CN111849809B - Strain capable of degrading inulin levan and application thereof - Google Patents
Strain capable of degrading inulin levan and application thereof Download PDFInfo
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- 229920001202 Inulin Polymers 0.000 title claims abstract description 24
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Abstract
The invention relates to a strain capable of degrading inulin fructan and application thereof, wherein the strain is obtained by separating and purifying strains from rotten jerusalem artichoke tubers. The strain provided by the invention is proved to have higher exoinulase enzyme activity through enzyme activity determination, and has the performance of well degrading inulin in jerusalem artichoke.
Description
Technical Field
The invention relates to Glutaminibacter mishrai NJAU-1 capable of degrading inulin fructan and application thereof.
Background
Jerusalem artichoke (Helianthus tuberosus.l) originally originated in north america and was introduced into the continental europe in the 17 th century, while it has become a more common crop in the early 20 th century (Seiler et al, 2006). The jerusalem artichoke is a plant with extremely wide adaptability and extremely strong viability, and can be planted in a large range in saline-alkali soil and coastal mud flat where the salt concentration is high and most plants cannot grow (Long et al, 2010; Longxiaohua, etc., 2005). The developed root system of jerusalem artichoke enables the area where the jerusalem artichoke stretches to be wider, and the jerusalem artichoke can still grow normally under the severe dry and rainless conditions of 45 days even if the jerusalem artichoke is planted in sand with great wind and sand hazards without any measures (Kongtao et al, 2009). Besides the salt stress resistance and sand resistance, the jerusalem artichoke also has good adaptability to high-temperature environment, and is a plant with particularly strong reproductive capacity. These characteristics have led to its introduction into our country where it is widely cultivated in relatively barren areas. The high salt tolerance of the plant is one of the selected saline-soil plants with good development prospect, and the plant can be planted in large area in saline-alkali soil of Jiangsu coastal beaches.
Inulin (Inulin, also called Inulin) is mainly present in the roots or stems of plants such as jerusalem artichoke, chicory, dandelion, burdock, artichoke and the like, and is a renewable resource with rich sources. By the catalytic action of inulase, inulin is used as a raw material to produce fine chemicals such as high fructose syrup, fructo-oligosaccharide, lactic acid, citric acid, ethanol, single-cell protein, single-cell grease, butanol and the like. The enzymatic method for producing the fructose and the fructo-oligosaccharide has the advantages of mild conditions, simple process, low cost, high safety and no pollution. Therefore, the enzymatic method for producing fructose and fructo-oligosaccharide is more suitable for industrial production.
Inulase is a natural fructan hydrolase, exo-inulase is the only important enzyme for producing high fructose syrup by one-step method, and endo-inulase is the important enzyme for producing fructo-oligosaccharide. The method for producing fructose and fructo-oligosaccharide by hydrolyzing jerusalem artichoke with inulase has the advantages of simple process, high conversion rate, low cost, no pollution and the like. Inulase may also be used in the fermentative production of bioethanol. Therefore, the development and utilization of inulinase have important significance for developing new food industry raw materials, solving the food crisis, the energy crisis and the like.
Because of the rapid propagation speed of the microorganism, the vigorous metabolism, the rich sources and the low cost, the microorganism is used for the production of the inulase in industry. At present, research on inulinase at home and abroad mainly focuses on development and utilization of microbial resources for producing inulinase. A wide variety of microorganisms have been found to synthesize inulinase, including molds, bacteria, yeast, etc., all of which produce inulinase. However, the activity of the existing strains capable of secreting inulase is low at present, and the requirement of further industrial application cannot be realized, so that the screening of the high-yield inulase microorganisms has important industrial production significance.
Disclosure of Invention
The invention aims to solve the problem of industrial large-scale fructose production, and provides a degradable inulin levan strain Glutamicibacter mishrai NJAU-1 which is a new strain and has the characteristic of exoinulase with high enzyme production activity.
The purpose of the invention can be realized by the following technical scheme:
a strain NJAU-1 capable of degrading inulin levan is a strain fatty glutamic acid bacillus (Glutamicibacter mishrai), is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, has a preservation date of No. 4/28 (2020 years), and has a preservation number of CGMCC No. 19750.
The application of the strain NJAU-1 in preparing the exoinulase is disclosed.
The application of the strain NJAU-1 in the production of fructose is provided.
Compared with the prior art, the invention has the following beneficial effects:
the strain NJAU-1 of the strain of the degradable inulin fructosan has the characteristic of higher inulase activity, can improve inulin conversion efficiency and fructose yield, and can be used for actual production.
