KR20130071320A - CHARACTERIZATION OF α-GALACTOSIDASE FROM BACILLUS SP. LX-1 AS A POTENTIAL FEED ADDITIVE AND OPTIMIZATION OF SOLID STATE FERMENTATION FOR THE ENZYME PRODUCTION - Google Patents

Abstract

PURPOSE: LX-1-derived alpha-galactosidase is provided to ensure strict substrate specificity by exclusively decomposing alpha-galactooligosaccharides, protease resistance, and smooth activity under an animal intestine-like environment, and to be used as an additive for soybean cake processing. CONSTITUTION: Bacillus sp. LX-1(deposit number KACC91695P) has 16S rDNA sequence of sequence number 1. An enzyme composition contains alpha-galactosidase with resistance to trypsin, pronase, or pancrease, derived from the strain. A method for hydrolyzing raffinose and stachyose in soybean cake comprises a step of treating the soybean cake with the strain-derived alpha-galactosidase. A feed additive composition contains the strain or the strain-derived alpha-galactosidase as an active ingredient.

Description

Characterization of alpha-galactosidase derived from Bacillus sp.L.-1 as a potential feed additive and optimization of solid fermentation for enzyme production {Characterization of α-galactosidase from Bacillus sp. LX-1 as a potential feed additive and Optimization of solid state fermentation for the enzyme production}

The present invention is a potential feed additive Bacillus sp. The invention relates to the optimization of solid fermentation for the production of LX-1 derived α-galactosidase and enzyme production.

Soybeans are a class of legumes classified as oilseed and are an important source of vegetable proteins and vegetable oils used around the world. It is also considered a source of complete protein because it contains enough essential amino acids for humans and animals. The major producers of soybeans are the United States (35%), Brazil (27%), Argentina (19%), China (6%) and India (4%). Soybeans can be fed to animals in the form of full-fat soybeans or soybean meal (SBM), which is a by-product left after oil removal.

Soybean meal is the most economical and high-quality vegetable protein feedstock and is the primary protein source for the poultry and livestock feed industry worldwide (Ghazi, S., Rooke, JA, Galbraith, H. 2003. Br. Poult Sci. 44: 410-418; Stein, HH, Berger, LL, Drackley, JK, Fahey, Jr. GC, Hernot, DC, Parsons, CM 2008. Johnson A, White PJ, Galloway R. ed. AOCS press, Urbana, IL, USA), accounting for more than 50% of the global use of protein resources. Raw soy contains harmful ingredients, which are anti-nutritive factors that inhibit the absorption and absorption of nutrients such as trypsin inhibitors or proteins and other minerals. Most anti-nutritive factors are heat-stable and processed into soybean meal. (Liener, IE 1994. Crit. Rev. Food Sci. Nutr. 34: 31-67). However, excessive heat treatment may reduce the nutritional value of soy because it reduces the availability of Lys (Parsons, CM 2000. Assessment of nutritional quality of soy product.pp 90-105 in Soy in Animal Nutrition; Drackley JK, ed. (Federation of Animal Science Societies: Savoy, IL, USA), and α-galactooligosaccharides such as raffinose and stachyose still exist after processing with soybean meal (Hartwig, EE, Kuo, TM, Kenty, MM 1997. Crop Sci. 37 Rackis, JJ 1981. J. Am. Oil Chem. Soc. 58: 503-511.

Soybean meal contains about 1% and 6% raffinose and stachyose, respectively, depending on building standards (Grieshop, CM, Kadzere, CT, Clapper, GM, Flickinger, EA, Bauer, LL, Frazier, RL, Fahey, Jr.). GC 2003. J. Agric.Food Chem. 51: 7684-7691). These α-galactooligosaccharides increase the viscosity of digesta and decrease the interaction between nutrients and digestive enzymes in the small intestine, thereby reducing the digestibility and availability of nutrients (Smits, CHM, Annison, G. 1996. World's Poult. Sci. J. 52 (204-221), which are considered anti-trophic factors (Anderson, RL, Wolf, WJ 1995. J. Nutr. 125: 581-588). When alpha-galactooligosaccharides such as Raffinose and stachyose reach the large intestine without digestion, they are decomposed by anaerobic bacteria in the large intestine, generating gases such as N ₂ , CO ₂ , and CH ₄ , causing flatulence. Falkoski, DL, Guimaraes, VM, Callegari, CM, Reis, AP, De, Barros.EG, De, Rezende.ST 2006.J. Agric.Food Chem. 54: 10184-10190; Yoon, MY, Hwang, HJ 2008 Food Microbiol. 25: 815-823), and gas production in the intestine is reported to cause nausea, abdominal pain and diarrhea (Calloway, DH, Colasito, DJ, Mathews, RD 1966. Nature 212: 1238- 1239).

Feed additives, on the other hand, refer to substances added in trace amounts for nutritional or specific purposes. These feed additives are very diverse, such as growth promoters, palatability enhancers, feed preservatives, quality improvers and feed efficiency enhancers.

At present, due to the rise in grain prices, the use of less available raw materials, such as barley, palm and palm leaves, is increasing to reduce feed costs. Therefore, various digestive enzymes are added to the feed to improve feed efficiency. Many commercial enzymes have been reported to be effective in addition to poultry feed rich in non-starch polysaccharides (NSP) (Wang, H., Guo, Y., Shih, JCH 2008. Anim. Feed Sci. Technol. 140: 376-384), and has also been reported to be used as feed additives in pigs to improve weight gain and nutrient digestibility (Kim, SW, Knabe, DA, Hong, KJ, Easter, RA 2003. J. Anim. Sci. 81 : 2496-2504). Grandhi (Grandhi, RR 2001. Can. J. Anim. Sci. 81: 125-132) and Olukosi et al. (Olukosi, OA, Sands, JS, Adeola, O. 2007. J. Anim. Sci. 85: 1702- 1711) reported that addition of carbohydrates in feed did not always improve the growth efficiency of animals. As a feed additive, digestive enzymes are used to improve the environment by reducing emissions of pollutants such as phosphorus or ammonia (Zakaria, HAH, Jalal, MAR, Ishmais, MAA 2010. Int. J. Poult. Sci. 9 126-133). Therefore, the development of enzymes can be expected not only to improve the productivity of livestock but also to improve the rearing environment.

As a related prior patent, Korean Patent Publication No. 1020070050873 relates to 'Feed additive containing alkaloid, livestock feed comprising the same and its use', and the feed or feed additive is preferably a protopene alkaloid, in particular α-allocriptopin. For example, it is described in combination with one or more benzophenandridine alkaloids in an effective amount as a growth promoter and appetite enhancer for livestock livestock.

In another related prior art, Korean Patent Publication No. 1020010032381 relates to a 'pectin degrading enzyme obtained from Bacillus rickeniformis', which has an optimal activity at a pH higher than 8, or preferably arranged Pectin degrading enzymes derived from or endogenous to other Bacillus species that are at least 99% homologous to Bacillus rickeniformis based on a 16S rDNA sequence are described.

The present invention has been made in view of the above necessity, and an object of the present invention is to provide a novel strain.

Another object of the present invention is to provide novel strain derived enzymes as potential feed additives.

Another object of the present invention is to provide an optimization of solid fermentation for the enzyme production.

The present invention for achieving the above object is a Bacillus (Bacillus) sp. Provides LX-1.

In one embodiment of the present invention, the Bacillus strain preferably has a 16S rDNA sequence of SEQ ID NO: 1, but is not limited thereto.

The present invention also provides an enzyme composition comprising α-galactosidase having resistance to trypsin, pronase, or pancreatin derived from the strain of the present invention as an active ingredient.

The present invention also provides a method of hydrolyzing raffinose and stachyose present in soybean meal by treating soybean meal with α-galactosidase derived from the strain of the present invention.

In another aspect, the present invention provides a method for producing α-galactosidase by inoculating the strain of the present invention in a solid medium using soybean meal.

In another aspect, the present invention provides a feed additive composition comprising the strain of the present invention or α-galactosidase derived from the strain as an active ingredient.

Hereinafter, the present invention will be described.

The present invention is potentially applied to feed industry and animal agriculture through the characterization of a novel micro-alpha-galactosidase from polar microorganisms capable of degrading α-galactooligosaccharides acting as an anti-nutritive factor in soybean meal, a protein source widely used as feedstock. Bacillus sp. Solid fermentation was used to enhance LX-1 derived α-galactosidase production.

