Use of a tannase in the preparation of gallic acid

Abstract

The application discloses application of tannase in preparation of gallic acid, and belongs to the technical field of enzyme engineering. The application comprises the following steps: 1) preparing a tannic acid solution with a concentration of 25%-35% (w / v), and adjusting the pH of the tannic acid solution to 4.0-5.0; 2) adding the tannic acid solution obtained in step 1) into a reaction kettle, adding tannase according to a substrate addition amount of 36-54 U / g, and hydrolyzing to generate gallic acid. The tannase used in the application is derived from Aspergillus niger ATCC13496, and has at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 13 or a mature polypeptide thereof. The application further discloses a method for preparing tannase by using an amino acid sequence of tannase expressed by Aspergillus niger CICC2462 strain.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology and relates to the application of a tanninase in the preparation of gallic acid. Background Technology

[0002] Tanninase (EC 3.1.1.20), also known as tannic acidase, is a tannic acid acyl hydrolase. This enzyme hydrolyzes acids with two phenolic groups, such as tannic acid, and can hydrolyze galloyltannins and alkyl gallate esters to produce gallic acid, glucose, and alkyl alcohols, among other small molecules. Due to its highly efficient hydrolysis of tannins, tanninase has been widely applied in the food, beverage, brewing, pharmaceutical, and chemical industries.

[0003] The sources of microorganisms capable of producing tannins are abundant, mainly fungi such as *Aspergillus*, *Penicillium*, and *Rhizopus*, especially *Aspergillus niger*, *Aspergillus oryzae*, and *Aspergillus flavus* (Sunny D, Gunjan M, Kumar SA. Recent trends and advancements in microbial tannase-catalyzed biotransformation of tannins: a review. International Microbiology, 2018, 21.). Current research on tannin fermentation production focuses primarily on *Aspergillus* and *Penicillium*, often employing methods such as mutagenesis breeding to select and improve producing strains, and optimizing fermentation conditions to enhance enzyme activity (LV Rodríguez-Durán, Valdivia-Urdiales B, Contreras-Esquivel JC, et al. Novel strategies for upstream and downstream processing of tannin acyl hydrolase. Enzyme research, 2011, 2011: 823619.). However, tanninases produced by this method generally suffer from low production efficiency and high production costs, and the mixed composition of enzyme components significantly limits their large-scale industrial application. Because tanninases are diverse and their properties vary greatly from source to source, finding a suitable tanninase for a specific application is extremely difficult. While some literature and patents report the use of genetic engineering to construct recombinant bacteria for tanninase production, these enzymes show limited effectiveness in hydrolyzing tararatanine, and the industrial application of using tanninase to hydrolyze tararatanine for efficient gallic acid production has not yet been reported.

[0004] Gallic acid, as an important fine chemical, is widely used in food, chemical, and pharmaceutical fields. Currently, industrial methods for preparing gallic acid (acid and alkali methods), while possessing advantages such as mature processes, also generate large amounts of wastewater and waste salts, leading to serious environmental pollution (Chinese patent documents CN106242966 B and CN108003012A). With increasingly stringent environmental regulations, the chemical methods for preparing gallic acid are no longer adequate for current development needs. Therefore, in recent years, research on tannin-enzymatic methods has been conducted both domestically and internationally. Some literature reports the use of microbial fermentation to prepare gallic acid, but this method has a long production cycle and incomplete hydrolysis. Chinese patent document CN1083532A improved upon this method by first culturing microorganisms and then adding raw materials to prepare gallic acid. However, this method also suffers from long production cycles, low product yields, and complex operations, making it unsuitable for large-scale production and unable to compete with chemical methods (Lokeswari N, JayaRK. Optimization of Gallic Acid Production from Terminalia Chebula by Aspergillus niger. Journal of Chemistry, 2007, 4(2): 287-293.). To date, there are no reports of industrial applications of directly using tanninase to prepare gallic acid. The main reasons are threefold: 1) No suitable enzyme and corresponding sequence for efficiently hydrolyzing tararatanin have been found; 2) No corresponding production strain has been developed that can effectively reduce production costs; and 3) No corresponding gallic acid preparation process has been developed. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide an application of a novel tanninase in the efficient preparation of gallic acid.

[0006] The amino acid sequence of the tanninase has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:13 or its mature polypeptide.

[0007] In a preferred embodiment, the amino acid sequence of the tanninase is the mature polypeptide of the polypeptide shown in SEQ ID NO:13 or SEQ ID NO:13, the tanninase is derived from Aspergillus niger ATCC 13496, the optimal reaction temperature is 65-75℃, and the optimal reaction pH is 4.0-5.0.

[0008] The polynucleotide sequence of the tanninase includes: 1) the polynucleotide sequence of SEQ ID NO:5; and / or, 2) the cDNA sequence of SEQ ID NO:5; and / or, 3) a polynucleotide sequence hybridizing with the polynucleotide sequence of 1) or 2) or its full-length complementary strand under very stringent conditions. Those skilled in the art will understand that, due to the well-known degeneracy in genetic coding, different polynucleotides encoding the same amino acid sequence can be designed and prepared using conventional techniques, and the optimization of codons for specific host cells is also well known in the art.

[0009] Tens of thousands of tanninase coding sequences have been disclosed in existing technologies. For example, more than 18,000 tanninase coding genes from different strains have been disclosed on NCBI. There are more than a dozen tanninases from Aspergillus nigerATCC 13496 alone. However, no research has yet discovered a tanninase variety with excellent performance that can be used for the large-scale industrial production of gallic acid. Literature reports that Aspergillus niger ATCC 13496 can produce tannase (Sharma S, Bhat TK, Dawra RK. Isolations, purification and properties of tannase from Aspergillus niger van Tieghem. World Journal of Microbiology & Biotechnology, 1999). However, this literature also discloses that the tannase derived from Aspergillus niger ATCC 13496 is an intracellular enzyme. Due to the low yield of intracellular enzymes, they cannot be industrialized. Therefore, this literature provides a reverse lesson for those skilled in the art, and there has been no further research on tannase derived from Aspergillus niger ATCC 13496 to date. Through extensive screening and verification experiments, the inventors have provided a tanninase derived from Aspergillus nigerATCC 13496, which is an extracellular secreted tanninase (designated Tan3 in this invention). The amino acid sequence of this enzyme is shown in SEQ ID NO:13. The properties of this tanninase are significantly superior to those of tanninases in the prior art.

[0010] The tanninase Tan3 has the following chemical properties:

[0011] 1) Function: It acts on the phenolic acid bond to carry out hydrolysis;

[0012] 2) Molecular weight: Approximately 800,000 Da (determined by SDS-PAGE);

[0013] 3) Temperature stability: The stability remains above 90% up to 65℃ (pH 4.0, 30-minute heat treatment);

[0014] 4) Optimal temperature: approximately 70℃;

[0015] 5) Optimal pH: Approximately 4.0;

[0016] 6) Acid resistance: Under pH 2.5 conditions, the enzyme activity loss is less than 2% (30℃) after 5 hours;

[0017] 7) Substrate specificity: It acts well on tannic acid and gallic acid esters.

[0018] In one specific embodiment of the present invention, the present invention utilizes Aspergillus niger CICC 2462 and the above sequence to prepare a tanninase with high enzyme activity (up to 1560.0 U / mL) and good thermal stability. This tanninase can maintain its enzyme activity for a long time in a high temperature range, and it can continuously hydrolyze tararatanine at a concentration as high as 30%, showing good substrate tolerance.

[0019] This invention also proposes a method for efficiently preparing gallic acid using the aforementioned tanninase, specifically comprising the following steps:

[0020] 1) Prepare a tannic acid solution with a concentration of 25%-35% (w / v) and adjust the pH of the tannic acid solution to 4.0-5.0;

[0021] 2) Add the tannic acid solution obtained in step 1) to the reaction vessel, add tanninase at a substrate addition rate of 36-54 U / g, and hydrolyze to generate gallic acid.

[0022] In one specific embodiment, the gallic acid is prepared as follows:

[0023] 1) Weigh 1-2 parts by weight of tara powder raw material and place it in a raw material extraction tank. Add 3-5 parts by weight of purified water and stir thoroughly to dissolve.

[0024] 2) At 30-35℃, stir and extract for 6-8 hours, then centrifuge and filter to obtain tannic acid extract;

[0025] 3) Heat and concentrate the tannic acid extract until the tannic acid concentration reaches 25%-35% (w / v), and adjust the pH of the concentrate to 4.0-5.0;

[0026] 4) Take the tannic acid extract with a concentration of 25%-35% (w / v) from step 3) and add it to the reaction vessel. Add the tannin enzyme of the present invention according to the substrate addition amount of 36 U / g-54 U / g to hydrolyze and generate gallic acid. The reaction temperature is controlled at 35℃-40℃ throughout the hydrolysis process.

[0027] In another specific embodiment, the method for preparing gallic acid is as follows:

[0028] 1) Weigh 2 kg of tara powder raw material and place it in a raw material extraction tank, then add 10 kg of purified water;

[0029] 2) At room temperature (35℃), stir and extract for 6 hours, centrifuge and filter to obtain approximately 9L of tannic acid extract;

[0030] 3) Concentrate 9L of tannic acid extract to a tannic acid concentration of 30%;

[0031] 4) Take 500 ml of the concentrated tannic acid extract with a substrate concentration of about 30% (w / v) into the reaction vessel, adjust the pH to 4.0, add tanninase solution, the amount of enzyme solution added per gram of substrate is 36 U, control the reaction temperature in a water bath at 40℃, after 8 hours of reaction, detect the amount of reduction of substrate tannic acid and the amount of product gallic acid generated, and calculate the conversion rate.

[0032] The tanninase described in this invention can continuously hydrolyze tannins to produce gallic acid at a tannin substrate concentration of up to 30% (w / v). Moreover, the hydrolysis cycle is short, requiring only 8 hours for complete hydrolysis, with a conversion rate as high as 99.5%. The prepared gallic acid has a purity of 99%, making this tanninase suitable for industrial-scale production of gallic acid. It also has significant advantages over chemical methods for preparing gallic acid.

[0033] The chemical method for preparing gallic acid reported in patent document CN108003012A is complex, requiring high temperature, high pressure, and large amounts of acids and alkalis. The method for efficiently preparing gallic acid using the aforementioned tanninase, proposed in this invention, is simpler to operate, does not require high temperature and pressure, and avoids the use of large amounts of acids and alkalis, thus preventing significant wastewater and waste salt pollution. The microbial method for preparing gallic acid reported in patent document CN1083532A is complex, with a reaction cycle exceeding 35 hours, a conversion rate of only about 73%, and a hydrolysate concentration of only about 8%, making it unsuitable for large-scale production. The method for preparing gallic acid proposed in this invention is simpler and more controllable, achieving a hydrolysate concentration of up to 30%, a conversion rate as high as 99.5%, and a cycle of only about 8 hours. In summary, the method for efficiently preparing gallic acid using the aforementioned tanninase proposed in this invention has significant advantages and can be well applied to the industrial-scale production of gallic acid.

[0034] This invention also proposes a method for preparing tanninase, wherein the method utilizes *Aspergillus niger* strain CICC 2462 to express the amino acid sequence of the tanninase, wherein the amino acid sequence of the tanninase has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:13 or its mature polypeptide; preferably, the amino acid sequence of the tanninase is SEQ ID NO:13 or the mature polypeptide of the polypeptide shown in SEQ ID NO:13. Using *Aspergillus niger* strain CICC 2462 to express the tanninase of this invention results in a product with high enzyme activity and low production cost, which is beneficial for industrial production.

[0035] Tanninase (EC3.1.1.20): Also known as tannic acidase. A tannic acid hydrolase that hydrolyzes acids containing two phenolic groups, such as tannic acid. This enzyme can be produced by molds, such as Aspergillus niger and Aspergillus oryzae. Preferably, the tanninase is derived from the genus Aspergillus, such as Aspergillus niger; more preferably, the amino acid sequence of the tanninase is shown in SEQ ID NO:13.

[0036] The term "amino acid sequence" is synonymous with and interchangeable with the terms "polypeptide," "protein," and "peptide." When such amino acid sequences exhibit activity, they are called "enzymes." Conventional single-letter or three-letter codes for amino acid residues are used, where the amino acid sequence is presented with a standard amino-to-carboxyl terminal orientation (i.e., N→C).

[0037] Mature polypeptide: The term "mature polypeptide" refers to a polypeptide in its final form after translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. It is known in the art that host cells can produce mixtures of two or more different mature polypeptides (i.e., with different C-terminal and / or N-terminal amino acids) expressed by the same polynucleotide. In a specific example of the invention, the protein encoded by SEQ ID NO:5 was predicted to be 583 amino acids. Using SignalP program version 3.0 (Nielsen et al., 1997, Protein Engineering 10:1-6), a 19-residue signal peptide was predicted, and the predicted mature protein contained 564 amino acids, with a predicted molecular weight of 61.8 kDa and an isoelectric point of 4.64.

[0038] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared from mature, spliced ​​mRNA molecules derived from eukaryotic cells via reverse transcription. cDNA lacks the intron sequences normally present in the corresponding genomic DNA. Initial, primary RNA transcripts are precursors to mRNA, which are processed through a series of steps, including splicing, to become mature, spliced ​​mRNA.

[0039] Sequence identity: The correlation between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity". When aligned using the CLUSTALW algorithm with preset parameters, a specific sequence has at least a certain percentage of amino acid residues that are identical to a specified reference sequence. The preset parameters for the CLUSTALW algorithm are: deletion count is the number of residues that are not identical to the reference sequence. This includes deletions occurring at any end. For example, a 500-amino acid peptide variant that omits five amino acid residues at the C-terminus has a 99% (495 / 500 identical residues × 100) sequence identity percentage relative to the parent peptide; such variants are covered by the language "variants with at least 99% sequence identity to the parent".

[0040] Very stringent conditions: This means that for probes at least 100 nucleotides in length, standard Southern blotting procedures are followed, including pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. The vector material is then washed three times at 65°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0041] The term "about" refers to ±10% of the value.

