JPS63146782A - Production of microbial cell having high lipase activity - Google Patents

Abstract

PURPOSE:To produce microbial cells having high lipase activity, by cultivating a microorganism having the ability to produce lipase with a microbial holding material and triglyceride or fatty acid and sticking the microorganism to the above-mentioned holding material for multiplication. CONSTITUTION:A microorganism of the genus Rhizopus, Aspergillus, Mucor, etc., and having high ability to produce lipase is cultivated in a culture fluid containing >=20vol./vol% holding material, consisting of a porous material having preferably 60-99% voids, 2-50 pores/cm based on the unit straight line length or metallic worked material having 60-99% voids and <=20mm diameter for a spherical shape and <=20mm length of one side for a block form and containing 2-5% added glyceride, e.g. olive oil, etc., or fatty acid, e.g. oleic acid, etc., adsorbed on the holding material and multiplied to afford the aimed microbial cells having increased lipase activity. The cultivation is preferably carried out in a nonstirring type reactor in aspects of operation and cost.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field 1] The present invention relates to a method for producing lipase using a microorganism. More specifically, when producing lipase using a microorganism capable of producing lipase, the growth ecology of the microorganism is adhered to the holding material by culturing it together with the holding material.
The present invention relates to a method for producing microorganisms with high lipase activity, which is characterized by dramatically and stably improving the lipase production ability of the microorganisms by forming them into a grown biofilm. Bacterial cells with high lipase activity suitable for hydrolysis reactions are produced.

[Prior Art] Lipase is originally a general term given to enzymes that have a catalytic effect on the hydrolysis reaction of fats and oils.
It is found in liver, etc.; in the plant world, it is found in chili peppers and oats; and in the microbial world, it is found in molds, yeasts, and bacteria. However, from the standpoint of enzyme resources, enzymes sourced from animal organs and plant seeds cannot be obtained inexhaustibly.
It also has the disadvantage of being unstable and difficult to extract into water. On the other hand, microbial-derived lipases are made by selecting lipases that produce the desired lipase from among the wide variety of microorganisms that exist in nature, and artificially increasing the productivity of the enzyme by variously changing the culture conditions. This is advantageous for industrial production. Against this background,
For example, lipase, which is used in transesterification reactions of fats and oils, which is an important technology along with hydrogenation in the production of processed fats such as margarine and shortening, is mostly industrially produced by microorganisms.

In the industrial production of lipase using microorganisms, microorganisms are screened with the goal of producing extracellular lipase from the viewpoint of ease of enzyme purification, and each microorganism is cultured under optimal culture conditions before being released outside the microbial cell. The secreted lipase is separated, purified and sold commercially.

Most of the currently reported techniques for enzymatic fat and oil transesterification reactions utilize commercially available enzymes obtained in this way, immobilized on carriers such as Celite.

In the enzymatic transesterification reaction, controlling the amount of water in the reaction system is considered to be an important point in order to avoid the production of 17'l such as diglycerides due to hydrolysis of fats and oils, or a decrease in the exchange capacity yield. There is. Under these circumstances, the following methods, for example, have been reported in order to suppress the hydrolysis of fats and oils and to efficiently carry out the transesterification reaction.

b) A method of using a lower polyhydric alcohol as a lipase activator instead of water during transesterification of fats and oils (Japanese Patent Publication No. 57-6480).

(01 Method of adding a surfactant (emulsifier) to the transesterification reaction system of fats and oils to efficiently bring the fats and oils into contact with lipase at the interface (Japanese Patent Application Laid-open No. 198798/1983).

A method of increasing the transesterification reaction rate by controlling the water content using a tetrahydrophobic resin
116689).

b) A method in which the reaction is carried out more uniformly by using a lower alcohol ester of a fatty acid with a low melting point instead of a fatty acid with a high melting point (Japanese Patent Publication No. 51-27159).

(e) A method of controlling moisture in the reaction system by drying and circulating solvent vapor (Japanese Patent Laid-Open No. 58-500638).

However, these methods have problems such as slow reaction rate, insufficient suppression of hydrolysis reaction, complicated process, and obstacles to recovery and reuse of lipase, making it difficult to consider for industrial use. I have to say that this is also insufficient.