The present invention for the development of a potential feed additive Bacillus sp. Enzymatic properties of α-galactosidase derived from LX-1 were investigated. Extracellular α-galactosidase-producing strains were isolated from the Antarctic soil, and Bacillus sp. It was named LX-1. The optimum pH and temperature for LX-1 derived α-galactosidase activity were 7.0 and 40 ° C, respectively. LX-1-derived α-galactosidase specifically hydrolyzed α-galactoside such as p -nitrophenyl-α-D-galactopyranoside, melibiose, raffinose, and stachyose. Ag ⁺ , Hg ^{2 +} , Cu ^{2 +} , and SDS at 2 mM concentrations strongly inhibited α-galactosidase activity, while Co ^{2 +} , Fe ^{2 +} , and PMSF slightly decreased enzyme activity. In contrast, β-mercaptoethanol and EDTA had little effect on α-galactosidase activity. Moreover, in more industries, the resistance of protease to enzymes for the application of LX-1-derived α-galactosidase has been shown to be resistant to some proteases such as trypsin, pronase and pancreatin. When soybean meal was treated with LX-1-derived α-galactosidase, α-galactooligosaccharides such as raffinose and stachyose in the soybean meal were hydrolyzed at 37 ° C. The addition of 2% galactose, 1% peptone, and 0.001% MnSO ₄ in liquid fermentation (SmF) increased the production of LX-1-derived α-galactosidase, which was selected as the best carbon source, nitrogen source, and mineral among the tested materials, respectively. It became.

Second experiment of the present invention using solid fermentation (SSF) Bacillus sp. Optimal culture conditions for LX-1-derived α-galactosidase production were investigated. Cereals, soybean meal, and corn flour were used as solid substrates for enzyme production. Substrate-to-moisture ratio 1: 2-1: 3 (w / v), pH 8.0, incubation time of 72 hours, and 4% inoculation conditions in soybean meal showed maximum α-galactosidase activity. The addition of galactose, a carbon source, increased the production of α-galactosidase by 1.5-fold and increased the enzyme activity to 5.843 U / g when the addition of peptone or tryptone as a nitrogen source. The use of solid media with low water content to produce high concentrations of α-galactosidase may have the advantage of minimizing the risk of contamination. In addition, the use of small amounts of distilled water allows the extraction of enzymes, which significantly reduces waste disposal costs.

The majority of α-galactosidase isolated from the existing microorganisms is relatively inaccurate to decompose α-galactooligosaccharide substrates contained in soybean meal, a vegetable protein resource widely used as feedstock. Promiscuous substrate specificity. However, LX-1-derived α-galactosidase of the present invention exhibited a strict substrate specificity that exclusively degrades α-galactooligosaccharide (raffinose, stachyose) contained in soybean meal in substrate specificity, and trypsin , pronase and pancreatin) and smooth activity under conditions similar to those of the animal's intestinal environment. This suggests a potential for use as an additive for soybean meal processing in the feed industry.

Hereinafter, the present invention will be described in detail.

Strain Isolation and Identification

To investigate the identification and phylogenetic relationship of the isolated strain LX-1, a baseline of 1446 bp was determined from the 16S rRNA gene, and the phylogenetic tree was prepared, and the results are shown in FIG. 1. As a result of BLAST search of NCBI, the isolate LX-1 was the closest to Bacillus megaterium . Therefore, LX-1 is considered to be a species of Bacillus sp. As a result of comparison with Bacillus sp. Strains registered on GenBank using MEGA 4.0 program, LX-1 is the closest to Bacillus 99.4% homology with megaterium DSM 32T. Based on this, the strain isolated from the present invention is Bacillus sp. LX-1 was named, and the base sequence of 16S rRNA gene was identified as Accession No. 1 in GenBank database. It is registered as HQ660811.

Bacillus sp. LX-1 strain was deposited with KACC91695P on December 16, 2011 to the Institute of Agricultural Biotechnology, Suwon-si, Gyeonggi-do.

Bacillus sp . LX -1 derived α- galactosidase Production of

Bacillus sp. According to the incubation time. Growth and L-galactosidase productivity of LX-1 is shown in FIG. 2. Bacillus sp. LX-1 strains grew somewhat slowly until the first 36 hours of incubation, and then increased sharply thereafter, reaching a peak at 72 hours. The OD ₆₀₀ value was 10.23 ± 0.07. On the other hand, the enzyme activity increased rapidly until the first 28 hours of fermentation, after which it reached a stable phase and no longer increased. Maximum activity was obtained at 28 hours, and the enzyme activity was 0.465 ± 0.009 U / mL.

Bacillus sp., Which produces high concentrations of extracellular α-galactosidase. It is believed that there may be an advantage in using the LX-1 strain as an industrial strain.

Partial purification of enzymes and zymogram  analysis

　　 Bacilllus sp. Purification profiles of the culture supernatant and coenzyme solution of LX-1 are shown in Table 1. The specific activity of the culture supernatant was 0.35 ± 0.02 U / mg, and it was purified 1.46 ± 0.11 times by ammonium sulfate precipitation, and the yield was 59.77 ± 1.03%.

A zymogram was performed for qualitative analysis of alpha-galactosidase activity, and the results of the zymogram analysis are shown in FIG. 3. In the BSA-injected lane (lane 1) used as a negative control, blue bands did not appear, but in the lanes injected with the coenzyme solution (lane 2), X-α-Gal was decomposed to show a blue band. This is Bacillus sp. It suggests that LX-1 strain produced extracellular α-galactosidase.

Table 1 shows Bacillus sp. Summary table of partial purification profiles for extracellular α-galactosidase produced by LX-1. ^a unit (U) of α-galactosidase activity is defined as the amount of enzyme that produces 1 mmol of p- nitrophenol from the substrate for p -nitrophenyl-α-D-galactopyranoside (Sigma) per minute at 40 ° C and pH 6.0 do. This value is the mean ± standard deviation of three replicates.

pH  Α- according to the change galactosidase  activation

As a result of investigating the change of α-galactosidase activity according to pH, Bacillus sp. LX-1-derived α-galactosidase exhibited maximum activity at pH 7.0, and over 50% of maximum activity remained at pH 6.0-7.0. However, no activity was observed in the acidic pH range of 3.0-5.0.

Soymilk's pH ranges from neutral (pH 6.2-6.4), but as the pH decreases, soy protein precipitates and becomes sour, so acidic α-galactosidase is not suitable for hydrolysis of soymilk (Patil, AG). , K, PK, Mulimani, VH, Veeranagouda, Y., Lee, K. 2010. J. Microbiol. Biotechnol. 20: 1546-1554). Therefore, Bacillus sp. Since LX-1-derived α-galactosidase exhibits maximum activity at pH 7.0, it is considered to be suitable for hydrolysis of α-galactooligosaccharide present in soymilk. In addition, the animal's intestinal environment (pH 6.0-7.0) is expected to function as a feed additive without losing activity.

Α- with temperature change galactosidase  Active and thermal stability

Α-galactosidase activity results according to the temperature change are as shown in FIG. Maximum activity was seen at 40 ° C. and maintained at least 50% of maximum activity in the 25-45 ° C. range. However, the enzyme activity was inactivated at a high temperature above 50 ° C.

In order to investigate thermal stability, the crude enzyme solution was left in the range of 40-70 ° C. for 30 minutes and the residual activity was measured. As a result, about 88% of the activity was maintained at 45 ° C., but the enzyme activity was completely lost at a temperature higher than 50 ° C. ( Data not shown). Thermal stability of enzymes is an advantage in industrial applications because most industrial processes are performed at high temperatures which result in thermally labile enzyme denaturation. However, it is not preferred for the application of soy milk and other food processes (King, MR, Yernool, DA, Eveleigh, DE, Chassy, BM 1998. FEMS Microbiol. Lett. 163: 37-42). For example, fermentation of soy milk using lactic acid bacteria showing α-galactosidase activity is mainly carried out at 25-45 ° C (Yoon, MY, Hwang, HJ 2008. Reduction of soybean oligosaccharides and properties of α-D-galactosidase from Lactobacillus curvatus R08 and Leuconostoc mesenteriodes JK55. Food Microbiol. 25: 815-823). In addition, if the feed is first processed through a process such as pelleting and then mixed in the form of a liquid or powder product to feed the livestock, it may be possible to overcome the heat resistance problem.

Therefore, LX-1-derived α-galactosidase is suitable as an additive for soybean fermentation. In addition, the optimum activity temperature is within the typical range identified in the bacterial α-galactosidase, and is very similar to the intestinal temperature of the animal (37-40 ° C.) (Yoon, MY, Hwang, HJ 2008. Food Microbiol. 25: 815-823). Therefore, it is considered that industrial applications such as poultry or pig feed additives have possible advantages.

Substrate specificity

Substrate specificity of α-galactosidase derived from LX-1 was investigated using synthetic substrates, natural substrates, and saccharogenic substrates. The results are shown in Table 2. Synthetic substrate showed high specificity only for p -nitrophenyl-α-D-galactopyranoside and the enzyme activity was 1.622 ± 0.063 U / mL. In contrast, p -nitrophenyl-β-D-xylopyranoside, p -nitrophenyl-β-D-cellobioside, p -nitrophenyl-α-D-glucopyranoside, o -nitrophenyl-β-D-glucopyranoside and p -nitrophenyl-α-L Other synthetic substrates, such as -arabinofuranoside, did not hydrolyze at all. This may be because LX-1 derived α-galactosidase has specificity for galactose residue.