[0042] The beneficial effects of this invention include:

[0043] 1) This invention provides a tanninase that can efficiently hydrolyze taratanin. This tanninase has superior enzymatic properties. Under pH 2.5 conditions, the enzyme activity loss is less than 2% (30°C) for 5 hours. It can continuously hydrolyze tannin to gallic acid at a tannin substrate concentration of up to 30% (w / v) with a conversion rate greater than 99.0%, making it applicable to large-scale industrial enzymatic preparation of gallic acid.

[0044] 2) This invention proposes a method for producing tanninase. The *Aspergillus niger* CICC 2462 strain of this invention can be used to prepare a tanninase with high activity (up to 1560.0 U / mL) and good thermal stability using the tanninase sequence of this invention. This tanninase can maintain its activity for a relatively long time within a high temperature range, and it exhibits good substrate tolerance. Compared with direct fermentation using natural production strains, this method is simpler, more stable, and has higher production efficiency, but also higher production costs. Furthermore, the enzyme component is singular. While some patents report the use of *Pichia pastoris* to express tanninase, this process requires the continuous addition of large amounts of methanol, which is toxic and difficult to separate. Clearly, the method for preparing tanninase in this invention avoids these problems.

[0045] 3) Current industrial methods for preparing gallic acid (acid and alkaline methods), while possessing advantages such as mature processes, also generate large amounts of wastewater and waste salts, leading to severe environmental pollution. Some literature and patents report the use of microbial methods to prepare gallic acid, but this method suffers from long production cycles, low product yields, and complex operations, making it unsuitable for large-scale production and unable to compete with chemical methods. To date, there are no reports of processes directly utilizing tanninases to prepare gallic acid. This invention discloses an enzymatic hydrolysis method for preparing gallic acid, which can continuously hydrolyze tannins to generate gallic acid at concentrations up to 30% (w / v) of tannin substrate. It is comparable to chemical methods in terms of production cycle and conversion rate, and is simple to operate, operates under mild conditions, and is environmentally friendly, making it highly suitable for large-scale production applications. Attached Figure Description

[0046] Figure 1 This is the pGla-amds plasmid map of the present invention.

[0047] Figure 2 The present invention is based on the SDS-PAGE of the shake-flask culture fermentation broth of the recombinant Aspergillus niger expression strain expressing tannins.

[0048] Figure 3 This invention relates to the determination of the optimal temperature for tanninases from different sources.

[0049] Figure 4 This invention relates to the determination of the optimal pH for tanninases from different sources.

[0050] Figure 5 This invention relates to the temperature stability of the tannin enzyme Tan3.

[0051] Figure 6 This is an acid resistance test of the tanninase Tan3 of this invention.

[0052] Figure 7 This is the curve of gallic acid production from tannic acid by enzymatic hydrolysis of tannic acid using recombinant tanninase. Detailed Implementation

[0053] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. Except for the contents specifically mentioned below, the processes, conditions, and experimental methods for implementing the present invention are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0054] This invention discloses numerous different embodiments or examples for implementing various methods of the invention. To simplify the disclosure, specific embodiments or examples are described below. These are merely examples and are not intended to limit the invention. Furthermore, examples of various specific processes and materials provided by this invention will allow those skilled in the art to recognize the applicability of other processes and / or the use of other materials. Unless otherwise stated, implementation of this invention will employ conventional techniques in fields such as chemistry and molecular biology, within the capabilities of those skilled in the art. Additionally, unless otherwise stated, nucleic acids are written from left to right in a 5' to 3' orientation, and amino acid sequences are written from left to right in a direction from the amino terminus to the carboxyl terminus.

[0055] Example 1: Construction of Tanninase Expression Plasmid

[0056] The method for constructing the tanninase expression plasmid includes the following parts:

[0057] 1) Linearize the pUC57 plasmid using vector-F and vector-R primers;

[0058] 2) Select the amdS expression box, which was synthesized by GenScript, and the sequence of the amdS expression box is shown in SEQ ID NO:1;

[0059] 3) A DNA fragment containing the gla promoter and terminator of the Aspergillus niger glucoamylase gene, synthesized by GenScript, with the sequence shown in SEQ ID NO:2;

[0060] 4) The tanninase genes were derived from Aspergillus niger CBS 513.88, Aspergillus niger An76, Aspergillus niger ATCC 13496, Aspergillus skawachii IFO 4308, Aspergillus niger FJ0118, Aspergillus oryzae RIB40, Aspergillus fumigatus Af293, and Penicillium rubens Wisconsin 54-1255 (nucleotide sequences SEQ ID NO: 3-10, amino acid sequences SEQ ID NO: 11-18, respectively). These genes were named tan1, tan2, tan3, tan4, tan5, tan6, tan7, and tan8, respectively, and were synthesized by GenScript.

[0061] First, using primers amdS-F and amdS-R, and gla-F and gla-R, respectively, PCR amplified the amdS expression cassette with recombinant arms and a DNA fragment containing the gla promoter and terminator. The linearized pUC57 vector, amdS expression cassette, and gla promoter and terminator DNA fragment were then recombined using a Gibson Master Mix Kit (E2611, New England Biolabs) to obtain the pGla-amdS plasmid. Sequencing confirmed the correct sequence. This plasmid can be linearized at the AflII and XhoI sites for insertion into tanninase genes.

[0062] Furthermore, the tanninase expression cassette was constructed as follows: Using primer pairs tan1F / tan1R, tan2F / tan2R, tan3F / tan3R, tan4F / tan4R, tan5F / tan5R, tan6F / tan6R, tan7F / tan7R, and tan8F / tan8R, tan1–tan8 genes with recombinant arms were amplified by PCR. Then, using a Gibson Master Mix Kit (E2611, New England Biolabs), the tan1–tan8 genes were recombined with the linearized pGla-amdS plasmid to obtain eight plasmids: ptan1-amdS, ptan2-amdS, ptan3-amdS, ptan4-amdS, ptan5-amdS, ptan6-amdS, ptan7-amdS, and ptan8-amdS, respectively. The sequences were confirmed by sequencing. These plasmids can be linearized at the HindIII site for protoplast transformation.

[0063] The relevant primer sequences are as follows:

[0064] Table 1 Primers used in Example 1 of this invention

[0065]

[0066]

[0067] Example 2: Transformation and Integration of Tanninase Expression Kits

[0068] The tanninase expression cassette from Example 1 of this invention was introduced into Aspergillus niger CICC 2462 strain (purchased from China Industrial Microbial Culture Collection Center CICC) using the protoplast transformation method. The specific operation steps are as follows:

[0069] (1) Preparation of protoplasts: Aspergillus niger mycelia were inoculated in nutrient-rich TZ liquid medium (0.8% beef extract, 0.2% yeast extract, 0.5% peptone, 0.2% NaCl, 3% sucrose, pH 5.8). After culturing for 48 h, the mycelia were collected by filtration using a Mira-cloth (Calbiochem) and washed with 0.7M NaCl (pH 5.8). After the mycelia were dried, they were transferred to an enzymatic hydrolysate (pH 5.8) containing 1% cellulase (Sigma), 1% snailase (Sigma), and 0.2% lysozyme (Sigma). The hydrolysate was incubated at 30℃ and 65 rpm for 3 h. The hydrolysate containing protoplasts was then placed on ice and filtered through four layers of lens paper. The filtrate was centrifuged at 3000 rpm and 4℃ for 10 min. The supernatant was discarded, and the protoplasts attached to the tube wall were treated with STC solution (1MD-Sorbitol, 50 mM CaCl2, 10 mM... Wash once with Tris (pH 7.5), and finally resuspend the protoplasm in an appropriate amount of STC solution.

[0070] (2) Protoplast transformation: 10 μl of DNA fragment containing a tannin expression cassette obtained by HindIII linearization was added to 100 μl of protoplast suspension. After mixing, the mixture was incubated at room temperature for 25 min. Then, 900 μl of PEG solution was added in three portions, and after mixing, the mixture was incubated at room temperature for 25 min. The mixture was then centrifuged at 3000 rpm for 10 min at room temperature. The supernatant was discarded, and the protoplasts attached to the tube wall were resuspended in 1 ml of STC solution. This was then mixed with acetamide medium (3% sucrose, 0.05% KCl, K2HPO4·3H2O) pre-cooled to about 45°C. Mix 0.1% FeSO4, 0.001% MgSO4, 0.0244% acetamide, 0.06% CsCl, and 0.34% CsCl and spread the mixture on a plate. After the plate solidifies, incubate it in a 34°C incubator for 4-5 days. Transfer the transformants to a new acetamide medium plate and incubate it in a 34°C incubator for another 4-5 days. The transformed transformants that grow are called positive transformants.

[0071] Example 3: Shake-flask culture and enzyme activity assay of recombinant tanninase expression strain

[0072] 1. Determination of tanninase activity

[0073] 1) Definition of enzyme activity unit

[0074] Under conditions of 30°C and pH 5.0, the amount of enzyme required to degrade propyl gallate (PG) solution and release 1 μmol of gallic acid per minute is defined as one unit of enzyme activity (U).

[0075] 2) Reagents

[0076] Unless otherwise specified, all reagents refer to analytical grade reagents and Class II water as specified in GB / T 6682.

[0077] 2.1 0.1 mol / L disodium hydrogen phosphate-0.05 mol / L citrate buffer

[0078] Accurately weigh 73.56g of disodium hydrogen phosphate dodecahydrate and 20.45g of citric acid monohydrate, add 800ml of water to dissolve, then bring the volume to 2000ml, adjust the pH to 5.0, and store at 4℃ for one month.

[0079] 2.2 0.5 mol / L KOH solution

[0080] Accurately weigh 28.055g of KOH, add 800ml of water to dissolve, and then bring the volume up to 1000ml. Store at room temperature.

[0081] 2.3 0.667% (w / v) Rhodanine solution (methanol-rhodanine)

[0082] Accurately weigh 0.667g of rhodanine, dissolve it in 80ml of methanol, and then dilute to 100ml with methanol. It is effective for 15 days when stored at room temperature.

[0083] 2.4 Gallic acid standard solution, concentration 10 mmol / L

[0084] Weigh 0.1701g of anhydrous gallic acid, dissolve it in phosphoric acid-citric acid buffer solution, and bring the volume to 100ml. Store at 4℃ for 15 days.

[0085] 2.5% propyl gallate (PG) solution, concentration 10 mmol / L

[0086] Weigh 0.2122 g of propyl gallate, add 80 ml of disodium hydrogen phosphate-0.05 mol / L citrate buffer, stir magnetically and heat until completely dissolved, cool, and bring the volume to 100 ml with buffer. Shake well before use, checking for crystals. If necessary, heat to dissolve before use. Refrigerate at 4°C for up to 10 days.

[0087] 3) Measurement steps

[0088] 3.1 Plotting the standard curve of gallic acid

[0089] Gallic acid standard solutions of different concentrations were prepared using a pH 5.0 disodium hydrogen phosphate-citric acid (0.1-0.05M) buffer solution, resulting in nine concentration gradients from 40 to 240 μmol / L. 0.5 mL of each diluted gallic acid standard solution was mixed with 0.3 mL of methanol-toluene-tannin solution (0.667% w / v) and added to all test tubes. The mixture was incubated at 30°C for 5 min, followed by 4.2 mL of KOH solution, and then incubated at 30°C for 10 min. A buffer solution was used as a blank control instead of the standard solution, and the absorbance was measured at 520 nm (A520). A standard curve was plotted with concentration (mmol / L) on the x-axis and A520 on the y-axis.

[0090] 3.2 Preparation of Sample Solution

[0091] Liquid samples can be directly diluted with buffer to a tannin activity of 0.03-0.15 U / ml in the enzyme solution to be tested.

[0092] 3.3 Sample Determination

[0093] 1) Take 4 clean test tubes, 1 blank tube and 3 test tubes. Add 0.25 ml of PG solution to each tube and incubate in a water bath at 30°C for 5-10 min. Then add 0.25 ml of the enzyme solution to be tested to the test tube at 15 s intervals and react in a water bath at 30°C for 5 min.

[0094] 2) Add 0.3 ml of methanol-tannin solution (0.667%, W / V) to all test tubes at 15 s intervals and keep warm for 5 min;

[0095] 3) Add 4.2 ml of KOH solution (0.5 M) to all test tubes and 0.25 ml of enzyme solution to the blank tube. After placing the tubes at 30 °C for 10 min (starting from the time when the last sample was added with KOH solution), reset the time to zero with the blank tube and measure the absorbance of each solution at 520 nm.

[0096] Calculation of enzyme activity in the sample:

[0097] XD=(△A520-b)×0.5×n / 0.25 / t / k

[0098] XD—Tanylase activity in the sample dilution, U / ml;

[0099] △A520—Absorbance of enzyme reaction solution - Absorbance of enzyme blank sample;

[0100] K—the slope of the standard curve;

[0101] b—the intercept of the standard curve;

[0102] 1 / 0.25 — converted to 1 ml of enzyme solution;

[0103] t—Enzymatic hydrolysis reaction time, 5 min;

[0104] 0.5-0.5 ml enzyme solution

[0105] n—the dilution factor of the sample.

[0106] Enzyme activity assay and protein electrophoresis analysis of recombinant Aspergillus niger expressing tanninase:

[0107] Positive transformants from the recombinant Aspergillus niger expressing tanninase in Example 2 of this invention were inoculated into shake flasks containing 50 ml of YPM medium (0.2% yeast extract, 0.2% peptone, 2% maltose) and cultured at 34°C and 220 rpm for 6 days. The supernatant of the fermentation broth was collected by centrifugation, and the tanninase activity of the obtained samples was determined according to the above-described method. The results are shown in Table 2 below. The results show significant differences in tanninase activity from different sources. Protein electrophoresis (SDS-PAGE) was performed on the samples after fermentation broth treatment, and the results are shown below. Figure 2 In this study, the molecular weights of tanninases from different sources were all around 80 kDa.

[0108] Table 2 Enzyme activity assays of recombinant tannin expression strains from different sources.

