CN111826308B - Marine sediment-derived chitin efficient degrading bacterium and application thereof - Google Patents

Marine sediment-derived chitin efficient degrading bacterium and application thereof Download PDF

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CN111826308B
CN111826308B CN202010540702.4A CN202010540702A CN111826308B CN 111826308 B CN111826308 B CN 111826308B CN 202010540702 A CN202010540702 A CN 202010540702A CN 111826308 B CN111826308 B CN 111826308B
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chitin
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bacteria
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CN111826308A (en
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陈吉刚
沈旭东
朱四东
杨季芳
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Zhejiang Wanli University
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2442—Chitinase (3.2.1.14)
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    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01014—Chitinase (3.2.1.14)
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    • Y02W10/00—Technologies for wastewater treatment
    • Y02W10/10—Biological treatment of water, waste water, or sewage

Abstract

The invention relates to a chitin high-efficiency degrading bacterium from marine sediments and application thereof, which is characterized in that: the chitin efficient degrading bacteria are rhinestone bacteria SX46, and the rhinestone bacteria SX46 are preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number as follows: CGMCC No.19597, the preservation date is 2020, 04 and 22 days; compared with the prior art, the invention has the following advantages: the strain SX46 provided by the invention is a new species of Rhizomyidae, the strain SX46 provided by the invention can grow well in a low-cost culture medium, the produced chitinase has high activity, colloidal chitin can be efficiently converted into N-acetyl-D-glucosamine (GlcNac), and the conversion rate is up to 90%.