Drawings
FIG. 1, colony morphology of Glutaminibacter mishrai NJAU-1 degrading inulin levan strain on ZS medium;
FIG. 2 is a phylogenetic dendrogram of 16S rDNA partial sequence analysis of a strain Glutaminibacter mishrai NJAU-1 degrading inulin levan; numbers in parentheses indicate GenBank sequence No
FIG. 3, sugar HPLC charts of resting cell NJAU-1 after incubation with inulin for 1h (A), and inulin blank control (B); f, fructose; FOSs: fructo-oligosaccharide
Biological material preservation information
NJAU-1, classified and named as fatty acid bacillus (Glutaminobacter mishrai), is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and has the preservation address as follows: the preservation date of No. 3 Xilu Beijing, Chaoyang, and the institute of microbiology of the Chinese academy of sciences is No. 4/28, 2020 and the preservation number is CGMCC No. 19750.
Detailed Description
Example 1
1. Separation and purification of Glutaminibacter mishrai NJAU-1 strain for degrading inulin levan
Grinding the rotten Jerusalem artichoke tuber tissue into slurry by using a mortar, standing for a period of time, sucking 100 mu l of supernatant into 100ml of ZS liquid culture medium, sealing, carrying out shake culture at 37 ℃ at 200r/min for 3 d. Sucking bacterial liquid, diluting in gradient 10-3-10-5Then, 100. mu.L of each was applied to ZS medium and cultured at 37 ℃ for 1 to 2 days. A single colony was streaked on a ZS medium of the formula shown in Table 1, which was then inverted and cultured in the dark at 37 ℃. And (4) screening out strains with better growth vigor, and judging the strains according to the strain forms and the color change of the culture medium.
TABLE 1 ZS Medium formulation (/ L)
By using the separation and purification method, a plurality of pure strains are obtained by streak culture in a solid culture medium ZS (table 1) after repeated purification for a plurality of generations, and NJAU-1 can be separated and purified in the implementation.
NJAU-1 morphological characteristics: the bacterial colony is smooth in colony morphology, small in circle, bright, translucent, white and light yellow. The diameter of the crystal after growing on the ZS plate for 3-5 days is 0.1-0.5 mm; culture characteristics: the optimal growth conditions of the strain are as follows: the pH value is 7, the temperature is 37 ℃, and the rotating speed is 200 r/min; physiological properties: the cells are gram-positive and strictly aerobic.
2. 16S rDNA sequence sequencing for degrading inulin levan strain Glutaminicibacter mishrai NJAU-1
Screening single colonies of NJAU-1, performing PCR specific amplification, inspecting PCR amplification products on 1.0% agarose gel electrophoresis, and using the PCR amplification products for sequence determination, wherein the 16SrDNA sequence of the strain is shown as SEQ ID NO. 1.
Obtaining a reference strain from a GenBank database, analyzing partial 16S rDNA sequences of the separated strain and the reference strain by using software MegAlign, constructing a phylogenetic relationship diagram of the separated strain and a standard strain, and obtaining a phylogenetic tree reflecting the evolution and development relationship between the strain to be detected and the reference strain, as shown in figure 2, thereby determining that the separated degraded inulin levan strain Glutamicibacter mishara NJAU-1 is a new strain of Glutamicibacter.
Example 2 determination of inulinase-producing enzyme Activity of Glutaminobacter mishrai NJAU-1
Inoculating separated and purified Glutaminibacter mishrai NJAU-1 strain to a 100mL triangular flask containing 30mLZS liquid culture medium, and shake culturing at 37 deg.C and 200r/min for 24h to make OD > 1. Centrifuging 3ml suspension (3 times) at 12000r/min at 4 deg.C for 10min, discarding supernatant, collecting thallus precipitate, washing with phosphate buffer solution with pH of 7.0, washing off culture solution, centrifuging, collecting thallus precipitate, washing for three times until supernatant is transparent, and discarding supernatant.
And (3) measuring the activity of endo-inulase: the centrifuged cell pellet was suspended and mixed by 3mL of phosphate buffer (2% inulin substrate was added) at pH 7.0. Culturing at 37 deg.C and 200r/min for 1 d. Centrifuging the suspension at 4 ℃ and 12000r/min for 10min, collecting supernatant, taking 200 μ L of supernatant, adding 1.5mL of DNS reagent and 1.8mL of water, mixing uniformly, reacting in a boiling water bath for 5min (accurately timing), cooling with cold water, diluting with distilled water to 25mL in a test tube, and measuring the absorbance at 520 nm. The content of sugar in the sample solution can be calculated by a fructose standard curve. The control group was treated identically except that no strain was inoculated.