[0109] strains Sequence source Enzyme activity U / ml Ptan1-amdS AspergillusnigerCBS513.88(SEQ ID NO:3) 1342 Ptan2-amdS AspergillusnigerAn76(SEQ ID NO:4) 50 Ptan3-amdS AspergillusnigerATCC13496(SEQ ID NO:5) 1560 Ptan4-amdS AspergilluskawachiiIFO4308(SEQ ID NO:6) 1804 Ptan5-amdS AspergillusnigerFJ0118(SEQ ID NO:7) 239 Ptan6-amdS AspergillusoryzaeRIB40(SEQ ID NO:8) 3099 Ptan7-amdS AspergillusfumigatusAf293(SEQ ID NO:9) 348 Ptan8-amdS PenicilliumrubensWisconsin54-1255(SEQ ID NO:10) 37 CICC2462 (control) 0

[0110] Example 4: Determination of Enzymatic Properties of Tanninases from Different Sources

[0111] 1) Optimal reaction temperature

[0112] Following the above method for determining tanninase activity, the reaction was carried out at temperatures of 20℃, 30℃, 35℃, 40℃, 50℃, 60℃, 70℃, and 80℃. The temperature at which the highest activity was observed was used as 100% relative activity to represent each measurement result. Figure 3 The results showed that the optimal reaction temperatures for Tan1, Tan3, Tan4, and Tan6 were around 70℃, while those for Tan2, Tan5, and Tan7 were around 60℃, and the optimal reaction temperature for Tan8 was around 50℃.

[0113] 2) Optimal reaction pH

[0114] Following the above method for determining tanninase activity, 10 mM propyl gallate was used as the substrate. The assay was performed in various buffer solutions (disodium hydrogen phosphate-citrate buffer pH 2.5, 3.0, 4.0, 5.0; phosphate buffer pH 6.0, 7.0, 8.0) at 30°C for 5 minutes. The pH value showing the maximum activity was used as 100% relative activity to represent each assay result. Figure 4 The results showed that the optimal reaction pH for Tan1 was around 7.0, for Tan2 and Tan4 it was around 6.0, for Tan5, Tan6, Tan7 and Tan8 it was around 5.0, and for Tan3 it was around 4.0.

[0115] Example 5: Hydrolysis of tannic acid by tanninases from different sources

[0116] 10% tannic acid was used as the substrate. 500 ml of the 10% tannic acid solution was added to the reaction vessel, and 900 U of tanninases Tan1, Tan2, Tan3, Tan4, Tan5, Tan6, Tan7, and Tan8 were added to the reaction vessel respectively. All reactions were controlled only at an initial pH of 4.0; pH was not controlled during the reaction. The temperature was maintained at approximately 40℃ throughout the reaction. Samples were taken every 2 hours to detect the reduction in tannic acid in the reaction solution, and the reaction was stopped after 18 hours. The conversion rate was calculated based on the reduction in tannic acid, and the degree of hydrolysis of tannic acid by tanninases from different sources was determined based on the conversion rate. The results are shown in Table 3. The results show that the hydrolytic activity of tanninases from different sources differed significantly. Tan3 exhibited the highest hydrolytic activity for tannic acid, achieving a conversion rate of 98.5% without controlling the pH during the reaction. The hydrolysis of tannic acid by Tan3 shows that this reaction process does not require large amounts of acid or alkali, the reaction conditions are mild, and the conversion rate can reach over 90%. Therefore, Tan3 is very suitable for preparing gallic acid using enzymatic hydrolysis.

[0117] Table 3 Hydrolysis reactions of tannic acid by tanninases from different sources

[0118]

[0119]

[0120] In Example 3, the substrate used for enzyme activity assay was propyl gallate, a simple small molecule compound synthesized chemically; the substrate used for the hydrolysis reaction was tannic acid, a complex natural macromolecule. Different enzymes have different affinities for different substrates, resulting in differences in enzyme activity. The higher enzyme activity of some enzymes in Example 3 compared to Tan3 is relative to the propyl gallate substrate, indicating that these enzymes have a higher affinity for propyl gallate than Tan3. However, in this example, the hydrolysis reaction used tannic acid, and the hydrolysis rate of Tan3 was significantly higher than that of other enzymes, indicating that Tan3 has a significantly higher affinity for tannic acid than other enzymes.

[0121] Example 6: Determination of the enzymatic properties of tanninase Tan3

[0122] 1) Temperature stability

[0123] The enzyme was heat-treated for 30 minutes at various temperatures (30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, and 80°C) in 50 mM disodium hydrogen phosphate-citrate buffer (pH 4.0), and the residual activity was determined using the above-described tanninase activity assay. The activity without heat treatment was considered as 100% residual activity, and each assay result was expressed as such. Figure 5It retains over 90% of its residual activity after heat treatment at 65℃ for 30 minutes and remains stable up to 65℃.

[0124] 2) Acid resistance test

[0125] After treatment at 30°C for 1, 2, 3, 4, and 5 hours in disodium hydrogen phosphate-citrate buffer (pH 2.5), the enzyme was diluted 5-fold with 0.2 M disodium hydrogen phosphate-citrate buffer (pH 4.0) and the activity was determined using the tanninase activity assay described above. The pH value at which the maximum activity occurred was taken as 100% relative activity to represent each measurement result. Figure 6 The results show that under pH 2.5 conditions, the enzyme activity loss was less than 2% (30℃) after 5 hours, indicating that tanninase Tan3 is very stable under acidic conditions. The preparation of gallic acid by hydrolyzing tannic acid with tanninase is carried out under acidic conditions, and as gallic acid is generated, the reaction pH gradually decreases and then tends to stabilize (gallic acid gradually becomes saturated). The acid resistance of tanninase Tan3 fully demonstrates that the enzyme Tan3 of this invention is very suitable for the enzymatic preparation of gallic acid.

[0126] 3) Effects of metal ions and inhibitors

[0127] In 50 mM disodium hydrogen phosphate-citrate buffer (pH 4.0), 20 mM of various metal ions and EDTA were added respectively. After treatment at 30°C for 30 minutes, the activity of tanninase was measured using the above-described method. The results are shown in Table 4. The activity without the addition of any additives is taken as 100% relative activity for each measurement. The results show that tanninase Tan3 is not inhibited by metal ions and EDTA.

[0128] Table 4. Effects of metal ions and inhibitors on Tan3

[0129]

[0130]

[0131] Example 7: Preparation of gallic acid using tanninases from different sources

[0132] 1) Weigh 2 kg of tara powder raw material and place it in a raw material extraction tank, then add 10 L of purified water;

[0133] 2) At room temperature (35℃), stir and extract for 6 hours, centrifuge and filter to obtain approximately 9L of tannic acid extract;

[0134] 3) Heat and concentrate 9L of tannic acid extract until the tannic acid concentration reaches 30% (w / v), and adjust the pH of the concentrate to 4.0. Tannic acid content and purity were determined by HPLC: Column: C18 (5μm, 4.6mm×250mm); Column temperature: 25℃; Mobile phase: Acetonitrile / water = 2:98 (water contains 0.065% trifluoroacetic acid, acetonitrile contains 0.05% trifluoroacetic acid); Flow rate: 1.0ml / min; Injection volume: 10μl; Detection wavelength: 278nm. Gallic acid content and purity were determined by HPLC: Column: C18 (5μm, 4.6mm×250mm); Column temperature: 25℃; Mobile phase: Acetonitrile / water = 2:98 (water contains 0.065% trifluoroacetic acid, acetonitrile contains 0.05% trifluoroacetic acid); Flow rate: 0.8ml / min; Injection volume: 10μl; Detection wavelength: 274nm.

[0135] 4) Add 500 ml of the 30% (w / v) tannic acid extract concentrate to the reaction vessel. Add 5400 U of tanninases Tan3, Tan5, Tan7, and Tan8 respectively, according to a substrate addition rate of 36 U / g. Do not control the pH during the reaction; maintain the temperature at approximately 40℃. Sample the reaction solution every 2 hours to detect the decrease in tannic acid. Calculate the conversion rate based on the decrease in tannic acid. After the reaction, detect the gallic acid content in the sample. Determine the final conversion rate based on the amount of gallic acid generated and the decrease in tannic acid. See the results below. Figure 7 .

[0136] The results showed that tanninases Tan5, Tan7, and Tan8 had very poor hydrolytic effects on tarattanine, with a maximum conversion rate of only about 30%. In contrast, tanninase Tan3 could continuously hydrolyze tarattanine to gallic acid under the same conditions with a tannin substrate concentration as high as 30% (w / v). Moreover, the hydrolysis cycle was short, requiring only 8 hours for complete hydrolysis, and the conversion rate was as high as 99.5%. After decolorization with activated carbon and vacuum drying, the gallic acid purity of the hydrolyzed sample was found to be 99%. This demonstrates that this tanninase can be well applied to the industrial mass production of gallic acid, and it has significant advantages over the chemical method for preparing gallic acid.

