WO2015143960A1

WO2015143960A1 - High xylanase yield aspergillus niger and application thereof

Info

Publication number: WO2015143960A1
Application number: PCT/CN2015/072766
Authority: WO
Inventors: 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
Original assignee: 中国科学院广州能源研究所
Priority date: 2014-03-28
Filing date: 2015-02-11
Publication date: 2015-10-01
Also published as: CN103992954A; CN103992954B

Abstract

Provided is a high xylanase yield Aspergillus niger strain SM24/a, accession number CGMCC No. 8671. The Aspergillus niger strain SM24/a can produce xylanase having a high tolerance to ethanol, acetic acid, furfural, vanillin, ferulic acid, and mixtures thereof, the enzyme activity of the resulting xylanase being very high, having solid state fermentation activity of up to 10801 IU/g, the optimum reaction pH value is 5.6, the optimum reaction temperature is 37ºC, and at 50ºC the residual rate of enzyme activity is 79.01%. In addition, 5-hydroxymethyl furfural has an inhibiting effect on the enzyme. Whether individually or in a mixture, the strength of 5-hydroxymethyl furfural shows a definite linear relationship with the rate of inhibition. Thus, also provided is an application of the xylanase as a qualitative and quantitative indicator of 5-hydroxymethyl furfural strength.

Description

Aspergillus niger with high yield of xylanase and application thereof

Technical field:

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.

Background technique:

Xylanase, the English name xylanase, referred to as XYL, is an enzyme that catalyzes the hydrolysis of β-1,4 xylan glycosidic linkages to release xylose and xylooligosaccharides. In the processing of hemicellulose raw materials and even cellulose raw materials, 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. Even, Billard H (2012) believes that the addition of cellulase by adding enzymes such as xylanase is a decisive factor in reducing the cost of enzymes in the field of lignocellulose and increasing the efficiency of enzymatic hydrolysis.

However, in the field of xylanase application to lignocellulosic biorefining, xylanases are subject to intricate enzymatic conditions and environments. Of note is the effect of inhibitors produced after pretreatment on the enzymatic properties of xylanase. However, up to now, the effects of inhibitors on xylanase have rarely been studied, and the types of inhibitors studied have been less.

Not all 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. For example, 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.

Summary of the invention:

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). 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.

The 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. After comparative analysis and using the BIOLOG identification system, 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. Up to 10801 IU / g (the amount of 1 μmol xylose catalyzed in 1 min is defined as one enzyme unit), the optimum reaction pH is 5.6, the optimum reaction temperature is 37 ° C, and the enzyme activity residual rate at 79 ° C is 79.01%.

Accordingly, 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 μmol of xylose catalyzed in 1 minute is defined as one enzyme unit), and the optimum pH value is determined. For 5.6, The optimum reaction temperature was 37 ° C, and the residual rate of enzyme activity at 50 ° C was 79.01%. Further, 5-hydroxymethylfurfural has an inhibitory effect on the enzyme. When 5-hydroxymethylfurfural is present alone or in combination, its concentration has a linear relationship with the inhibition rate. Therefore, 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.

BRIEF DESCRIPTION OF THE DRAWINGS:

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;

2 is a pH enzymatic characteristic of a xylanase produced by 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.

detailed description:

The following examples are intended to further illustrate the invention and not to limit the invention.

Example 1: Screening of strains

The screening samples are materials such as dead leaves and soil. After the screening sample is properly treated, 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. MgSO ₄ ·7H ₂ O 0.25g, xylan (purchased from Shanghai Yuanju Bio) 5.0g, agar 18 ~ 20g, the balance is water, pH 5.6. Cultured at 30 ° C for 4 ~ 7d, picking hydrolyzed transparent circle Large colonies were purified on PDA medium, and the purified strain was stored on a PDA slope.

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. Take 0.9mL 1% xylan substrate in 15mL calibration tube, preheat for 5min at 50 °C, accurately add 0.2mL of the appropriately diluted enzyme solution (the fermentation broth of the purified strain), accurately time the reaction with a stopwatch for 30min, and immediately add The reaction was terminated by 2 mL of DNS, and the absorption of light (OD) was measured at 540 nm in a boiling water bath for 5 min. Then, strains with higher xylanase activity were selected for proper preservation. 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.

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.