Under these circumstances, the present inventors cultivated lipase-producing microorganisms and encapsulated the lipase within the microbial cells as a method for suppressing the hydrolysis of oils and fats and efficiently performing the transesterification reaction. We have already reported that we can increase the rate of transesterification reaction and sustain the reaction stably for a long time by using dried bacterial cells in the above-mentioned state as a substitute for the lipase enzyme preparation in the above-mentioned method.
(Japanese Unexamined Patent Publication No. 60-34189).

[Problems to be Solved by the Invention] However, in such a transesterification reaction using dried bacterial cells, a culture method that increases the lipase activity of the dried bacterial cells, that is, the lipase productivity of a microorganism having lipase-producing ability, is required. Since the culture is used as dried bacterial cells, it is important to determine culture conditions that will prevent the secretion of the produced lipase from the bacterial cells as much as possible.

As a result of intensive research into determining the above-mentioned culture conditions, the present inventors found that there are amino acid groups that have an inhibitory effect on lipase production in the medium components, and an organic nitrogen source with a low content of these amino acid groups that have a large inhibitory effect has been found. By using it as a main component of the culture medium, it is possible to enhance the lipase production ability of microorganisms capable of producing lipase, and by feeding a substrate mainly composed of amino acids or amino M and peptides, the amino acid concentration in the culture solution can be reduced. It has already been found that the lipase-producing ability of microorganisms capable of producing lipase can be increased by culturing while maintaining low turbidity.

However, even with the above two methods, the number of stirring, ventilation, l! The influence of various physical manipulation factors such as the position of the itchypella, the geometrical shape of the culture tank, and the culture medium on the lipase production ability of microorganisms is complex, and even the method of controlling the medium composition described above does not affect the lipase activity. Although it can increase
It is very difficult to determine operating conditions that give a stable and constant level of high activity.

In particular, when producing lipase by culturing microorganisms capable of producing lipase, it is essential to use a natural organic nitrogen source.
Coupled with the instability of the composition, it is extremely difficult to determine culture conditions and to scale up the process for industrial use.

[Means for Solving the Problems] As a result of intensive research from the above-mentioned viewpoint, the present inventor has found that when culturing lipase-producing microorganisms, if the microorganisms are cultured together with a microorganism-retaining material, the microorganisms adheres to and proliferates on the holding material, forming a relatively dense biofilm near the surface layer of the holding material (hereinafter referred to as "biofilm growth"),
We found that the lipase activity within the bacterial cells increased dramatically. That is, the present invention provides a high-quality microorganism, which is characterized in that, when producing lipase by culturing microorganisms capable of producing lipase, the microorganisms are cultured together with a microorganism holding material and glyceride or fatty acid, thereby allowing the microorganisms to adhere to and proliferate on the holding material. Provides a method for producing microbial cells having lipase activity, which is characterized by producing biofilm-like growth that adheres to and proliferates on a microorganism-retaining material. It is.

In particular, when a microorganism-retaining material is used, the effects of medium composition, culture operating factors, etc. that affect the growth form of microorganisms capable of producing lipase can be minimized. Therefore, the method of the present invention can stably increase the lipase productivity of microorganisms capable of producing lipase, and can suppress the effects of medium composition and culture operation conditions. Industrial production of lipase enzyme preparations made of microbial cells with high lipase activity that can be used for decomposition reactions is made possible.

[Function and Examples] Next, the method of the present invention will be explained in more detail.

The method of the present invention uses a microorganism that produces lipase, such as Rhizopus chinensis, a heat-resistant strain of the genus Rhizopus (RtliZOl IIs).
) to produce lipase, the lipase-producing ability of the microorganism can be improved by culturing the microorganism into a biofilm-like form (hereinafter simply referred to as "biofilm") in which the microorganism is grown and attached to the microorganism-retaining material. The goal is to dramatically improve lipase activity and produce a lipase enzyme preparation made from dried bacterial cells with high lipase activity.

