WO2015143960A1 - Aspergillus niger à rendement élevé en xylanase et son application - Google Patents

Aspergillus niger à rendement élevé en xylanase et son application Download PDF

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WO2015143960A1
WO2015143960A1 PCT/CN2015/072766 CN2015072766W WO2015143960A1 WO 2015143960 A1 WO2015143960 A1 WO 2015143960A1 CN 2015072766 W CN2015072766 W CN 2015072766W WO 2015143960 A1 WO2015143960 A1 WO 2015143960A1
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xylanase
aspergillus niger
enzyme
activity
furfural
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PCT/CN2015/072766
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English (en)
Chinese (zh)
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袁振宏
许敬亮
何敏超
张宇
孔晓英
梁翠谊
陈小燕
刘云云
郭颖
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中国科学院广州能源研究所
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Publication of WO2015143960A1 publication Critical patent/WO2015143960A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C12R2001/685—Aspergillus niger
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)

Definitions

  • the invention belongs to the field of microbial fermentation and its enzymatic engineering application, in particular to a high-yield xylanase Aspergillus niger SM24/a and the xylanase for rapid detection of 5-hydroxyl in the preparation of an enzyme inhibition method Application in formulations based on furfural concentration.
  • Xylanase the English name xylanase, referred to as XYL, is an enzyme that catalyzes the hydrolysis of ⁇ -1,4 xylan glycosidic linkages to release xylose and xylooligosaccharides.
  • XYL xylanase
  • the addition of xylanase can significantly accelerate the reaction process, improve the use characteristics of the product, and reduce the cost, energy consumption and environmental pollution of the production process. Therefore, xylanase is widely used in traditional fields such as pulp and paper, food, feed and textile, and lignocellulosic ethanol.
  • Xylanase has important research significance and significant application prospects in the field of lignocellulosic biorefining.
  • Pryor S W (2012) believes that the addition of xylanase can promote the enzymatic hydrolysis of sugarcane bagasse after soaking in liquid nitrogen.
  • Sills DL (2012) can further increase the yield of reducing sugar by adding xylanase to switchgrass after alkali treatment.
  • the role and effect of xylanase in promoting cellulase enzymatic pretreatment has been widely recognized.
  • xylanases are subject to intricate enzymatic conditions and environments.
  • the effect of inhibitors produced after pretreatment on the enzymatic properties of xylanase is important.
  • the effects of inhibitors on xylanase have rarely been studied, and the types of inhibitors studied have been less.
  • xylanases have better or better tolerance to a variety of single inhibitors or mixtures of multiple inhibitors. Nor is it a well tolerated xylanase whose strain can produce high xylanase.
  • de Souza Moreira (2013) showed that vanillin and ferulic acid have a certain inhibitory effect on xylanase derived from Aspergillus terreus, but the strain of Aspergillus terreus used has extremely low xylanase activity.
  • a first object of the present invention is to provide a high-yield xylanase-producing Aspergillus niger SM24/a, which was deposited on December 31, 2013 at the General Microbiology Center of the China Microbial Culture Collection Management Committee (CGMCC).
  • CGMCC China Microbial Culture Collection Management Committee
  • the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with the preservation number: CGMCC No.8671.
  • the Aspergillus niger SM24/a of the present invention is screened and cultured from materials such as dead leaves and soil. Its fungal taxonomy shows that the mycelium of the fungus is white, the colony is small and dense, and it can produce black spores.
  • the PDA medium was cultured for 1 day to start long white hyphae, and the long black spores were started after about 36 hours of culture.
  • taxonomic status of Aspergillus niger SM24/a of the present invention is determined according to the following method:
