AU2015237100A1

AU2015237100A1 - High xylanase yield Aspergillus niger and application thereof

Info

Publication number: AU2015237100A1
Application number: AU2015237100A
Authority: AU
Inventors: Xiaoyan Chen; Ying GUO; Minchao HE; Xiaoying KONG; Cuiyi LIANG; Yunyun LIU; Jingliang XU; Zhenhong Yuan; Yu Zhang
Original assignee: Guangzhou Institute of Energy Conversion of CAS
Current assignee: Guangzhou Institute of Energy Conversion of CAS
Priority date: 2014-03-28
Filing date: 2015-02-11
Publication date: 2016-11-10
Anticipated expiration: 2035-02-11
Also published as: CN103923840A; CN103923840B; AU2015237100B2; WO2015143961A1

Abstract

Provided are a high xylanase yield Aspergillus niger and application thereof, the Aspergillus niger being Aspergillus niger SM751, accession number CGMCC No. 8670. The enzyme activity of the xylanase produced by said strain is activated under the effect of a mix of various inhibitors, and compared to blank controls, the activation rate of the enzyme activity can reach up to 33.43%.

Description

1

High Xylanase Yield Aspergillus Niger and Application Thereof

Technical Field

The present invention relates to the field of microbiological fermentation and enzyme engineering applications thereof, specifically to Aspergillus niger SM751 having high xylanase yield, xylanase produced there from, and applications of the xylanase in enzymatic hydrolysis of hemicellulose in lignocellulose hydrolysate.

Background of the Invention

With a global annual yield of 6X1010 tons, hemicellulose is an important reproducible carbon resource ranking only after cellulose, and has a wide use in production of biofuel and various biochemical refined products. During the processing of plant materials rich in hemicellulose, addition of xylanase can significantly speed up the reactions, enhance the hydrolysis effects of other enzymes such as cellulase, pectase and mannase, and significantly improve performance of the products. Therefore, xylanase has been widely applied in traditional fields such as paper-making, food, feed and textile, and in fields related to the lignocellulose alcohol.

Xylanases are of great practical value in enzymatic hydrolysis of lignocellulose. Some researchers believe that cellulase being mixed with enzymes such as xylanase is a decisive factor in reducing the cost and enhancing the hydrolysis of lignocellulose. Study results of Vamai A (2011) showed that, for lignocellulose from softwood, hardwood and agricultural crops, xylanase as the coenzyme of cellulase can significantly reduce the cost and increase enzymatic hydrolysis efficiency of cellulase. A possible mechanism is that, enzymatic hydrolysis of the cellulase is enhanced by eliminating the obstruction caused by xylanase or obstacles of xylo-oligomers against absorption of the cellulase. Study results of Alvira (2011) showed that the degree of synergism between xylanase and cellulase can reach 29.9%, and an increase of hydrolysis yield of 10% was obtained. Some researchers even believe that xylanases can be used to partially replace cellulases (Hu J, 2011).

Xylanases are of great practical value in producing cellulosic ethanol with lignocellulose as the raw material. However, not all xylanases are applicable to pretreated lignocellulose. Fermentation inhibitors, such as acetic acid, furfuraldehyde, 5-hydroxymethyl furfural, vanillin and ferulic acid, are more or less found in the solution of pretreated lignocellulose. Among thousands of research reports on xylanases, only a few involve the influences of fermentation inhibitors on xylanases. Study results of de Souza Moreira (2013) showed that vanillin and ferulic acid had certain inhibitory effect or activating effect on xylanases from Aspergillus terreus, but the xylanase from Aspergillus terreus in the study showed very low activity. Meanwhile, the influences of furfuraldehyde, acetic acid and 5-hydroxymethyl furfural on enzymatic hydrolysis activity of xylanases on pretreated lignocellulose have not yet been reported. Besides, alcohol generated during the fermentation also has an inhibitory effect on enzymatic hydrolysis activity of xylanases.

Studies on the influences of fermentation inhibitors, which are generated during the pretreatment of lignocelluloses, on enzymatic hydrolysis activity of xylanase are barely reported at present.

