JP5867954B2 - Antarctic basidiomycetous yeast having milk fat resolving ability and method of use thereof - Google Patents

Description

  The present invention relates to an Antarctic basidiomycetous yeast and a method for treating wastewater containing milk fat with a lipase produced by the microorganism.

  Currently, industrial wastewater generated at agricultural and livestock product production sites has been subjected to inadequate treatment such as infiltration, dilution, and discharge, and is a source of contamination of groundwater and rivers. In particular, parlor drainage (milking cowshed drainage) generated from dairy farming is mainly composed of milking line washing water, waste milk, detergent, manure, disinfectant, etc., and has a significant impact on the surrounding environment. It has become a serious environment and fishery problem, such as having an adverse effect on the ascent of the salmon.

  In Hokkaido, there were approximately 8,310 dairy farmers in 2007, and 34,400 tons / day of parlor drainage was discharged (Ministry of Agriculture, Forestry and Fisheries “Statistics on Livestock”). The treatment methods for parlor wastewater that have been developed so far include microbe utilization method and membrane separation method or ozone oxidation decomposition method. However, the membrane separation method has a high running cost for cleaning and replacing the membrane. In addition, the ozone oxidative decomposition method requires an ozone generator and thus has disadvantages such as high initial cost and running cost. In addition, a purification method using plants has been developed, but it requires a very large site and takes time to process, so it is not practical. Therefore, it can be said that the microbial treatment method that can make the treatment system inexpensive is the most effective. The BOD (biochemical oxygen demand) of parlor wastewater is usually as high as 2,000 to 4,000 mg / l, and it contains a lot of persistent substances such as milk fat. Even if the biological treatment method is applied, it is never easy, and in a cold region such as Hokkaido, a decrease in the efficiency of microbial treatment due to a decrease in the water temperature during the winter period is a further problem.

  The use of polar microorganisms has been studied as a wastewater treatment in such a low temperature environment, and a reduction in total phosphorus, phosphoric acid, nitrate nitrogen, and total carbon in wastewater has been reported. However, there is little research on the treatment of fat that solidifies at low temperatures and is less susceptible to microbial degradation.

Yoshikazu Sato et al: Purification treatment of parlor and paddock wastewater by membrane-separated activated sludge method. Abstracts of Annual Meeting of the Agricultural Facility Society, 134-135, 2002 Kunihiko Kato et al .: Domestic information Underground flow reed filter artificial wetland system for dairy parlor wastewater treatment. Livestock technology (649), 32-37, 2009 Keiko Hirayama et al .: Treatment of organic wastewater with Antarctic low temperature fermentative yeast. Water treatment technology, 38, 1-4, 1997 E.P.Tang et al: Polar cyanobacteriaversus algae for tertiary waste-water treatment in cool climates. Journal of Applied Phycology, 9, 371-381, 1997 K. Katayama-Hirayama et al .: Nitrate removal by Antarctic psychrophilic yeast cells under high salt conditions. Proceedings of NIPR Symposium. Polar Biolology, 11, 92-97, 1998 P. Chevalier et al: Nitrogen and phosphorus removal by high latitude mat-forming cyanobacteria for potential use in tertiary wastewater treatment. Journal of Applied Phycology, 12, 105-112, 2000 K. Katayama-Hirayama et al .: Removal of nitrogen by Antarctic yeast cells at low temperature. Polar Bioscience, 16, 43-48, 2003 S. Brizzio et al: Extracellular functional activities of basidiomycetous yeasts isolated from glacial and subglacial waters of northwest Patagonia (Argentina). Canadian Journal of Microbiology, 53, 519-525, 2007 V. de Garcia et al: Biodiversity of cold-adapted yeasts from glacial meltwater rivers in Patagonia, Argentina. FEMS Microbiological Ecology, 59, 331-341, 2008 B. Turchetti et al .: Psychrophilic yeasts in glacial environments of Alpine glaciers. FEMS Microbiological Ecology, 63, 73-83, 2008 A.A.K.Pathan et al: Diversity of yeasts from puddles in the vicinity of Midre LovenbreenGlacier, Arctic and bioprospecting for enzymes and fatty acids. Current Microbiology, 60, 307-314, 2010 N.R.Kamini, T. Fujii, T. Kurosu, H. Iefuji (2000) Production, purification and characterization of an extracellular lipase from the yeast, Cryptococcus sp. S-2.Process Biochemistry 36, 317-324

  An object of the present invention is to provide a microorganism capable of decomposing fat such as milk fat in parlor drainage at a low water temperature or an enzyme capable of decomposing at a low temperature derived from a microorganism.