Description

Chitin efficient degrading bacterium derived from marine sediments and application thereof
Technical Field
The invention belongs to the technical field of applied microorganisms, and particularly relates to a chitin efficient degrading bacterium suitable for marine sediment sources and application thereof.
Background
Chitin (chitin), also called chitin, is a linear polymer formed by connecting N-acetyl-beta-D-glucosamine (GlcNac) through beta-1, 4-glycosidic bonds, is the most abundant renewable resource in marine environment, is formed by more than 1011 tons of chitin every year, and is mainly degraded by a chitinase (chitinase) system degradation system secreted by marine microorganisms. Chitinase degrades chitin to generate chitin oligosaccharide, chitin monosaccharide and derivatives thereof with high added value, and the like, has good histocompatibility and biodegradability, biological activities of regulating immunity, resisting bacteria, inducing plant disease resistance, promoting plant growth, resisting cancer and the like, and has wide application prospect in various fields of medicine industry, food industry, environment, agriculture, breeding industry and the like.
Although marine environments are a huge pool of microbial resources, the number of chitinase-producing bacteria derived from marine environments is much lower than that of terrestrial environments. The lack of efficient separation method is the main reason for the low yield of chitinase-producing bacteria from marine environment sources. At present, China is lack of efficient separation of chitinase-producing bacteria suitable for marine sediment environments and strains of high-yield and high-activity chitinase, so that related patents for preparing high value-added products by utilizing chitinase from microorganisms are rare.
Disclosure of Invention
The first technical problem to be solved by the invention is to provide a chitin high-efficiency degrading bacterium from marine sediment aiming at the current situation of the prior art.
The second technical problem to be solved by the present invention is to provide an application of the above chitin-degrading bacteria in view of the current state of the prior art.
The technical scheme adopted by the invention for solving the first technical problem is as follows: the chitin efficient degrading bacterium derived from marine sediments is characterized in that: the chitin efficient degrading bacteria are Rheinheimera sp SX46, and the Rheinheimera sp SX46 is preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms with the preservation number as follows: CGMCC No.19597, preservation date of 22 days 04.2020 Rhizomenon reinhardtii.
The invention also provides an enrichment method for enriching the chitin high-efficiency degrading bacteria, which is characterized by comprising the following steps of: enrichment of chitin-degrading bacteria in high efficiency is carried out by adopting enrichment medium, and nitrogen source in the enrichment medium is (NH)4)2SO4、KNO3And yeast extract, wherein the carbon source is chitin powder, and the formula of the enrichment medium is as follows: na (Na)2HPO4，6.0g/L；KH2PO4，3.0g/L；NH4Cl, 1.0 g/L; NaCl, 0.5 g/L; yeast extract, 0.05 g/L;chitin powder, 10.0g/L, and adjusting pH of the culture medium to 7.0.
The invention also provides a screening method for screening the chitin high-efficiency degrading bacteria, which is characterized by comprising the following steps of: screening of chitin-degrading bacteria is carried out by adopting a screening culture medium, wherein a nitrogen source in the screening culture medium is (NH)4)2SO4、KNO3And yeast extract, carbon source is chitin powder, and colloidal chitin is added.
In order to solve the second technical problem, the invention provides an application of chitin efficient degrading bacteria in producing chitinase, which is characterized in that: inoculating the rhymenia reinhardtii SX46 into a fermentation culture medium with colloidal chitin as a unique carbon source, wherein the content of the colloidal chitin is 10-15 g/L.
Compared with the prior art, the invention has the following advantages: the strain SX46 provided by the invention is a new species of Rhizomyidae, the strain SX46 provided by the invention can grow well in a culture medium with low cost, the produced chitinase has high activity, colloidal chitin can be efficiently converted into N-acetyl-D-glucosamine (GlcNac), the conversion rate is up to 90%, the invention conforms to the environmental protection concept of green and safety, the maximum utilization of marine microorganism resources is realized, and the high-yield and high-activity chitinase strain which is not reported in marine environment is obtained by innovatively designing a conventional method; chitinase produced by the strain can efficiently convert chitin into N-acetyl-D-glucosamine (GlcNac) with application value.
Deposit description
1. The Lymphenium reinhardtii SX46 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is the microorganism research institute of China academy of sciences No. 3 of the Xilu No.1 of Beijing, Chaozhou, Chaoyang, the preservation date is 2020, 04, 22 days, and the preservation number is CGMCC No. 19597.
Drawings
FIG. 1 is a graph showing the results of the enrichment culture system for the 3 rd generation chitin-degrading bacteria in example 1;
FIG. 2 is a graph showing the results of the transparent circles produced by the strain SX46 on the screening plate in example 1;
FIG. 3 is a graph showing the results of production of GlcNac in example 4.
Detailed Description
The invention is further illustrated by the following figures, sequence listing and examples.
Example 1
(1) Enrichment culture of chitin-degrading bacteria
Transferring 5g of the sediment into a conical flask containing 100mL of enrichment medium, and culturing at 8 ℃ and 150 rpm for 30 days to obtain a 1 st generation enrichment culture; taking 1mL of the 1 st generation enrichment non-inoculated culture medium to a conical flask containing 100mL of enrichment culture medium, and continuing shaking culture for 14 days to obtain a 2 nd generation enrichment culture; inoculating 1mL of the enriched culture of the generation 2 into a conical flask containing 100mL of the enriched culture medium, and continuing shaking culture for 14 days to obtain the enriched culture of the generation 3, wherein the sediment is derived from marine sediment collected by 50 voyages in the ocean.
The formula of the enrichment medium is (g/L): na (Na)2HPO4，6.0；KH2PO4，3.0；NH4Cl, 1.0; 0.5 parts of NaCl; yeast extract, 0.05 g; chitin powder, 10.0g, pH of the medium was adjusted to 7.0.
As shown in FIG. 1, after 3 generations of enrichment culture, the enrichment liquid turns turbid, and the powdered chitin is partially degraded, indicating that the chitin-degrading bacteria in the enrichment have reached a certain amount.
(2) Screening and purifying chitin degrading bacteria
The 3 rd generation enrichment culture is diluted by an enrichment culture medium in a series of gradient, coated on a solid screening culture medium and cultured in an incubator at 20 ℃. And (3) selecting colonies with transparent circles around the colonies, and carrying out streaking purification for 3 times to obtain a pure strain SX46 capable of degrading chitin.