[0137] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0138] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of this invention are included in this invention and are protected by the appended claims. <110> Nanjing BestGene Biotechnology Co., Ltd. <120> Application of a tanninase in the preparation of gallic acid <160> 40 <170> PatentIn version 3.5 <210> 1 <211> 2724 <212> DNA <213> Artificial synthesis <400> 1 ctagatctac gccaggaccg agcaagccca gatgagaacc gacgcagatt tccttggcac 60 ctgttgcttc agctgaatcc tggcaatacg agatacctgc tttgaatatt ttgaatagct 120 cgcccgctgg agagcatcct gaatgcaagt aacaaccgta gaggctgaca cggcaggtgt 180 tgctagggag cgtcgtgttc tacaaggcca gacgtcttcg cggttgatat atatgtatgt 240 ttgactgcag gctgctcagc gacgacagtc aagttcgccc tcgctgcttg tgcaataatc 300 gcagtgggga agccacaccg tgactcccat ctttcagtaa agctctgttg gtgtttatca 360 gcaatacacg taatttaaac tcgttagcat ggggctgata gcttaattac cgtttaccag 420 tgccgcggtt ctgcagcttt ccttggcccg taaaattcgg cgaagccagc caatcaccag 480 ctaggcacca gctaaaccct ataattagtc tcttatcaac accatccgct cccccgggat 540 caatgaggag aatgaggggg atgcggggct aaagaagcct acataaccct catgccaact 600 cccagtttac actcgtcgag ccaacatcct gactataagc taacacagaa tgcctcaatc 660 ctgggaagaa ctggccgctg ataagcgcgc ccgcctcgca aaaaccatcc ctgatgaatg 720 gaaagtccag acgctgcctg cggaagacag cgttattgat ttcccaaaga aatcggggat 780 cctttcagag gccgaactga agatcacaga ggcctccgct gcagatcttg tgtccaagct 840 ggcggccgga gagttgacct cggtggaagt tacgctagca ttctgtaaac gggcagcaat 900 cgcccagcag ttagtagggt cccctctacc tctcagggag atgtaacaac gccaccttat 960 gggactatca agctgacgct ggcttctgtg cagacaaact gcgcccacga gttcttccct 1020 gacgccgctc tcgcgcaggc aagggaactc gatgaatact acgcaaagca caagagaccc 1080 gttggtccac tccatggcct ccccatctct ctcaaagacc agcttcgagt caaggtacac 1140 cgttgcccct aagtcgttag atgtcccttt ttgtcagcta acatatgcca ccagggctac 1200 1260 acaaccatgc tccgcaaagc cggtgccgtc ttctacgtca agacctctgt cccgcagacc 1320 ctgatggtct gcgagacagt caacaacatc atcggcgca ccgtcaaccc acgcaacaag 1380 aactggtcgt gcggcggcag ttctggtggt gagggtgcga tcgttgggat tcgtggtggc 1440 gtcatcggtg taggaacgga tatcggtggc tcgattcgag tgccggccgc gttcaacttc 1500 ctgtacggtc taaggccgag tcatgggcgg ctgccgtatg caaagatggc gaacagcatg 1560 gagggtcagg agacggtgca cagcgttgtc gggccgatta cgcactctgt tgagggtgag 1620 tccttcgcct cttccttctt ttcctgctct ataccaggcc tccactgtcc tcctttcttg 1680 ctttttatac tatatacgag accggcagtc actgatgaag tatgttagac ctccgctctct 1740 tcaccaaatc cgtcctcggt caggagccat ggaaatacga ctccaaggtc atccccatgc 1800 cctggcgcca gtccgagtcg gacattattg cctccaagat caagaacggc gggctcaata 1860 tcggctacta caacttcgac ggcaatgtcc ttccacaccc tcctatcctg cgcggcgtgg 1920 aaaccaccgt cgccgcactc gccaaagccg gtcacaccgt gaccccgtgg acgccataca 1980 agcacgattt cggccacgat ctcatctccc atactcgc ggctgacggc agcgccgacg 2040 taatgcgcga tatcagtgca tccggcgagc cggcgattc aaattcaaa gacctactga 2100 acccgaacat caaagctgtt aacatgaacg agctctggga cacgcatctc cagaagtgga 2160 attaccagat ggagtacctt gagaaatggc gggaggctga agaaaaggcc gggaaggaac 2220 tggacgccat catcgcgccg attacgccta ccgctgcggt acggcatgac cagttccggt 2280 actatgggta tgcctctgtg atcaacctgc tggatttcac gagcgtggtt gttccggtta 2340 cctttgcgga taagaacatc gataagaaga atgagagttt caaggcggtt agtgagcttg 2400 atgccctcgt gcaggaagag tatgatccgg aggcgtacca tggggcaccg gttgcagtgc 2460 aggttatcgg acggagactc agtgaagaga ggacgttggc gattgcagag gaagtgggga 2520 agttgctggg aaatgtggtg actccatagc taataagtgt cagatagcaa tttgcacaag 2580 aaatcaatac cagcaactgt aaataagcgc tgaagtgacc atgccatgct acgaaagagc 2640 agaaaaaaac ctgccgtaga accgaagaga tatgacacgc ttccatctct caaaggaaga 2700 atcccttcag ggttgcgttt ccag 2724 <210> 2 <211> 997 <212> DNA <213> Synthetic <400> 2 gccattggcg gaggggtccg gacggtcagg aacttagcct tatgagatga atgatggacg 60 tgtctggcct cggaaaagga tatatgggga tcatgatagt actagccata ttaatgaagg 120 gcatatacca cgcgttggac ctgcgttata gcttcccgtt agttatagta ccatcgttat 180 accagccaat caagtcacca cgcacgaccg gggacggcga atccccggga attgaaagaa 240 attgcatccc aggccagtga ggccagcgat tggccacctc tccaaggcac agggccattc 300 tgcagcgctg gtggattcat cgcaatttcc cccggcccgg cccgacaccg ctataggctg 360 gttctcccac accatcggag attcgtcgcc taatgtctcg tccgttcaca agctgaagag 420 cttgaagtgg cgagatgtct ctgcaggaat tcaagctaga tgctaagcga tattgcatgg 480 caatatgtgt tgatgcatgt gcttcttcct tcagcttccc ctcgtgcaga tgaggtttgg 540 ctataaattg aagtggttgg tcggggttcc gtgaggggct gaagtgcttc ctccctttta 600 gacgcaactg agagcctgag cttcatcccc agcatcatta cacctcagca cttaagacta 660 gtacgcgtct cgagatctag agggtgactg acacctggcg gtagacaatc aatccatttc 720 gctatagtta aggatgggg atgagggcaa ttggttatat gatcatgtat gtagtgggtg 780 tgcataatag tagtgaaatg gaagccaagt catgtgattg taatcgaccg acggaattga 840 ggatatccgg aaatacagac accgtgaaag ccatggtctt tccttcgtgt agaagaccag 900 acagacagtc cctgatttac ccttgcacaa agcactagaa aattagcatt ccatccttct 960 ctgcttgctc tgctgatatc actgtcattc aatgcat 997 <210> 3 <211> 1725 <212> DNA <213> Aspergillus niger CBS 513.88 <400> 3 atgcgttcac ccgcctgggc tcccatagcc accacagcct ttgcggcatt ggcaaatgct 60 gcaactccct ccacgttggc agagctttgc actgattcca tcgtgaaggc agctctacca 120 ccatctgagt tcatccaagg cataacaatt gactcagact ccgtgacgac agaagtcgta 180 acaaacagca gtgtctccag cgagttctac cccagcgcca caatcaatta ttgcaatgtc 240 acattcgcct actcccacga tggcattgac ggtgaccaag tccttttgga aatctggctt 300 cctgcaccca cggatttcca aaaccgctgg ctctccactg gtggaggagg ttatgccatt 360 aactccggag accagtcgct gccgggaggc gtaatgtacg gtgctgcgtc aggtatgacg 420 gatggtggtt ttggaggatt ttcaaacaat gcggacacgg ctatgctgtt ggcaaatggc 480 acccttgact acgagacgct ttacatgttt gcatacaaag cgcatcggga gcttagtttg 540 attggaaaag ccttgacccg caatgtatac gggatgagcg acagcgataa gctgtatgca 600 tattatcaag gctgctctga aggaggccgt gaaggttgga gtcaagtgca gcgcttcggt 660 gatgaatggg atggagccat tattggcgct ccagctttcc gatggtcatt ccaacagacg 720 cagcatcttt attccaacgt cgtcgagaag acactggatt actacccacc cccctgtgag 780 ctggacaaga tcgtcaacga gaccatcgct gcctgtgatg ccatggacgg aaaggtagat tgggtggttg cacggaccga tctctgcttg ctcgacttcg acattagtac catcgagggt aagccctact cgtgcgctgc atcaaggggt acccctgcac agaatggcac ggtctccgcc 960 aagggtatcg aagtcgcaaa aaccatcatc aatggattgc atgattcgca gggtcgccgg gtctactttt cctaccagcc aacggccgcc ttcgatgacg ctgagacgca gtacaactcc 1080 acgacaggtc agtgggggct ggatatcgat cagctcggag gcgaatat tgctctcttg gtagacaaga acggcactac actagacagc ctggatggtg tcacctatga taccctcaag gactggatga tctcggggcct gcaggaatac tacagcacct tgcagaccac ttggccggac ctcacgccct tccacgaagc aggaggcaaa gtcatccatt tccacggtga tgccgacttc 1320 agtattccca ccgccgcatc catccgctat tgggaatcag tacgcagcat catgtacccc aatcaagact ataactccag tgccgaggcg cttaacgagt ggtaccgtct gtacactgtc ccaggagcgg gtcattgtgc gaccaacgat gctatgccca acggcccctt cccacagacg 1500 aacatggctg tgatgatcga ctgggtggag aatggagtag tacctacaac gctcaatgcg 1560 accgtgctcc agggagagaa cgaaggacag aaccaacagc tttgtgcctg gccactgcgg 1620 cccttgtgga ccaacaacgg aaccaccatg gagtgcgtgt acaaccagcg ttcgattgat 1680 agctggcatt atgacttgga tgcggttcct atgcctgtgt actaa 1725 <210> 4 <211> 1746 <212> DNA <213> Aspergillus niger An76 <400> 4 atgcgcaagt ccactcgcgc agttgtcgct gctctggcag cagccactgc tcaggctagt 60 tccctgtccg atctctgcac tgtttccaat gttcagtccg cccttccttc caacggcacc 120 ctcctgggaa tcaacttgat cccctccgcc gtcactgcca acaccgtcac cgatgccagc 180 tctggcatgg gtagctccgg ctcctacgac tactgcaatg tcactgttac ctacacccac 240 accgggaagg gtgacaaggt tgtcgtgaag tacgccctgc ccgctccttc cgacttcaag 300 aatcgtttct acgttgccgg tggtggtggt ttctccctgt ccagcgatgc tactggcggt 360 ctcgagtacg gtgctgcctc cggcgccacc gacgccggct acgacgcctt ctcctacagc 420 480. tcgtcctcta cggcaacggc tcgatcaact gggatgctac ttacatgttc 540. tcctaccagg ctcttggtga aatgaccaag atcgccaagc ccctgacccg tggtttctac ggtctctcca gcgacaagaa gatttacacc tactacgagg gctgttccga tggtggtcgt gagggtatga gtcaggtcca gcgctgggga gatgaatg acggtgtcat tgccggtgcc 660. cctgccttcc gctttgctca gcagcaggtc caccatgtct tccctgccac tattgaacac 720 accatggact actaccctcc cccttgcga cttgacaaga tcgtcaacgc taccattga gcctgtgacc ctctcgacgg ccgtaccgat ggtgttgtct cccgtactga cctttgcatg 840 ctgaacttca acctcacctc catcattggc gagtcctact actgcgctga acagaactac acctccctgg gctttggctt cagcaagcgt gctgaaggca gcactactag ctaccagccc 960 gcccagaacg gcagcgtcac cgctgagggt gttgctctcg cccaggccat ctacgacggt 1020 cttcacgact ctaacggcaa gcgtgcctac ctctcgtggc agatcgccgc tgagctgtcc 1080 gatggtgaca ccgagtacga ctccaccact gactcctgga ctctgagcat cccctccacc 1140 ggtggcgagt acgtgaccaa gttcgtgcag ctcctcaaca tcgataacct ggagaacctt 1200 gacaacgtca cctacgacac cctggttgac tggatgaaca tcggtatgat tcgctacatt 1260 gacagtctcc agaccaccgt catcgacctc accaccttca aggagtccgg tggcaagatg 1320 atccactacc acggtgaatc cgaccccagt atccccaccg cctcgtccgt ccactactgg 1380 caggctgtcc gtcaggccat gtaccccaac accacctaca cccagtccct gcaggatatg 1440 tccaactggt accagctcta cctcgtcccc ggcgctgctc actgcggtac caactccctc 1500 cagcctggtc cttaccccga ggacaacatg gagatcatga tcgactgggt tgaaaacggc 1560 aacaagcctt cccgcctcaa cgccaccgtc tcctccggta cctatgctgg tgagacccag 1620 atgctttgcc aatggccttc tcgtcctctc tggaacagca actccagctt ctcttgtgtt 1680 cacgactcca agtcccttgc tacttgggac tacacttttg atgctttcaa gatgcccgtt 1740 ttctaa 1746 <210> 5 <211> 1752 <212> DNA <213> Aspergillus niger ATCC 13496 <400> 5 atgagaaccg ccctcggggt cctggccggg gccacgacca ttgctctatc caaggcagcc 60 agtctggcgg atgtctgtac taccagccac gccaagtcgt ccttgcctag cagcgatgag 120 attgaaggtc tcgagatcga cacttcatcc atcaccgctg gtgctgtcta caatgcttcc 180 acatcgggta gctacttctt cccggctgct tcctacagct actgcaacgt gaccctctcg 240 tacactcggc ctggtcttga tacccccttc ctgttggagc tctggttgcc cgctcccgag 300 gactaccaaa accggtggct gtccacaggt ggtggtggct ttgccatcaa ctctggatcc 360 agctccctgc cgggtggtgt gatgtatgga gctgcagcag gtatcacgga tggtggcttc 420 ggaggcttcg acacacagtt cgacgctgtc ttcctcgaag ccaacggtac catcaactac 480 gaggcgttgt attcatttgg