In the α-galactooligosaccharides such as melibiose, raffinose and stachyose, melibiose was most effectively hydrolyzed, followed by raffinose and stachyose. At this time, the enzyme activities were 1.384 ± 0.073 U / mL, 14.563 ± 0.146 U / mL, and 0.777 ± 0.064 U / mL, respectively. The lack of enzymatic activity for lactose (galactose-β-1,4-glucose) suggests that LX-1-derived α-galactosidase has specificity for anomer carbon.

In addition, LX-1-derived α-galactosidase did not hydrolyze polysaccharides such as CMC, xylan, locust bean gum (galactomannan) and starch. α-galactosidase is classified into family 36 group and family 27 group according to substrate specificity. The family 36 group has specificity only for low molecular weight α-galactosides such as p NPGal, melibiose and oligosaccharides, while the family 27 group has specificity for galactomannan as well as low molecular weight α-galactoside. Therefore, LX-1-derived α-galactosidase, which has no hydrolytic capacity against galactomannan, appears to belong to the family 36 group.

Table 2 shows Bacillus sp. Table shows the substrate specificity of LX-1 derived α-galactosidase.

Data are shown as mean and standard deviation from three replicates, with ND, meaning not detected

To investigate the effects of enzymes on metal ions and chemicals, each metal ion and chemical were added to the reaction solution containing p -nitrophenyl-α-D-galactopyranoside ( p NPG) to a final concentration of 2 mM. Ag ⁺ as in the result of measuring the enzyme activity, and Table 3, Hg ^{+ 2,} was inhibited strongly the enzymatic activity by a metal such as Cu ^{+ 2.}

In some α-galactosidase sulfhydryl reactive metal ions such as Ag ^+, Hg ^{2 +} have been reported to thiol-specific inhibitor to interfere with the interaction of the substrate and enzyme in combination with the thiol group of a cysteine residue present in the active site (Cao, Y., Yang, P., Shi, P., Wang, Y., Luo, H., Meng, K., Zhang, Z., Wu, N., Yao, B., Fan, Y. 2007. Enz Microbial.Tech. 41: 835-841). Therefore, cysteine residues appear to be present near the active site of LX-1-derived α-galactosidase. Copper, which inhibits the activity of LX-1-derived α-galactosidase, is an essential mineral and is added to feed to reduce livestock growth and lower cholesterol (Bakalli, RI, Pesti, GM, Ragland, WL, Konjufca, V. 1995). Poult.Sci. 74: 360-365). In particular, the addition of copper to pig feed has the effect of preventing anemia (Osaki, S., Johnson, DA, Frieden, E. 1996. J. Biol. Chem. 241: 2746-2750). However, due to environmental pollution, the allowable amount of copper in feed decreased from less than 135 ppm in piglets and weaned pigs to less than 135 ppm, from 130 ppm and 60 ppm in early and late piglets, respectively, and below 25 ppm in finishing pigs and pigs (Kang, JM, Cho, SB, Kim, SK, Lee, SS, Lee, SK 2010. J. Life Sci. 20: 717-722). Therefore, since the concentration of copper (2 mM) used in the present invention is 135 ppm level allowed in the feed for pigs, it may be determined that the problem of inhibiting the enzyme activity may be partially solved in the late feed, finishing and pig feed.

Enzyme of the present invention inhibited the Zn ^{+ 2} and Ni ^{+ 2} were each reduced enzyme activity in the presence ^{48%, 63%, Co 2} + and Fe ^{2 +} is 21% and 19% of enzyme activity, respectively. However, ^{Ca 2 +, Mg 2+, Mn} 2 +, Ba 2 +, K +, the change in enzyme activity was hardly appear under Na ⁺ present. Zn ^{2 +,} Ni ^{2 +,} transition metals such as Co ^{2 +,} Fe ^{2 +} with a tendency for the oxidation of sulfhydryl group has the effect of inhibiting the α-galactosidase activity.

In addition, LX-1-derived α-galactosidase was strongly inhibited by SDS, an anionic surfactant, and partially inhibited by PMSF (phenylmethylsulfonyl fluoride), a serine protease inhibitor. This resulted in the loss of function due to the complete or partial destruction of α-galactosidase by tertiary or partial structure of α-galactosidase.

Β-mercaptoethanol, which breaks disulfide bonds, and EDTA (ethylenediaminetetraacetic acid), a chelating agent, have little effect on enzyme activity. LX-1-derived α-galactosidase does not have disulfide bonds in the active site and releases metal ions during enzyme activity. It is not considered to be a metalloenzyme.

Table 3 shows the effects of metal ions and compounds on LX-1 α-galactosidase activity.

Alpha - galactosidase  Effect of Protease on Activity

The resistance of LX-1 derived α-galactosidase to protease is shown in FIG. 6. Enzyme activity was completely inhibited by proteinase K and 97% by subtilisin carlsberg. On the other hand, trypsin, pronase and pancreatin treatments showed 94.7%, 93% and 98.7% of enzyme activity, respectively. Earlier reports showed that Rhizopus sp. F78 ACCC 30795 and Streptomyces sp. 27-derived α-galactosidase inhibited the enzyme activity by 30% and 50% by trypsin treatment (Cao, Y., Yang, P., Shi, P., Wang, Y., Luo, H., Meng, K). , Zhang, Z., Wu, N., Yao, B., Fan, Y. 2007. Enz. Microbial.Tech. 41: 835-841).

Recently, α-galactosidase and proteolytic enzymes are often added together as additives in the food and animal feed industries to remove anti-nutritive factors to improve digestibility and increase nutritional value. In the pulp and paper industry, α-galactosidase is also used with other enzymes including proteases (Cao, Y., Yang, P., Shi, P., Wang, Y., Luo, H., Meng, K., Zhang, Z., Wu, N., Yao, B., Fan, Y. 2007. Enz. Microbial.Tech. 41: 835-841). Therefore, the resistance of enzymes to proteolytic enzymes is an important criterion for use as an industrial enzyme (Liu, X., Meng, K., Wang, Y., Shi, P., Yuan, T., Yang, P. , Luo, H., Bai, Y., Yao, B. 2009. GWorld.J. Microbiol.Biotechnol. 25: 1633-1642). It must also be unaffected by the intestinal protease in animals to function as a feed additive. Therefore, LX-1-derived α-galactosidase, which has strong resistance to trypsin, pronase and pancreatin, appears to have attractive characteristics in industrial applications.

Soybean meal  Within α- galactooligosaccharide Decomposition of

In order to investigate the hydrolysis capacity of α-galactosidase derived from LX-1 to α-galactooligosaccharide in soybean meal, the amount of free galactose was measured by adding coenzyme solution to soybean meal, and the results are shown in FIG. 7.

Galactose was not released at 0 hours, and 0.650 ± 0.013 μmole / g of galactose was released after 1 hour of culture. The amount of galactose released gradually increased to 4.906 ± 0.049 μmole / g after 9 hours of culture. This shows that LX-1 derived α-galactosidase can hydrolyze α-galactooligosaccharides present in soybean meal even in the intestinal environment of animals (37-40 ℃).

Falkoski et al. (2006) reported soybean seed, Aspergillus terreus , Penicillium Degradation of α-galactooligosaccharides in soy molasses and soy flour by griseoroseum-derived α-galactosidase treatment was reported. Gote et al. (2004) also reported Bacillus against α-galactooligosaccharide in soymilk. The hydrolytic capacity of α-galactosidase derived from stearothermophilus was reported. To enhance the nutritional value of soy foods, microbial or plant-derived α-galactosidase can be used, but microorganism-derived α-galactosidase is generally used. However, microbial-derived enzymes have problems with stability, and soy-derived enzymes have a small amount and are difficult to purify (Falkoski, DL, Guimaraes, VM, Callegari, CM, Reis, AP, De, Barros.EG) , De, Rezende.ST 2006. J. Agric.Food Chem. 54: 10184-10190).

Therefore, Bacillus sp. Studies on cloning genes encoding LX-1-derived α-galactosidase and building a recombinant overexpression system are necessary.

Alpha - galactosidase  For production Carbonic  effect

The effect of carbon source on alpha-galactosidase production is shown in FIG. In the basal medium, enzyme production was not achieved. When 1% and 2% of the wheat flour were added, enzyme production was induced but showed extremely low productivity. Enzyme activity was 0.008 U / mL and 0.011 U / mL, respectively. On the other hand, the media added with 1% galactose showed 0.168 U / mL enzyme activity, and the treatment with 2% galactose added 0.558 U / mL, which was about 3.3 times higher than that with 1% galactose. -galactosidase production was shown.