  • the fungal 18s rDNA was extracted by a conventional method, and its nucleotide sequence is shown in SEQ ID NO.
  • the Aspergillus niger SM24/a of the present invention belongs to the genus Aspergillus, and is named as Aspergillus niger SM24/a, which was deposited on December 31, 2013. China Microbial Culture Collection Management Committee General Microbiology Center (CGMCC), the preservation number is: CGMCC No.8671.
  • the xylanase activity was determined by using xylan (Beech wood, SIGMA), and it was found that the Aspergillus niger SM24/a of the present invention can produce high xylanase, and the xylanase can have the highest solid fermentation activity.
  • xylan Beech wood, SIGMA
  • the optimum reaction pH is 5.6
  • the optimum reaction temperature is 37 ° C
  • the enzyme activity residual rate at 79 ° C is 79.01%.
  • a second object of the present invention is to provide a xylanase which is obtained by fermentation using Aspergillus niger SM24/aW2 as a fermentation strain.
  • a third object of the present invention is to provide the use of Aspergillus niger SM24/a for the production of xylanase.
  • a fourth object of the present invention is to provide a xylanase for use in the preparation of a preparation for rapidly detecting the concentration of 5-hydroxymethylfurfural by an enzyme inhibition method.
  • the present invention provides Aspergillus niger SM24/a which is capable of producing a xylanase which is well tolerated to ethanol, acetic acid, furfural, vanillin, ferulic acid and mixtures thereof, and the production
  • the xylanase activity of the enzyme strain is extremely high, and the solid fermentation enzyme activity of the xylanase is up to 10801 IU/g (the amount of 1 ⁇ mol of xylose catalyzed in 1 minute is defined as one enzyme unit), and the optimum pH value is determined.
  • the optimum reaction temperature was 37 ° C, and the residual rate of enzyme activity at 50 ° C was 79.01%.
  • 5-hydroxymethylfurfural has an inhibitory effect on the enzyme.
  • the xylanase of the present invention can also be used as a qualitative or quantitative indicator of the concentration of 5-hydroxymethylfurfural.
  • the Aspergillus niger SM24/a of the present invention was deposited at the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee on December 31, 2013, and the deposit address was No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.
  • the deposit number is: CGMCC No.8671.
  • Figure 1 is a graph showing the effect of pH as a fermentation parameter on the xylanase activity of Aspergillus niger SM24/a of the present invention
  • Figure 3 is a temperature enzymatic property of a xylanase produced by Aspergillus niger SM24/a of the present invention
  • Figure 4 is a graph showing the relationship between different concentrations of mixed inhibitors containing 5-hydroxymethylfurfural and xylanase enzymatic retention
  • Figure 5 is a phylogenetic tree constructed using the 18S rDNA of Aspergillus niger SM24/a of the present invention.
  • the screening samples are materials such as dead leaves and soil.
  • the gradient dilution coating is applied to the xylan screening medium, which is formulated to contain KH 2 PO 4 0.5 g, (NH 4 ) 2 SO 4 2.0 g per liter of the medium.
  • Fermentation activity of xylanase-producing strains The purified strains preserved on the PDA slope were inoculated into an equal amount of fermented rescreening medium, and cultured at 30 ° C, 120 rpm for 6 days. According to Bailey (1992) et al. The method of determination is appropriately modified. The specific method is as follows: 1 g of xylan is accurately weighed, and the solution is adjusted to a 1% substrate after stirring at a low speed of pH 4.8 (0.2 mol/L) in an acetic acid-sodium acetate buffer for 2.5 hours.
  • the fermented rescreening medium is a modified Mandel's nutrient solution, which is based on the original Mandel's nutrient solution and the yeast powder and peptone are removed, and the corn cob powder is added to a final concentration of 30 g/L.
  • strain SM24/a A strain with extremely high xylanase activity was obtained by screening, and the strain was named as strain SM24/a.