Measuring the activity of the inulase exonuclease: the centrifuged cell pellet was suspended in 3mL of phosphate buffer (2% sucrose substrate was added) at pH 7.0 and then mixed with air. Culturing at 37 deg.C and 200r/min for 1 d. Centrifuging the suspension at 4 ℃ and 12000r/min for 10min, collecting supernatant, taking 200 μ L of supernatant, adding 1.5mL of DNS reagent and 1.8mL of water, mixing uniformly, reacting in a boiling water bath for 5min (accurately timing), cooling with cold water, diluting with distilled water to 25mL in a test tube, and measuring the absorbance at 520 nm. The content of sugar in the sample solution can be calculated by a fructose standard curve. The control group was treated identically except that no strain was inoculated.
One unit of enzyme activity is defined as the amount of enzyme required to produce 1. mu. mol fructose per hour. Calculating the I/S <1, and judging that the inulase is the exoinulase. As shown in Table 4, the strain has high activity of exoinulase.
TABLE 2 phosphate buffer (/ L)
TABLE 3 DNS reagent (/ L)
TABLE 4 enzymatic Activity of inulinase by DNS method
Example 3
The centrifuged cell pellet was suspended and mixed by 3mL of phosphate buffer (added with 2% inulin substrate) with pH 7.0, and cultured at 37 ℃ for 1 hour at 200 r/min. The suspension was centrifuged at 12000r/min at 4 ℃ for 10min, and the supernatant was collected and subjected to sugar content measurement by high performance liquid chromatography, the results of which are shown in FIG. 3.
One unit of enzyme activity is defined as the amount of enzyme required to produce 1. mu. mol fructose per hour. As shown in Table 5, the strain has stronger activity of exoinulase.
TABLE 5 HPLC method inulinase enzyme Activity
Sequence listing
<110> Nanjing university of agriculture
<120> bacterial strain capable of degrading inulin levan and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1448
<212> DNA
<213> Glutaminobacter lipolyticus mishrai)
<400> 1
gatgaacgct ggcggcgtgc ttaacacatg caagtcgaac gatgaagccc tgcttgcagg 60
gtggattagt ggcgaacggg tgagtaacac gtgagtaacc tgcccccgac tctgggataa 120
gcccgggaaa ctgggtctaa taccggatat gacttcctac tgcatggtgg gttgttgaaa 180
gatttatcgg tgggggatgg actcgcggcc tatcagcttg ttggtgaggt aatggctcac 240
caaggcgacg acgggtagcc ggcctgagag ggtgaccggc cacactggga ctgagacacg 300
gcccagactc ctacgggagg cagcagtggg gaatattgca caatgggcgg aagcctgatg 360
cagcgacgcc gcgtgaggga tgacggcctt cgggttgtaa acctctttca gtagggaaga 420
agcgaaagtg acggtacctg cagaagaagc gccggctaac tacgtgccag cagccgcggt 480
aatacgtagg gcgcaagcgt tatccggatt tattgggcgt aaagagctcg taggcggttt 540
gtcgcgtctg ccgtgaaagt ccgaggctca acctcggatc tgcggtgggt acgggcagac 600
tagagtgatg taggggagac tggaattcct ggtgtagcgg tgaaatgcgc agatatcagg 660
aggaacaccg atggcgaagg caggtctctg ggcatttact gacgctgagg agcgaaagca 720
tggggagcga acaggattag ataccctggt agtccatgcc gtaaacgttg ggcactaggt 780
gtgggggaca ttccacgttt tccgcgccgt agctaacgca ttaagtgccc cgcctgggga 840
gtacggccgc aaggctaaaa ctcaaaggaa ttgacggggg cccgcacaag cggcggagca 900
tgcggattaa ttcgatgcaa cgcgaagaac cttaccaagg cttgacatgt gccagaccgc 960
tccagagatg gggtttccct tcggggctgg ttcacaggtg gtgcatggtt gtcgtcagct 1020
cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca accctcgttc catgttgcca 1080
gcacgtagtg gtggggactc atgggagact gccggggtca actcggagga aggtggggat 1140
gacgtcaaat catcatgccc cttatgtctt gggcttcacg catgctacaa tggccggtac 1200
aatgggttgc gatactgtga ggtggagcta atccctaaaa gccggtctca gttcggattg 1260
gggtctgcaa ctcgacccca tgaagtcgga gtcgctagta atcgcagatc agcaacgctg 1320
cggtgaatac gttcccgggc cttgtacaca ccgcccgtca agtcacgaaa gttggtaaca 1380
cccgaagccg atggcctaac caccttgtgt ggggggagtc gtcgaaggtg ggactggcga 1440
ttgggact 1448
Claims (3)
1. A strain fatty glutamic acid bacillus (Glutaminobacter mishrai) NJAU-1 capable of degrading inulin levan is preserved in China general microbiological culture Collection center (CGMCC), wherein the preservation date is No. 4/28 at 2020, and the preservation number is CGMCC No. 19750.
2. Use of NJAU-1 as claimed in claim 1 for the preparation of an exoinulase.
3. Use of NJAU-1 according to claim 1 for the production of fructose.
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