  As a result of intensive studies to achieve the above-mentioned problems, the present inventors found that basidiomycetous yeasts such as Antarctic Murakia blollopis decompose milk milk at low water temperature. It came to be completed.

That is, the present invention includes the following inventions.
(1) Microorganisms belonging to Mulakia broropis or Mulakia fridida that have high milk lipolytic properties.
(2) The microorganism according to (1), wherein the Mirakia brolopis having high milk lipolytic property is Murakia brolopis SK-4 strain (FERM AP-22126).
(3) The microorganism according to (1), wherein the Mirakia freedida having a high milk lipolytic property is Mrakia Freedida SK-2 strain (FERM AP-22153).
(4) A method for treating fat-containing wastewater using the microorganism according to any one of (1) to (3).
(5) The method for treating fat-containing wastewater according to (4), wherein activated sludge treatment is performed at 0 ° C to 15 ° C.
(6) The processing method according to (4) or (5), wherein the fat-containing wastewater is parlor wastewater.
(7) The following physicochemical properties produced by microorganisms belonging to the genus Murachia (i) Action: Hydrolyzes lipids containing saturated fatty acids having carbon chain lengths of 4 to 18 to produce fatty acids.
(B) Optimal temperature: 60-65 ° C
(C) Thermal stability: When kept warm in 50 mM Tris-HCl buffer (pH 8.5) for 30 minutes, it is stable up to 65 ° C.
(D) Optimal pH: 7.5-9
(E) Stable pH range: 4-10
(F) Molecular weight: Approximately 60,000
A lipase.
(8) The lipase according to (7), wherein the microorganism belonging to the genus Murakia is Murakia broropis.
(9) A method for producing a lipase, comprising culturing a strain belonging to the genus Murakia and having the ability to produce the lipase according to (7) or (8), and collecting the lipase from the culture.

  According to the present invention, it is possible to provide a microorganism that produces a lipase capable of decomposing milk fat content in parlor drainage at a low water temperature.

The phylogenetic tree of the Murakia brolopis SK-4 strain, the Murakia freedida SK-2 strain and other Murachia strains of the present invention is shown. The numerical value in the figure indicates the bootstrap value. (A) lipase-producing bacterium Murachia broropis SK-4 strain of the present invention, (B) milk lipolytic bacterium Janibacter anoferis AC-16 strain isolated from activated sludge in a parlor wastewater treatment facility in Hokkaido, and (C FIG. 4 is a diagram showing milk lipolysis at a culture temperature of 10 ° C. for Pseudomonas alkalines SB3-4. Milk fat degradation was evaluated from a halo around the colony (in which the milk fat content was degraded and the medium became translucent). The lipase-producing bacterium Murachia brolopis SK-4 strain (indicated by a circle) of the present invention and the milk lipolytic bacterium Janibacter anoferis AC-16 strain (indicated by a circle) isolated from activated sludge in a parlor wastewater treatment facility in Hokkaido It is a figure which shows the amount of oxygen consumption in the case of milk lipolysis. It is the result of Coomassie brilliant green staining of purified Murakia brolopis SK-4 strain lipase (lipase of the present invention) after dodecyl sulfate polyacrylamide electrophoresis. The molecular weight of the lipase of the present invention was determined to be 60 kDa. It is a figure which shows the substrate specificity of this invention lipase. A fatty acid p-nitrophenyl derivative was used as a substrate, an enzyme was added to 50 mM phosphate buffer pH 7.0 containing 1 mM substrate, and an enzyme reaction was performed at 30 ° C. for 30 minutes. It is a figure which shows the optimal temperature of this invention lipase. The enzyme was added to 50 mM phosphate buffer pH 7.0 containing 1 mM p-nitrophenyl palmitate, and the enzyme reaction was carried out at each temperature for 30 minutes. It is a figure which shows the temperature stability of this invention lipase. The enzyme solution was heated at each temperature for 30 minutes, Tris-HCl buffer pH 8.5 containing 1 mM p-nitrophenyl palmitate was added, and the enzyme reaction was performed at 65 ° C. for 30 minutes. It is a figure which shows the optimum pH of this invention lipase. The enzyme was added to a 50 mM buffer containing 1 mM p-nitrophenyl palmitate, and the enzyme reaction was performed at 30 ° C. for 30 minutes. It is a figure which shows pH stability of this invention lipase. 25 mM various buffer solutions were added to the enzyme solution, heated at 30 ° C. for 15 hours, pH was adjusted to 8.5, 1 mM p-nitrophenyl palmitate was added, and the enzyme reaction was carried out at 65 ° C. for 30 minutes.