Summary of the Invention

The first objective of the present invention is to provide Aspergillus niger SM751 having high xylanase yield. The Aspergillus niger SM751 has been deposited in the China General

Microbiological Culture Collection Center (CGMCC, address: Institute of Microbiology, Chinese Academy of Sciences, No.3, Yard l,Beichen West Road, Chaoyang District, Beijing) numbered as CGMCC No.8670 on December 31, 2013. 2

The Aspergillus niger SM751 of the present invention was isolated from materials such as deadwoods, rotten leaves and soil in the Mulun Conservation Area of Guangxi Province. Taxonomic characteristics of the fungus were observed as follows: mycelial color was white at the beginning, colony color became grey-black after about 36-48 hours and finally at the 48th hour, thick dense black spores were produced. 18s rDNAof the fungus, with a nucleotide sequence as SEQ ID NO.l, was extracted by using a conventional method. Sequence analysis of the 18s rDNA showed that the fungus was Aspergillus niger, and thus we named it Aspergillus niger SM751.The Aspergillus niger SM751 was deposited in the China General Microbiological Culture Collection Center (CGMCC, address: Institute of Microbiology, Chinese Academy of Sciences, No.3, Yard l,Beichen West Road, Chaoyang District, Beijing) numbered as CGMCC No.8670 on December 31, 2013.

Enzyme activity of xylanase was determined by using xylan (Beech wood, SIGMA), and the result showed that Aspergillus niger SM751 had high xylanase yield. The enzyme activity of the xylanase in solid state fermentation could reach 10446 IU/g, and the pH value most suitable for the reaction was 5.0. The remaining enzyme activity was 93-99.8% under pH of 3.0-6.0. When the pH was 6.5, the enzyme activity was 78% of that at the most suitable pH. Accordingly, the xylanase has very high pH stability at pH of 3.0-6.0. The temperature most suitable for the reaction was 45°C; at the temperatures of 37°C, 50°C and 55°C, the remaining enzyme activity was respectively 80.41%, 98.96% and 79.01% at 37°C, 50°C and 55°C

Thus, the second objective of the present invention is to provide a xylanase, characterized in that, the xylanase is prepared by fermentation using the Aspergillus niger SM751 as a fermenting strain.

The third objective of the present invention is to provide applications of the Aspergillus niger SM751 in the production of xylanase.

The xylanase of the present invention has a tolerance to the fermentation inhibitors in the pretreated solution. The enzyme activity of the xylanase produced by the Aspergillus niger SM751 was activated under the influence of the various inhibitors. The maximum activation ratio could reach 33.43% compared with a control group.

Therefore, the fourth objective of the present invention is to provide applications of the xylanase in the enzymatic hydrolysis of hemicellulose in lignocellulose hydrolysate.

The Aspergillus niger SM751 of the present invention can produce xylanase, which has a high enzyme activity and a tolerance to fermentation inhibitors, at high yield, and thus has a wide application in enzymatic hydrolysis of lignocellulose.

The Aspergillus niger SM751 of the present invention has been deposited in the China General Microbiological Culture Collection Center (CGMCC, address: Institute of Microbiology, Chinese Academy of Sciences, No.3, Yard l,Beichen West Road, Chaoyang District, Beijing) numbered as CGMCC No.8670 on December 31, 2013.

Description of the Drawings

Figure 1 shows the effect of pH value, as a fermentation parameter, on the enzyme activity of xylanase produced by the Aspergillus niger SM751 of the present invention.

Figure 2 shows enzyme activity of the xylanase produced by the Aspergillus niger SM751 of the present invention under different pH values.

Figure 3 shows enzyme activity of the xylanase produced by the Aspergillus niger SM751 of 3 the present invention at different temperatures.

Figure 4 shows the effects of different fermentation inhibitors on the xylanase produced by the Aspergillus niger SM751 of the present invention.

Figure 5 shows a phylogenetic tree constructed based on the 18S rDNAof the Aspergillus niger SM751 of the present invention.

Detailed Description of the Embodiments

Embodiment below is provided for further illustration but without limiting the present invention.

Embodiment 1: Screening of strain

Samples for selection included deadwoods, rotten leaves and soil. After being properly treated, each of the samples was subjected to gradient dilutions and inoculated onto a xylan medium for primary screening. Each litre of the medium contained 0.5 g of KH2PO4, 2.0 g of (NFLt)2SC>4, 0.25 g of MgS04 □ 7H2O, 5.0 g of xylan (purchased from Shanghai Yuanju Biotech), 18-20 g of agar and the balance of water, with a pH value of 5.6. The samples were then cultured at 30°C for 4-7 days; colonies with a larger transparent zone of hydrolysis were selected and purified on PDA medium, and the purified strains were preserved in PDA slants.