The formula of the solid identification medium is (g/L): na (Na)2HPO4，6.0g；KH2PO4，3.0g；NH4Cl, 1.0 g; NaCl, 0.5 g; yeast extract, 0.05 g; chitin powder, 5 g; colloidal chitin, 10 g. Agar powder, 15 g. RegulatingThe pH of the culture medium is 7.0, and the chitin in the culture medium is identified to form a transparent ring after being degraded by the strain SX46 (figure 2).
Example 2: molecular identification and named preservation of chitin degrading bacteria SX46
(1) Molecular characterization of strains
The amplification and sequencing of rRNA gene of the strain SX 4616S are carried out by adopting a conventional molecular cloning method, and the sequencing result is submitted to an EzTaxon website to determine the classification status of the strain.
(2) Strain 16S rDNA gene sequence and strain preservation information
The 16S rRNA gene sequence of the strain SX46 is shown in a sequence table, and the total length of the gene is 1453 bp. Homology analysis of the EzTaxon website shows that the strain has the highest homology (98.55%) with Rheinheimeria aquimaris (Rhizomyidae), and is a potential new species.
Example 3 preparation and Activity determination of crude chitinase of Strain SX46
(1) Strain SX46 fermentation culture
Selecting SX46 single colony, inoculating the single colony in 10mL of fermentation medium, and performing shake culture at 20 ℃ and 150r/min until the OD600 of the bacterial liquid is 0.8-1.0 as seed liquid; inoculating the seed liquid into a fermentation culture medium according to a ratio of 1:100, performing shake culture at 20 ℃ and 150r/min for 5d, centrifuging at 3000 r/min for 5min, and collecting clear liquid.
The culture of the fermentation medium is (g/L): na (Na)2HPO4，6.0g；KH2PO4，3.0g；NH4Cl, 1.0 g; NaCl, 0.5 g; yeast extract, 0.1 g; 10g of colloidal chitin, and adjusting the pH value of the culture medium to 7.0.
(2) Preparation of chitinase crude enzyme
Adding ammonium sulfate with final concentration of 80% into the supernatant, shaking at 4 deg.C for 30min, centrifuging at 4 deg.C and 10000g for 20min, and collecting precipitate; 2ml of 50mM KH was used for precipitation2PO4After the buffer solution (pH5.0) is fully dissolved, transferring the solution to a dialysis bag (MWVO: 6-8 kDa); placing the dialysis bag in a container with 50mM KH2PO4Dialyzing at 4 deg.C for 12-24h in a buffer solution container, and replacing dialysate every 2h during dialysis. Collecting the solution in the dialysis bag after dialysis to obtain chitinCrude enzyme solution of the enzyme.
(3) Chitinase crude enzyme activity determination
And (3) determining the activity of the crude chitinase solution by using a DNS method. The specific operation steps for determining the chitinase activity by the DNS method are as follows: mixing 1.5mL of chitin crude enzyme solution with 1.5mL of phosphate buffer (0.1M, pH 5.5) containing 1.0% colloidal chitin, incubating at 40 ℃ for 30min, boiling for 5min to terminate the enzymatic reaction, cooling in an ice bath, centrifuging at 10000 rpm for 5min, taking 1mL of supernatant and 1mL of DNS reagent, mixing uniformly, carrying out boiling water bath for 10min, cooling in an ice bath, centrifuging, taking the supernatant, and measuring the absorbance at the wavelength of 540 nm. N-acetylglucosamine was used as a standard control, and the procedure was as described above, except that boiling inactivation was performed before the reaction. The enzyme activity unit (U) is defined as: the amount of enzyme required to produce 1. mu. mol reducing sugar per minute at 40 ℃. The enzyme activity calculation formula is as follows: the enzyme activity (U/mL) is m.times.Nx.1000/(M.times.T.times.V). In the formula: m is the amount of N-acetyl-D-glucosamine, N is the dilution multiple, M is the molecular weight of N-acetyl-D-glucosamine, T is the reaction time, and V is the volume of the enzyme solution.
The result shows that after the strain is cultured for 5 days, the activity of the crude chitinase reaches 150U/mL, and the crude chitinase has strong enzyme activity.
Example 4 production of GlcNac Using chitinase produced by Strain SX46
(1) GlcNAc conversion reaction system
The transformation system contained 2% (w/v) colloidal chitin at final concentration, 0.5U/mL chitinase crude enzyme solution, and 50mM KH2PO4 buffer solution (pH 5.0). And (3) placing the reaction system in a constant temperature environment of 37 ℃ for shaking incubation for 96h, sampling for 1 time every 2 hours, and determining the concentration of GlcNAc in the reaction system.
(2) GlcNAc production assay
The concentration of GkcNac in the above reaction system was measured using a high performance liquid chromatography. A30 cm X7.8 mm format column (Aminex HPX-87H) was used. After loading, the column was eluted with 5mM H2SO4 at a flow rate of 0.8 m/min. The GlcNAc content of the GlcNAc conversion system was obtained by comparing the ratio of the area of the GlcNAc peak in the sample to the area of the standard GlcNAc peak (product of Sigma). The GlcNAc production (%) was calculated as: the amount of GlcNAc released in solution (mg). times.100/initial concentration of colloidal chitin (mg). times.100.
The results show (fig. 3), that colloidal alpha-chitin can be converted to GlcNac by chitin crude enzyme solution prepared by strain SX46, and 75% of colloidal chitin is converted to GlcNa within 12 h; the highest conversion efficiency for GlcNac occurred at 24h after the reaction, when 90% of the colloidal chitin was converted to GlcNac.
Sequence listing
<110> Zhejiang university college
<120> chitin high-efficiency degrading bacterium derived from marine sediments and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1452
<212> DNA
<213> SX46
<400> 1
gtggccgggc agggccttac acatgcaagt cgagcgaatg agggtagctt gctacctgat 60
ttagcggcgg acgggtgagt aatgtatagg gagctgcccg atagaggggg ataccagttg 120
gaaacgactg ttaataccgc ataatgtcta cggaccaaag tgtgggacct tcgggccaca 180
tgctatcgga tgcacctata tgggattagc tagttggtgg ggtaacggct caccaaggcg 240
acgatcccta gctggtttga gaggatgatc agccacactg gaactgagac acggtccaga 300
ctcctacggg aggcagcagt ggggaatatt ggacaatggg cgcaagcctg atccagccat 360
gccgcgtgtg tgaagaaggc cttcgggttg taaagcactt tcagcgagga ggaagggtgt 420
tgtgttaata gcacagcatt ttgacgttac tcgcagaaga agcaccggct aactccgtgc 480
cagcagccgc ggtaatacgg agggtgcaag cgttaatcgg aattactggg cgtaaagcgc 540
acgtaggcgg tgtgttaagt tggatgtgaa agccccgggc tcaacctggg aattgcattc 600
aaaactggca cgctagagta tgtgagaggg gggtagaatt ccaagtgtag cggtgaaatg 660
cgtagagatt tggaggaata ccagtggcga aggcggcccc ctggcacaat actgacgctc 720
aggtgcgaaa gcgtggggag caaacaggat tagataccct ggtagtccac gccgtaaacg 780
atgtctacta gctgttcgtg gtcttgtact gtgagtagcg cagctaacgc actaagtaga 840
ccgcctgggg agtacggtcg caagattaaa actcaaatga attgacgggg gcccgcacaa 900
gcggtggagc atgtggttta attcgacgca acgcgaagaa ccttacctac tcttgacatc 960
tagcgaagat tgcagagatg cagttgtgcc ttcgggaacg ctaagacagg tgctgcatgg 1020
ctgtcgtcag ctcgtgttgt gaaatgttgg gttaagtccc gcaacgagcg caacccttat 1080
ccttagttgc cagcacgtaa tggtgggaac tctagggaga ctgccggtga taaaccggag 1140
gaaggtgggg acgacgtcaa gtcatcatgg cccttacgag tagggctaca cacgtgctac 1200
aatggtacgt acagagggag gcaagctggc gacagtgagc ggatctctta aagcgtatcg 1260
tagtccggat tggagtctgc aactcgactc catgaagtcg gaatcgctag taatcgcaaa 1320
tcagaatgtt gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccatggg 1380
agtgggttgc aaaagaagta ggtagcttaa ccttcgggag ggcgccttac ccacctttgg 1440
gatttccagg tg 1452