  In the present specification, lipase means an enzyme that hydrolyzes using lipid as a substrate, as usual meaning in the art.

1. The present invention relates to a microorganism belonging to the genus Murchia having a milk fat resolving ability at low temperatures, a method for decomposing milk fat under the low temperature by the microorganism, a lipase related thereto, and a culture of the microorganism belonging to the genus Murcia. Is included.

  The microorganism belonging to the genus Murachia used in the present invention may be any strain as long as it can degrade milk fat at low temperatures, but is preferably a strain capable of growing at a low temperature of 10 ° C or lower. Specific examples of such strains include the fungi of the genus Basidiomycota agaricus Amanita shiroki jellyfish cystofilobasidium cystofirobasididae Murchia, such as Murakia blollopis, and Murachia fridida ( Mrakia frigida). Murakia brolopis SK-4 strain (FERM P-22126 strain) and Murakia freedida SK-2 strain (FERM AP-22153) are most preferred.

  In addition, the present invention includes microorganisms belonging to Murachia brolopis or Murachia fridida, which are highly milk-decomposable at low temperatures. High milk lipolytic property means rapid halo formation around colonies when cultured in a solid medium containing milk fat. More specifically, it means an agar medium containing fresh cream, a culture temperature of 10 ° C., and a culture in which halo formation of 5 mm or more is observed around the colony when cultured for 7 days. More specifically, examples include, but are not limited to, Murakia brolopis SK-4 strain (FERM P-22126 strain) and Murakia Fridida SK-2 strain (FERM AP-22153 strain).

  In the present invention, as microorganisms belonging to Mulakia brolopis having high milk lipolytic property at low temperatures, for example, Mulakia brolopis SK-4 strain (FERM P-22126 strain) and Mulakia Fridida SK-2 strain (FERM AP) -22153 strain). Here, low temperature means 0 ° C. to 20 ° C., more preferably 4 ° C. to 10 ° C.

  Murakia brolopis SK-4 strain is a newly isolated strain from nature and exhibits the following microbiological properties.

  When cultivated on a potato-dextrose agar medium at a culture temperature of 10 ° C., white mycelia are seen around the white yeast flora. DNA was prepared from the cultured strain by a conventional method, PCR reaction was performed using typical PCR primers ITS1F and ITS4 used for fungal lineage analysis, and the gene sequence of the obtained PCR product was determined (see Sequence Listing). However, since the ITS sequence of this strain has 99% or more homology with the ITS sequence of Murachia brolopis in the database, it was considered Murachia brolopis. This strain is “Mrakia blollopis SK-4 strain”, dated June 13, 2011, National Institute of Advanced Industrial Science and Technology, Patent Microorganism Deposit Center, East 1-chome, Tsukuba City, Ibaraki, Japan (postal mail) No. 305-8566) is deposited under the accession number FERM P-22126.

  In addition, Murakia Fridida SK-2 strain is a newly isolated strain from the natural world and exhibits the following microbiological properties.

  When cultivated on a potato-dextrose agar medium at a culture temperature of 10 ° C., white mycelia are seen around the white yeast flora. DNA was prepared from the cultured strain by a conventional method, PCR reaction was performed using typical PCR primers ITS1F and ITS4 used for fungal lineage analysis, and the gene sequence of the obtained PCR product was determined (see Sequence Listing). However, the ITS sequence of this strain had a homology of 99% or more with the ITS sequence of Murachia Stokeshi in the database. In the current classification system by Index Fungorum (http://www.indexfungorum.org/), Murakia Stokesi was considered a Mirakia freedida synonym, and therefore this strain was regarded as a Murchia freedida.