Carbon sources are one of the most important factors in the production of enzymes derived from microorganisms, and the type and concentration of carbon sources are known as nutritional factors that regulate the synthesis of α-galactosidase from bacteria. Carbon sources such as galactose, melibiose and raffinose are Lactobacillus fermentum and Bacillus While stearothermophilus is known to be a significant factor in the production of α-galactosidase derived from NCIM 5146, glucose has been reported to inhibit α-galactosidase production due to catabolite repression (Garro, MS, Valdez, GF, Oliver, G). Giori, GS 1996. Curr. Microbiol. 33: 302-305. Yoon and Hwang (2008), on the other hand, were Lactobacillus curvatus R08 and Leuconostoc. It was reported that mesenteriodes JK55 inhibited the production of α-galactosidase by galactose. This may be because galactose is an inappropriate substrate due to the lack of galactokinase activity in most lactic acid bacteria.

Cereals are often used as a carbon source because they are inexpensive and contain growth factors. Liu et al (Liu, CQ, Ruan, H. , Shen, H., Chen, QH, Zhou, B., Li, Y., He, G. 2007. J. Food Sci 72:. 120-125) is Aspergillus It was reported that foetidus ZU-G1-derived α-galactosidase increased the productivity by wheat vegetation , but in this experiment, wheat vegetation did not affect α-galactosidase production.

Therefore, in the present invention, Bacillus sp. Α-galactosidase biosynthesis by LX-1 seems to be closely correlated with the concentration of galactose and galactose.

Alpha - galactosidase  Effect of Nitrogen Sources on Production

To investigate the effect of nitrogen source on the production of alpha-galactosidase, enzyme activity was measured by adding 1% organic nitrogen source and inorganic nitrogen source to 2% galactose-fixed culture medium which showed good effect in the experiment of carbon source effect. The effect of nitrogen sources on α-galactosidase production is shown in FIG. 9.

Since only one component of yeast extract and tryptone was excluded, α-galactosidase production was not induced, so 0.5% yeast extract and 1% tryptone were added to the basal medium (data not shown). In basal medium containing 0.5% yeast extract and 1% tryptone, the enzyme activity was 0.667 U / mL. However, the addition of 1% yeast extract to basal medium inhibited the production of α-galactosidase with 0.430 U / mL of enzyme activity. The addition of 1% tryptone did not affect enzyme production at 0.642 U / mL, which is similar to the control. It did not appear to give. When 1% of soybean meal was added, enzyme production was strongly inhibited at 0.064 U / mL, which was about 10 times lower than that of the control, and 0.403 U / mL was lower than that of the control when the ammonium sulfate, an inorganic nitrogen source, was added. On the other hand, when 1% peptone was added, α-galactosidase production tended to increase to 0.856 U / ml, which is 1.2 times higher than the control.

Therefore, in the present invention, organic nitrogen sources such as yeast extract, tryptone, and peptone are Bacillus sp. It is considered to be an important nitrogen source to increase LX-1-derived α-galactosidase production.

Alpha - galactosidase Effect of Essential Elements on Production

In addition to carbon and nitrogen sources, essential elements such as magnesium and calcium, or trace elements such as copper, iron, manganese and zinc are required to maintain cellular functions. However, there is little information on the effect of essential elements on α-galactosidase production (Shivam, K., Tripathi, CPM, Mishra, SK 2009. Culture conditions for the production of α-galactosidase by Aspergillus parasiticus MTCC 2796: a novel source. Electronic. J. Biotechnol. 12: 1-9). Therefore, in the present invention, in order to investigate the effect of essential elements on the production of α-galactosidase, the control material in which the best carbon source 2% galactose and the best nitrogen source 0.5% yeast extract, 1% tryptone, 1% peptone was fixed Enzyme activity was measured by adding each essential element. Bacillus sp. The effect of essential elements on LX-1 derived α-galactosidase production is shown in FIG. 10.

Enzyme activity was 0.312 U / mL in the control group containing only carbon and nitrogen sources. The production medium added 0.07% KH ₂ PO ₄ showed little difference from the control, and the addition of 0.01% MgSO ₄ showed 0.256 U / mL of the enzyme activity, which was slightly lower than the control. NaCl, CaCl ₂ and FeSO ₄ slightly increased α-galactosidase production but did not appear to have a significant effect on enzyme production. Enzyme activity was 0.4 U / mL, 0.437 U / mL and 0.415 U / mL, respectively. On the other hand, medium with 0.001% MnSO ₄ showed the highest α-galactosidase production, 0.901 U / mL, which was 2.9 times higher than the control.

Therefore, it is determined that the present invention, the α-galactosidase was selected for Mn ^{+ 2,} an essential element to the element for the optimization of the production medium components, the optimization of the production medium must be made primarily to increase the α-galactosidase production. However, the one factor at a time method does not consider the interactions between factors, and thus has a limit in obtaining an optimal result. Therefore, in order to analyze the effects on the media components found by the one factor at a time method, further experiments to find the optimal conditions using the response surface method are necessary.

Alpha - galactosidase  Screening of Solid Substrates for Production

In solid fermentation, the selection of a suitable solid substrate for the fermentation process is an important factor, which involves the selection of many agricultural raw materials for the growth and production of microorganisms (Selvakumar and Pandey, 1999). Bacillus sp. The effect of substrate on LX-1-derived α-galactosidase production is shown in Table 6.

Cereals and corn flour induced low α-galactosidase production, and enzyme activity was 0.014 ± 0.001 U / g and 0.012 ± 0.002 U / g, respectively. Soybean meal, on the other hand, induced an enzyme production of 0.529 ± 0.018 U / g, about 44 times higher than other substrates. It is believed that abundant α-galactooligosaccharides present in soybean meal induce α-galactosidase synthesis. Therefore, soybean meal was used as a substrate for α-galactosidase production in a later experiment.

 In solid fermentation, the price and availability of substrates play an important role in enzyme production, so the selection of farmers' products is worth investigating. Since soybean meal is readily available as a carbon and nitrogen source, it is very attractive to develop α-galactosidase production using soybean meal as a solid substrate.

Therefore, Bacillus sp. The use of solid fermentation for the production of α-galactosidase derived from LX-1 seems to have an economical advantage over the use of purified oligosaccharides for fermentation since the soybean meal is used directly as a solid substrate.

Table 4 shows Bacillus sp. Table shows the synthesis of Alpha-galactosidase by LX-1. ^a unit (U) of α-galactosidase activity is defined as the amount of enzyme that produces 1 mmol of p -nitrophenol from the substrate for p -nitrophenyl-α-D-galactopyranoside (Sigma) per minute at 30 ° C and pH 6.0 do. This value is the mean ± standard deviation of three replicates.

Alpha - galactosidase  Effect of Water Content on Production

The effect of water content on α-galactosidase production in solid fermentation is as shown in FIG. The soybean meal and water content ranged from 1: 2 to 1: 3 (w / v) and showed the maximum α-galactosidase production. The enzyme activity was 3.805 ± 0.038 U / g. Soybean meal and moisture ratios were not significantly different in α-galactosidase production from 1: 1.5 to 1: 4 (w / v).

Unlike the results of this study, Anisha et al. (Anisha, GS, Rojan, PJ, Nicemol, J., Niladevi, KN, Prema, P. 2008. Food Chem. 111: 631-635) had a maximum content of 40%. Α-galactosidase production was reported, Liu et al. (Liu, CQ, Chen, QH, Cheng, QJ, Wang, JL, He, GQ 2007. J. Zhejiang Univ. Sci. B. 8: 371-376) Reported maximum α-galactosidase activity at 60% moisture content. This may be because the type of substrate used, the culture temperature, and the type of strain are different. In solid fermentation, water content is generally considered as a fundamental factor for microbial growth, metabolite formation and secretion, and is known to affect hydrolytic enzyme production (Battalino, RA, Huergo, M., Pilosuf, AMR). , Bartholomai, GB 1991. Appl. Microbiol. Biotechnol. 35: 292-296). This may be because water content affects the physical properties of the substrate.

Low moisture content is advantageous because it reduces the likelihood of contamination in fermentation broth. However, less than the optimum content reduces the solubility and availability of nutrients. It also reduces the swelling of the substrate and increases the water tension (Archana, A., Satyanarayana, T. 1997. Xylanase production by thermophilic Bacillus lichenifomis A99 in solid-state fermentation. Enz. Microbial. Tech. 21: 12-17). On the other hand, if the moisture content is higher than the optimum content, the decrease in porosity, the change in the structure of the substrate particles, and the development of adhesion occur, resulting in low oxygen supply and heat dissipation (Krishna, C. 2005. Solid-state fermentation system- An overview.Crit. Rev. Biotechnol. 25: 1-30).

In the case of solid fermentation, the water content of the substrate depends on the water activity of the substrate. As a result, it depends on the nature of the substrate that binds to water (Krishna, C. 2005. Solid-state fermentation system-An overview. Crit. Rev. Biotechnol. 25: 1-30). Substrate such as soybean meal has a very high water activity, so even if the water content is slightly increased when the inorganic salt solution is added, aggregation of the substrate occurs (Anisha et al., 2008).