It is not clear why lipase productivity increases dramatically by creating a biofilm-like growth form, but as mentioned above, in order to promote lipase production, amino acids or all amino acids that inhibit lipase production can be used. The reason for this is thought to be that it is necessary to keep the WI concentration low, and that this condition is promoted by growing in a biofilm-like form, or that secretion of lipase to the outside of the bacterial cell is suppressed.

As the vine microorganism that can be applied to the method of the present invention, any microorganism that has the ability to produce lipase can be used.
us)fi, Aspergillus
Representative examples include the genus S) and Mucor B.

As the microorganism-retaining material, any material that enables adsorption and proliferation of microorganisms due to the adhesive and adsorption power of microorganisms can be used. For example, polymeric porous materials include polyolefins such as polyethylene or polypropylene; dienes such as butadiene or isoprene; vinyl polymers such as polyurethane, polyvinyl chloride, acrylamide or polystyrene; polyethers, polyesters, polycarbonates or nylon, etc. Condensed system of:
Materials such as silicon and fluororesin, and inorganic materials include ceramics, glass, activated carbon, pumice, and metals. In order to properly immobilize microorganisms on the microorganism-retaining material in any material, the void ratio should be 60 to 99% and the number of holes should be 2 to 50 per car position line length.
As the porous material in the range of α, it is preferable to use a metal processed material having a porosity of 60 to 99%.

By applying a retention material with the above characteristics,
During the process of microorganisms repeating their growth, the microorganisms are adsorbed and immobilized by the adhesive force inherent in the microorganisms and the enveloping action of the holding material, resulting in a biofilm-like growth form that is effective for lipase production.

Such various holding materials can be appropriately selected depending on the type of microorganism and culture conditions, and can be processed into shapes such as spheres, blocks, or sheets. The dimensions can be determined depending on the type of microorganism, culture conditions, type of reactor, etc. If it is roughly spherical, it has a diameter of 1 to 100 m, and if it is block-shaped, it has a side of 1 to 100 m.

However, if it is too large, the filling rate of microbial cells within the microorganism-retaining material (volume occupied by microorganisms/apparent volume of the microorganism-retaining material) will decrease, which is undesirable as it will increase the dead space within the microorganism-retaining material. So, if it is spherical, the diameter is 2o! If it is less than In, and it is block-shaped, it is more preferable that the quality of one side is 20M or less.

In order to adsorb and multiply microorganisms on the above microorganism holding material,
They can be easily adsorbed and propagated using commonly known batch, half-batch, and continuous culture methods.

In the method of the present invention, air, oxygen, or a mixture of both gases are used for aeration, and the reactor can be of any type, including a stirred wire type, a non-stirrer type, and a bubble column type. For adsorption and biofilm growth, a non-stirrer type reactor is more preferable in terms of operation and cost.

Furthermore, when determining the culture conditions when culturing with the microorganism-retaining material by the method of the present invention, the reduction in lipase productivity due to changes in the growth form due to the culture conditions is not considered, and microorganisms are adsorbed to the microorganism-retaining material. The selection may be made within the range of growth, focusing on conditions that maximize the growth rate of microorganisms within the microorganism-retaining material from the standpoint of industrial production.

However, as a result of research by the present inventors, microorganisms with high lipase-producing ability, such as Rhizopus chinensis (Rh) belonging to the genus Rhizopus, have been found.

chlnensis), Rhizopus preN/- (Rh
, deleear), Rhizopus javanicus (Rh,
Rhizopus japonictls), Rhizopus oligosporus (Rh, oliaosporus), Rhizopus niveus (Rh, n1veuS), Rhizopus javanicus (Rh, jaVanic13), and even Mucor javanicus (HuCOr javanicus)
, Aspergillus niger
In order to make microorganisms produce lipase suitable for transesterification, it is necessary to add glycerides such as olive oil or fatty acids such as oleic acid to the culture medium as inducers. The same applies when using a microorganism-retaining material according to the method. However, when a lipophilic polymeric porous material such as polyurethane is used as the microorganism-retaining material, these inducers are absorbed into the microorganism-retaining material. Therefore, if a large amount of the inducer is absorbed into the microorganism-retaining material, the resistance to movement of oxygen and substrate into the microorganism-retaining material increases, which becomes a negative factor for the growth of microorganisms within the microorganism-retaining material. As a result, since proliferation within the microorganism-retaining material is impossible, growth outside the microorganism-retaining material becomes dominant, the biofilm-like growth effect of the microorganism-retaining material is lost, and lipase production is no longer promoted.