  This strain is called “Mrakia frigida SK-2 strain”, which was dated July 20, 2011, the National Institute of Advanced Industrial Science and Technology Patent Microbiology Deposit Center, Higashi 1-chome, Tsukuba City, Ibaraki, Japan ( Deposited under the postal code 305-8566) under the accession number FERM AP-22153.

  The above-mentioned Murakia Broropis SK-4 strain and Murakia Fridida SK-2 strain are under the control of the present inventors before filing this application, and are not distributed at all. There is no intention to sell until after filing this application.

2. 2. Production of microbial cells and lipases of microorganisms belonging to the genus Murakia 2-1. Production of microbial cells and lipases of microorganisms belonging to the genus Murakia It can be produced by culturing and recovering bacterial cells and lipase from the culture solution. The medium used for culturing the microorganism of the present invention is not particularly limited as long as it contains an appropriate amount of carbon source, nitrogen source, inorganic substance, and other necessary nutrients necessary for fungi to assimilate. Any synthetic medium can be used. Specific examples of the medium include a potato / dextrose medium, corn meal medium, and malt broth medium commonly used for fungal culture. As the carbon source of the synthetic medium, for example, fresh cream, olive oil, Tween 80 and the like are used. As the nitrogen source, for example, nitrogen-containing natural products such as peptones, yeast extract and meat extract, and inorganic nitrogen-containing compounds such as sodium nitrate and ammonium chloride are used. As the inorganic substance, for example, potassium phosphate, sodium phosphate, magnesium sulfate, calcium chloride, ferric chloride and the like are used. Although the culture method is not particularly limited, it is usually carried out by shaking culture, aeration stirring culture or stationary culture. The culture temperature is not particularly limited, but is preferably at a low temperature, for example, in the range of -1 to 15 ° C, more preferably 10 ° C or less. Alternatively, the cells can be sufficiently grown at the optimum growth temperature, then transferred to a new medium and cultured at 10 ° C. or lower. The culture period is usually about 2 weeks.

  For purification of the lipase of the present invention, purification methods generally used in the art can be used. For example, centrifugation, filtration, ultrafiltration, or the like can be used for separating the cells. The lipase contained in the culture supernatant obtained after separation of the bacterial cells can be salted out with ammonium sulfate or sodium sulfate, organic solvent precipitation with acetone or ethanol, cation exchanger (eg, CM, S, SP) or anion. It can be isolated and purified by column chromatography using an ion exchanger (for example, DEAE, Q, QAE) or the like, gel filtration using an agarose derivative or the like.

  The crude lipase and purified lipase obtained by these methods can be used in liquid form with the addition of a stabilizer such as glycerol, sucrose, ethylene glycol, or a drying method such as spray drying or freeze drying. It can also be used as a powder.

  As described above, the cells and lipase of the present invention can be produced by culturing a microorganism belonging to the genus Murachia, preferably Murakia brolopis, and collecting it from the culture solution. Since the present fungi can be easily cultured in large quantities using an inexpensive medium, the lipase and lipase producing ability of the present invention can be obtained in a large amount at a low cost.

2-2. The lipase of the present invention The present invention includes a lipase produced by a microorganism belonging to the genus Murchia. The lipase of the present invention has the following physicochemical properties. (1) As an action, lipids containing saturated fatty acids having a carbon chain length of 4 to 18 are hydrolyzed to produce fatty acids. (2) The optimum temperature is 60 to 65 ° C. (3) It has lipid resolution at low temperature. (4) The thermal stability is stable up to 65 ° C. when kept in a 50 mM Tris-HCl buffer (pH 8.5) for 30 minutes. (5) The optimum pH is 7.5-9. (6) The stable pH range is 4-10. (7) The molecular weight is about 60,000.

3. Wastewater treatment method using microorganisms belonging to Murakia Murachia microorganisms For example, microorganisms belonging to Murakia broropis or Murakia freedida can be included in the activated sludge. Thus, by including the microorganisms of the present invention in activated sludge, wastewater containing milk fat at a high concentration can be efficiently treated even at low temperatures.