ctacctcgct catcacgagc tcactgtcgt cggcaaagcc 540 ttcagccgca acttctacag cgtcggtgac tccaagatct acgcctacta ccaaggttgt 600 tcggaaggtg gccgcgaagg ttggagccag gtgcaacgct atgcagacca gtgggatgga 660 gccgttatcg gtgctcctgc cttccggtac ggtcaacaac aagtgaacca cctgtacccc 720 gacgtggttg agcacactct gggctactac cctcccactt gcgaattgga aaagatcgtc aacttgacca ttgcggcgtg cgatgccctg gacggccgtg tcgatggcgt ggtggctcgt 840 accgacctgt gcaagctgca cttcaacgtg aactcgaccg tcggtgcccc ttactcctgc 900 ccggcctcta cctccacgac agtcctcaag aagcgcactt cgatgtctaa caccaccccc gcccagaacg gcaccgtttc tgctatcggt gccgccgcag ccagcaagat gctcgacggc 1020 ctgcgcaccc tggacggccg ccgtgcttac atctggtacc agccctcctc cacctttgac 1080 gatgcccaga cccagtacaa ctacgacacc ggaaaatggg agttggatat tacctccctg ggcggtgaat gggtcgcccg cttcctggag ctgcgcgatg cagacaatct ctccagcctg gacaacgtca cgtacgatac cctgaaggag tggatggagc tcggctggca gcgctacgag gacagcctcc agaccacctg gcccgacttg accccgttcc aatcagccgg tggaagatt attcacgtgc atggcgagtc cgaccccagt atcccgaccg gttcgtccgt gcattatcac 1380 gagtccgtcc gccagatcat gtaccccggc atgtcgttca acgagagctc cgaggctctg 1440 aatgaatgga accgattgta tctgatccct ggtgcggcgc actgcgcgtc tagcactacc 1500 cagcctaatg ggccgttccc gcagactacc ctcgagactc tgatccaatg ggtcgaggga 1560 ggcgtcttcc cgacccgctt gaatgccacc gtgctggctg gtgagcgtga gggtgagcag 1620 cagcagttgt gcgcgtggcc cttgcgtccc ctgtggaaga acaacggcac cgtcatggac 1680 tgcgtgtttg atgaggcgtc gtataagacc tgggtttatg attttgatgc gtacaagctg 1740 cccctgtact ag 1752 <210> 6 <211> 1725 <212> DNA <213> Aspergillus kawachii IFO 4308 <400> 6 atgcgctccc ccgcctgggc ctccatcgcc atcaccgcct tcgccgccct ggccaacgcc 60 ggtaccccct ccaccctggc cgagctgtgc accgactcca tcgtcaagc cgccctgccc 120 ccctccgagt tcatcaaggg tatcaccatc gactccgact ccgtcaccac cgaggtcgtc 180 240. accaactcct ccttctcctc cgagttctac ccctccgcca cctcgacta ctgcaacgtc accttcgcct actcccacga cggtatcgac ggtgaccagg tcctgctgga gatctggctg 300 360. cccgccccca ccaacttcaa gaaccgctgg ctgtccaccg gtggtggtgg ttacgccatc aactccggtg accagtccct gcccggtggt gtcatgtacg gtgccgcctc cggtatgacc 420 gacggtggtt tcggtggttt ctccaacaac gccgacaccg ccatgctgct ggccaacggt 480 accctgaact accgagaccct gtacatgttc gcctacaagg cccaccgcga gctgtccctg 540 ctgggtaagg ccctgacccg caacgtctac ggtatgtccg actccgacaa gctgtacgcc tactaccagg gttgctccga gggtggtcgc gaggttggt cccaggtcca gcgcttcggt 660 gacgagtggg acggtgccat catcggtgcc cccgccttcc gctggtcctt ccagcagacc 720 cagcacctgt actccaacat cgtcgagaag accctggact actacccccc cccctgcgag 780 ctggacaaga tcgtcaacga gaccatcgcc gcctgcgacg ccatggacgg taaggtcgac tgggtcgtcg cccgcaccga cctgtgcctg ctggacttcg acatctccac catcgagggt aagccctact cctgcgccgc ctcccgcggt acccccgccc agaacggtac cgtctccgcc 960 aagggtatcg aggtcgccaa gaccatcatc aacggtctgc acgactccca gggtcgccgc 1020 gtctacttct cctaccagcc caccgccgcc ttcgacgacg ccgagaccca gtacaactcc 1080 accaccggtc agtggggtct ggacatcgac cagctgggtg gtgagtacat cgccctgctg 1140 gtcgacaaga acggtaccac cctggactcc ctggacggta tcacctacga caccctgaag 1200 gactggatga tctccggtct gcaggagtac tactccaccc tgcagaccac ctggcccgac 1260 ctgaccccct tccacaacgc cggtggtaag gtcatccact tccacggtga cgccgacttc 1320 tccatcccca ccgccgcctc catccgctac tgggagtccg tccgctccat catgtacccc 1380 aacaaggact acaactcctc cgccgaggcc ctgaacgagt ggtaccgcct gtacaccgtc 1440 cccggtgccg gtcactgcgc caccaacgac gccatgccca acggtccctt cccccagacc 1500 aacatggccg tcatgatcga ctgggtcgag aacggtgtcg tccccaccac cctgaacgcc 1560 accgtcctgc agggtgagaa cgagggtcag aaccagcagc tgtgcgcctg gcccctgcgc 1620 cccctgtgga ccaacaacgg taccaccatg gagtgcgtct acaaccagcg ctccatcgac 1680 tcctggcact acgacctgga cgccgtcccc atgcccgtct actaa 1725 <210> 7 <211> 1713 <212> DNA <213> Aspergillus niger FJ0118 <400> 7 atgtccaagt tcctccttg gaccgtctc gcggcggcc tcgcgcgc cgcgactctc 60 gagcaggtct gtaccgttgc tcacgcaaag gccgccgtgc ccgcatccgg cctggtgcag 120 ggtgtggtca ccaacccttc cagtgtcact gcggttcccg tctacaatga atccacctct 180 ggatccgact actacccggc cggaacctat gattactgca atatcaccat gacttattct 240 catgctggca aggacgacag cgtcctgctg caattctgga tgcccactcc cgatgacttc 300 cagaaccgct gggtgtctac cggtggcttt ggctttgcca tcaacgttgc ctccaacgtg 360 cctgctggtc ttccctacgg tgccgtcgcc ggccggaccg acggtggctt tggcagcttc 420 gacactcagt tctcctctgt gtaccccctg gtgaatggta ccgccaacta cgatgccctc 480 tacatgttcg gctaccaggc ccaccacgag atgtcctcca tcggcaaggc gttcaccaag 540 aacttctaca gcatgtctga caagctctac gcctactacc agggttgctc cgagggtggt 600 cgtgagggct ggagtcaggt gcagcgcttc cccgaggagt gggatggtgc cgtcattggt 660 gctcctgcca tccgctatgg ccagcagcag gtcaaccacc tggtgcccca ggtcgtggaa 720 cagaccctcg attactaccc tgaaaactgc gagttctccg agatggtgac tctgattatc 780 gatgcctgtg atgaactcga tggccgtaag gatggcgtca tcggccgcac cgatctgtgc 840 cagctcaact tcgacctcaa gtccaccatc ggtaagcctt acgcctgcaa cgccaccacc 900 agcatgggtg tgaccacccc ggcccagaac ggcaccatca ctgcggaggg tgtgcgcgtc 960 gctcagacca tcctggacgg attgaaggac tctcagggtc gtcaggccta cttctggtac 1020 cgcattggct cggagttcag cgatgcagag actacctacg actcgaccac cggcacctac 1080 ggtatcgaca tctccgagct gggtggtgag tgggtgactc acggactgca gctgctggac 1140 ctcgacaacc tctccactct ggacaacgtc acctacgaca ccctgcgtga ctggatgctc 1200 gagggtctcc agcgctacga ggatgtcctg cagaccacct ggcccgacct gtcccgcttc 1260 caggctgcag gtggcaaggt catccactac cacggtgaat cggacaacag catccctccc 1320 gcgtcttcgg tgcgctactt cgagtccgtc cgcgacacca tgttccccaa caagacctac 1380 aacgcctcgg ttgaagccct gaacgagttc taccgtctct acctggttcc cggtgcctcc 1440 cactgcaaca ccaactctct gcagcccaac ggaccctacc cccagaactc ccttggcgac 1500 ctgatgaatt gggtcgagaa gggcatcgag ccggtgactc tgaacggcac ggtgctgagc 1560 ggcgatctcg agggagagga gcagaagatc tgctcgtggc ctctgcgtcc tttgtggaag 1620 aacaacggca cggagatgga gtgcgtgtac gaccagaagt cgattgacac atggttgtat 1680 gatctggatg cgtatgacat tccgatctat tag 1713 <210> 8 <211> 1722 <212> DNA <213> Aspergillus oryzae RIB40 <400> 8 atgcgctccg ccctgaacat cctggccggt gccgccaccc tgaccgtcgc ccaggccgcc 60 tccctgtccg acgtctgcac caactcccac gtcaagtccg ccctgccctc catcgacctg 120 atcaacggtc tggtcatcga cccctcctcc atcaccacca acgccgtcta caacgcctcc acccccggtg gtgactactt ccccgccgcc tccgcctacg acttctgcaa cgtcaccctg 240 acctacgccc accccggtcg caacgaccgc gtccacctga agctgtggat gcccgccccc 300 360. gaccagttcc agaaccgctg gctgtccacc ggtggtggtg gtttcgccat caaccacgac gagcagcagc tgcccggtgg tgtccagtac ggtgccgccg ccggtatcac cgacggtggt 420 ttcggttcct tctacaccca gttcgaccag gtcttcctgc tggccaacgg taccatcaac 480 tacgaggccc tgtacatgtt cggttaccag gcccaccacg agctgtccgt catcggtaag 540 gccctgacca agaacttcta cggtaccggt gacgccaagc tgtacgccta ctggcagggt 600 tgctccgagg gtggtcgcga gggtttctcc caggtccagc gcttccagga gttcgacggt 660 gccgtcatcg gtgccccgc cctgcgctac ggtcagcagc aggccaacca cctgtacggt 720 aacctggtcg agcacaccct gaagtactac ccccccccct gcgagctgga gaagatcgtc 780 aacctgacca tcaccgcctg cgaccgcctg gacggtcgct ccgacggtgt cgtctcccgc 840 accgacctgt gcaagctgca cttcaacatc aactccacca tcggtgcccc ctactcctgc 900 cccgcctcca ccaccaccac cggtaccacc cccgcccaga acggtaccgt ctccgccctg 960 ggtgccgccg ccgcccgcaa gatgctggac ggtctgcgca ccctggactc ccgccgcgcc 1020 tacatcttct accagccctc cgccaccttc gacgacgccc agaccaagta caaccccgag 1080 accaagcagt tcgagctgga ggtctccgcc tacgccgccg agtggatccc ccgcttcctg 1140 cagctgcaga actacaccct gtcctccctg gagaacgtca cctacgacac cctgaaggac 1200 tggatggagc tgggttggca gcgctacgag gacgtcctgc agaccacctg gcccgacctg 1260 acccccttcc agtccgccgg tggtaaggtc ctgcactacc acggtgagtc cgacccctcc 1320 atccccgccg gttcctccgt ccactaccac gagtccgtcc gcaagaccat gtaccccaac 1380 atgtccttca acgagtccaa ccaggccctg aacgagtgga accgcctgtt cctggtcccc 1440 ggtgccgccc actgcgcctc ctccaccgac cagcccaacg gtcccttccc ccaggccacc 1500 ctgaagaccc tgatcgagtg ggtcgagaac tccatcgtcc ccgagaccct gaacggtacc 1560 gtcctggacg gtgaccacaa gggtgagcag cagcagatct gcgcctggcc cctgcgcccc 1620 ctgtggaccg agaacggtac cgtcatgaac tgcgtctacg accaggcctc cctggacacc 1680 tgggactacg agttcgacgc ctaccgcatc cccctgtact aa 1722 <210> 9 <211> 1767 <212> DNA <213> Aspergillus fumigatus Af293 <400> 9 atgcgcatct cctacggttc cgccgtcgcc gccctggccg ccgccgccaa cgccgcctcc 60 ctggccgacg tctgcaccat ctcccacgtc cagtccgtcc tgccctccaa cggtaccctg 120 ctgggtatca acgtcatccc ctccgccgtc accgcctccg ccgtctacaa ctccacctcc 180 tccggtggta tgggtggtat gggtggttcc aactccgcca actaccccta ctgcaacgtc 240 accgtcacct acacccaccc cggtaagggt gacaaggtcg tcgtcaagta cgccttcccc 300 cagccctccg acttcaagaa ccgcttctac gtcgccggtg gtggtggtta ctccctgtcc 360 tccgacgcca ccggtggtct ggagtacggt gccgcctccg gtgccaccga cgccggttac 420 gacgccttct cctactccta cgacgaggtc gtcctgtacg gtaacggttc catcaactgg 480 gacgccacct acatgttcgc ctaccaggcc ctgggtgaga tgaccaccct gggtaagacc 540 ctgacccgca acttctacgg tctgtcctcc gacgccaaga tctacaccta ctacgagggt 600 tgctccgacg gtggtcgcga gggtatgtcc caggtccagc gctacggtga cctgtacgac 660 ggtgccatca ccggtgcccc cgccttccgc tacgcccagc agcaggtcca ccacgtcttc 720 tcctccgtcg tcgagaagac cctggactac tacccccccc cctgcgagct ggccaagatc 780 gtcaacgcca ccatcgaggc ctgcgacccc ctggacggtc gcaccgacgg tgtcgtctcc 840 cgcaccgacc tgtgcaagct gcacttcgac ctgtccaaga tcatcggtga gccctactac 900 tgcgccgcca agacctccac ctccctgggt ttcggtttct ccaagcgcca ggccgccggt 960 tccaccacct cctaccagcc cgcccagaac ggtaccgtca ccaaggaggg tgtcgccgtc 1020 gccaaggcca tctacgacgg tctgcacaac acccagggtc agcgcgccta cctgtcctgg 1080 cagatcgcct ccgagttctc cgacgccacc accgagtgga acaacgacac cggttcctgg 1140 gagctgtcca tcccctccac cggtggtgag ttcgtcacca agttcgtcca gctgctggac 1200 ctggacaacc tgtccaccct ggacaacgtc acctacgaca ccctggtcga gtggatgaac 1260 accgccatgg tccgctacat ggactccctg cagaccaccg tccccgacct gaccaccttc 1320 aagtcctccg gtggtaagct gctgcactac cacggtgagt ccgacccctc catccccgcc 1380 gcctcctccg tccactactg gcagtccgtc cgctccatca tgtaccccgg tgtctccgcc 1440 gccaagtccc tgaaggagct gcaggagtgg taccagttct acctgatccc cggtgccgcc 1500 cactgcggtg ccaactccct gcagcccggt ccctaccccc