The total DNA of the strain was extracted by the modified CTAB method, and the universal primers for amplifying the fungal ITS sequence were selected: ITS1: 5'-TCC GTA GGT GAA CCT GCG G-3' and ITS4: 5'-TCC TCC GCT TAT TGA TAT GC-3' Sequence amplification of Aspergillus niger SM24/a total DNA. In a 20 μL PCR reaction system, 10×Buffer 2 μL (containing MgCl ₂ , 2.5 mmol/L), dNTP (10 mmol/L) 0.4 μL, upstream and downstream primers of 10 pmol, rTag (5 U/μL) 0.2 μL, about 50 ng. 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. After comparison and analysis, combined with BIOLOG identification results, the strain It belongs to Aspergillus niger and is named as Aspergillus niger SM24/a. It was deposited on December 31, 2013 at the General Microbiology Center (CGMCC) of China Microbial Culture Collection Management Committee. Address: Beichen, Chaoyang District, Beijing No. 3, West Road No. 1, Institute of Microbiology, Chinese Academy of Sciences, with the preservation number: CGMCC No.8671.

Example 2: Preparation of xylanase and determination of its enzyme activity and enzymatic properties

1. Preparation of 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 . ₄ · 7H ₂ O, 5 mg / L; CoCl ₂ , 20 mg / L; MnSO ₄ , 1.6 mg / L; ZnSO ₄ , 1.4 mg / L) is configured as a nutrient solution, the solvent is water, the initial pH of the nutrient solution is 5.0 (as shown in Figure 1, the pH is determined after a series of single factor optimization experiments), after the solid-state fermentation medium is set to 1:3.5 in the ratio of feed to water (mixture of corn cob and bran: nutrient solution) bacteria. The inoculum amount was 7.5% v/v for fermentation. After fermentation for 92-108 hours, the fermentation broth is a xylanase preparation, and the xylanase activity assay is performed.

Add 40 mL of acetic acid buffer solution of pH 4.8 to the flask, smash the agglomerated solid fermentation medium (same as above) with a glass rod, shake at 110 rpm and 30 ° C for 60 min, and directly draw 0.4 mL of the extract to carry out gradient dilution until it reaches The appropriate multiple is then measured for xylanase activity.

2. Determination of enzyme activity of xylanase: according to the assay method of Bailey (1992) et al. and appropriate modification. After weighing 1 g of beech xylan into a 100 mL beaker, it was poured into 60 mL of acetic acid buffer of pH 4.8 and stirred at low speed for 2.5 h, and then the volume of the 100 mL volumetric flask was set to 1% beech xylan solution. Pipette 0.9mL 1% beech xylan solution (Beech wood, SIGMA) as a substrate, preheat for 5min and then add 0.2mL of appropriately diluted xylanase preparation, react at 50 ° C for 30min, immediately add 2mL DNS solution to terminate the reaction. The light absorption value (OD) was measured at 540 nm in a boiling water bath for 5 min. It was determined that the xylanase activity of the xylanase preparation produced by Aspergillus niger SM24/a obtained in the step 1 reached 10801 IU/g, and the enzyme activity was extremely high.

Enzyme activity definition: The amount of 1 μmol of reduced xylose catalyzed by 1 min was defined as one enzyme unit.

3. Determination of the enzymatic properties of xylanase: According to the above determination method, the other conditions are unchanged, the buffer is adjusted to different pH and different temperature to carry out the enzyme activity measurement reaction, and the highest enzyme activity is measured. 100%, the condition is the optimum reaction condition for measuring xylanase. The gradient was diluted with the corresponding buffer and the optimum pH was determined at the same temperature. At an optimum pH of 50 ° C, different temperatures were set to determine the optimum temperature of the enzyme.

The results showed that the optimal reaction pH of the xylanase produced by Aspergillus niger SM24/a of the present invention was 5.6 (Fig. 2), and the enzyme activity was stable under the condition of pH 5.6, and the optimum reaction temperature was obtained. It is 37 ° C (as shown in Figure 3).

Example 3: Tolerance of xylanase to fermentation inhibitors

1. Tolerance of xylanase to single inhibitor

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. At concentrations of ethanol and acetic acid of 5g/L, 15g/L and 30g/L, as well as furfural (2.5g/L, 1.25g/L and 0.4g/L), ferulic acid (1.2g/L, 0.6g) 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).

2. Tolerance of xylanase to mixed inhibitors supplemented with 5-hydroxymethylfurfural:

As shown in Figure 4, 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). g/L, 1.2 g/L, 1.27 g/L and 1.24 g/L), 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.

The detailed description of the present invention is intended to be illustrative of the preferred embodiments of the present invention, and is not intended to limit the scope of the invention. .

Claims

An Aspergillus niger SM24/a, the preservation number is: CGMCC No.8671.
A xylanase obtained by fermenting the Aspergillus niger SM24/aW2 according to claim 1 as a fermentation strain.
Use of Aspergillus niger SM24/a according to claim 1 for producing the xylanase of claim 2.
The use of the xylanase according to claim 2 for the preparation of a preparation for rapidly detecting the concentration of 5-hydroxymethylfurfural by an enzyme inhibition method.