  The present invention includes a wastewater treatment method by an active water sludge method, wherein the activated sludge contains the microorganisms belonging to the above-mentioned Murakia brolopis or Murachia fridida. Preferably, microorganisms belonging to the above-mentioned Murachia brolopis or Murachia fridida can be used for treatment of wastewater containing milk fat at a high concentration by an active water sludge method. More specifically, wastewater containing milk fat such as parlor wastewater at a high concentration is treated at a low temperature of 0 to 15 ° C., for example, about 10 ° C., using activated sludge using microorganisms belonging to the Murachia brolopis. be able to.

  The activated sludge method mainly focuses on decomposing pollutants such as wastewater with activated sludge containing microorganisms in the aeration tank, but the microorganisms belonging to Murakia Brolopis of the present invention should be added to the activated sludge in this aeration tank. Can do.

  As the activated sludge method, any commonly used method or process can be adopted. For example, as the activated sludge method that can be used in the present invention, there are various methods such as a batch activated sludge method, a continuous activated sludge method, and various separation steps and precipitation steps before the activated sludge treatment in the aeration tank. It is also possible to provide a pretreatment process such as an activated sludge after the aeration tank, or to provide a posttreatment process such as an activated sludge precipitation process or a disinfection process.

  More specifically, in addition to the activated sludge method, this modified method can be used for the contact stabilization method, the long-time aeration method, the step aeration method, the high concentration oxygen activated sludge method, and the membrane separation activated sludge method. As a typical aerobic biological treatment method other than the activated sludge method, there is a biofilm method. The present invention can also be used in the present method. This method uses plastic pieces, plastic nets, shells, gravel, etc. as a carrier, and can purify wastewater while ventilating with microorganisms adhering to the surface of the membrane.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited to these examples.

[Example 1] Comparison of milk fat resolution (Comparison with Antarctic basidiomycetous yeast)
In January 2007, the sediment collected from the lake of Sukarubunesu Soya Coast was directly inoculated into potato dextrose agar medium containing 50 μg / ml chloramphenicol in April of the same year, and cultured at a temperature of 10 ° C. The yeast was used thereafter. Molecular biological techniques were used to identify the isolates. In other words, the ITS region (Internal Transcribed Spacer region; a sequence containing 5.8S rDNA and ITS1 and ITS2 non-transcribed regions among the transcription regions from 18S rDNA to 25S rDNA) is used as a molecular marker. It was. The bacterial cells were scraped from the medium, and DNA was extracted using a commercially available DNA extraction kit. Using this DNA as a template, White et al. Using fungal primers ITS-1F (5'-TCCGTAGGTGAACCTGCGG-3 ', Gardes and Bruns, 1993) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', White et al., 1990) The ITS region was amplified according to the conditions of (1990). The annealing temperature was 50 to 55 ° C. After the PCR product was purified, direct sequencing was performed using ITS1F as a probe to obtain the base sequence of the ITS region. This was compared with homologous sequences in the database to search for related species. As a result of the above identification, as shown in Table 1, the isolated yeast was found to be isolated from Murachia brolopis, Cryptococcus aqaticus, Cryptococcus sp. , Dioszegia fristingensis, Rhodotorula glaciallis, and Rhodotorula sp. It was roughly divided into 6 groups with high homology. The resolution of milk fat between these strains was cultured on an agar medium containing fresh cream and evaluated by the size of halo formed on the medium (Table 1).

  The Mulakia brolopis strain SK-4, which is highly homologous to the Mulakia brolopis strain DBVPG4974, formed the largest halo, confirming that the Mulakia strain has high milk fat resolution. Therefore, the milk fat resolving power of all isolates of the genus Murachia was examined (Table 2).

  Milk fat resolution was greatly different depending on the test strain. As a result of culturing at 10 ° C. for 10 days, all 75 strains grew on the fresh cream agar medium, but 19 strains did not form halos. 56 strains formed clear halos under the same conditions, 30 of which formed halos with a width of 1 cm or more.