Therefore, the optimal water content to be added to the substrate should be considered when using solid fermentation. Since LX-1-derived α-galactosidase is synthesized even with a small water content, it is expected to minimize contamination and reduce waste disposal costs.

Α- according to incubation time galactosidase  production

Incubation time and Bacillus sp. The relationship between LX-1 derived α-galactosidase productivity is shown in FIG. 12. LX-1-derived α-galactosidase showed low productivity up to 24 hours at the beginning of fermentation and then increased rapidly from then to 72 hours. Maximum productivity was obtained at 72 hours, and the enzyme activity was 3.218 ± 0.003 U / g. After 72 hours it reached a plateau and no longer increased.

Liu (Liu, CQ, Chen, QH, Cheng, QJ, Wang, JL, He, GQ 2007. J. Zhejiang Univ. Sci. B. 8: 371-376) and Anisha et al. (Anisha, GS, Rojan, PJ, Nicemol, J., Niladevi, KN, Prema, P. 2008. Food Chem. 111: 631-635), Aspergillus foetidus ZU-G1 and Streptomyces It was reported that griseoloalbus- derived α-galactosidase showed the maximum productivity at 144 and 96 hours, respectively.

Maximum enzyme production does not occur until sufficient biomass is formed, and furthermore, long incubation time is not effective to improve enzyme productivity (Liu, CQ, Chen, QH, Cheng, QJ, Wang, JL, He, GQ 2007. J. Zhejiang Univ. Sci. B. 8: 371-376). Therefore, Bacillus sp. Showed maximum α-galactosidase productivity in a short incubation time. The LX-1 strain is believed to have the advantage of being used as an industrial strain.

Alpha - galactosidase  Initial badge for production pH Influence of

In general, since the substrate used for solid fermentation has a low buffering capacity, the pH of the medium may change during the fermentation. Therefore, the initial pH of the medium is an important factor affecting the growth and enzyme production of microorganisms (Mahanta, N., Gupta, A., Khare, S. K. 2008. Bioresour. Technol. 99: 1729-1735). The effect of medium pH on LX-1 derived α-galactosidase production is as shown in FIG. 13.

Α-galactosidase synthesis increased as the initial pH of the medium increased, showing maximum productivity at pH 8.0. At this time, the enzyme activity was 6.874 ± 0.204 U / g. Assuming 100% relative activity at pH 8.0, LX-1-derived α-galactosidase showed more than 55% of its activity in a wide range of pH 6.5-8.5, and enzyme activity decreased rapidly above pH 8.5. The high activity in a wide range of pH is believed to be due to the excellent buffering capacity of solid substrates, and the change in enzyme production according to pH is believed to be due to the growth of bacteria controlled by pH.

Liu et al. (Liu, CQ, Chen, QH, Cheng, QJ, Wang, JL, He, GQ 2007. J. Zhejiang Univ. Sci. B. 8: 371-376) had an initial pH of pH 5.0-6.0 days. When Aspergillus It was reported that the largest α-galactosidase was produced from foetidus ZU-G1, and Shankar and Mulimani (Shankar, SK, Mulimani, VH 2007. Bioresour. Technol. 98: 958-961) were identified as Aspergillus at pH 5.5. It was reported that oryzae- derived α-galactosidase showed the highest productivity. Wang et al. (Wang, CL, Li, DF, Lu, WQ, Wang, YH, Lai, CH 2004. Lett. Appl. Microbiol. 39: 369-375) also described Penicillium sp. Derived α-galactosidase was reported to be maximally synthesized at pH 5.5. This is probably because most fungi and fungi grow well at pH in the acid range.

Most bacteria and fungi cannot grow well under alkaline conditions (Danesh, A., Mamo, G., Mattiasson, B. 2011. Production of haloduracin by Bacillus halodurans using solid-state fermentation.Biotechnol. Lett. 33: 1339- 1344). Therefore, from an industrial application point of view, Bacillus sp., Which exhibits the highest α-galactosidase productivity at pH, a weak alkaline condition. LX-1 strains are likely to provide sufficient benefits to reduce the risk of contamination.

Alpha - galactosidase  Influence of Inoculation on Production

Bacillus sp. The results of the effect of the inoculum on LX-1 derived α-galactosidase production are shown in FIG. 14. Α-galactosidase synthesis gradually increased as the inoculation amount increased. However, the enzyme activity decreased from 4% or more. At 4% inoculation, α-galactosidase synthesis peaked and the maximum enzyme activity was 3.007 ± 0.021 U / g.

Proper inoculation reduces contamination of other organisms by increasing microbial growth and product formation (Hashemi, M., Shojaosadati, SA, Razavi, SH, Mousavi, SM 2011. Iranian. J. Biotechnol. 9: 188-196 ). However, higher levels of inoculum can produce too much biomass and deplete the nutrients necessary for product formation, so the imbalance between nutrients and biomass reduces enzyme synthesis (Ramachandran, S., Roopesh, K., Nampoothiri, KM, Szakacs , G., Pandey, A. 2005. Process Biochem. 40: 1749-1754). In addition, even though the water holding capacity of the medium increases, heat transfer becomes difficult (Wang, CL, Li, DF, Lu, WQ, Wang, YH, Lai, CH 2004. Lett. Appl. Microbiol. 39: 369- 375). On the other hand, small amounts of inoculation can result in insufficient biomass, causing undesirable growth of the organism, and also slowing down the growth of the inoculation strain, thereby reducing enzyme productivity (Liu, CQ, Chen, QH, Cheng, QJ). Wang, JL, He, GQ 2007. J. Zhejiang Univ. Sci. B. 8: 371-376).

Therefore, studies on the determination of optimal inoculation conditions provide useful information to increase enzyme production. Therefore, this study increased LX-1-derived α-galactosidase production by determining the optimal inoculation necessary to improve enzyme productivity.

Α- in solid fermentation galactosidase  For production Carbon source  Effect of addition

The effect of carbon source addition on α-galactosidase production in solid fermentation is shown in Figure 15. α-galactosidase production increased with the addition of galactose and lactose, and showed the highest productivity with the addition of galactose. The enzyme activity was 4.898 ± 0.147 U / g. This is consistent with the increase of LX-1-derived α-galactosidase production by galactose in liquid fermentation. Although lactose, known as the inducer of β-galactosidase, has β-1,4-galactoside binding, many researchers It was used as an inducer of α-galactosidase (Srinivas, MRS, Chand, N., Losane, BK 1994. Bioprocess Engineering 10: 139-144). On the other hand, treatment with maltose and sucrose decreased 12% and 10%, respectively, and treatment with glucose decreased α-galactosidase production by more than 55%. This suggests that LX-1-derived α-galactosidase production is inhibited by catabolism.

But Ramesh and Lonsane (Ramesh, MV, Lonsane, BK 1991. Appl. Microbiol. Biotechnol. 35: 591-593) and Viniegra-Gonzalez and Favela-Torres (Viniegra-Gonzalez, G., Favela-Torres, E. 2006. 44.397-406) reported that solid fermentation is resistant to catabolism at high sugar concentrations. This seems to be because the permeability of microorganisms growing on the surface of the solid substrate changes. In contrast, the presence of monosaccharides in liquid fermentation strongly inhibits the productivity of inducible enzymes.

Although the soybean meal used as a substrate contains abundant α-galactooligosaccharides that induce α-galactosidase production, LX-1-derived α-galactosidase improved productivity by adding galactose and lactose. The results of this study are expected to enable an easier approach in improving the productivity of α-galactosidase derived from LX-1, and further experiments on the effects of galactose and lactose concentrations on enzyme production are needed.

Α- in solid fermentation galactosidase  Effect of Nitrogen Source Addition on Production

16 shows the effect of the addition of nitrogen source in solid fermentation on LX-1-derived α-galactosidase production. Yeast extract did not affect the production of α-galactosidase, but tryptone and peptone increased the production of α-galactosidase by about 1.8 times. Enzyme activity at this time was 5.816 ± 0.061 U / g and 5.843 ± 0.087 U / g, respectively. On the other hand, the inorganic nitrogen sources ammonium sulfate and sodium nitrate rather reduced α-galactosidase production. This may be because the growth of microorganisms is better when the organic nitrogen source is added than the inorganic nitrogen source.

Srinivas et al. (Srinivas, MRS, Chand, N., Losane, BK 1994. Bioprocess Engineering 10: 139-144) were used to treat Aspergillus by corn steep liquor in solid fermentation. α-galactosidase production was increased from niger MRSS 234, and corn steep liquor has been reported to be used for the industrial production of many enzymes and metabolites. In general, the best nitrogen source to induce α-galactosidase synthesis is soybean meal.

In the present invention, the addition of tryptone and peptone, which is an organic nitrogen source to soybean meal, further improved α-galactosidase production. The study of the effects of nitrogen sources on the production of α-galactosidase using solid fermentation is extremely rare and incomparable with the results of the present invention. Therefore, further experiments on the production of α-galactosidase according to the nitrogen source concentration are necessary to improve the production of LX-1-derived α-galactosidase, which is a potential feed additive.