agaacaacat ggacatcatg 1560 atcgactggg tcgagaacgg tgtccagccc tcccgcctga acaccaccgt ctcctccggt 1620 gactacgccg gtgagaccca gatgctgtgc cagtggccca cccgccccct gtggaaggac 1680 aactccacct tcgactgcgt caacgacgag aagtccatcg agtcctggac ctacaccttc 1740 cccgccttca aggtccccgt ctactaa 1767 <210> 10 <211> 1743 <212> DNA <213> Penicillium rubens Wisconsin 54‑1255 <400> 10 atgcgcctgt cctggggtgc ctccgccgcc gccctggccg cctccgcctc cgccgcctcc 60 ctggccgacg tctgcaccgt ctccaacgtc cagtccgccc tgccctccaa cggtaccctg 120 ctgggtatca acatgatccc ctccaccgtc accgcctccc ccgtctacaa cgcctccgcc 180 ggtatgggtt ccaccgagac ctacacctac tgcaacgtca ccgtcaccta cgagcacacc 240 ggtaagggtg actccgtcgt catcaagtac gccttcccca agccctccga cttcaagaag 300 cgcttctacg tcgccggtgg tggtggtttc tccctgtcct ccgacgccac cggtggtctg 360 gagtacggtg ccgtcggtgg tgccacctcc gccggttacg acgccttcaa caactcctac 420 gacgaggtcg tcctgtacgg taacggtacc atcaactggg acgccaccta catgttcgcc 480 taccaggccc tgggtgagat gaccaagatc ggtaaggtcc tgaccaaggg tttctacggt 540 atggcctcct ccgccaaggt ctacacctac tacgagggtt gctccgacgg tggtcgcgag 600 ggtatgtccc agatccagcg ctacggtgag gagtacgacg gtgccatcac cggtgccccc 660 gccttccgct tcgcccagca gcaggtccac cacgtcttct ccgccgccgc cgagcagacc 720 ctggactact acccccccccc ctgcgagctg gccaagatcg tcaacgccac catcgccgcc 780 tgcgaccccc tggacggtcg caccgacggt gtcatctccc gcaccgacct gtgcaagctg 840 aagttcaacc tgacctccat catcggtgag gagtactact gcgccgccgc cacctccacc 900 tccctgggtt tcggtttctc caagcgcgcc gacggttcct ccacctccac cacccccgag 960 cagtccggta aggtcaccgc caagggtgtc caggtcgccc aggccatcta cgacggtctg 1020 cacaactcca agggtgagcg cgcctacctg tcctggcagg tcggttccga gctgtccgac 1080 ggtgacacca cctggaacaa cgccacctcc aagtgggaga tgtccatccc ctccaccggt 1140 ggtgagtacg tcaccaagtt catccagctg ctggacctgg acaacctgtc cgacctggac 1200 aacgtcacct acgacaccct ggtcgactgg atgaacaccg gtatggtccg ctacatggac 1260 tccctgcaga ccaccctgcc cgacctgacc cccttccagt cctccggtgg taagctgctg 1320 cactaccacg gtgagtccga cccctccatc ccctccgcct cctccgtcca ctactggcag 1380 tccgtccgct ccatcatgta cccccacctg tcctcccagg actccctgaa ggagctggcc 1440 gactggtacc agttctacct ggtccccggt gccgcccact gcggtaccaa caagctgcag 1500 cccggtccct accccgagaa caacatgcag accatgatcg actgggtcga gaacgacgtc 1560 aagccctccc gcctgaacgc caccgtctcc tccggtacct acgagggtga gacccagatg 1620 ctgtgccagt ggcccacccg cccctgtgg aagtccaact ccaccttcca gtgcgtcgac 1680 gacaaggcct ccatcgagtc ctggacctac tccttccccg ccttcaaggt ccccgtctac 1740 city ​​1743 <210> 11 <211> 574 <212> PRT <213> Aspergillus niger CBS 513.88 <400> 11 Put Arg Ser Pro Ala Trp Ala Pro Ile Ala Thr Thr Ala Phe Ala Ala 1 5 10 15 Leu Ala Asn Ala Ala Thr Pro Ser Thr Leu Ala Glu Leu Cys Thr Asp 20 25 30 Ser Ile Val Lys Ala Ala Leu Pro Pro Ser Glu Phe Ile Gln Gly Ile 35 40 45 Thr Ile Asp Ser Asp Ser Val Thr Thr Glu Val Val Thr Asn Ser Ser 50 55 60 Val Ser Ser Glu Phe Tyr Pro Ser Ala Thr Ile Asn Tyr Cys Asn Val 65 70 75 80 Thr Phe Ala Tyr Ser His Asp Gly Ile Asp Gly Asp Gln Val Leu Leu 85 90 95 Glu Ile Trp Leu Pro Ala Pro Thr Asp Phe Gln Asn Arg Trp Leu Ser 100 105 110 Thr Gly Gly Gly Gly Tyr Ala Ile Asn Ser Gly Asp Gln Ser Leu Pro 115 120 125 Gly Gly Val Met Tyr Gly Ala Ala Ser Gly Met Thr Asp Gly Gly Phe 130 135 140 Gly Gly Phe Ser Asn Asn Ala Asp Thr Ala Met Leu Leu Ala Asn Gly 145 150 155 160 Thr Leu Asp Tyr Glu Thr Leu Tyr Met Phe Ala Tyr Lys Ala His Arg 165 170 175 Glu Leu Ser Leu Ile Gly Lys Ala Leu Thr Arg Asn Val Tyr Gly Met 180 185 190 Ser Asp Ser Asp Lys Leu Tyr Ala Tyr Tyr Gln Gly Cys Ser Glu Gly 195 200 205 Gly Arg Glu Gly Trp Ser Gln Val Gln Arg Phe Gly Asp Glu Trp Asp 210 215 220 Gly Ala Ile Ile Gly Ala Pro Ala Phe Arg Trp Ser Phe Gln Gln Thr 225 230 235 240 Gln His Leu Tyr Ser Asn Val Val Glu Lys Thr Leu Asp Tyr Tyr Pro 245 250 255 Pro Pro Cys Glu Leu Asp Lys Ile Val Asn Glu Thr Ile Ala Ala Cys 260 265 270 Asp Ala Met Asp Gly Lys Val Asp Trp Val Val Ala Arg Thr Asp Leu 275 280 285 Cys Leu Leu Asp Phe Asp Ile Ser Thr Ile Glu Gly Lys Pro Tyr Ser 290 295 300 Cys Ala Ala Ser Arg Gly Thr Pro Ala Gln Asn Gly Thr Val Ser Ala 305 310 315 320 Lys Gly Ile Glu Val Ala Lys Thr Ile Ile Asn Gly Leu His Asp Ser 325 330 335 Gln Gly Arg Arg Val Tyr Phe Ser Tyr Gln Pro Thr Ala Ala Phe Asp 340 345 350 Asp Ala Glu Thr Gln Tyr Asn Ser Thr Thr Gly Gln Trp Gly Leu Asp 355 360 365 Ile Asp Gln Leu Gly Gly Glu Tyr Ile Ala Leu Leu Val Asp Lys Asn 370 375 380 Gly Thr Thr Leu Asp Ser Leu Asp Gly Val Thr Tyr Asp Thr Leu Lys 385 390 395 400 Asp Trp Met Ile Ser Gly Leu Gln Glu Tyr Tyr Ser Thr Leu Gln Thr 405 410 415 Thr Trp Pro Asp Leu Thr Pro Phe His Glu Ala Gly Gly Lys Val Ile 420 425 430 His Phe His Gly Asp Ala Asp Phe Ser Ile Pro Thr Ala Ala Ser Ile 435 440 445 Arg Tyr Trp Glu Ser Val Arg Ser Ile Met Tyr Pro Asn Gln Asp Tyr 450 455 460 Asn Ser Ser Ala Glu Ala Leu Asn Glu Trp Tyr Arg Leu Tyr Thr Val 465 470 475 480 Pro Gly Ala Gly His Cys Ala Thr Asn Asp Ala Met Pro Asn Gly Pro 485 490 495 Phe Pro Gln Thr Asn Met Ala Val Met Ile Asp Trp Val Glu Asn Gly 500 505 510 Val Val Pro Thr Thr Leu Asn Ala Thr Val Leu Gln Gly Glu Asn Glu 515 520 525 Gly Gln Asn Gln Gln Leu Cys Ala Trp Pro Leu Arg Pro Leu Trp Thr 530 535 540 Asn Asn Gly Thr Thr Met Glu Cys Val Tyr Asn Gln Arg Ser Ile Asp 545 550 555 560 Ser Trp His Tyr Asp Leu Asp Ala Val Pro Met Pro Val Tyr 565 570 <210> 12 <211> 581 <212> PRT <213> Aspergillus niger An76 <400> 12 Met Arg Lys Ser Thr Arg Ala Val Val Ala Ala Leu Ala Ala Ala Thr 1 5 10 15 Ala Gln Ala Ser Ser Leu Ser Asp Leu Cys Thr Val Ser Asn Val Gln 20 25 30 Ser Ala Leu Pro Ser Asn Gly Thr Leu Leu Gly Ile Asn Leu Ile Pro 35 40 45 Ser Ala Val Thr Ala Asn Thr Val Thr Asp Ala Ser Ser Gly Met Gly 50 55 60 Ser Ser Gly Ser Tyr Asp Tyr Cys Asn Val Thr Val Thr Tyr Thr His 65 70 75 80 Thr Gly Lys Gly Asp Lys Val Val Val Lys Tyr Ala Leu Pro Ala Pro 85 90 95 Ser Asp Phe Lys Asn Arg Phe Tyr Val Ala Gly Gly Gly Gly Phe Ser 100 105 110 Leu Ser Ser Asp Ala Thr Gly Gly Leu Glu Tyr Gly Ala Ala Ser Gly 115 120 125 Ala Thr Asp Ala Gly Tyr Asp Ala Phe Ser Tyr Ser Tyr Asp Glu Val 130 135 140 Val Leu Tyr Gly Asn Gly Ser Ile Asn Trp Asp Ala Thr Tyr Met Phe 145 150 155 160 Ser Tyr Gln Ala Leu Gly Glu Met Thr Lys Ile Ala Lys Pro Leu Thr 165 170 175 Arg Gly Phe Tyr Gly Leu Ser Ser Asp Lys Lys Ile Tyr Thr Tyr Tyr 180 185 190 Glu Gly Cys Ser Asp Gly Gly Arg Glu Gly Met Ser Gln Val Gln Arg 195 200 205 Trp Gly Asp Glu Tyr Asp Gly Val Ile Ala Gly Ala Pro Ala Phe Arg 210 215 220 Phe Ala Gln Gln Gln Val His His Val Phe Pro Ala Thr Ile Glu His 225 230 235 240 Thr Met Asp Tyr Tyr Pro Pro Pro Cys Glu Leu Asp Lys Ile Val Asn 245 250 255 Ala Thr Ile Glu Ala Cys Asp Pro Leu Asp Gly Arg Thr Asp Gly Val 260 265 270 Val Ser Arg Thr Asp Leu Cys Met Leu Asn Phe Asn Leu Thr Ser Ile 275 280 285 Ile Gly Glu Ser Tyr Tyr Cys Ala Glu Gln Asn Tyr Thr Ser Leu Gly 290 295 300 Phe Gly Phe Ser Lys Arg Ala Glu Gly Ser Thr Thr Ser Tyr Gln Pro 305 310 315 320 Ala Gln Asn Gly Ser Val Thr Ala Glu Gly Val Ala Leu Ala Gln Ala 325 330 335 Ile Tyr Asp Gly Leu His Asp Ser Asn Gly Lys Arg Ala Tyr Leu Ser 340 345 350 Trp Gln Ile Ala Ala Glu Leu Ser Asp Gly Asp Thr Glu Tyr Asp Ser 355 360 365 Thr Thr Asp Ser Trp Thr Leu Ser Ile Pro Ser Thr Gly Gly Glu Tyr 370 375 380 Val Thr Lys Phe Val Gln Leu Leu Asn Ile Asp Asn Leu Glu Asn Leu 385 390 395 400 Asp Asn Val Thr Tyr Asp Thr Leu Val Asp Trp Met Asn Ile Gly Met 405 410 415 Ile Arg Tyr Ile Asp Ser Leu Gln Thr Thr Val Ile Asp Leu Thr Thr 420 425 430 Phe Lys Glu Ser Gly Gly Lys Met Ile His Tyr His Gly Glu Ser Asp 435 440 445 Pro Ser Ile Pro Thr Ala Ser Ser Val His Tyr Trp Gln Ala Val Arg 450 455 460 Gln Ala Met Tyr Pro Asn Thr Thr Tyr Thr Gln Ser Leu Gln Asp Met 465 470 475 480 Ser Asn Trp Tyr Gln Leu Tyr Leu Val Pro Gly Ala Ala His Cys Gly 485 490 495 Thr Asn Ser Leu Gln Pro Gly Pro Tyr Pro Glu Asp Asn Met Glu Ile 500 505 510 Met Ile Asp Trp Val Glu Asn Gly Asn Lys Pro Ser Arg Leu Asn Ala 515 520 525 Thr Val Ser Ser Gly Thr Tyr Ala Gly Glu Thr Gln Met Leu Cys Gln 530 535 540 Trp Pro Ser Arg Pro Leu Trp Asn Ser Asn Ser Ser Phe Ser Cys Val 545 550 555 560 His Asp Ser Lys Ser Leu Ala Thr Trp Asp Tyr Thr Phe Asp Ala Phe 565 570 575 Lys Met Pro Val Phe 580 <210> 13 <211> 583 <212> PRT <213> Aspergillus niger ATCC 13496 <400> 13 Met Arg Thr Ala Leu Gly Val Leu Ala Gly Ala Thr Thr Ile Ala Leu 1 5 10 15 Ser Lys Ala Ala Ser Leu Ala Asp Val Cys Thr Thr Ser His Ala Lys 20 25 30 Ser Ser Leu Pro Ser Ser Asp Glu Ile Glu Gly Leu Glu Ile Asp Thr 35 40 45 Ser Ser Ile Thr Ala Gly Ala Val Tyr Asn Ala Ser Thr Ser Gly Ser 50 55 60 Tyr Phe Phe Pro Ala Ala Ser Tyr Ser Tyr Cys Asn Val Thr Leu Ser 65 70 75 80 Tyr Thr Arg Pro Gly Leu Asp Thr Pro Phe Leu Leu Glu Leu Trp Leu 85 90 95 Pro Ala Pro Glu Asp Tyr Gln Asn Arg Trp Leu Ser Thr Gly Gly Gly 100 105 110 Gly Phe Ala Ile Asn Ser Gly Ser Ser Ser Leu Pro Gly Gly Val Met 115 120 125 Tyr Gly Ala Ala Ala Gly Ile Thr Asp Gly Gly Phe Gly Gly Phe Asp 130 135 140 Thr Gln Phe Asp Ala Val Phe Leu Glu Ala Asn Gly Thr Ile Asn Tyr 145 150 155 160 Glu Ala Leu Tyr Ser Phe Gly Tyr Leu Ala His His Glu Leu Thr Val 165 170 175 Val Gly Lys Ala Phe Ser Arg Asn Phe Tyr Ser Val Gly Asp Ser Lys 180 185 190 Ile Tyr Ala Tyr Tyr Gln Gly Cys Ser Glu Gly Gly Arg Glu Gly Trp 195 200 205 Ser Gln Val Gln Arg Tyr Ala Asp Gln Trp Asp Gly Ala Val Ile Gly 210 215 220 Ala Pro Ala Phe Arg Tyr Gly Gln Gln Gln Val Asn His Leu Tyr Pro 225 230 235 240 Asp Val Val Glu His Thr Leu Gly Tyr Tyr Pro Pro Thr Cys Glu Leu 245 250 255 Glu Lys Ile Val Asn Leu Thr Ile Ala Ala Cys Asp Ala Leu Asp Gly 260 265 270 Arg Val Asp Gly Val Val Ala Arg Thr Asp Leu Cys Lys Leu His Phe 275 280 285 Asn Val Asn Ser Thr Val Gly Ala Pro Tyr Ser Cys Pro Ala Ser Thr 290 295 300 Ser Thr Thr Val