  Of the above 75 strains, the sequences of the obtained ITS regions for 23 strains are shown in the Sequence Listing. Using the isolated strain and the ITS region of the genus Murachia strain registered in the database, a system diagram was created by the neighborhood joining method (Fig. 1). As an outer group, the Murakialla cryoconiti DBVPG5180 strain belonging to the genus Murakiara, which is closely related to the genus Murakia, was used as the outer group. The phylogenetic tree creation was repeated 1000 times to obtain the bootstrap value. The strain SK-4 of the present invention has high milk fat resolution (agar medium containing fresh cream, culture temperature 10 ° C., and when cultivated for 7 days, a halo of 5 mm or more is formed around the colony). Nine ponds and six ponds formed the same clade as Murakia broropis (Fig. 1). The only other strains with high milk fat resolving power are 2 Sonohachi Ponds (Murakia Fridida SK-2 strain) contained in the Murachia Fridida clade.

  Conventionally, Murakia Fridida, Murakia sp. (A strain having 98% homology to Murakia Fridida), and Murakia sp. YSAR9 strain (a strain having 99.6% homology to Murakia cyclophila) have been The activity of degrading extracellular Tween80 or tributyrin has been reported (Non-patent Documents 8-11). On the other hand, Murakia sp. YSAR11 strain (a strain having 99.3% homology to Murakia fridida) and Murakia sp. YSAR12 strain (a strain having 100% homology to Murakia fridida) are extracellular lipases. It is said that there is no activity (Non-patent Document 11). These reports suggest that the production amount or production conditions of extracellular lipase produced by Murachia may vary greatly at the species level. Of the 23 strains identified by ITS sequence, 4 out of 14 strains in Mr. brolopis, 1 out of 6 strains in Mr. frida, and 0 out of 2 strains in Mr. It was. In the present invention, it has been clarified for the first time that milk fat resolving power of strains belonging to Murachia brolopis or Murakia Fridida is systematically high.

[Example 2] Comparison of milk fat resolution (comparison with bacteria in parlor drainage facility)
Mulakia broropis SK-4 strain of the present invention and milk lipolytic bacterium Janibacter anophelis AC-16 strain, Shudomonas obtained from an aeration tank treating activated sludge of parlor drainage at a farm in Kamishihoro, Hokkaido -Alkagenes (Pseudomonas alcaligenes) SB3-4 strain was inoculated on a fresh cream agar medium, and milk lipolysis was observed at a culture temperature of 10 ° C. (FIG. 2). Although a clear halo was confirmed around the Mulakia brolopis SK-4 strain, cell growth was confirmed in the Janibacter anoferis AC-16 strain, but halo formation during the culture period could not be confirmed. In addition, the cell growth of Pseudomonas alkaligenes SB3-4 could not be visually confirmed under the above culture conditions.

  Furthermore, the microbial degradability of milk fat was evaluated by oxygen consumption using Mulakia brolopis SK-4 strain and Janibacter anopheris strain AC-16. Each specimen to be measured as a test strain was prepared as follows. In other words, add 500 ml of inorganic nutrient salt, bacterial cells and tap water to 100 ml of a 500 ml culture bottle diluted with fresh cream 1,000 times with distilled water to adjust the total volume to 200 ml. Oxygen consumption was measured using an aerobic microbial respirometer (Kitakai trial coulometer, manufactured by Okura Electric Co., Ltd.). Inorganic nutrient salt is 1.9g potassium dihydrogen phosphate, 2.6g disodium monohydrogen phosphate, 0.19g ammonium chloride, 0.037g calcium chloride, 0.03g dibasic water, 0.1g magnesium sulfate and seven water, and 0.015g ferrous chloride per liter. did.

  Murakia brolopis SK-4 strain had a delay time of about 15 hours until the start of oxygen consumption, and then showed rapid oxygen consumption (Fig. 3). On the other hand, the delay time of the Janibacter anoferris AC-16 strain was about 45 hours, and the subsequent oxygen consumption rate was slower than that of the Murakia brolopis SK-4 strain. From the above, it was proved that Mulakia brolopis SK-4 strain has high milk lipolysis ability at low water temperature.