Claims (4)

1. A chitin high-efficiency degrading bacterium derived from marine sediments is characterized in that: the chitin efficient degrading bacteria are Rheinheimera sp SX46, and the Rheinheimera sp SX46 is preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms with the preservation number as follows: CGMCC No.19597, preservation date of 22 days 04.2020 Rhizomenon reinhardtii.
2. An enrichment method for the chitin-degrading bacterium according to claim 1, wherein: enrichment of chitin-degrading bacteria with enriched culture medium containing (NH) as nitrogen source4)2SO4、KNO3And the carbon source of the yeast extract is chitin powder, and the formula of the enrichment medium is as follows: na (Na)2HPO4，6.0g/L；KH2PO4，3.0g/L；NH4Cl, 1.0 g/L; NaCl, 0.5 g/L; yeast extract, 0.05 g/L; chitin powder, 10.0g/L, and adjusting pH of the culture medium to 7.0.
3. The use of the chitin-degrading bacterium of claim 1 for producing chitinase, wherein: inoculating the rhymenia reinhardtii SX46 into a fermentation culture medium with colloidal chitin as a unique carbon source, wherein the content of the colloidal chitin is 10-15 g/L.
4. The use of the chitin-degrading bacterium of claim 3 for producing chitinase, wherein: the formula of the fermentation medium is Na2HPO4，6.0g/L；KH2PO4，3.0g/L；NH4Cl, 1.0 g/L; NaCl, 0.5 g/L; yeast extract, 0.1 g/L; 10g/L of colloidal chitin, and adjusting the pH value of the culture medium to 7.0.
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