Leu Lys Lys Arg Thr Ser Met Ser Asn Thr Thr Pro 305 310 315 320 Ala Gln Asn Gly Thr Val Ser Ala Ile Gly Ala Ala Ala Ala Ser Lys 325 330 335 Met Leu Asp Gly Leu Arg Thr Leu Asp Gly Arg Arg Ala Tyr Ile Trp 340 345 350 Tyr Gln Pro Ser Ser Thr Phe Asp Asp Ala Gln Thr Gln Tyr Asn Tyr 355 360 365 Asp Thr Gly Lys Trp Glu Leu Asp Ile Thr Ser Leu Gly Gly Glu Trp 370 375 380 Val Ala Arg Phe Leu Glu Leu Arg Asp Ala Asp Asn Leu Ser Ser Leu 385 390 395 400 Asp Asn Val Thr Tyr Asp Thr Leu Lys Glu Trp Met Glu Leu Gly Trp 405 410 415 Gln Arg Tyr Glu Asp Ser Leu Gln Thr Thr Trp Pro Asp Leu Thr Pro 420 425 430 Phe Gln Ser Ala Gly Gly Lys Ile Ile His Val His Gly Glu Ser Asp 435 440 445 Pro Ser Ile Pro Thr Gly Ser Ser Val His Tyr His Glu Ser Val Arg 450 455 460 Gln Ile Met Tyr Pro Gly Met Ser Phe Asn Glu Ser Ser Glu Ala Leu 465 470 475 480 Asn Glu Trp Asn Arg Leu Tyr Leu Ile Pro Gly Ala Ala His Cys Ala 485 490 495 Ser Ser Thr Thr Gln Pro Asn Gly Pro Phe Pro Gln Thr Thr Leu Glu 500 505 510 Thr Leu Ile Gln Trp Val Glu Gly Gly Val Phe Pro Thr Arg Leu Asn 515 520 525 Ala Thr Val Leu Ala Gly Glu Arg Glu Gly Glu Gln Gln Gln Leu Cys 530 535 540 Ala Trp Pro Leu Arg Pro Leu Trp Lys Asn Asn Gly Thr Val Met Asp 545 550 555 560 Cys Val Phe Asp Glu Ala Ser Tyr Lys Thr Trp Val Tyr Asp Phe Asp 565 570 575 Ala Tyr Lys Leu Pro Leu Tyr 580 <210> 14 <211> 574 <212> PRT <213> Aspergillus kawachii IFO 4308 <400> 14 Met Arg Ser Pro Ala Trp Ala Ser Ile Ala Ile Thr Ala Phe Ala Ala 1 5 10 15 Leu Ala Asn Ala Gly Thr Pro Ser Thr Leu Ala Glu Leu Cys Thr Asp 20 25 30 Ser Ile Val Lys Ala Ala Leu Pro Pro Ser Glu Phe Ile Lys Gly Ile 35 40 45 Thr Ile Asp Ser Asp Ser Val Thr Thr Glu Val Val Thr Asn Ser Ser 50 55 60 Phe Ser Ser Glu Phe Tyr Pro Ser Ala Thr Ile Asp Tyr Cys Asn Val 65 70 75 80 Thr Phe Ala Tyr Ser His Asp Gly Ile Asp Gly Asp Gln Val Leu Leu 85 90 95 Glu Ile Trp Leu Pro Ala Pro Thr Asn Phe Lys Asn Arg Trp Leu Ser 100 105 110 Thr Gly Gly Gly Gly Tyr Ala Ile Asn Ser Gly Asp Gln Ser Leu Pro 115 120 125 Gly Gly Val Met Tyr Gly Ala Ala Ser Gly Met Thr Asp Gly Gly Phe 130 135 140 Gly Gly Phe Ser Asn Asn Ala Asp Thr Ala Met Leu Leu Ala Asn Gly 145 150 155 160 Thr Leu Asn Tyr Glu Thr Leu Tyr Met Phe Ala Tyr Lys Ala His Arg 165 170 175 Glu Leu Ser Leu Leu Gly Lys Ala Leu Thr Arg Asn Val Tyr Gly Met 180 185 190 Ser Asp Ser Asp Lys Leu Tyr Ala Tyr Tyr Gln Gly Cys Ser Glu Gly 195 200 205 Gly Arg Glu Gly Trp Ser Gln Val Gln Arg Phe Gly Asp Glu Trp Asp 210 215 220 Gly Ala Ile Ile Gly Ala Pro Ala Phe Arg Trp Ser Phe Gln Gln Thr 225 230 235 240 Gln His Leu Tyr Ser Asn Ile Val Glu Lys Thr Leu Asp Tyr Tyr Pro 245 250 255 Pro Pro Cys Glu Leu Asp Lys Ile Val Asn Glu Thr Ile Ala Ala Cys 260 265 270 Asp Ala Met Asp Gly Lys Val Asp Trp Val Val Ala Arg Thr Asp Leu 275 280 285 Cys Leu Leu Asp Phe Asp Ile Ser Thr Ile Glu Gly Lys Pro Tyr Ser 290 295 300 Cys Ala Ala Ser Arg Gly Thr Pro Ala Gln Asn Gly Thr Val Ser Ala 305 310 315 320 Lys Gly Ile Glu Val Ala Lys Thr Ile Ile Asn Gly Leu His Asp Ser 325 330 335 Gln Gly Arg Arg Val Tyr Phe Ser Tyr Gln Pro Thr Ala Ala Phe Asp 340 345 350 Asp Ala Glu Thr Gln Tyr Asn Ser Thr Thr Gly Gln Trp Gly Leu Asp 355 360 365 Ile Asp Gln Leu Gly Gly Glu Tyr Ile Ala Leu Leu Val Asp Lys Asn 370 375 380 Gly Thr Thr Leu Asp Ser Leu Asp Gly Ile Thr Tyr Asp Thr Leu Lys 385 390 395 400 Asp Trp Met Ile Ser Gly Leu Gln Glu Tyr Tyr Ser Thr Leu Gln Thr 405 410 415 Thr Trp Pro Asp Leu Thr Pro Phe His Asn Ala Gly Gly Lys Val Ile 420 425 430 His Phe His Gly Asp Ala Asp Phe Ser Ile Pro Thr Ala Ala Ser Ile 435 440 445 Arg Tyr Trp Glu Ser Val Arg Ser Ile Met Tyr Pro Asn Lys Asp Tyr 450 455 460 Asn Ser Ser Ala Glu Ala Leu Asn Glu Trp Tyr Arg Leu Tyr Thr Val 465 470 475 480 Pro Gly Ala Gly His Cys Ala Thr Asn Asp Ala Met Pro Asn Gly Pro 485 490 495 Phe Pro Gln Thr Asn Met Ala Val Met Ile Asp Trp Val Glu Asn Gly 500 505 510 Val Val Pro Thr Thr Leu Asn Ala Thr Val Leu Gln Gly Glu Asn Glu 515 520 525 Gly Gln Asn Gln Gln Leu Cys Ala Trp Pro Leu Arg Pro Leu Trp Thr 530 535 540 Asn Asn Gly Thr Thr Met Glu Cys Val Tyr Asn Gln Arg Ser Ile Asp 545 550 555 560 Ser Trp His Tyr Asp Leu Asp Ala Val Pro Met Pro Val Tyr 565 570 <210> 15 <211> 570 <212> PRT <213> Aspergillus niger FJ0118 <400> 15 Met Ser Lys Phe Leu Leu Trp Thr Val Ser Ala Ala Ala Leu Ala Arg 1 5 10 15 Ala Ala Thr Leu Glu Gln Val Cys Thr Val Ala His Ala Lys Ala Ala 20 25 30 Val Pro Ala Ser Gly Leu Val Gln Gly Val Val Thr Asn Pro Ser Ser 35 40 45 Val Thr Ala Val Pro Val Tyr Asn Glu Ser Thr Ser Gly Ser Asp Tyr 50 55 60 Tyr Pro Ala Gly Thr Tyr Asp Tyr Cys Asn Ile Thr Met Thr Tyr Ser 65 70 75 80 His Ala Gly Lys Asp Asp Ser Val Leu Leu Gln Phe Trp Met Pro Thr 85 90 95 Pro Asp Asp Phe Gln Asn Arg Trp Val Ser Thr Gly Gly Phe Gly Phe 100 105 110 Ala Ile Asn Val Ala Ser Asn Val Pro Ala Gly Leu Pro Tyr Gly Ala 115 120 125 Val Ala Gly Arg Thr Asp Gly Gly Phe Gly Ser Phe Asp Thr Gln Phe 130 135 140 Ser Ser Val Tyr Pro Leu Val Asn Gly Thr Ala Asn Tyr Asp Ala Leu 145 150 155 160 Tyr Met Phe Gly Tyr Gln Ala His His Glu Met Ser Ser Ile Gly Lys 165 170 175 Ala Phe Thr Lys Asn Phe Tyr Ser Met Ser Asp Lys Leu Tyr Ala Tyr 180 185 190 Tyr Gln Gly Cys Ser Glu Gly Gly Arg Glu Gly Trp Ser Gln Val Gln 195 200 205 Arg Phe Pro Glu Glu Trp Asp Gly Ala Val Ile Gly Ala Pro Ala Ile 210 215 220 Arg Tyr Gly Gln Gln Gln Val Asn His Leu Val Pro Gln Val Val Glu 225 230 235 240 Gln Thr Leu Asp Tyr Tyr Pro Glu Asn Cys Glu Phe Ser Glu Met Val 245 250 255 Thr Leu Ile Ile Asp Ala Cys Asp Glu Leu Asp Gly Arg Lys Asp Gly 260 265 270 Val Ile Gly Arg Thr Asp Leu Cys Gln Leu Asn Phe Asp Leu Lys Ser 275 280 285 Thr Ile Gly Lys Pro Tyr Ala Cys Asn Ala Thr Thr Ser Met Gly Val 290 295 300 Thr Thr Pro Ala Gln Asn Gly Thr Ile Thr Ala Glu Gly Val Arg Val 305 310 315 320 Ala Gln Thr Ile Leu Asp Gly Leu Lys Asp Ser Gln Gly Arg Gln Ala 325 330 335 Tyr Phe Trp Tyr Arg Ile Gly Ser Glu Phe Ser Asp Ala Glu Thr Thr 340 345 350 Tyr Asp Ser Thr Thr Gly Thr Tyr Gly Ile Asp Ile Ser Glu Leu Gly 355 360 365 Gly Glu Trp Val Thr His Gly Leu Gln Leu Leu Asp Leu Asp Asn Leu 370 375 380 Ser Thr Leu Asp Asn Val Thr Tyr Asp Thr Leu Arg Asp Trp Met Leu 385 390 395 400 Glu Gly Leu Gln Arg Tyr Glu Asp Val Leu Gln Thr Thr Trp Pro Asp 405 410 415 Leu Ser Arg Phe Gln Ala Ala Gly Gly Lys Val Ile His Tyr His Gly 420 425 430 Glu Ser Asp Asn Ser Ile Pro Pro Ala Ser Ser Val Arg Tyr Phe Glu 435 440 445 Ser Val Arg Asp Thr Met Phe Pro Asn Lys Thr Tyr Asn Ala Ser Val 450 455 460 Glu Ala Leu Asn Glu Phe Tyr Arg Leu Tyr Leu Val Pro Gly Ala Ser 465 470 475 480 His Cys Asn Thr Asn Ser Leu Gln Pro Asn Gly Pro Tyr Pro Gln Asn 485 490 495 Ser Leu Gly Asp Leu Met Asn Trp Val Glu Lys Gly Ile Glu Pro Val 500 505 510 Thr Leu Asn Gly Thr Val Leu Ser Gly Asp Leu Glu Gly Glu Glu Gln 515 520 525 Lys Ile Cys Ser Trp Pro Leu Arg Pro Leu Trp Lys Asn Asn Gly Thr 530 535 540 Glu Met Glu Cys Val Tyr Asp Gln Lys Ser Ile Asp Thr Trp Leu Tyr 545 550 555 560 Asp Leu Asp Ala Tyr Asp Ile Pro Ile Tyr 565 570 <210> 16 <211> 573 <212> PRT <213> Aspergillus oryzae RIB40 <400> 16 Met Arg Ser Ala Leu Asn Ile Leu Ala Gly Ala Ala Thr Leu Thr Val 1 5 10 15 Ala Gln Ala Ala Ser Leu Ser Asp Val Cys Thr Asn Ser His Val Lys 20 25 30 Ser Ala Leu Pro Ser Ile Asp Leu Ile Asn Gly Leu Val Ile Asp Pro 35 40 45 Ser Ser Ile Thr Thr Asn Ala Val Tyr Asn Ala Ser Thr Pro Gly Gly 50 55 60 Asp Tyr Phe Pro Ala Ala Ser Ala Tyr Asp Phe Cys Asn Val Thr Leu 65 70 75 80 Thr Tyr Ala His Pro Gly Arg Asn Asp Arg Val His Leu Lys Leu Trp 85 90 95 Met Pro Ala Pro Asp Gln Phe Gln Asn Arg Trp Leu Ser Thr Gly Gly 100 105 110 Gly Gly Phe Ala Ile Asn His Asp Glu Gln Gln Leu Pro Gly Gly Val 115 120 125 Gln Tyr Gly Ala Ala Ala Gly Ile Thr Asp Gly Gly Phe Gly Ser Phe 130 135 140 Tyr Thr Gln Phe Asp Gln Val Phe Leu Leu Ala Asn Gly Thr Ile Asn 145 150 155 160 Tyr Glu Ala Leu Tyr Met Phe Gly Tyr Gln Ala His His Glu Leu Ser 165 170 175 Val Ile Gly Lys Ala Leu Thr Lys Asn Phe Tyr Gly Thr Gly Asp Ala 180 185 190 Lys Leu Tyr Ala Tyr Trp Gln Gly Cys Ser Glu Gly Gly Arg Glu Gly 195 200 205 Phe Ser Gln Val Gln Arg Phe Gln Glu Phe Asp Gly Ala Val Ile Gly 210 215 220 Ala Pro Ala Leu Arg Tyr Gly Gln Gln Gln Ala Asn His Leu Tyr Gly 225 230 235 240 Asn Leu Val Glu His Thr Leu Lys Tyr Tyr Pro Pro Pro Cys Glu Leu 245 250 255 Glu Lys Ile Val Asn Leu Thr Ile Thr Ala Cys Asp Arg Leu Asp Gly 260 265 270 Arg Ser Asp Gly Val Val Ser Arg Thr Asp Leu Cys Lys Leu His Phe 275 280 285 Asn Ile Asn Ser Thr Ile Gly Ala Pro Tyr Ser Cys Pro Ala Ser Thr 290 295 300 Thr Thr Thr Gly Thr Thr Pro Ala Gln Asn Gly Thr Val Ser Ala Leu 305 310 315 320 Gly Ala Ala Ala Ala Arg Lys Met Leu Asp Gly Leu Arg Thr Leu Asp 325 330 335 Ser Arg Arg Ala Tyr Ile Phe Tyr Gln Pro Ser Ala Thr Phe Asp Asp 340 345 350 Ala Gln Thr Lys Tyr Asn Pro Glu Thr Lys Gln Phe Glu Leu Glu Val 355 360 365 Ser Ala Tyr Ala Ala Glu Trp Ile Pro Arg Phe Leu Gln Leu Gln Asn 370 375 380 Tyr Thr Leu Ser Ser Leu Glu Asn Val Thr Tyr Asp Thr Leu Lys Asp 385 390 395 400 Trp Met Glu Leu Gly Trp Gln Arg Tyr Glu Asp Val Leu Gln Thr Thr 405 410 415 Trp Pro Asp Leu Thr Pro Phe Gln Ser Ala Gly Gly Lys Val Leu His 420 425 430 Tyr His Gly Glu Ser Asp Pro Ser Ile Pro Ala Gly Ser Ser Val His 435 440 445 Tyr His Glu Ser Val Arg Lys Thr Met Tyr Pro Asn Met Ser Phe Asn 450 455 460 Glu Ser Asn Gln Ala Leu Asn Glu Trp Asn Arg Leu Phe Leu Val Pro 465 470 475 480 Gly Ala Ala His Cys Ala Ser Ser Thr Asp Gln Pro Asn Gly Pro Phe 485 490 495 Pro Gln Ala Thr Leu Lys Thr Leu Ile Glu Trp Val Glu Asn Ser Ile 500 505 510 Val Pro Glu Thr Leu Asn Gly Thr Val Leu Asp Gly Asp His Lys Gly 515 520 525 Glu Gln Gln Gln Ile Cys Ala Trp Pro Leu Arg Pro Leu Trp Thr Glu 530 535 540 Asn