Fermentation for secondary screening: each of the above preserved purified strains was respectively inoculated into a liquid medium for secondary screening, cultured at 30°C, 120 rpm for 6 days, and thereby fermentation liquor was obtained. Activity of the xylanase was measured using the method of Bailey (1992) with appropriate modifications, which was as below: 1 g of xylan was added into a buffer solution (acetic acid/sodium acetate, pH 4.8, 0.2mol/L), and then stirred for 2.5 h at a low speed, and then added with water to obtain a 1% substrate. 0.9 mL of the 1% xylan substrate was transferred into a 15 mL graduated test tube, and then preheated for 5 minutes at 50°C. 0.2mL of properly diluted liquid medium was added into the substrate. After reacted for 30 minutes using a stopwatch for timing, the mixture was added with 2 mL of DNS to stop the reaction, and subjected to a boiling bath for 5 minutes. Optical density (OD) at 540 nm was measured. Strains that produced xylanase having relatively high activity were carefully preserved. The liquid medium for secondary screening was a modified Mandeb s medium, which was prepared by removing the yeast powder and peptone in the Mendel’s medium and adding with corncob powder to obtain a concentration of 30 g/L.

After screening, one strain, the xylanase produced from which had extremely high activity, was obtained and named Aspergillus niger SM751.

The total DNAof the Aspergillus niger SM751 was extracted by using improved CTAB method. The total DNA of the Aspergillus niger SM751 was sequentially amplified by using universal primers, ITS1: 5'-TCC GTA GGT GAA CCT GCG G-3' and ITS4: 5'-TCC TCC GCT TAT TGA TAT GC-3', which are used for amplifying the ITS sequence of fungi. 20 pL of a PCR reaction system contained 2 pL of lOx Buffer (containing 2.5 mmol/L of MgCh), 0.4 pL of dNTP (10 mmol/L), 10 pmol of primers, 0.2pL of rTag (5 U/pL), about 50ng of template DNA, and the balance of sterile super pure water. Condition for the PCR amplification was as follows: pre-denaturation at 95°C for 3 minutes, denaturation at 94°C for 1 minute, annealing at 52°C for 50 seconds, extension at 72°C for 50 seconds, 35 cycles, and then extension at 72°C for 10 minutes. The PCR products were collected by using a DNA gel extraction kit and sequenced, and thereby SEQ ID NO.l was obtained. The above sequence was blasted against known sequences in GenBank database, and a phylogenetic tree as shown in Figure 5 was constructed based on the 18s rDNA sequences obtained from the database. The fungus was identified as Aspergillus niger based 4 on analysis and a BIOLOG identification result, and named as Aspergillus niger SM751.

Embodiment 2: Preparation of xylanase, and determination of enzyme activity and enzymatic properties thereof 1. Preparation of xylanase: the Aspergillus niger SM751 was seed cultured and then activated. Medium for seed culture was a modified Mandefs medium (identical with that in embodiment 1), added with 5 g/L of xylan, with an initial pH value of 5.6. The medium was then sterilized for 30 minutes at 115°C. Seed culture was then performed on a shaker at 30°C, 120 rpm for three days to obtain seed culture liquor. The seed culture liquor was inoculated onto a solid fermentation medium with an inoculation amount of 10% (v/w). The solid fermentation medium was prepared by mixing a substrate and a nutrient solution in a mass ratio of 1:3.5 and then sterilized. Corncob and bran were mixed in a mass ratio of 1:5 to obtain a mixture as the substrate and supporting material. The nutrient solution with water as the solvent was prepared with the following ingredients: (NH4)2SC>4, 1.4 g/L; KH2PO4, 2.0 g/L; CaCl2, 0.3 g/L; MgSCL, 0.3 g/L; a proper amount of microelements (FeS04-7H20, 5 mg/L; C0CI2, 20 mg/L; MnSCL, 1.6 mg/L; ZnS04, 1.4 mg/L). The initial pH value of the nutrient solution was 3.5 (as shown in Figure 1, the pH value was determined by a series of single factor optimizations). Fermentation was performed with an inoculation amount of 7.5% (v/v) for 92-108 hours to obtain a fermentation liquid, i.e., the xylanase preparation. And then determination of enzyme activity of the xylanase was carried out. 40 mL of acetic acid buffer solution (pH 4.8) was added into a Erlenmeyer flask. The caked solid fermentation medium was smashed with a glass rod, added into the buffer solution, and then vibrated for 60 minutes at 30°C and 110 rpm. Then 4.0 mL of extract was collected without centrifugation and filtration, and subjected to gradient dilutions to obtain a proper concentration for determination of enzyme activity of the xylanase. 2. Determination of the enzyme activity of the xylanase: The determination was performed using the method of Bailey (1992) with proper modifications. 1 g of xylan from beechwood was added into a 100 mL beaker, followed by the addition of 60 mL of acetic acid buffer solution (pH 4.8), stirred for 2.5 hours at a low speed, and then added with water to obtain 100 mL of 1% xylan solution. 0.9 mL of the 1% xylan solution (Beech wood, SIGMA) was preheated for 5 minutes, added with 0.2 mL of the properly diluted xylanase, and reacted at 50°C. After 30 minutes of reaction, the solution was immediately added with 2 mL of DNS solution to terminate the reaction, and subjected to boiling bath for 5 minutes, and then the optical density (OD) at 540 nm was measured. Results showed that the activity of the xylanase produced by the Aspergillus niger SM751 obtained in step 1 reached 10446 IU/g, indicating a very high activity.