  • the strain SM24/a mycelium is white, the colony is small and dense, and can produce black spores.
  • the PDA medium was cultured for 1 day to start long white hyphae, and the long black spores were started after about 36 hours of culture.
  • ITS1 5'-TCC GTA GGT GAA CCT GCG G-3'
  • ITS4 5'-TCC TCC GCT TAT TGA TAT GC-3' Sequence amplification of Aspergillus niger SM24/a total DNA.
  • 10 ⁇ Buffer 2 ⁇ L containing MgCl 2 , 2.5 mmol/L
  • dNTP 10 mmol/L
  • Template DNA the remaining volume is complemented with sterile ultrapure water.
  • the PCR amplification conditions were: pre-denaturation at 95 ° C for 3 min; denaturation at 94 ° C for 1 min, annealing at 52 ° C for 50 s, extension at 72 ° C for 50 s, 35 cycles; extension at 72 ° C for 10 min.
  • the PCR amplification product was subjected to tapping recovery using a DNA gel recovery kit, and sequenced, and the sequence thereof is shown in SEQ ID NO. The sequence was compared with the known sequence in the GenBank database, and the 18S rDNA sequence of the relevant species was obtained from the database to construct a phylogenetic tree, as shown in Figure 5.
  • Example 2 Preparation of xylanase and determination of its enzyme activity and enzymatic properties
  • xylanase preparation Aspergillus niger SM24/a is activated, seed cultured, the seed medium is modified Mandel's nutrient solution (same as in Example 1), and then xylan is added to make The final concentration was 5 g/L, pH 5.6, and sterilized at 115 ° C for 30 min.
  • the parameters of the shaker were set to 30 ° C, 120 rpm, and the seed culture solution was obtained after 3 days of culture.
  • the seed culture was inoculated into a solid fermentation medium at a 7.5% (v/w) inoculum.
  • the solid fermentation medium was formulated with a corn kernel to bran mass ratio of 1:5 to corn cob and bran.
  • the mixture is a substrate and a support material, and an appropriate amount of trace elements (FeSO) is added at 1.4 g/L (NH 4 ) 2 SO 4 , 2.0 g/L KH 2 PO 4 , 0.3 g/L CaCl 2 , 0.3 g/L MgSO 4 .
  • Enzyme activity definition The amount of 1 ⁇ mol of reduced xylose catalyzed by 1 min was defined as one enzyme unit.
  • Example 3 Tolerance of xylanase to fermentation inhibitors
  • the xylanase has different relative enzyme activities for different concentrations of the same fermentation inhibitors (such as ethanol, acetic acid, furfural, vanillin and ferulic acid), but overall, it retains a higher Enzyme activity residual rate.
  • the same fermentation inhibitors such as ethanol, acetic acid, furfural, vanillin and ferulic acid
  • the enzyme activity retention rate of the xylanase of the present invention is 93 to 108% in the concentration range of /L and 0.2 g/L) and vanillin (1.3 g/L, 0.65 g/L and 0.2 g/L).
  • the concentrations of ethanol, acetic acid, furfural, vanillin, ferulic acid, and 5-hydroxymethylfurfural were higher in the higher concentration of the mixed inhibitor system (30 g/L, 30 g/L, 2.1, respectively).
  • the enzyme activity retention rate of the xylanase of the invention is 83.30%; in a medium concentration mixed inhibitor system (ethanol, acetic acid,
  • the concentrations of furfural, vanillin, ferulic acid and 5-hydroxymethylfurfural are (15g/L, 15g/L, 1.1g/L, 0.6g/L, 0.6g/L and 0.6g/L), respectively.
  • the enzymatic activity retention rate of the xylanase of the invention is 90.10%; in the low concentration mixed inhibitor system (ethanol, acetic acid, furfural, vanillin, ferulic acid and 5-hydroxymethylfurfural are respectively 5 g/ L, 5 g/L, 0.35 g/L, 0.2 g/L, 0.2 g/L, and 0.2 g/L), the enzyme activity retention rate of the xylanase of the present invention is 95.59%. Therefore, xylan can be utilized.
  • the enzyme inhibition method indicates a mixed inhibitor containing 5-hydroxymethylfurfural.
  • the tolerance of the xylanase of the present invention to an inhibitor is completely different from that of the xylanase of the prior art, and this tolerated xylanase has not been reported.
  • the xylanase of the present invention has a linear indication of 5-hydroxymethylfurfural in the mixed inhibitor, and this linear indication has not been reported. Therefore, the application of the xylanase of the present invention belongs to a new field.