Gly Thr Val Met Asn Cys Val Tyr Asp Gln Ala Ser Leu Asp Thr 545 550 555 560 Trp Asp Tyr Glu Phe Asp Ala Tyr Arg Ile Pro Leu Tyr 565 570 <210> 17 <211> 588 <212> PRT <213> Aspergillus fumigatus Af293 <400> 17 Met Arg Ile Ser Tyr Gly Ser Ala Val Ala Ala Leu Ala Ala Ala Ala 1 5 10 15 Asn Ala Ala Ser Leu Ala Asp Val Cys Thr Ile Ser His Val Gln Ser 20 25 30 Val Leu Pro Ser Asn Gly Thr Leu Leu Gly Ile Asn Val Ile Pro Ser 35 40 45 Ala Val Thr Ala Ser Ala Val Tyr Asn Ser Thr Ser Ser Gly Gly Met 50 55 60 Gly Gly Met Gly Gly Ser Asn Ser Ala Asn Tyr Pro Tyr Cys Asn Val 65 70 75 80 Thr Val Thr Tyr Thr His Pro Gly Lys Gly Asp Lys Val Val Val Lys 85 90 95 Tyr Ala Phe Pro Gln Pro Ser Asp Phe Lys Asn Arg Phe Tyr Val Ala 100 105 110 Gly Gly Gly Gly Tyr Ser Leu Ser Ser Asp Ala Thr Gly Gly Leu Glu 115 120 125 Tyr Gly Ala Ala Ser Gly Ala Thr Asp Ala Gly Tyr Asp Ala Phe Ser 130 135 140 Tyr Ser Tyr Asp Glu Val Val Leu Tyr Gly Asn Gly Ser Ile Asn Trp 145 150 155 160 Asp Ala Thr Tyr Met Phe Ala Tyr Gln Ala Leu Gly Glu Met Thr Thr 165 170 175 Leu Gly Lys Thr Leu Thr Arg Asn Phe Tyr Gly Leu Ser Ser Asp Ala 180 185 190 Lys Ile Tyr Thr Tyr Tyr Glu Gly Cys Ser Asp Gly Gly Arg Glu Gly 195 200 205 Met Ser Gln Val Gln Arg Tyr Gly Asp Leu Tyr Asp Gly Ala Ile Thr 210 215 220 Gly Ala Pro Ala Phe Arg Tyr Ala Gln Gln Gln Val His His Val Phe 225 230 235 240 Ser Ser Val Val Glu Lys Thr Leu Asp Tyr Tyr Pro Pro Pro Cys Glu 245 250 255 Leu Ala Lys Ile Val Asn Ala Thr Ile Glu Ala Cys Asp Pro Leu Asp 260 265 270 Gly Arg Thr Asp Gly Val Val Ser Arg Thr Asp Leu Cys Lys Leu His 275 280 285 Phe Asp Leu Ser Lys Ile Ile Gly Glu Pro Tyr Tyr Cys Ala Ala Lys 290 295 300 Thr Ser Thr Ser Leu Gly Phe Gly Phe Ser Lys Arg Gln Ala Ala Gly 305 310 315 320 Ser Thr Thr Ser Tyr Gln Pro Ala Gln Asn Gly Thr Val Thr Lys Glu 325 330 335 Gly Val Ala Val Ala Lys Ala Ile Tyr Asp Gly Leu His Asn Thr Gln 340 345 350 Gly Gln Arg Ala Tyr Leu Ser Trp Gln Ile Ala Ser Glu Phe Ser Asp 355 360 365 Ala Thr Thr Glu Trp Asn Asn Asp Thr Gly Ser Trp Glu Leu Ser Ile 370 375 380 Pro Ser Thr Gly Gly Glu Phe Val Thr Lys Phe Val Gln Leu Leu Asp 385 390 395 400 Leu Asp Asn Leu Ser Thr Leu Asp Asn Val Thr Tyr Asp Thr Leu Val 405 410 415 Glu Trp Met Asn Thr Ala Met Val Arg Tyr Met Asp Ser Leu Gln Thr 420 425 430 Thr Val Pro Asp Leu Thr Thr Phe Lys Ser Ser Gly Gly Lys Leu Leu 435 440 445 His Tyr His Gly Glu Ser Asp Pro Ser Ile Pro Ala Ala Ser Ser Val 450 455 460 His Tyr Trp Gln Ser Val Arg Ser Ile Met Tyr Pro Gly Val Ser Ala 465 470 475 480 Ala Lys Ser Leu Lys Glu Leu Gln Glu Trp Tyr Gln Phe Tyr Leu Ile 485 490 495 Pro Gly Ala Ala His Cys Gly Ala Asn Ser Leu Gln Pro Gly Pro Tyr 500 505 510 Pro Gln Asn Asn Met Asp Ile Met Ile Asp Trp Val Glu Asn Gly Val 515 520 525 Gln Pro Ser Arg Leu Asn Thr Thr Val Ser Ser Gly Asp Tyr Ala Gly 530 535 540 Glu Thr Gln Met Leu Cys Gln Trp Pro Thr Arg Pro Leu Trp Lys Asp 545 550 555 560 Asn Ser Thr Phe Asp Cys Val Asn Asp Glu Lys Ser Ile Glu Ser Trp 565 570 575 Thr Tyr Thr Phe Pro Ala Phe Lys Val Pro Val Tyr 580 585 <210> 18 <211> 580 <212> PRT <213> Penicillium rubens Wisconsin 54‑1255 <400> 18 Met Arg Leu Ser Trp Gly Ala Ser Ala Ala Ala Leu Ala Ala Ser Ala 1 5 10 15 Ser Ala Ala Ser Leu Ala Asp Val Cys Thr Val Ser Asn Val Gln Ser 20 25 30 Ala Leu Pro Ser Asn Gly Thr Leu Leu Gly Ile Asn Met Ile Pro Ser 35 40 45 Thr Val Thr Ala Ser Pro Val Tyr Asn Ala Ser Ala Gly Met Gly Ser 50 55 60 Thr Glu Thr Tyr Thr Tyr Cys Asn Val Thr Val Thr Tyr Glu His Thr 65 70 75 80 Gly Lys Gly Asp Ser Val Val Ile Lys Tyr Ala Phe Pro Lys Pro Ser 85 90 95 Asp Phe Lys Lys Arg Phe Tyr Val Ala Gly Gly Gly Gly Phe Ser Leu 100 105 110 Ser Ser Asp Ala Thr Gly Gly Leu Glu Tyr Gly Ala Val Gly Gly Ala 115 120 125 Thr Ser Ala Gly Tyr Asp Ala Phe Asn Asn Ser Tyr Asp Glu Val Val 130 135 140 Leu Tyr Gly Asn Gly Thr Ile Asn Trp Asp Ala Thr Tyr Met Phe Ala 145 150 155 160 Tyr Gln Ala Leu Gly Glu Met Thr Lys Ile Gly Lys Val Leu Thr Lys 165 170 175 Gly Phe Tyr Gly Met Ala Ser Ser Ala Lys Val Tyr Thr Tyr Tyr Glu 180 185 190 Gly Cys Ser Asp Gly Gly Arg Glu Gly Met Ser Gln Ile Gln Arg Tyr 195 200 205 Gly Glu Glu Tyr Asp Gly Ala Ile Thr Gly Ala Pro Ala Phe Arg Phe 210 215 220 Ala Gln Gln Gln Val His His Val Phe Ser Ala Ala Ala Glu Gln Thr 225 230 235 240 Leu Asp Tyr Tyr Pro Pro Pro Cys Glu Leu Ala Lys Ile Val Asn Ala 245 250 255 Thr Ile Ala Ala Cys Asp Pro Leu Asp Gly Arg Thr Asp Gly Val Ile 260 265 270 Ser Arg Thr Asp Leu Cys Lys Leu Lys Phe Asn Leu Thr Ser Ile Ile 275 280 285 Gly Glu Glu Tyr Tyr Cys Ala Ala Ala Thr Ser Thr Ser Leu Gly Phe 290 295 300 Gly Phe Ser Lys Arg Ala Asp Gly Ser Ser Thr Ser Thr Thr Pro Glu 305 310 315 320 Gln Ser Gly Lys Val Thr Ala Lys Gly Val Gln Val Ala Gln Ala Ile 325 330 335 Tyr Asp Gly Leu His Asn Ser Lys Gly Glu Arg Ala Tyr Leu Ser Trp 340 345 350 Gln Val Gly Ser Glu Leu Ser Asp Gly Asp Thr Thr Trp Asn Asn Ala 355 360 365 Thr Ser Lys Trp Glu Met Ser Ile Pro Ser Thr Gly Gly Glu Tyr Val 370 375 380 Thr Lys Phe Ile Gln Leu Leu Asp Leu Asp Asn Leu Ser Asp Leu Asp 385 390 395 400 Asn Val Thr Tyr Asp Thr Leu Val Asp Trp Met Asn Thr Gly Met Val 405 410 415 Arg Tyr Met Asp Ser Leu Gln Thr Thr Leu Pro Asp Leu Thr Pro Phe 420 425 430 Gln Ser Ser Gly Gly Lys Leu Leu His Tyr His Gly Glu Ser Asp Pro 435 440 445 Ser Ile Pro Ser Ala Ser Ser Val His Tyr Trp Gln Ser Val Arg Ser 450 455 460 Ile Met Tyr Pro His Leu Ser Ser Gln Asp Ser Leu Lys Glu Leu Ala 465 470 475 480 Asp Trp Tyr Gln Phe Tyr Leu Val Pro Gly Ala Ala His Cys Gly Thr 485 490 495 Asn Lys Leu Gln Pro Gly Pro Tyr Pro Glu Asn Asn Met Gln Thr Met 500 505 510 Ile Asp Trp Val Glu Asn Asp Val Lys Pro Ser Arg Leu Asn Ala Thr 515 520 525 Val Ser Ser Gly Thr Tyr Glu Gly Glu Thr Gln Met Leu Cys Gln Trp 530 535 540 Pro Thr Arg Pro Leu Trp Lys Ser Asn Ser Thr Phe Gln Cys Val Asp 545 550 555 560 Asp Lys Ala Ser Ile Glu Ser Trp Thr Tyr Ser Phe Pro Ala Phe Lys 565 570 575 Val Pro Val Tyr 580 <210> 19 <211> 23 <212> DNA <213> Synthetic <400> 19 aagcttggcg taatcatggt cat 23 <210> 20 <211> 51 <212> DNA <213> Synthetic <400> 20 cggacccctc cgccaatggc ggcgcgccaa gcttgcatgc aggcctctgc a 51 <210> 21 <211> 34 <212> DNA <213> Synthetic <400> 21 tgtcattcaa tgcatctgga aacgcaaccc tgaa 34 <210> 22 <211> 40 <212> DNA <213> Synthetic <400> 22 accatgatta cgccaagctt ctagatctac gccaggaccg 40 <210> 23 <211> 20 <212> DNA <213> Synthetic <400> 23 gccattggcg gaggggtccg 20 <210> 24 <211> 40 <212> DNA <213> Synthetic <400> 24 gggttgcgtt tccagatgca ttgaatgaca gtgatatcag 40 <210> 25 <211> 38 <212> DNA <213> Synthetic <400> 25 agcatcatta cacctcagca atgcgttcac ccgcctgg 38 <210> 26 <211> 43 <212> DNA <213> Artificial synthesis <400> 26 gtcaccctct agatctcgag ttagtacaca ggcataggaa ccg 43 <210> 27 <211> 38 <212> DNA <213> Artificial synthesis <400> 27 agcatcatta cacctcagca atgcgcaagtccactcgc 38 <210> 28 <211> 44 <212> DNA <213> Artificial synthesis <400> 28 gtcaccctct agatctcgag ttagaaaacg ggcatcttga aagc 44 <210> 29 <211> 38 <212> DNA <213> Artificial synthesis <400> 29 agcatcatta cacctcagca atgagaaccgccctcggg 38 <210> 30 <211> 40 <212> DNA <213> Artificial synthesis <400> 30 gtcaccctct agatctcgag ctagtacagg ggcagcttgt 40 <210> 31 <211> 38 <212> DNA <213> Artificial synthesis <400> 31 agcatcatta cacctcagca atgcgctccc ccgcctgg 38 <210> 32 <211> 42 <212> DNA <213> Artificial synthesis <400> 32 gtcaccctct agatctcgag ttagtagacg ggcatgggga cg 42 <210> 33 <211> 42 <212> DNA <213> Artificial synthesis <400> 33 agcatcatta cacctcagca atgtccaagt tcctcctttg ga 42 <210> 34 <211> 45 <212> DNA <213> Artificial synthesis <400> 34 gtcaccctct agatctcgag ctaatagatc ggaatgtcat acgca 45 <210> 35 <211> 38 <212> DNA <213> Artificial synthesis <400> 35 agcatcatta cacctcagca atgcgctccgccctgaac 38 <210> 36 <211> 40 <212> DNA <213> Artificial synthesis <400> 36 gtcaccctct agatctcgag ttagtacagg gggatgcggt 40 <210> 37 <211> 38 <212> DNA <213> Artificial synthesis <400> 37 agcatcatta cacctcagca atgcgcatct cctacggt 38 <210> 38 <211> 43 <212> DNA <213> Artificial synthesis <400> 38 gtcaccctct agatctcgag ttagtagacg gggaccttga agg 43 <210> 39 <211> 38 <212> DNA <213> Artificial synthesis <400> 39 agcatcatta cacctcagca atgcgcctgt cctggggt 38 <210> 40 <211> 43 <212> DNA <213> Artificial synthesis <400> 40 gtcaccctct agatctcgag ttagtagacg gggaccttga agg 43

Claims

1. A method for preparing gallic acid using tanninase, characterized in that, The method includes: 1) Prepare a tannic acid solution with a concentration of 25%-35% w / v, and adjust the pH of the tannic acid solution to 4.0-5.0; 2) Take the tannic acid solution obtained in step 1) and add it to the reaction vessel. Add tanninase at a dosage of 36-54 U / g substrate to hydrolyze and generate gallic acid. The amino acid sequence of the tanninase is shown in SEQ ID NO:

13.

2. The method as described in claim 1, characterized in that, The tanninase was derived from Aspergillus niger ATCC 13496.

3. The method as described in claim 1, characterized in that, The optimal reaction temperature for the tanninase is 65-75℃, and the optimal reaction pH is 4.0-5.

0.

4. The method as described in claim 1, characterized in that, The polynucleotide sequence of the tanninase consists of the polynucleotide sequence of SEQ ID NO:

5.

5. The method as described in claim 1, characterized in that, The concentration of the tannic acid solution in step 1) is 30%.

6. The method as described in claim 1, characterized in that, In step 1), adjust the pH of the tannic acid solution to 4.

0.

7. The method as described in claim 1, characterized in that, In step 2), the amount of tanninase added is 36 U / g substrate.

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