIG. 1, the β-glucosidase activity did not decrease at all after repeated use of 13 times, but only slightly decreased after 14 times. In Fig. 1, the activity in the first reaction is 10
As 0, the activity at each number of times is shown by relative activity.

(ii) Recovery of β-glucosidase activity by reculturing The immobilized yeast having a decreased β-glucosidase activity was able to recover the activity by reculturing in the cellobiose medium at 30 ° C. for 48 hours. One example is shown in FIG. FIG. 2 shows the recovery of the activity of the immobilized yeast (Lipomyces starkey HUT 7263) whose activity was reduced by repeated use 15 times.

(iii) Continuous use of immobilized cells by column Next, the possibility of continuous use of the immobilized cells of Lipomyces Starkey HUT 7263 (hereinafter referred to as immobilized Lipomyces) prepared in this Example was investigated. That is,
2 ml of immobilized Lipomyces was packed in a glass column and 40 ℃
The cellobiose solution having a concentration of 100 mM was fed at 0.7 ml / hr, and the amount of residual cellobiose in the solution flowing out and the amount of glucose produced were measured with time. As a result, as shown in FIG. 3, the glucose production yield was kept at 95% for 20 days, and it was shown that stable and continuous glucose production was possible for a long period by immobilized Lipomyces.

It should be noted that the results were similar to those described above, although there was a difference in the activity levels of the other lipomyces yeasts, IFO 0678 and IFO 1288.

Example 3 Saccharification of Cellulose by Immobilized Lipomyces The saccharification efficiency of cellulose by immobilized Lipomyces was examined using an apparatus as shown in FIG.

That is, in FIG. 4, a solution (pH of cellulose (44 g /) derived from corn and cellulase derived from Aspergillus (manufactured by Shin Nippon Kagaku Kogyo Co., Ltd .: 230 units /) in reaction tank A (pH).
Add 5), hold at 50 ° C and stir. Next, a part of the transparent reaction solution from this reaction tank A was kept at 35 ° C. in a immobilized Lipomyces (1) 20% packed fluidized bed reactor (reaction tank B).
The mixture was introduced into (pH 5) while being recycled, and changes with time of cellobiose and glucose concentrations in the mixture (2) in the reaction tank A were measured. On the other hand, the cellobiose and glucose concentrations in the reaction tank A in the recycle system in which the reaction tank B was not passed were measured to evaluate the effect of immobilized lipomyces.
As a result, as shown in FIG. 5, when recycled through the reaction tank B, the glucose (− ○ −) concentration increased and cellobiose (−Δ−) was maintained at a low concentration. It was shown that the glucose (-●-) production amount was low and the cellobiose (-▲-) concentration was high when there was no glucose. This is because, by passing through the reaction tank B, the saccharification activity of cellobiose in the reaction solution (β-glucosidase activity) is improved, and as a result, the cellobiose concentration is decreased, and accordingly, the inhibition of glucose products by cellobiose is decreased,
It is considered that the activity of the entire saccharification system of cellulose was improved.

Example 4 Production of Genipin from Geniposide by Immobilized Lipomyces (i) Microbial Geniposide Degrading Activity in Non-Proliferation System: Lipomyces Starkey HUT 7263 One platinum loop of cellobiose medium (cellobiose 1.0%, yeast extract 0.5%, pH 5.
7) Inoculate 90 ml and cultivate with shaking at 30 ℃ for 48 hours.
l was spun down and washed with sterile water, and the bacterial cells were added to 5 ml of a 130 mM geniposide solution, reacted at 35 ° C. for 30 minutes, and the amount of genipin produced was measured by HPLC to determine β-glucosidase activity. The activity of producing 1 μmol of genipin in 1 minute was defined as 1 unit.

As a result, the number of bacteria after shaking culture for 48 hours was 6.24 × 10 ⁷ cells / m 2.
The β-glucosidase activity of this bacterium was 2.45 × 1
^0-7 units / cell was shown.

(ii) Repeated Geniposide Degrading Activity of Immobilized Lipomyces Starkey in Non-Proliferation System; 2% sodium alginate solution of Lipomyces Starkey collected from 90 ml of the culture cultivated by the method of (i) above
Immobilized yeast was prepared by suspending it in 20 ml of 1% CaCl ₂ solution. Fifty immobilized yeasts were added to 5 ml of a 195 mM geniposide solution, reacted at 35 ° C. for 30 minutes for 24 hours, and the amount of genipin produced was measured by HPLC. The activity was determined from the amount of genipin produced after 30 minutes, and the yield of genipin production was determined from the amount of genipin produced 24 hours later. After 24 hours, the reaction solution was removed, a new substrate was added, and the above operation was repeated 8 times.

As a result, the activity of immobilized yeast was 1.11 units / ml gel, 1.
00 × 10 ^-9 units / cell was shown. FIG. 6 shows the activity of the immobilized yeast repeatedly used 8 times and the change in the amount of genipin produced after 24 hours. Regarding the activity, the activity at the first time was 100%. This immobilized yeast did not show a decrease in activity after repeated use eight times. Furthermore, the yield of genipin production after 24 hours was as high as 90%.