The majority of α-galactosidase isolated from the existing microorganisms is relatively inaccurate to decompose α-galactooligosaccharide substrates contained in soybean meal, a vegetable protein resource widely used as feedstock. Although it showed promiscuous substrate specificity, the LX-1-derived α-galactosidase of the present invention has a strict substrate specificity that exclusively degrades α-galactooligosaccharide (raffinose, stachyose) contained in soybean meal in substrate specificity. It is characterized by resistance to proteases (trypsin, pronase and pancreatin) and smooth activity under conditions similar to those of the intestinal environment of animals. This suggests a potential for use as an additive for soybean meal processing in the feed industry. It can be used as a future industrial feed additive that can reduce the cost of feed production by replacing soybean meal, which is an expensive protein resource, by enhancing the energy price of low-availability feedstocks (palm, palm, and lupine seeds). It seems to be. Bacillus sp. The results of solid fermentation for the production of extracellular α-galactosidase from LX-1 suggested the possibility of reducing the overall cost of enzyme production by providing basic information on the expansion to industrial scale.

1 is Bacillus. Other strain types and B acillus sp. Phylogenetic bootstrap values (16 trials and values> 50%) of 16S rRNA of LX-1 are shown in the node. GenBank accession numbers in parentheses. Bar , 5-base substitution / 1,000 nucleotide position. B. stands for Bacillus .,
2 is Bacillus sp. Α-galactosidase by LX-1 and time balance of cell growth. The symbol represents α-galactosidase activity (filled square with dotted line) and growth (filled triangle with solid line). Data are expressed as mean and standard deviation from three iterations,
3 shows a Zymogram analysis of α-galactosidase activity in enzyme preparation. Lane 1, Bovine serum albumin (negative control); Lane 2, the LX-1 α-galactosidase
4 is Bacillus sp. PH effect on the activity of α-galactosidase derived from LX-1. Data are expressed as mean and standard deviation from three iterations,
5 is Bacillus sp. Shows the temperature effect on the activity of α-galactosidase derived from LX-1. Data are expressed as mean and standard deviation from three iterations,
Figure 6 shows the protease effect on the activity of partially purified α-galactosidase. Shown as 100% of the activity of the untreated control. Data is expressed as mean and standard deviation from three replicates, N. A: No activity.
7 is Bacillus sp. Shows the amount of galactose released during α-galactooligosaccharide hydrolysis of soybean meal by LX-1, data expressed in mean and standard deviation with three replicates, N. A: No activity.
8 is Bacillus sp. Shows the effect of carbon source on α-galactooligosaccharide production by LX-1, basal medium was 0.5% yeast extract, 1% tryptone, 1% NaCl, 0.01% CaCl ₂ 2H ₂ O, 0.01% MgSO ₄ 7H ₂ O, 0.07 % KH ₂ PO ₄ , 0.001% MnSO ₄ 4H ₂ O, and 0.001% FeSO ₄ 7H ₂ O (pH 7.0). Data is expressed as mean and standard deviation from three replicates, N. A: No activity.
9 is Bacillus sp. Effect of nitrogen source on α-galactooligosaccharide production by LX-1, basal medium was 2% galactose, 0.5% yeast extract, 1% tryptone, 1% NaCl, 0.01% CaCl ₂ 2H ₂ O, 0.01% MgSO ₄ 7H ₂ O, 0.07% KH ₂ PO ₄ , 0.001% MnSO ₄ 4H ₂ O, and 0.001% FeSO ₄ 7H ₂ O (pH 7.0). Data is expressed as mean and standard deviation from three replicates, N. A: No activity.
10 is Bacillus sp. Effect of essential elements on α-galactooligosaccharide production by LX-1, basal medium consists of 2% galactose, 0.5% yeast extract, 1% tryptone, 1% peptone (pH 7.0). Data are expressed as mean and standard deviation from three iterations,
11 shows Bacillus sp. In SSF (temperature: 28 ° C., incubation period: 96 h). Shows the effect of the ratio of substrate to moisturizing agent on α-galactooligosaccharide production by LX-1, data expressed in mean and standard deviation from three replicates,
12 shows Bacillus sp. In SSF using soybean meal as substrate (temperature: 28 ° C., substrate vs. wetting agent: 1: 3). Shows the time course of α-galactooligosaccharide production by LX-1; data are expressed as mean and standard deviation from three replicates,
FIG. 13 shows the effect of initial pH of fermentation medium on α-galactooligosaccharide production at SSF (temperature: 28 ° C., substrate to wetting agent: 1: 3, incubation period: 72 h); Expressed as standard deviation,
FIG. 14 shows Bacillus sp. In SSF (temperature: 28 ° C., substrate vs. humectant: 1: 3, incubation period: 72 h). Shows the effect of inoculation size on α-galactooligosaccharide production by LX-1; data are expressed as mean and standard deviation from three replicates,
15 is Bacillus sp. Shows the effect of several carbon sources on α-galactooligosaccharide production by LX-1, adopting fermentation medium without carbon as a control (temperature: 28 ° C, substrate to wetting agent: 1: 3, incubation period: 72 h) Expressed as mean and standard deviation by three iterations,
16 is Bacillus sp. Shows the effect of several nitrogen sources on α-galactooligosaccharide production by LX-1, adopting fermentation medium without nitrogen as a control (temperature: 28 ° C, substrate to wetting agent: 1: 3, incubation period: 72 h) Expressed as mean and standard deviation of three replicates.

The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.

Example  One: Bacillus sp . LX -1 derived α- galactosidase of Enzymatic  Characterization

Securing Polar Soil Resources Microorganisms

In cooperation with the Korea Polar Research Institute (KOPRI) of the Korea Institute of Ocean Research and Development, KFRI received microbial colonies that were cultured and separated from organic soil near the area where flowering plants around Antarctic Sejong Station grew. The microbial colonies received were used as samples for microbial separation to produce α-galactosidase of the present invention.

Alpha - galactosidase  Screening and Pure Separation of Production Strains

The inoculated fungi were inoculated into Luria Bertani (LB) containing 0.2% lactose [1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.2] medium and 220 rpm for 4 days in a 28 ° C shaking incubator. Shake culture at a rate of. After confirming the growth of the cells, the sample was taken and serial dilutions were made. The samples were diluted in 1 × 10 ⁰ ~ 1 × 10 ^-6 and 0.2% lactose and 5-bromo-4-chloro-3 at 32 ㎍ / ml Stained blue strains were plated on LB plate medium containing -indolyl-α-D-galactopyranoside (X-α-Gal, Sigma) and incubated in a 28 ° C. incubator for 2 days. The colonies of the isolated strains were purely separated by three passages in a streaking method on the LB plate medium as described above.

Taxonomic Identification of Isolated Strains

Strains were identified through 16S rRNA gene sequencing analysis of isolated bacteria. For 16S rRNA gene sequencing of the strain, genomic DNA was extracted from the isolate using FastDNA kit (Qbiogene) and then polymerase chain reaction (PCR) was performed to amplify the 16S rRNA gene. Primers for PCR were 27F (5'-AGAGTTTGATCCTGGCTCAG-3 ') and 1492R (5'-GGTTACCTTGTTACGACTT-3') (William, GW, Susan, MB, Dale, AP, David, JL 1991. J. Bacteriol 173: 697-703. The amplified fragment was sequenced using an ABI PRISM 3730 XL DNA analyzer (Applied Biosystems). The determined nucleotide sequence was subjected to homology test using the BLAST program on the information registered in the NCB GenBank database. Lineage analysis was sorted using the CLUSTAL W program (Thompson, JD, Higgins, DG, Gibson, TJ 1994. Nucleic Acids Res. 22: 4673-4680). Genetic distance between sequences was calculated using the Maximum Composite Likelihood method and 16S rRNA gene sequence information of Bacillus strains registered in GenBank database, and phylogenic tree was drawn using MEGA 4.0 program (Tamura, K., Nei). , M., Kumar, S. 2004. Proc. Nat. Aca. Sci. USA. 101: 11030-11035).

Strain Growth and α- galactosidase Production of

In order to investigate the growth and enzyme production of the strain, 200 ml of enzyme production medium (1% galactose, 1% tryptone, 0.5% yeast extract, 1% NaCl) was made in 1 L Erlenmeyer flask, 1% inoculation and sampling while shaking culture at 220 rpm in a 28 ℃ shaking incubator. The degree of growth of the strain was measured by using a spectrophotometer to measure the absorbance of the culture at 600 nm, the supernatant obtained by centrifugation (12,000 rpm, 5 min) to the enzyme activity was used as the crude enzyme solution.