Definition of enzyme activity: one enzyme unit is defined as the amount of the enzyme that catalyzes the production of 1 pmol of reduced xylan per minute. 3. Determination of enzymatic properties of the xylanase: Enzyme activity was measured using the above mentioned method under different pH values and different temperatures. Relative enzyme activity was obtained as a ratio between an enzyme activity and the maximum enzyme activity observed, wherein the maximum enzyme activity was assigned as 100%. Gradient dilution was performed using a corresponding buffer solution, and the optimal pH value at 50°C was determined. Under the optimal pH, the optimal temperature was determined by measuring enzyme activity at different temperatures. 5

Results showed that, the optimal pH value, for the xylanase produced by the Aspergillus niger SM751 of the present invention, in enzymatic hydrolysis was 5.0 (as shown in Figure 2), the enzyme activity was stable under this pH value, and the optimal temperature for enzymatic hydrolysis was 45°C (as shown in Figure 3). The xylanase had very high pH stability under the pH of 3.5-6.0, with a relative enzyme activity of 93.45%~99.84%.

Embodiment 3: Effects of fermentation inhibitors on xylanase 1. Inhibitory effect of a single fermentation inhibitor on the xylanase produced by the Aspergillus niger SM751 of the present invention:

Inhibitory effects of different fermentation inhibitors varied, as shown in Figure 4. With 10.00 g/L of ethanol, the remaining enzyme activity of the xylanase was 102.15%, and the enzyme activity was increased by about 2.15%. With 10.10 g/Lof acetic acid, the remaining enzyme activity of the xylanase was about 99.05%. With 1.40 g/L of furfuraldehyde, the remaining enzyme activity of the xylanase was 100.08%. With 1.20 g/L of 5-hydroxymethyl furfural, the remaining enzyme activity of the xylanase was 95.64%. With 1.10 g/L of vanillin, the remaining enzyme activity of the xylanase was 98.16%. With l.lOg/L of ferulic acid, the remaining enzyme activity of the xylanase was about 72.99%. The above results indicated that the xylanase of the present invention had high tolerance to fermentation inhibitors. 2. Inhibitory effects of mixed inhibitors on the xylanase produced by the Aspergillus niger SM751 of the present invention: A mixture of ethanol (11.90 g/L), acetic acid (13.60 g/L), furfuraldehyde (1.40 g/L), 5-hydroxymethyl furfural (1.10 g/L), ferulic acid (1.20g/L) and vanillin (1.30g/L) showed an activation effect on the xylanase. The remaining enzyme activity was 133.39%, i.e., under the effect of the mixed fermentation inhibitors, the enzyme activity of the xylanase was 11008 IU/g, and the activation ratio reached 33.39%.

As showed above, the xylanase produced by the Aspergillus niger SM751 of the present invention has a very high enzyme activity up to 10446 IU/g. When used in enzymatic hydrolysis in lignocellulose hydrolysate, the xylanase has high tolerance to various fermentation inhibitors or the mixture thereof.

The tolerance of the xylanase of the present invention to the inhibitors is completely different from those in the prior art. Xylanase with such tolerance has not yet been reported.

The xylanase produced by the Aspergillus niger SM751 was used in the enzymatic hydrolysis of bagasses which had been pretreated with high-temperature liquid water. Conditions for pretreatment of bagasses with high-temperature liquid water included: 180°C temperature, nitrogen pressurizing to 4MPA, 20min pretreating time, and 1:20 solid-liquid ratio. After 600IU of xylanase was added to perform enzymolysis for 24h, the yield of xylan was 75.63%.

The above detailed description is a specific explanation for feasible embodiments of the present invention. The embodiments are not used for limiting the scope of the present invention. Any equivalent or changes made on the basis of the present invention shall fall within the scope of the present invention.

Claims

Claims

1. Aspergillus niger SM751, having an accession number CGMCC No.8670.
2. A xylanase, characterized in that, it is prepared by fermentation using the Aspergillus niger SM751 according to claim 1 as a fermentation strain.
3. An application of the Aspergillus niger SM751 according to claim 1 in production of the xylanase according to claim 2.
4. An application of the xylanase according to claim 2 in enzymatic hydrolysis of hemicellulose in lignocellulose hydrolysate.