(iii) Continuous hydrolysis of geniposide by immobilized Lipomyces starkey: 2 ml of immobilized Lipomyces starkey prepared in Example (4)-(ii) was packed in a glass column and kept at 40 ° C., 195
The mM geniposide solution was delivered at 0.7 ml / hr. The hydrolyzed liquid of geniposide that flowed out was collected over time, and the concentration of residual geniposide and the concentration of generated genipin were measured.

As a result, the yield of genipin production was kept at 95% for 20 days. After that, the yield of genipin production gradually decreased to 80% after 40 days.
Fell to.

(iv) Reactivation of geniposide-degrading activity by reculturing immobilized Lipomyces starkey: In Example (4)-(iii), immobilized Lipomyces starkey that was continuously operated for 40 days was taken out from the column reactor, and )-(Ii) was used to measure the β-glucosidase activity of immobilized Lipomyces starkey (FIG. 7). As a result, the activity of the immobilized yeast was reduced to 64% at the time of preparation, which showed 0.71 unit / ml gel. Therefore, 2 ml of this immobilized yeast was placed in 20 ml of cellobiose medium (cellobiose 1.0%, yeast extract 0.5%, pH 5.65), and cultured by shaking at 30 ° C for 24 hours, and the β-glucosidase activity of the immobilized yeast in the culture solution was measured. As a result, the activity of the gel was 1.30 units / ml, which was higher than the activity at the time of preparation.

(v) Crystallization of Genipin from Continuous Hydrolysis Solution of Geniposide: Hydrolysis solution of geniposide obtained by the method of Example (4)-(iv) 5
0 ml was left at 5 ° C. for 24 hours. As a result, needle-like crystals appeared. This crystal was separated and dried to obtain 1.2 g of a dried product. The dried product was dissolved in methanol and analyzed by HPLC (column YMC-ODS A-312, solvent: 43% methanol 1 ml / hr, detection: UV-240 nm), and it was confirmed to be genipin with a purity of 98%. did.

The amount of genipin in 50 ml of the hydrolyzed solution of geniposide was 2.05 g, and 1.2 g of it could be collected as crystals very easily.

For the remaining genipin in the mother liquor after removing the crystals, concentrate the mother liquor under reduced pressure to 1.5 times and then leave it at 5 ° C. for 24 hours to precipitate genipin crystals again to obtain 0.3 g of genipin crystals. I was able to.

EFFECTS OF THE INVENTION As described above, the present invention provides a β-glucosyl compound β-
It provides efficient production of a reaction product obtained by cleaving the glucoside bond, and has great industrial utility.

[Brief description of drawings]

Fig. 1 shows β in repeated use of immobilized Lipomyces.
-Is a graph showing changes in glucosidase activity. FIG. 2 is a graph showing reactivation of β-glucosidase by reculturing immobilized Lipomyces. FIG. 3 is a graph showing a pattern of continuous saccharification of cellobiose by immobilized Lipomyces. FIG. 4 is a schematic diagram showing an apparatus for saccharification of cellulose. FIG. 5 is a graph showing the production process of glucose and cellobiose in a cellulose saccharification experiment. FIG. 6 is a graph showing the geniposide degrading activity in repeated use of immobilized Lipomyces. FIG. 7 is a graph showing continuous hydrolysis of geniposide by immobilized Lipomyces. 1 ... Immobilized Lipomyces 2 ... Mixture in reaction tank A A ... Reaction tank B ... Reaction tank filled with immobilized Lipomyces

Claims (12)

[Claims] 1. An immobilized microorganism in which yeast belonging to the genus Lipomyces, which exhibits β-glucosidase activity even in a growth quiescent state, is immobilized on a carrier. 2. The yeast is Lipomyces starkey (Lipomyc
es starkeyi) or Lipomyces lipophers (Lipomyc
The immobilized microorganism according to claim 1, which is an es lipofer). 3. The deposit number of Lipomyces Starkey is FE.
The immobilized microorganism according to claim 2, which is RMP-9037 or IFO 0678. 4. The deposit number of Lipomyces Lipopher is IF
The immobilized microorganism according to claim 2, which is O 1288. 5. The immobilized microorganism according to any one of claims 1 to 4, which is immobilized with calcium alginate. 6. A β-glucosyl compound is contacted with an immobilized microorganism obtained by immobilizing a yeast belonging to the genus Lipomyces, which exhibits β-glucosidase activity even in a growth quiescent state, on a carrier, and β-glucosyl compound A method of cleaving the glucosidic bond and obtaining the resulting decomposition product. 7. The method according to claim 6, wherein the contact between the immobilized microorganism and the β-glucosyl compound is carried out under the growth quiescent condition of the yeast. 8. The method according to claim 7, wherein the β-glucosyl compound is cellooligosaccharide or geniposide. 9. The method according to claim 7, wherein the decomposition product is glucose or genipin. 10. The method according to claim 8, wherein the yeast is Lipomyces starkey or Lipomyces lipophore. 11. The method according to claim 6, wherein the growth suspension condition of yeast is a condition substantially free of nutrients as a nitrogen source. 12. The method according to claim 6, wherein the immobilized microorganism is used by being packed in a column, and the contact with the β-glucosyl compound is continuously performed.