Therefore, the amount of inducer added to the culture medium must be 1 to maintain stable growth within the microorganism-retaining material, promote lipase production, and suppress growth outside the microorganism-retaining material.
-8%, preferably 2-5%.

Furthermore, in order to maintain stable growth within the microorganism-retaining material and to suppress the detachment of microorganisms from the microorganism-retaining material, the turbulence intensity of the culture solution and the concentration of the microorganism-retaining material in the culture solution are important factors. . If the turbulence strength is too strong, the microorganisms will separate from the microorganism-retaining material, and if the turbulence strength is too weak, the microorganisms will separate from the microorganism-retaining material, and if the turbulence strength is too weak, the gas-liquid will separate from the solid-liquid interface between the microorganism-retaining material and the culture solution, or between the culture solution and the aeration gas. The resistance to movement of oxygen, substrates, etc. increases at the interface, and the growth rate of microorganisms within the microorganism-retaining material becomes extremely slow.

Therefore, the strength of turbulence required to maintain stable growth of lipase-producing microorganisms in the microorganism holding material is 10 at the stirring Reynolds number in a stirred rock-shaped reactor.
It is operated under stirring conditions of 2 to 107, preferably 103 to 105.

In a non-stirrer type bubble column, the problem of detachment of microorganisms from the microorganism-retaining material is not observed particularly in the range up to the line velocity of venting where vent gas blow-through occurs. Therefore, from the point of view of rapid I11 growth of microorganisms within the microorganism-retaining material, at an aeration linear velocity of 2C! It is operated at a linear aeration velocity range of R/sec, preferably 3 cta/Sec or more, until the aeration gas is blown out according to the type of each bubble column.

On the other hand, regarding the amount (concentration) of the microorganism-retaining material added to the culture solution, if the amount (concentration) is too small, the possibility (opportunity) of microorganisms adhering to the microorganism-retaining material and multiplying will be reduced. In order to ensure stable growth within the microorganism-retaining material, the concentration thereof is 10 V/V% or more, preferably 20 V/V% or more.

Next, the present invention will be explained in more detail using Examples, but the present invention is not limited to these.

In the following examples, the bacterial cells and the bacterial cells adsorbed to the microorganism holding material were separated from the culture solution, washed twice with tap water, then washed twice with an 80% acetone aqueous solution, and then incubated at room temperature for 24 hours. Vacuum dried.

Since a general method for measuring the transesterification activity of lipase contained in bacterial cells has not yet been established, in the method of the present invention, the reaction substrate is olive oil: methyl stearate: hexane - 1:4:2. Using a mixture consisting of 5, an appropriate amount of dried bacterial cells was added, and the transesterification reaction was carried out at 40°C for a certain period of time. The amount of enzyme that produces 1 g of SO3 per sample was defined as 1 unit (1 u).

The hydrolysis activity of lipase was measured by a known method by neutralization titration of oleic acid produced by decomposing olive oil.

Example 1 Various microorganisms were mixed with 7% polypeptone (manufactured by Daigoei 11 flavor) and N.
aNO30, 1%, KH2POao, 1%, 8030
Initial pH in a medium consisting of 40.05% and 2% oleic acid.
5, 6, IH [i) culture Lr= for 4 days at a temperature of 28°C.

Polyurethane foam (6 on each side) is used as a microorganism retention material.
M block-shaped ones (manufactured by Bridgestone @ Everlight Scotto HR-4011 100 pieces/100af!
It was added to form a medium (17.8 V/V%). As a control, shaking culture was also conducted without adding any microorganism-retaining material, and Table 1 shows the activity in the dried cells for comparison.

By culturing microorganisms capable of producing lipase together with microorganism-retaining materials, both transesterification and hydrolysis activities in dried bacterial cells increased.