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Abstract

L'invention concerne une souche d'Aspergillus niger SM24/a à rendement élevé en xylanase dont le numéro d'accès est CGMCC 8671. La souche d'Aspergillus niger SM24/a peut produire une xylanase présentant une forte tolérance à l'éthanol, à l'acide acétique, au furfural, à la vanilline, à l'acide férulique, et leurs mélanges, l'activité enzymatique de la xylanase résultante est très élevée, présente une activité de fermentation à l'état solide jusqu'à 10801 IU/g, la valeur du pH de réaction optimale est 5,6, la température de réaction optimale est de 37 °C, et à 50 °C le taux résiduel de l'activité enzymatique est de 79,01 % En outre, le 5-hydroxyméthyl furfural pésente un effet inhibiteur sur l'enzyme. Que ce soit individuellement ou dans un mélange, la résistance au 5-hydroxyméthyl furfural présente une relation linéaire définie avec le taux d'inhibition. L'invention concerne également une application de la xylanase en tant qu'indicateur qualitatif et quantitatif de la résistance au 5-hydroxyméthyl furfural.
PCT/CN2015/072766 2014-03-28 2015-02-11 Aspergillus niger à rendement élevé en xylanase et son application WO2015143960A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018032797A1 (fr) * 2016-08-18 2018-02-22 青岛蔚蓝生物集团有限公司 Souche mutante d'aspergillus niger et ses applications
CN114437938A (zh) * 2022-01-14 2022-05-06 山东隆科特酶制剂有限公司 一株高产耐高温酸性β-甘露聚糖酶的菌株及其应用

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103992954B (zh) * 2014-03-28 2016-05-25 中国科学院广州能源研究所 一种高产木聚糖酶的黑曲霉及其应用
CN105063170B (zh) * 2015-08-03 2018-11-13 中国热带农业科学院海口实验站 一种产木聚糖酶微生物甄别培养基及其培养方法
CN110004070B (zh) * 2019-04-10 2020-11-03 南京工业大学 一株产木聚糖酶黑曲霉基因工程菌及其构建方法与应用
CN113913305B (zh) * 2021-11-22 2023-10-13 山东隆科特酶制剂有限公司 一株高产酸性木聚糖酶的突变菌株及其应用

Citations (3)

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CN1651569A (zh) * 2004-02-05 2005-08-10 中国农业大学 一种黑曲霉菌株及其用途
CN100999713A (zh) * 2006-12-20 2007-07-18 浙江省农业科学院 一种黑曲霉菌株及其nsp酶的制备方法
CN103992954A (zh) * 2014-03-28 2014-08-20 中国科学院广州能源研究所 一种高产木聚糖酶的黑曲霉及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1651569A (zh) * 2004-02-05 2005-08-10 中国农业大学 一种黑曲霉菌株及其用途
CN100999713A (zh) * 2006-12-20 2007-07-18 浙江省农业科学院 一种黑曲霉菌株及其nsp酶的制备方法
CN103992954A (zh) * 2014-03-28 2014-08-20 中国科学院广州能源研究所 一种高产木聚糖酶的黑曲霉及其应用

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018032797A1 (fr) * 2016-08-18 2018-02-22 青岛蔚蓝生物集团有限公司 Souche mutante d'aspergillus niger et ses applications
CN114437938A (zh) * 2022-01-14 2022-05-06 山东隆科特酶制剂有限公司 一株高产耐高温酸性β-甘露聚糖酶的菌株及其应用
CN114437938B (zh) * 2022-01-14 2023-06-06 山东隆科特酶制剂有限公司 一株高产耐高温酸性β-甘露聚糖酶的菌株及其应用

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