[Example 3] Microbial treatment of parlor drainage Using a diluted milk solution as a model drainage of parlor drainage, treatment with activated sludge added with Murakia brolopis SK-4 strain of the present invention was performed. The activated sludge used in the experiment was obtained from an aeration tank in which the parlor wastewater was actually treated with activated sludge at a farm in Kamishihoro, Hokkaido. This was acclimatized for about one month at room temperature (20-23 ° C.) using milk as a substrate in a 2.5 L aeration tank and adding a small amount of inorganic nutrient salts.

  Activated sludge (MLSS: approx. 3,000 mg / l, Mixed Liquor Suspended Solid mixture suspended matter) acclimatized at room temperature to Murachia brolopis SK-4 strain (dry weight approx. 1 g) cultured in potato-dextrose liquid medium at 10 ° C Concentration) was added to 700 ml and cultured at 10 ° C. Up to the 7th day of culture, aeration was stopped once a day and sludge was allowed to settle, and then the supernatant was removed. The precipitated sludge was acclimated to a low temperature by a fill and draw method in which 2.8 ml of commercially available milk (BOD: about 14,000 mg / l) and tap water were added. On the 7th and 12th days of culture, MLSS was adjusted again to about 3,000 mg / l, 5.6 ml and 8.4 ml of milk stock solutions were added, and BOD after 24 hours was measured. The culture for 8 to 11 days was carried out in the same manner as the 7th day of culture. Moreover, the same process was performed using the activated sludge acclimatized at room temperature to which no Murakia brolopis SK-4 strain was added, and the results were compared.

The BOD removal rate of activated sludge added with Murakia brolopis SK-4 strain was improved compared to activated sludge not added (Table 3). BOD sludge load (kg-BOD / kg-MLSS / d, BOD treatment amount per MLSSkg) in general wastewater treatment is 0.2 to 0.4, BOD volumetric load (kg-BOD / m ³ / d, per 1m3 aeration tank BOD processing amount) is 0.4 to 0.8. The activated sludge to which 5.6 ml and 8.4 ml of milk stock solution were added after 7 days and 12 days of culture, respectively, had a BOD sludge load of about 0.35 and 0.52, and a BOD volume load of 1.0 and 1.5. From these results, it was confirmed that the addition of Murakia broropis SK-4 strain producing the lipase of the present invention to activated sludge improves the BOD removal rate of wastewater with relatively high load.

  From the above results, it was revealed that the treatment efficiency of parlor wastewater containing milk fat was improved even at low water temperature by using the strain of the present invention for parlor wastewater treatment.

[Example 4] Manufacture of lipase derived from Mulachia brolopis The high milk lipolytic activity at low temperature exhibited by Mulakia bloropis SK-4 strain of the present invention is considered to be caused by an enzyme involved in lipolysis, and an extracellular lipase Was purified. 3 liter Erlenmeyer flask containing 1 liter of liquid medium containing 1% Tween 80, 0.5% yeast extract, 0.2% potassium dihydrogen phosphate, 0.29% disodium phosphate, 0.02% ammonium chloride, 0.04% calcium chloride, 0.001 ferric chloride And autoclaved at 121 ° C. for 20 minutes. Murachia brolopis SK-4 strain (FERM P-22126) was inoculated as an inoculum and cultured at 10 ° C. for 2 weeks to obtain a culture solution. The culture broth was centrifuged, and the resulting supernatant was concentrated by ultrafiltration, and then dialyzed against 10 mM Tris-HCl buffer pH 8.5 using a dialysis membrane having a molecular weight cut off of 10,000 to 8,000. Fractionation was performed with butyl Toyopearl-650M column chromatography equilibrated with the same buffer, and it was confirmed that a single band was obtained by sodium dodecyl sulfate-polyacrylamide electrophoresis. The obtained protein had the following properties.

  The molecular weight of the protein was about 60 kDa (FIG. 4) as measured by sodium dodecyl sulfate-polyacrylamide electrophoresis.

[Example 5] Measurement of lipolytic activity Substrate specificity: Using fatty acid p-nitrophenyl derivatives with carbon chain lengths of 4, 5, 8, 10, 14, 16, and 18 as substrates, add enzyme to 50 mM phosphate buffer pH 7.0 containing 1 mM substrate, and bring the temperature to 30 ° C. The enzyme reaction was performed for 30 minutes. The lipase derived from the Antarctic basidiomycetous yeast Murachia brolopis SK-4 strain (hereinafter referred to as the lipase of the present invention) was confirmed to hydrolyze all substrates with different carbon chain lengths (FIG. 5). This property seems to enable efficient degradation of various fatty acids contained in milk fat.