[Example 2 in the margin below Example 1 using Rhizopus chinensis as the microorganism
Instead of polypeptone, yeast peptone (manufactured by Food Springer@), which is an organic nitrogen source with an amino acid composition unsuitable for lipase production, and in which microorganisms exhibit a pulpy growth form in normal batch culture, and Proextract P (manufactured by Banshu Seasoning ■), meat extract (manufactured by Wako Pure Condiments)
Shaking culture was performed using each of the microorganisms in the same manner as in Example 1, and the activities of the obtained microorganisms were compared. Second result
Shown in the table.

Even when an organic nitrogen source unsuitable for lipase production was used, both transesterification and hydrolysis activities were increased by culturing with the microorganism-retaining material.

[Left below] Example 3 Using Rhizopus delemer as a microorganism, glucose 1
%, polypeptone 7%, NaNO30.1%, to'th
A seed mother was prepared by preculturing at 28° C. for 24 hours in a medium containing 0.1% POa and 40.05% HQSO.

Batch culture was carried out under the following conditions using a 5f111 Ikudan Jar Furmentor (manufactured by Iwashiya Biochemical Co., Ltd., HB-c-5, stirring blade outer diameter = 81).

Medium composition used: yeast peptone 7%, NaNO30,
1%, 82PO40.1%, HO3O40.05%,
Olive oil 2%, pH 5, 6, stirring number 40 rpm.

Ventilation to, svv+ Temperature: 30°C Block-shaped nylon (6M on each side) as microorganism retention material
Porosity 95-98%, number of pores 30/ctx) 15
00 cells/IJI medium (7) 13 II (24,5V
/V%).

In addition, we conducted a control experiment in which no microbial retention material was added.
As shown in FIG. 1 for comparison, the transesterification activity of dried bacterial cells was measured over time of culture.

Transesterification activity was increased by culturing with microbial retention material.

Example 4 A seed mother was prepared in the same manner as in Example 3 using Rhizopus oligosporus as the microorganism.

Using a bubble column with the shape shown in Figure 2, the aeration linear velocity a3.2
+71/SeC (15ctxφ cross section reference), temperature 30
℃.

Batch culture was performed at pH 5 and 6.

The medium used was the same as in Example 3 except that meat extract was used instead of yeast peptone.

As a microorganism-retaining material, spherical stainless steel processed material (diameter 5 m, porosity 80-85%) was added at a ratio of 1,500 pieces/1 (15.8 V/V%), and culture was performed without using the microorganism-retaining material. A comparison with the activity of dried bacterial cells in a control experiment is shown in FIG.

Transesterification activity was also increased by culturing with microorganism-retaining material in a bubble column.

[Effects of the Invention] According to the present invention, the lipase-producing ability of microorganisms can be effectively and stably brought out by adhering to and growing a microorganism-retaining material to form a biofilm. Furthermore, according to the present invention, it is possible to eliminate the influence of culture medium conditions and culture operation conditions on the growth form, which not only makes it possible to use inexpensive organic nitrogen sources in culture and to ease stirring and aeration conditions, etc. Since the microorganisms are adsorbed on the microorganism-retaining material, it improves the operability of each process such as spreading, washing with water, washing with acetone, and drying during the production of dried microorganisms.
It has lipase activity and is very convenient for industrial production of microbial cells containing lipase used in transesterification reactions and fat and oil hydrolysis reactions.

[Brief explanation of the drawing]

Figure 1 is a graph showing a comparison over time of the transesterification activity of dried bacterial cells in the case of Rhizopus deremer treated with microorganism retention material at I8 yen and the control case in Example 3. 3 is a schematic diagram without showing the bubble column used for batch culture in Example 4, and FIG. It is a graph showing a comparison of transesterification activity over time. 12 Ventilation power

Claims (1)

[Scope of Claims] 1. When producing lipase by culturing microorganisms capable of producing lipase, the microorganisms are cultured together with a microorganism holding material and glyceride or fatty acid to cause them to adhere to and proliferate on the holding material. A method for producing bacterial cells with high lipase activity. 2 A microorganism capable of producing lipase is Rhizopus (Rhi).
zopus genus, Aspergillus genus or Mucor (Muc
The production method according to claim 1, wherein the microorganism belongs to the genus Or).