Crude enzyme solution  Produce

Pure inoculated colonies were inoculated in LB medium containing 0.2% lactose, shaken and cultured at a speed of 220 rpm for 48 hours in a 28 ° C shake incubator, followed by centrifugation at 5,000 rpm for 20 minutes at low temperature (4 ° C). Got it. Then, 75% ammonium sulfate was added to the culture supernatant, stirred for 6 hours at a constant rate in a magnetic stirrer, and centrifuged again (10,000 × g; 30 min; 4 ° C.) to obtain a precipitate. After that, the precipitate was dissolved in 25 mM Tris-HCl buffer (pH 8.0) and dialyzed in the same buffer for 12 hours to prepare a crude enzyme (crude enzyme). All procedures were carried out at 4 ℃, this coenzyme solution was used for the experiment on the enzyme properties.

zymogram  analysis

After preparing 6.5% non-denaturing polyacrylamide gel, 20 μl of coenzyme solution and 5 μl of sample buffer were injected into the groove, and Modular Mini-Protein II Electrophoresis System (Bio-Rad) was used at 50 V at 4 ° C. Electrophoresis was performed for 5-6 hours. After electrophoresis, the gel was placed on a 1.5% bacto agar plate containing 4 mg / ml X-α-Gal and incubated at 40 ° C. for 12 hours. The activity of alpha-galactosidase was confirmed by the appearance of a blue band. Bovine serum albumin (BSA) was used as a negative control.

Alpha - galactosidase  Active measurement

In the activity measurement of alpha-galactosidase, p- nitrophenyl-α-D-galactopyranoside ( p NPG) was used as a substrate. Enzyme was added to the reaction solution containing 1 mM p NPG and 50 mM Na-phosphate buffer (pH 7.0) so that the final reaction volume was 1 ml, and reacted at 40 ° C. for 15 minutes, followed by 1 ml of 1 M Na ₂ CO ₃ solution. The reaction was terminated by addition, and the color development was measured for absorbance at 405 nm. Enzyme activity was calculated from p -nitrophenol ( p NP) standard curve. The quantitative enzyme activity unit 1 U was defined as the amount of enzyme that releases 1 μmol of p NP from p NPG per reaction time (minutes) under the above conditions (Patil, AG, K, PK, Mulimani, VH, Veeranagouda, Y., Lee, K. 2010. J. Microbiol. Biotechnol. 20: 1546-1554).

Alpha - galactosidase  Active pH And temperature effects and thermal stability

In order to investigate the effect of pH on the activity of alpha-galactosidase, the activity of α-galactosidase was measured in the range of pH 3.0 to pH 8.5 at a reaction temperature of 40 ℃. pH 3.0 was 50 mM glycine-HCl buffer, 50 mM sodium acetate buffer at pH 4.0-5.5, 50 mM sodium phosphate buffer at pH 6.0-7.0, and 50 mM Tris-HCl buffer at pH 7.4-8.5. In addition, to investigate the effect of reaction temperature on the activity, 100 μl of 10 mM p NPG solution and 800 μl of 50 mM sodium phosphate buffer (pH 7.0) were added to 5 μl of coenzyme solution in the range of enzyme reaction temperature from 0 to 70 ℃. The enzyme activity was measured and then expressed as relative activity.

In order to investigate the thermal stability of alpha-galactosidase, the crude enzyme solution was left at 40 to 70 ° C. for 30 minutes and the enzyme activity was measured under the conditions of temperature (40 ° C.) and pH (7.0).

Substrate specificity

To investigate α-galactosidase activity on the substrate, p -nitrophenyl-α-D-galactopyranoside, p -nitrophenyl-β-D-xylopyranoside, p -nitrophenyl-β-D-cellobioside, and p -nitrophenyl -α-D-glucopyranoside, o -nitrophenyl-β-D-glucopyranoside and p -nitrophenyl-α-L-arabinofuranoside were each added at a concentration of 1 mM and each substrate was subjected to optimum pH (7.0) and temperature (40 ° C) conditions. The activity of the enzyme against was determined. The quantitative enzyme activity unit 1 U at this time was defined as the amount of enzyme that liberates 1 μmol of p -nitrophenol or o -nitrophenol from the substrate per reaction time (minutes) under the above conditions (Patil, AG, K, PK, Mulimani). , VH, Veeranagouda, Y., Lee, K. 2010. J. Microbiol. Biotechnol. 20: 1546-1554).

In addition, natural substrates such as melibiose, raffinose, stachyose, and lactose were added at a concentration of 1 mM and reacted for 3 hours under the optimum pH (7.0) and temperature (40 ° C) conditions, followed by the galactose test kit (Boehringer Mannheim GmbH). The amount of D-galactose liberated from was determined by the manual specified in the kit and the enzyme activity was calculated. At this time, 1 U quantitative enzyme activity unit was defined as the amount of enzyme that releases 1 μmol of galactose from the substrate per reaction time (minutes) under the above conditions.

Saccharogenic substrates such as carboxymethylcellulose (CMC), xylan, mannan and starch were measured for enzyme activity under optimal conditions by dinitrosalicylic acid (DNS) method (Miller, GL 1959. Anal. Chem. 31: 426-428). The concentration of the substrate was 0.4% and the enzyme activity unit 1 U was defined as the amount of enzyme that liberates 1 μmol of reducing sugar from the substrate per reaction time (minutes) under the above conditions (Park, IK, Cho, JS 2010. African J Microbiol. 4: 1257-1264).

Alpha - galactosidase  Metal Ions and Chemicals on Activity

To investigate the effects of metal ions and chemicals on alpha-galactosidase activity, 2 mM Ca ^{2 +} , Co ^{2 +} , Fe ^{2 +} , Mg ^{2 +} , Mn ^{2 +} , Ba ^{2 +} , Cu ^{2 +} , Zn ^{2 + , Ni 2 +, Hg 2 +} , K +, Na +, Ag +, β-mercaptoethanol, EDTA, SDS , and PMSF was added to a crude enzyme solution in 50 mM sodium phosphate buffer (pH 7.0 ) that contains, and for 20 minutes at 25 ℃ After incubation, the enzyme activity was measured under standard conditions (pH 7.0, 40 ℃).

Α- for proteolytic enzymes galactosidase Resistance

In order to investigate the resistance of alpha-galactosidase to protease, Cao method (Cao et al., 2010) was performed with a slight modification. Proteolytic enzymes were trypsin (Sigma), pronase (Sigma), pancreatin (Sigma), subtilisin carlsberg (Sigma) and proteinase K (Sigma). The protein amounts of the crude enzyme and protease were measured by the bradford method (Bradford, MM 1976. Anal. Biochem. 72: 248-254), and then added to 50 mM sodium phosphate buffer (pH 7.0) at a ratio of 10: 1 37 After culturing for 30 minutes at ℃, the enzyme activity under standard conditions (pH 7.0, 40 ℃) was measured and expressed as relative activity.

Soybean meal  Within α- galactooligosaccharide Decomposition of

In order to investigate the degradation of α-galactooligosaccharide in soybean meal, the amount of free D-galactose was measured using a galactose test kit (Boehringer Mannheim GmbH). A total of 0.5 U coenzyme solution was added to 10 ml of 50 mM sodium phosphate buffer (pH 7.0) containing 1.25 g of soybean meal, and 0, 1, 3, 6 and 9 hours at 130 rpm in a 37 ° C shaking water bath. Samples were taken hourly during incubation to determine the amount of free galactose.

Alpha - galactosidase  Best for production Medium component  Selection

In order to optimize the media composition, one factor at a time (OFAT) method, in which all other components are fixed to a certain level and only one element is changed (Liu et al., 2007c), is effective for α-galactosidase production. An experiment was performed to select (Table 5).

The carbon sources used in the carbon source selection experiments were galactose and wheat bran. Each carbon source was added to basal medium 1 at 1% and 2% concentrations, and the enzyme activity was measured after culturing at 220 rpm for 24 hours at 28 ℃ shaking incubator. .

In the nitrogen source selection experiment, yeast extract, tryptone, peptone, soybean meal, and ammonium sulfate were used, and a medium prepared with 10 g / L concentration of each nitrogen source in basal medium 2 was prepared at a speed of 220 rpm in a shaker at 28 ° C. After incubation for 24 hours, enzyme activity was measured.

Finally, essential element screening experiments were performed using sodium chloride, calcium chloride, magnesium sulfate, potassium phosphate monobasic, manganese sulfate, and ferrous sulfate. Enzyme activity was measured after 24 hours of incubation at 220 rpm in a shaker incubator. Sodium chloride was added at 1%, calcium chloride and magnesium sulfate at 0.01%, potassium phosphate monobasic at 0.07%, and manganese sulfate and ferrous sulfate at 0.001%.

Table 5 is a table of the basal medium composition of one factor at a time (OFAT) experiment.