  2. Effect of temperature: Enzyme activity at each temperature of the lipase of the present invention was measured (FIG. 6). The optimum temperature was at 60-65 ° C. The specific activity at low temperature was 0.8% at 0 ℃, 2.8% at 10 ℃, and 11.5% at 20 ℃. The residual activity of the lipase of the present invention hardly changed after heat treatment at 65 ° C. for 30 minutes (FIG. 7). Cryptococcus sp., Which has been studied for similar uses. In the lipase derived from the two strains, the optimum temperature is around 40 ° C. and the specific activity at a reaction temperature of 60 ° C. is 60% or less (Non-patent Document 12). From these results, the lipase of the present invention is It was confirmed that the enzyme shows activity at low temperatures while having high stability to heat.

  3. Effect of pH: The enzyme activity at each pH of the lipase of the present invention was measured. A citrate buffer solution was used from pH 3 to pH 5, a phosphate buffer solution was used from pH 6 to pH 8, a glycine-sodium hydroxide buffer solution was used for pH 9, and a carbonate buffer solution was used for pH 10. The optimum pH was 7.5 to 9 (Fig. 8), and high stability was exhibited in a wide pH range from 4 to 10 (Fig. 9).

  4). Effect of metal salt: Table 1 shows the effect of metal salt on the lipase of the present invention. The activity of the lipase of the present invention was not lost by the metal salt.

  From the above results, it was shown that the lipase of the present invention has a wide range of substrate specificities, is more stable than enzymes produced by conventional fungi adapted to low-temperature environments, and functions in a wide range of environments. .

  The Antarctic basidiomycetous yeast Murachia brolopis produces a highly stable lipase in a variety of environments, thereby exhibiting a higher milk lipolysis effect than conventional psychrophilic microorganisms. In addition, due to the biological characteristics of microorganisms belonging to the genus Murchia, a large amount of lipase and lipase producing ability can be prepared at low cost by culturing the microorganisms. Furthermore, since the tested strain has not been reported to be toxic to the human body, it is considered highly safe. Therefore, the present invention is a very practical and effective technique in, for example, biological treatment of wastewater containing lipids typified by parlor wastewater in cold regions.

FERM P-22126
FERM AP-22153

Claims (6)

Mulakia brolopis SK-4 strain (FERM P-22126) which is a microorganism belonging to Mulakia brolopis and has the ability to produce a lipase having the following physicochemical properties .
(1) Action: Hydrolyzes lipids containing saturated fatty acids with carbon chain lengths of 4 to 18 to produce fatty acids
(2) Optimal temperature: 60-65 ° C
(3) Thermal stability: stable to 65 ° C when incubated for 30 minutes in 50 mM Tris-HCl buffer (pH 8.5)
Is
(4) Optimum pH: 7.5-9
(5) Stable pH range: 4-10, and
(6) Molecular weight: Approximately 60,000 (measured by sodium dodecyl sulfate-polyacrylamide electrophoresis)
Fixed) The processing method of the fat containing waste water using the microorganisms of Claim 1 . The processing method of the fat containing waste water of Claim 2 which performs an activated sludge process at 0 degreeC-15 degreeC. The processing method according to claim 2 or 3 , wherein the fat-containing wastewater is parlor wastewater. Lipases having the following physicochemical properties producing microorganism belonging to Murakia genus of claim 1.
(1) Action: Hydrolyzes lipids containing saturated fatty acids with carbon chain lengths of 4 to 18 to produce fatty acids (2) Optimal temperature: 60-65 ° C
(3) Thermal stability: stable to 65 ° C when incubated for 30 minutes in 50 mM Tris-HCl buffer (pH 8.5) (4) Optimum pH: 7.5-9
(5) Stable pH range: 4 to 10, and (6) Molecular weight: about 60,000 (measured by sodium dodecyl sulfate-polyacrylamide electrophoresis)
Fixed) A method for producing a lipase, comprising culturing a strain belonging to the genus Murakia and capable of producing the lipase according to claim 1 , and collecting the lipase from the culture.

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