Example 2: Bacillus using solid fermentation sp . Explore the culture conditions for the LX -1 derived from α- galactosidase production

Used strain  And strain storage

Bacillus sp. Used in this experiment. LX-1 (HQ660811) was isolated from the Antarctic soil-derived microbial colonies and showed α-galactosidase production capacity. This strain was inoculated in tryptic soy broth (Difco) medium and incubated at 28 ° C. for 24 hours, suspended in 50% glycerol and stored at -70 ° C., and passaged at 28 ° C. using tryptic soy agar medium. It was.

Α- using solid fermentation galactosidase  production

The spawn medium was tryptic soy broth (Difco) and was incubated for 24 hours at 28 ℃ and 220 rpm in a shaker was used as spawn medium. Soybean meal and 10 g of distilled water were added to 250 ml Erlenmeyer flask to adjust the water content to 50%. After sterilization and cooling at 121 ° C. for 15 minutes, the seed culture solution was inoculated at 4% and incubated at 28 rpm for 4 days at a speed of 220 rpm.

Alpha - galactosidase And Crude enzyme solution  Ready

40 mL of distilled water was added to the fermented solid substrate, and the culture supernatant obtained by centrifugation at 4 ° C. at 5000 rpm for 10 minutes was stirred at 28 ° C. and 220 rpm for 1 hour in a shake incubator.

Alpha - galactosidase Activity measurement of

Unless stated otherwise, the activity measurement of α-galactosidase was determined using p- nitrophenyl-α-D-galactopyranoside ( p NPG) as substrate, 800 μl 50 mM sodium phosphate buffer (pH 7.0), 10 mM p NPG The reaction solution consisting of 100 µl, 20 µl of crude enzyme solution and 80 µl of distilled water was incubated at 40 ° C for 15 minutes, and then 1 ml of 1 M Na ₂ CO ₃ was added to terminate the reaction. Color development was measured by absorbance at 405 nm and enzyme activity was calculated from p -nitrophenol ( p NP) standard curve. At this time, 1 U quantitative enzyme activity unit was defined as the amount of enzyme releasing 1 μmol p NP from p NPG per reaction time (minutes) under the above conditions.

Alpha - galactosidase  Screening of Solid Substrates for Production

For the experiment of substrate selectivity, wheat flour, soybean meal, corn flour were used as a solid substrate. 10 g of distilled water was added to 10 g of each solid substrate, sterilized for 15 minutes at 121 ° C., and inoculated with 4% of the seed culture. The inoculated solid medium was incubated for 4 days at a speed of 220 rpm in a 28 ° C. shaking incubator, and the enzyme was extracted to measure the enzyme activity. At this time, the reaction solution for measuring enzyme activity was composed of 800 μl of 50 mM sodium phosphate buffer (pH 6.0), 100 μl of 10 mM p NPG and 100 μl of coenzyme solution and incubated for 25 minutes in a 30 ° C. constant temperature water bath.

Alpha - galactosidase  Effect of Water Content on Production

In order to investigate the effect of water content on the production of alpha-galactosidase, soybean meal and water ratio were prepared in the range of 1: 0.5 to 1: 4 (w / v). 4% inoculation of the spawn culture was incubated at a speed of 220 rpm in a shaker at 28 ° C. for 4 days, and the enzyme was extracted to measure the enzyme activity.

Α- according to incubation time galactosidase  production

To investigate the enzyme production according to the incubation time, 10 g of soybean meal and 30 g of distilled water were put into a 250 ml Erlenmeyer flask to sterilize for 15 minutes at 121 ° C. to prepare a medium. After spawning 4% of the spawn culture solution, the culture was incubated at a speed of 220 rpm in a shaker at 28 ° C., and enzyme activity was measured by extracting enzyme according to the culture time.

Alpha - galactosidase  For production Solid medium pH Influence of

In order to investigate the effect of the pH of the solid medium, 10 g of soybean meal and 30 g of distilled water were added to a 250 ml Erlenmeyer flask, and the pH of the solid medium was adjusted from pH 5.0 to pH 9.0 using 1 M NaOH and 1 N HCl. The medium was prepared by sterilization at 121 ° C. for 15 minutes. 4% inoculation of the spawn culture was incubated for 72 hours at a speed of 220 rpm in a shaker at 28 ° C., and the enzyme was extracted to measure the enzyme activity.

Alpha - galactosidase  Effect of spawn volume inoculation on production

In order to investigate the effect of spawn culture solution inoculation on α-galactosidase production, 10 g of soybean meal and 30 g of distilled water were added to a 250 ml Erlenmeyer flask and sterilized at 121 ° C. for 15 minutes to prepare a medium. Each solid medium was inoculated with 1, 2, 4, 6, 8, and 10% seed cultures incubated for 24 hours in tryptic soy broth, incubated for 72 hours at a speed of 220 rpm in a 28 ° C shake incubator, followed by enzyme extraction. The enzyme activity was measured.

Alpha - galactosidase  For production Carbon source  Of nitrogen and nitrogen sources

Galactose, lactose, glucose, maltose, and sucrose were used as carbon sources, and yeast extract, tryptone, peptone, ammonium sulfate, and sodium nitrate were used as carbon sources. 10 g of soybean meal and 1% of each carbon source and nitrogen source were added to a 250 ml Erlenmeyer flask, distilled water was added so as to have a water content of 75%, and sterilized at 121 ° C. for 15 minutes to prepare a medium. 4% inoculation of the spawn culture was incubated for 72 hours at a speed of 220 rpm in a shaker at 28 ° C., and the enzyme was extracted to measure the enzyme activity.

Agricultural Biotechnology Research Institute KACC91695P 20111216

<110> Konkuk University Industrial Cooperation Corp. <120> Characterization of alpha-galactosidase from Bacillus sp. LX-1 as          a potential feed additive and Optimization of solid state          fermentation for the enzyme production <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 1446 <212> DNA <213> Bacillus sp. LX1 <400> 1 atgnaancag ctanacatgc aagtcgagcg aactgattag aagcttgctt ctatgacgtt 60 agcggcggac gggtgagtaa cacgtgggca acctgcctgt aagactggga taacttcggg 120 aaaccgaagc taataccgga taggatcttc tccttcatgg gagatgattg aaagatggtt 180 tcggctatca cttacagatg ggcccgcggt gcattagcta gttggtgagg taacggctca 240 ccaaggcaac gatgcatagc cgacctgaga gggtgatcgg ccacactggg actgagacac 300 ggcccagact cctacgggag gcagcagtag ggaatcttcc gcaatggacg aaagtctgac 360 ggagcaacgc cgcgtgaggt gatgaaggct ttcgggtcgt aaaactctgt tgttagggaa 420 gaacaagtac aagagtaact gcttgtacct tgacggtacc taaccagaaa gccacggcta 480 actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttatccggg aattattggg 540 cgtaaagcgc gcgcaggcgg tttcttaagt ctgatgtgaa agcccacggc tcaaccgtgg 600 agggtcattg gaaactgggg aacttgagtg cagaagagaa aagcggaatt ccacgtgtag 660 cggtgaaatg cgtagagatg tggaggaaca ccagtggcga aggcggcttt ttggtctgta 720 actgacgctg aggcgcgaaa gcgtggggag caaacaggat tagataccct ggtagtccac 780 gccgtaaacg atgagtgcta agtgttagag ggtttccgcc ctttagtgct gcagctaacg 840 cattaagcac tccgcctggg gagtacggtc gcaagactga aactcaaagg aattgacggg 900 ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag 960 gtcttgacat cctctgacaa ctctagagat agagcgttcc cctttcgggg gacagagtga 1020 caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttggggttaa gtcccgcaac 1080 gagcgcaacc cttgatctta gttgccagca tttagttggg cactctaagg tgactgccgg 1140 tgacaaaccg gaggaaggtg gggatgacgt caaatcatca tgccccttat gacctgggct 1200 acacacgtgc tacaatggat ggtacaaagg gctgcaagac cgcgaggtca agccaatccc 1260 ataaaaccat tctcagttcg gattgtaggc tgcaactcgc ctacatgaag ctggaatcgc 1320 tagtaatcgc ggatcagcat gccgcggtga atacgttccc gggccttgta cacaccgccc 1380 gtcacaccac gagagtttgt aacacccgaa gtcggtggag taaccgtaag gagctagccg 1440 ctatat 1446

Claims (6)

Bacillus (Bacillus) sp. LX-1 (Accession No. KACC91695P). The method of claim 1, wherein the Bacillus strain Bacillus ( Bacillus ) sp., Characterized in that having a 16S rDNA sequence of SEQ ID NO: 1. LX-1 (Accession No. KACC91695P). An enzyme composition comprising α-galactosidase having resistance to trypsin, pronase, or pancreatin derived from the strain of claim 1 as an active ingredient. A method of hydrolyzing raffinose and stachyose present in soybean meal by treating soybean meal with α-galactosidase derived from the strain of claim 1. A method of producing α-galactosidase by inoculating the strain of claim 1 in a solid medium using soybean meal. Feed additive composition comprising the strain of claim 1 or α-galactosidase derived from the strain as an active ingredient.

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